Epigenetic initiation of the TH17 differentiation program is promoted by Cxxc finger protein 1 – Science Advances

INTRODUCTION

T helper 17 (TH17) cells, a subset of CD4+ T cells, are characterized by the secretion of interleukin-17A (IL-17A), IL-17F, IL-21, IL-22, and the transcription factors (TFs) retinoic acid receptorrelated orphan receptor t (RORt) and ROR (13). RORt and ROR are critical drivers of autoimmune tissue inflammation in human autoimmune diseases (multiple sclerosis), mouse models [experimental autoimmune encephalomyelitis (EAE)], and other autoimmune conditions (4). TH17 cells also maintain mucosal tissue homeostasis and contribute to the host defense against bacterial and fungal infections (2, 5). The activation of naive TH cells in the presence of transforming growth factor1 (TGF-1) and IL-6 leads to the development of TH17 cells (6). Other cytokines such as IL-21, IL-1, and IL-23 are crucial for the expansion, stability, and functional maturation of TH17 cells (4, 6, 7). RORt and ROR are the master regulators of TH17 cells, and other TFs, c-Maf, IRF4 (interferon regulatory factor 4), BATF (basic leucine zipper transcription factor, ATF like), and IkB, are required for the induction of RORt, IL-17, IL-21, and IL-22 in vivo and in vitro (8). IL-6, IL-21, and IL-23 can all activate signal transducers and activators of transcription 3 (STAT3), and the activation of STAT3 is crucial for their effects on TH17 cell differentiation (1). The impairment of STAT3 skews TH17 differentiation toward anti-inflammatory Treg cells, and TAZ [transcriptional coactivator with postsynaptic density 65discs largezonula occludens 1binding (PDZ) motif]/TEAD1 (TEA-ATTS DNAbinding domain 1) regulates reciprocal RORt/Foxp3 expression downstream of STAT3 (9).

Epigenetic mechanisms such as DNA methylation and histone acetylation/methylation have been reported as key players in different T cell subsets (10). In TH1 cells, permissive histone modifications and DNA demethylation in interferon- (IFN-) and Tbx21 could promote IFN- expression and help TH1 lineage differentiation and stability (10, 11). The TH2-specific genes (IL4, IL5, and IL13) are repressed by suppressive histone modifications and DNA methylation in the process of TH1 cell differentiation (12). However, TH2 cells contain activated histone modifications and undergo DNA demethylation at the TH2-specific gene cluster, as well as contain repressive histone modifications and undergo DNA methylation in TH1-specific genes (IFN- and Tbx21) (13). Foxp3, the master TF for Treg cells, was marked with permissive H3K4me3 modification and underwent DNA demethylation at its gene locus, which contributes to the determination and commitment of the Treg cell lineage (14). Similarly, genome-wide epigenetic analysis of TH17 cells uncovered the enrichment of permissive histone modifications such as H3K4me3 and DNA demethylation in the promoters of cytokine and lineage-specific genes such as IL17a, IL17f, IL21, IL23r, and Rorc (15).

CXXC finger protein 1 (Cxxc1), defined as an epigenetic regulator, binds to DNA using its CXXC finger domain and recruits SETD1 to most CpG islands (CGIs) through its Setd1-interacting domain (16, 17). Clouaire et al. (16) showed that Cxxc1 is required for H3K4me3 modification in embryonic stem cell, and Thomson et al. (18) found a concordance of Cxxc1 binding with H3K4me3 and nonmethylated CGIs in mouse brain. Our recent studies also demonstrated that Cxxc1-dependent H3K4me3 plays a critical role in thymocyte development, phagocytosis, and the bactericidal activity of macrophages (19, 20). However, the role of Cxxc1-mediated H3K4 trimethylation and DNA methylation in TH cells remains unclear.

To explore the role of Cxxc1 in TH cell differentiation, we took advantage of T cellspecific Cxxc1 knockout (KO) mice. Here, we demonstrate that Cxxc1-deficient TH17 cells exhibited impaired differentiation and stability, which led to susceptibility to bacterial infection and protected against EAE in vivo. Fluorescence-activated cell sorting (FACS) and RNA sequencing (RNA-seq) analyses identified that Cxxc1 deficiency skews TH17 differentiation toward anti-inflammatory Treg cells both in vivo and in vitro.

Via chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) analysis, we revealed genome-wide Cxxc1-binding sites in TH17 cells and H3K4me3 modification changes in Cxxc1-deficient TH17 cells compared with the wild-type (WT) control. We found that Cxxc1 bound to IL-6R and other TFs (IRF4 and BATF) by maintaining the appropriate H3K4me3 modification of their promoter regions. Moreover, IL-6R, a direct target of Cxxc1, could partially rescue the differentiation and stability defects seen in Cxxc1-deficient TH17 cells. Cxxc1 thus reciprocally regulates the balance between TH17 and Treg cells by regulating IL-6/STAT3 signaling. This suggests that the Cxxc1-mediated epigenetic program is required for T cell differentiation and TH17-related autoimmune diseases.

To investigate the potential role of Cxxc1 in TH cell function, we generated conditional KO mice by crossing mice with loxP-flanked Cxxc1 alleles to mice with transgenic Cre driven by the distal Lck promoter (dLck-Cre mice), which mediated the deletion of genes on peripheral CD4+ and CD8+ T cells. dLckcreCxxc1fl/fl mice developed normally with no obvious difference in their T cell development in the thymus (fig. S1A). Further analysis of the peripheral T cells showed a decrease in CD8+ T cell numbers, especially in its effector/memory population, while CD4+ T cell numbers and phenotypes were normal (fig. S1, B to D). CellTrace dilution showed little impairment of the CD8+ T cell proliferation capacity of Cxxc1-deficient T cells cultured under T cell receptor (TCR) stimulation, while CD4+ T cells showed no influence of TCR stimulation (fig. S1E).

We then isolated naive CD4+ T cells from dLckcreCxxc1wt/wt and dLckcreCxxc1fl/fl mice and conducted T cell differentiation in vitro. We found that Cxxc1 ablation led to severely defective TH17 differentiation characterized by reduced IL-17A and IL-17F in two TH17 cellpolarizing conditions: (i) TGF-1 and IL-6 and (ii) IL-1, IL-6, and IL-23, while Foxp3 expression increased (Fig. 1, A and B). However, Cxxc1-deficient T cells exhibited no obvious difference in TH1 or TH2 differentiation and a moderate increase in induced T-regulatory cell (iTreg) differentiation (fig. S2, A to C), while the expression levels of Cxxc1 protein were consistent among these T helper subsets and in different stimulation time of TH17 cells (fig. S2, G and H). Furthermore, we also used CD4+ T cells from CreERT2+ Cxxc1fl/fl mice to test Cxxc1 function in an in vitro culture system and found a similar requirement for Cxxc1 in TH17 differentiation in the presence or absence of 4-hydroxytamoxifen treatment (which initiated the access of Cre recombinase to the nucleus and led to the deletion of Cxxc1 in vitro, as shown in fig. S2I) (Fig. 1, C and D) for 4 days, and we observed no obvious difference in TH1, TH2, or iTreg differentiation (fig. S2, D to F).

(A and B) Naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from dLckcreCxxc1fl/fl or WT mice were differentiated into TH17 cells with (A) IL-6 and TGF-1 or (B) IL-1, IL-6, and IL-23 for 96 hours and then restimulated for intracellular cytokine staining. One of five to seven experiments is shown. (C and D) Naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from ERT2creCxxc1fl/fl mice were differentiated into TH17 cells with (C) IL-6 and TGF-1 or (D) IL-1, IL-6, and IL-23 for 96 hours in the presence or absence of 4-OHT (4-Hydroxytamoxifen) and then restimulated for intracellular cytokine staining. One of five experiments is shown. (E and F) Naive CD4+ T cells from RORtcreCxxc1fl/fl or WT mice were differentiated into TH17 cells with (E) IL-6 and TGF-1 or (F) IL-1, IL-6, and IL-23 for 96 hours and then restimulated for intracellular cytokine staining. One of seven experiments is shown. (G) Intracellular staining of IL-17A in lipoprotein lipase (LPL) CD4+ T cells in the small intestines of RORtcreCxxc1fl/fl and WT mice. One of four experiments is shown. Error bars show the means SD. **P 0.01, ***P 0.001, ****P 0.0001 using the Students t test.

Next, we tested the function of Cxxc1 in TH17 cells by generating RORtcre Cxxc1fl/fl mice. Similar to the dLck-mediated deletion of Cxxc1, RORtcre Cxxc1fl/fl mice developed normally in terms of T cell development in the thymus (fig. S3A) and exhibited decreased CD8+ T cell numbers in the periphery (fig. S3, B and D), while CD4+ T cell numbers were normal (fig. S3, B and C). Then, we isolated naive CD4+ T cells from RORtcreCxxc1wt/wt and RORtcre Cxxc1fl/fl mice and conducted TH17 cell differentiation (TGF-1 and IL-6 or IL-1, IL-6, and IL-23) in vitro. Compared with the dLck-mediated deletion of Cxxc1, T cells in RORtcreCxxc1fl/fl mice differentiated under TH17 cellpolarizing conditions showed much less IL-17A and IL-17F production than Cxxc1-competent RORtcreCxxc1wt/wt cells and markedly increased Foxp3 production (Fig. 1, E and F). This is due to the sufficient deletion of Cxxc1 protein in TH17 cells in the RORtcre system compared with dLckcre system (fig. S2I). Under steady-state conditions, TH17 cells are preferentially located in the lamina propria (LP) of the small intestine (3). We also showed that CD4+ T cells in the LP of RORtcre Cxxc1fl/fl mice showed notably reduced IL-17 production in vivo (Fig. 1G), although we found a little increased frequency of Treg cells in LP and normal frequency of Treg cells in lymph nodes and spleen (fig. S3E).

To characterize IL-17A expression more specifically, we introduced IL-17AeGFP reporter mice into dLckcreCxxc1fl/fl and RORtcre Cxxc1fl/fl backgrounds separately, and the results also confirm defective TH17 differentiation in Cxxc1-deficient cells with enhanced green fluorescent protein expression (fig. S3, F and G). These results indicate that Cxxc1 is essential for TH17 cell differentiation in vitro.

To further evaluate whether defects in TH17 cells caused by Cxxc1 deficiency affect the development of TH17-dependent inflammatory diseases in vivo, we used a TH17-dependent autoimmune disease model, EAE, that mimics the human neuroinflammatory disease multiple sclerosis.

As we found decreased CD8+ T cell numbers in the periphery (fig. S3B), to exclude the effect of CD8+ T cell (21), we sorted naive CD4+ T cells (CD4+CD44loCD62L+) from RORtcreCxxc1fl/fl and RORtcreCxxc1wt/wt mice, transferred these cells into Rag1/ mice, and then monitored them for the induction of EAE. In agreement with the results showing the in vitro defects, transfer of RORtcreCxxc1fl/fl-naive CD4+T cells alleviated EAE, and significantly less mononuclear cell infiltration and demyelination of the spinal cord were observed (Fig. 2, A and B). Within the central nervous system (CNS)infiltrating draining lymph nodes and spleen CD4+ T cell population, the production of IL-17A+ T helper cells was reduced in the hosts that had received RORtcreCxxc1fl/fl cells, whereas the number of Treg cells increased in the CNS-infiltrating CD4+ T cells (Fig. 2, C and D). Cxxc1-deficient splenocytes isolated from myelin oligodendrocyte glycoprotein (MOG)immunized mice showed impaired production of IL-17 but normal production of IFN- upon restimulation with the MOG peptide compared with Cxxc1-sufficient splenocytes (Fig. 2E). We found similar results when the EAE model was induced in the Rag1/ hosts with dLckcreCxxc1fl/fl cells compared with dLckcreCxxc1wt/wt cells (fig. S4, A to C). We can find that Cxxc1 deficiency alleviated symptoms of autoimmunity in dLckcreCxxc1fl/fl (fig. S4, D to G) and RORtcreCxxc1fl/fl (fig. S4, H to K) mice compared with appropriate control mice as well. These data suggested that the Cxxc1-deficient cells were less susceptible to the EAE disease model than Cxxc1-sufficient cells, which was in accordance with the in vitro results showing defective IL-17 expression.

(A) Mean clinical scores for EAE in Rag1/ recipients of RORtcreCxxc1fl/fl (n = 13) or WT (n = 11) naive CD4+ T cells after being immunized with MOG3555, complete Freunds adjuvant (CFA), and pertussis toxin. Data are summed from three independent experiments. (B) Representative histology of the spinal cord of Rag1/ mice after EAE induction (day 25). Hematoxylin and eosin (H&E) staining (left), Luxol fast blue (F&B) staining (right). (C) On day 20 after the induction of EAE in Rag1/ hosts, CD4+ T cells were analyzed from leukocytes isolated from the CNS, draining lymph nodes (dLNs), and spleen and further analyzed for the frequency of IL-17A+ and IFN-+ T cells (left). Summary of CNS IL-17A+CD4+ and IFN-+CD4+ T cells in Rag1/ hosts (right). One representative of three experiments is depicted. (D) The frequency of Foxp3+ cells from CNS-infiltrating lymphocytes in Rag1/ EAE mice was determined at day 20 after immunization (top). Summary of CNS Foxp3+CD4+ cells in Rag1/ hosts (bottom). One representative of three experiments is depicted. (E) Splenocytes were rechallenged with the MOG peptide (0, 5, and 25 g/ml) for 3 days, and then, cytokine production was measured by enzyme-linked immunosorbent assay (ELISA). (F) Body weight changes of Rag1/ recipients of naive CD4+ T cells from RORtcreCxxc1fl/fl (n = 12) or WT (n = 11) mice after oral inoculation with C. rodentium at the indicated time points. Data are summed from three independent experiments. (G) Colon length for Rag1/ recipients of naive CD4+ T cells from RORtcreCxxc1fl/fl or WT mice after oral inoculation with C. rodentium at day 7. Summary of colon lengths in Rag1/ hosts (right). (H) Histological analysis of representative colons from Rag1/ hosts 7 days after inoculation. (Photo credit: Feng Lin, Institute of Immunology, Zhejiang University School of Medicine). (I) C. rodentium colony-forming units (CFUs) in the colon 7 days after inoculation. Data are summed from three independent experiments. (J) FACS analysis of IL-22 expression from isolated LPLs in Rag1/ hosts at day 7 after inoculation. One of five experiments is shown. Error bars show the means SD. *P 0.05, **P 0.01, ***P 0.001 using the Students t test.

IL-22 is produced by leukocytes, particularly TH17 cells, and has a crucial role in host defense against bacterial infections. Ouyang and his team (5) found that IL-22 has a crucial role in the early phase of host defense against Citrobacter rodentium. We sorted naive CD4+ T cells from RORtcreCxxc1fl/fl and RORtcreCxxc1wt/wt mice, transferred these cells into Rag1/ mice, and then inoculated them with C. rodentium. On day 7 after inoculation, mice that had undergone transfer of RORtcreCxxc1fl/fl-naive CD4+ T cells developed a more aggravated infection than mice that had undergone transfer of RORtcreCxxc1wt/wt-naive CD4+ T cells. The mice showed a significantly greater loss of body weight and shorter colon length than the WT mice (Fig. 2, F and G). Histological analysis of colons from C. rodentiuminfected RORtcreCxxc1fl/fl mice showed increased mucosal hyperplasia and submucosal inflammation compared to the RORtcreCxxc1wt/wt mice, suggesting that Cxxc1 deficiency leads to compromised epithelial barrier function (Fig. 2H). In addition, we found that the bacterial burdens in the feces of RORtcreCxxc1fl/fl mice were increased compared to those in the feces of RORtcreCxxc1wt/wt mice (Fig. 2I). Cytokine analysis showed reduced IL-22 production in CD4+ T cells and group 3 innate lymphoid cell (ILC3) from the LP of hosts with Cxxc1-deficient cells transfer (Fig. 2J and fig. S5A), and ILC3s IL-22 production impairment is possibly due to increased Treg cells (fig. S5B) (22). We found that IL-17 production was unaffected, which imply that cytokines other than IL-6 may regulate IL-17 in this model, and we found normal STAT3 phosphorylation by IL-21 stimulation of RORtcreCxxc1fl/fl cells (fig. S5, C and D). In addition, we found similar results when we performed C. rodentium model in RORtcreCxxc1fl/fl mice (fig. S5, E to H). These findings further support the conclusion that Cxxc1 contributes to TH17 differentiation and function in vivo.

To further analyze the genes regulated by Cxxc1, we performed RNA-seq analysis of RORtcreCxxc1fl/fl and RORtcreCxxc1wt/wt TH17 cells generated in vitro in the presence of TGF- and IL-6 for 72 hours. The RNA-seq results showed that Cxxc1-deficient TH17 cells almost completely lost their features and exhibited a Treg celllike expression profile. Key TH17 cellrelated cytokines and transcripts (e.g., Il17a, Il17f, Il21, Il22, Il6ra, Irf4, Cxcr4, Maf, and Satb1) were significantly down-regulated in RORtcreCxxc1fl/fl TH17 cells, and the master TFs Rorc and Rora also showed a decrease in expression (Fig. 3, A and B). Key Treg cellrelated transcripts (e.g., Foxp3, Ccl3, Itgae, Gpr83, Mgat5, Ikzf2, Ikzf4, Tigit, and Tnfsf10) were significantly up-regulated in RORtcreCxxc1fl/fl TH17 cells (Fig. 3, A and B). Using a fold change of 2 and P < 0.05 to threshold parameters, we found that 782 genes were down-regulated by Cxxc1 knockdown in Cxxc1-defcient cells, and 1411 genes were up-regulated. By pathway analysis, we found the significant enrichment of many genes associated with the inflammatory response and immune-related signaling pathways (e.g., the Janus kinaseSTAT cascade and tyrosine phosphorylation of STAT3 protein) (Fig. 3C). We then measured the expression of TH17 and Treg signature genes, including Il17a, Il17f, Il21, Il22, Rorc, Il6ra, Foxp3, Mgat5, Itgae, Gpr83, Tgfbr1, and Ccl3, from Cxxc1-deficient cells and Cxxc1-sufficient cells by quantitative polymerase chain reaction (qPCR) and obtained the same results as those from 72-hour RNA-seq analysis (Fig. 3D). We also verified these genes in dLCK-Creinduced Cxxc1-deficient TH17 cells by qPCR and got the same results (fig. S5I), which indicates that Cxxc1-deficient TH17 cells exhibit a Treg celllike expression profile.

(A) Naive CD4+ T cells (CD4+CD25CD62LhiCD44lo) from WT and RORtcreCxxc1fl/fl mice were differentiated in the presence of TGF-1 and IL-6 (TH17) for 72 hours, and the total RNA from the cells was analyzed by RNA-seq [STAR (structured transparent accessible reporting) method]. Scatter diagram showing changes in gene expression in WT and Cxxc1-deficient TH17 cells. Down-regulated genes are indicated in blue; up-regulated genes are indicated in red. (B) Heatmap of the fold change (log2) for differentially expressed genes (false discovery rate < 0.05 in TH17 cells is shown). (C) Pathway analysis of the down-regulated genes (left) and up-regulated genes (right). (D) The expression of the selected transcripts was quantified in TH17 cell samples differentiated from naive CD4+ T cells with TGF-1 and IL-6 for 72 hours by real-time qPCR. One of five experiments is shown. Error bars show the means SD. *P 0.05, **P 0.01, ***P 0.001 using the Students t test.

Cxxc1 has two main functional domains. One domain is the N-terminal domain, which interacts with unmethylated CpG DNA to mediate its interaction with DNA methyltransferases 1 (DNMT1) and stabilizes the DNMT1 protein to regulate DNA methylation (17, 18, 23). The other is the C-terminal domain, which interacts with the Setd1 H3K4 methyltransferase complex through the Smad interaction domain to regulate histone methylation. An N-terminal fragment of Cxxc1 (residues 1 to 367) and Cxxc1 containing the point mutation C375A retain their interaction with DNMT1, but the Setd1-interacting activity of Cxxc1 is interrupted. While the C-terminal fragment of Cxxc1 (residues 361 to 656) and Cxxc1 containing the point mutation C169A still have Setd1-interacting activity and can methylate H3K4, the DNA binding activity of Cxxc1 is interrupted. To explore which functional domains within Cxxc1 were necessary for its role in TH17 differentiation, we used an in vitro TH17 differentiation system, and different vectors expressing mutated Cxxc1 proteins were overexpressed in Cxxc1-deficient TH17 cells. The overexpression of full-length Cxxc1 notably rescued the TH17 differentiation defects seen in Cxxc1-deficient cells (fig. S6A). The overexpression of the two Cxxc1 fragments (residues 1 to 367 and residues 361 to 656) and Cxxc1 containing two point mutations (C169A and C375A) showed that the C-terminal domain of Cxxc1 (residues 361 to 656, C169A) rather than the N-terminal domain of Cxxc1 (residues 1 to 367, C375A) was able to rescue TH17 differentiation in Cxxc1-deficient TH17 cells (fig. S6, A and B). Similarly, the overexpression of the C-terminal domain of Cxxc1 reduced Foxp3 expression in Cxxc1-deficient TH17 cells, but the overexpression of the N-terminal domain of Cxxc1 did not (fig. S6C). We also checked gene expression changes by real-time PCR, such as TH17 and Treg signature genes, including Il17a, Il17f, Il21, Il22, Rorc, Il6ra, Foxp3, Mgat5, Itgae, Gpr83, Tgfbr1, and Ccl3, further elucidating the role of two CXXC1 functional domains (fig. S6D). These data showed that the Setd1-interacting domain but not the DNA binding domain in Cxxc1 is crucial for TH17 differentiation, indicating that Cxxc1 may function through regulating H3K4me3 rather than through DNA methylation in TH17 cells.

To investigate the direct targets of Cxxc1 in TH17 cells, we performed ChIP-seq to map genome-wide Cxxc1-binding sites in WT TH17 cells, as well as in Cxxc1-deficient TH17 cells serving as a negative control. Compared with Cxxc1-binding sites in the mouse genome, an obvious enrichment of Cxxc1-binding sites was found in gene promoters [5-kb upstream and downstream of the transcription start site (TSS), 25.56% of Cxxc1-binding sites versus 2% of the mouse genome], exons (3.52% versus 2%), introns (32.67% verus 20%), and intergenic regions (38.25% versus 76%) (Fig. 4A). This represented a relatively high degree of enrichment at promoter regions compared with the distribution of Cxxc1-binding sites in the mouse genome. Analysis of the average binding location also showed that Cxxc1 showed high binding activity at TSS (Fig. 4B).

(A) Naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from WT and RORtcreCxxc1fl/fl mice were differentiated in the presence of TGF-1 and IL-6 (TH17) for 24 hours, and ChIP-seq analysis was conducted to map genome-wide Cxxc1-binding sites in WT TH17 cells. Distribution of the genetic features across the whole mouse genome (mm10) (left) and the distribution of Cxxc1-binding peaks in TH17 cells (right). (B) Distribution of Cxxc1-binding peaks across extended gene bodies in TH17 cells. The tag density of Cxxc1 binding to gene bodies [between the transcription start site (TSS) and the transcription termination site (TTS)], as well as 3-kb upstream of the TSS and 3-kb downstream of the TTS regions of all RefSeq (mm10) genes, was calculated. (C) Enrichment of Cxxc1-binding peaks on CGIs. The tag density of Cxxc1 binding to CGIs and 5-kb flanking regions was calculated. (D) Overlapped regions between Cxxc1-binding sites, H3K4me3 sites, and RNA-seq down-regulated genes in WT and Cxxc1-deficient TH17 cells. (E) Integrative Genomics Viewer browser view of Cxxc1-binding peaks (red) in WT TH17 cells and H3K4me3 markers (blue) in WT and Cxxc1-deficient TH17 cells. (F) Naive WT CD4+ T cells were sorted and cultured under TH17 differentiation conditions (TGF- and IL-6) for 24 hours, and ChIP-qPCR analysis of Cxxc1 binding at the indicated gene loci was performed. (G) Naive CD4+ T cells from WT and RORtcreCxxc1fl/fl mice were differentiated into TH17 cells in the presence of TGF-1 and IL-6 for 24 hours, and H3K4me3 modifications at the indicated gene loci were detected by ChIP-qPCR. The statistical significance was determined by Students t test. Error bars show the means SD. *P 0.05, **P 0.01.

As shown in Fig. 4C, Cxxc1-binding sites were enriched at the center of CGIs, and about 31% of the Cxxc1-binding sites were found to colocalize with CGIs in TH17 cells (fig. S7A). We found that Cxxc1-dependent H3K4me3 modifications might be indispensable during TH17 differentiation (fig. S6); therefore, we carried out ChIP-seq analysis to map the genome-wide H3K4me3 modifications in TH17 cells. Similar to the locations of Cxxc1-binding sites, peaks indicating H3K4me3 modifications were also found mainly in gene promoters, and a reduction in the number of H3K4me3 peaks was found in Cxxc1-deficient TH17 cells (fig. S7B).

We mapped the genes with direct CXXC1 binding, genes with at least twofold difference of H3K4me3 modifications between WT and CXXC1 KO cells, and genes positively regulated by CXXC1 (at least twofold difference in gene expression between WT and KO cells). As shown in the Venn diagram (Fig. 4D), the loci of 1874 of 2346 (80%) genes with direct CXXC1 binding were associated with H3K4me3 changes, suggesting an important role of CXXC1 in mediating the histone modification of H3K4me3 in TH cells. Our data also identified 165 genes positively regulated by CXXC1 through positively changing the H3K4me3 modifications on their loci directly (Fig. 4D and table S1). On the other hand, we were able to identify more than 4000 genes whose expression was negatively regulated by CXXC1, although, in most of the cases, through indirect signaling pathways without directly affecting the H3K4me3 on their loci (fig. S7C). Together, our data suggest that CXXC1 plays a key role in regulating gene expressions through recruitment of H3K4me3 in TH cells.

ChIP-seq data showed that Cxxc1 bound upstream of or bound to the gene body of the Il6r, Il6st, Runx1, Satb1, Il21, Irf4, Rorc, Rora, and Batf gene loci was associated with a significant decrease in the H3K4me3 modification of the promoter regions of these genes in Cxxc1-deficient TH17 cells (Fig. 4E and fig. S7D). The direct binding of Cxxc1 in WT TH17 cells and the reduction in H3K4me3 modification in Cxxc1-deficient cells at these gene loci were confirmed by ChIP-PCR (Fig. 4, F and G, and fig. S7, E and F).

We then conducted 24-hour RNA-seq to determine potential key genes that are regulated by Cxxc1 at the early stage of differentiation and further confirmed the ChIP-seq results. We found that, even at the early stage, most of the key TH17 cellrelated transcripts (e.g., Il17a, Il17f, Il21, Il22, Il6ra, Il6st, Runx1, and Satb1) were significantly down-regulated in RORtcreCxxc1fl/fl TH17 cells and that the expression of key Treg cellrelated transcripts increased (e.g., Foxp3, Ccl3, Mgat5, Itgae, and Gpr83) (fig. S7G). We measured the expression of those genes by qPCR and obtained the same results as those obtained by 24-hour RNA-seq analysis (fig. S7H). In contrast to the 72-hour RNA-seq and qPCR results, we did not find a remarkable change in Rorc, Rora, or Irf4 expression at 24 hours. Although there are interactions between CXXC1 and loci of Rorc, Rora, and Irf4, these bindings only indicate enhanced accessibility of these loci. It still requires upstream TFs to promote the expression of Rorc, Rora, and IRF4. It may take 24 hours to open the gene locus, while it may take a longer time for essential TFs to promote their expression. Similar to the ChIP-seq results, we found a decline in Il6ra and Il6st (gp130), which organize the functional receptor for IL-6. The IL-6R subunit binds to IL-6 and the IL-6ST subunit, which are involved in signal transduction and play a vital role in TH17 cell differentiation (24).

We found that Il6r showed a significant decline in H3K4me3 modification levels and mRNA expression. Then, we detected the protein levels of IL-6R by flow cytometry and found a significant reduction in IL-6R in Cxxc1-deficient cells under TH17 cellpolarizing conditions (TGF-1 and IL-6) in different stages of differentiation (Fig. 5A). In addition, we found the same phenomenon in pathogenic TH17 cells (IL-1, IL-6, and IL-23), indicating that the reduction of IL-6R is independent of TGF- signaling (Fig. 5B). Moreover, we also detected a significant decline in IL-6R in dLckcre and ERT2cre deletion mice compared with that in WT mice (fig. S8A). Although IL-6ST expression was reduced at 24 hours, we found a moderate increase in IL-6ST expression at 72 hours (fig. S8B). IL-6R may be secreted in its soluble form, sIL-6R, which mediates the response by forming a complex with IL-6ST in a mechanism named trans-signaling (25). We therefore measured the level of sIL-6R in the supernatant by enzyme-linked immunosorbent assay (ELISA) and detected significantly lower levels of sIL-6R in Cxxc1-deficient cells than those in WT mice at all time points under TH17 cellpolarizing conditions (TGF-1 and IL-6) (Fig. 5C). These results indicated that IL-6R expression was significantly reduced in Cxxc1-deficient TH17 cells both in its membrane-bound and soluble forms.

(A and B) Naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from RORtcreCxxc1fl/fl and WT mice were differentiated into TH17 cells with IL-6 and TGF-1 (A) or IL-1, IL-6, and IL-23 (B). The expression of IL-6R was measured by flow cytometry (left), and the mean fluorescence intensity (MFI) of IL-6R at different time points was measured (right). One of six experiments is shown. (C) Naive CD4+ T cells from RORtcreCxxc1fl/fl and WT mice were differentiated into TH17 cells with IL-6 and TGF-1, and the supernatants from cell cultures were collected at indicated time points. The amounts of IL-6R were then measured by ELISA. One of four experiments is shown. (D and E) Purified naive CD4+ T cells were stimulated for the indicated times with IL-6 (10 ng/ml). Phosphorylated and total STAT3 proteins were detected by Western blot assays (D) or flow cytometry (E). One of five experiments is shown. (F) Naive CD4+ T cells from WT and RORtcreCxxc1fl/fl mice were polarized into TH17 cells in the presence of TGF- and IL-6, and varying concentrations of IL-6R antibody were added. The expression levels of IL-17A and IL-17F were then analyzed by intracellular staining after 72 hours. One of six experiments is shown. (G and H) Naive CD4+ T cells from WT and RORtcreCxxc1fl/fl mice were cultured in the presence of TGF-1 and varying concentrations of IL-6 for 72 hours, and then, the expression levels of IL-17A, IL-17F, and Foxp3 were analyzed by intracellular staining after restimulation. One of seven experiments is shown. The statistical significance was determined by Students t test. Error bars show the means SD. *P 0.05, **P 0.01, ***P 0.001.

IL-6 signaling is required for the differentiation of TH17 cells, and the activation of STAT3 is a vital component of the TH17 cell induction mechanism (26). The significant reduction of IL-6R in RORtcreCxxc1fl/fl TH17 cells indicated that IL-6 signaling was possibly affected by the loss of Cxxc1 during TH17 differentiation. To assess this, we sorted RORtcreCxxc1wt/wt and RORtcreCxxc1fl/fl naive CD4+ T cells and stimulated them with IL-6 for different time periods, and then, we detected the activation of the downstream signaling protein STAT3. Both Western blot analysis and flow cytometry results showed a significant reduction in STAT3 activation in Cxxc1-deficient cells compared to that in WT cells stimulated with IL-6 (Fig. 5, D and E).

To further confirm the role of IL-6R defects in Cxxc1-deficient TH17 cells, we added different concentrations of IL-6R blocking antibody to TH17 culture medium in vitro. WT TH17 cells showed a marked reduction in differentiation when IL-6R blocking antibody was added, and the higher the concentration of IL-6R blocking antibody was, the lower the level of WT TH17 cell differentiation was. However, there was only a slight impact in Cxxc1-deficient TH17 cells compared with WT cells when IL-6R blocking antibody was added (Fig. 5F), further indicating the defects of IL-6R in Cxxc1-deficient TH17 cells.

IL-6 binds to IL-6R and is required for TH17 differentiation by activating STAT3 and inhibiting TGF-driven Foxp3 expression. The defects in IL-6R expression in TH17 cells may be compensated if the level of IL-6 is increased. To assess this, we polarized naive RORtcreCxxc1wt/wt or RORtcreCxxc1fl/fl CD4+ T cells into TH17 cells with varying levels of IL-6. Although IL-17 expression was only slightly restored with low and moderate levels of IL-6, it was restored with a high level of IL-6 (Fig. 5G). Moreover, IL-6 inhibited Foxp3 expression in RORtcreCxxc1fl/fl cells in a dose-dependent manner (Fig. 5H). These data suggest that Cxxc1 may regulate TH17 differentiation dependent on the IL-6/STAT3 pathway in the modulation of early signaling events downstream of the IL-6 receptor.

IL-21 or IL-6 alone or in combination with TGF- resulted in the up-regulation of the IL-23 receptor (IL-23R), RORt, and the TH17 cytokines. Our flow cytometry results showed no change in IL-21R expression, while the IL-23R expression was decreased in RORtcreCxxc1fl/fl TH17 cells (fig. S8C). The IL-6induced expression of IL-21, a process that is dependent on STAT3 and IL-21, serves as an autocrine factor that promotes and sustains TH17 lineage commitment (1, 7). IL-21, in synergy with TGF-, induced IL-17 expression independent of IL-6 and induced naive IL-6/ T cells into TH17 cells (6, 27). We then cultured naive CD4+ T cells in vitro with varying concentrations of IL-21 along with TGF-. Similar to the results observed upon the addition of varying levels of IL-6, IL-21 restored inconspicuous IL-17A and IL-17F expression at low and moderate levels, while it fully restored IL-17A and IL-17F expression in Cxxc1-deficient cells at high levels (fig. S8D). IL-23 promotes maintenance of the TH17 lineage and maintains the IL-17secreting phenotype, but it does not promote commitment to an IL-17secreting lineage. IL-23 could also induce IL-17A and IL-17F expression independent of IL-6 in conjunction with TGF- in naive CD4+ T cells (6). When naive CD4+ T cells were cultured in vitro with TGF- and varying concentrations of IL-23, defective IL-17A and IL-17F expression could not be restored, even at the highest level of IL-23, in Cxxc1-deficient cells (fig. S8E). In addition, to eliminate the residue effect of IL-6dependent signaling, we added IL-6R blocking antibody in these cultures and found consistent results (fig. S8, F and G).

As TGF- receptors are important for both TH17 and Treg cell differentiation, we detected the expression of TGF- receptors I and II. Protein levels of TGF- receptors I and II detected by flow cytometry analysis did not show a significant change in naive Cxxc1-deficient CD4+ T cells but showed an increase in Cxxc1-deficient TH17 cells compared with WT cells (fig. S9, A and B). Smad3 and Smad2 are downstream of TGF- signaling, and Smad2 positively regulates the generation of TH17 cells (28), while Smad3 promotes iTreg and inhibits TH17 cell differentiation (29). Western blot results showed no significant change in the Smad2 phosphorylation level (fig. S9C), while Smad3 showed a slight increase in phosphorylation (fig. S9D). Furthermore, the TCR activationinduced phosphorylation of both ERK and JNK proteins was also normal in Cxxc1-deficient cells (fig. S9E). These results indicated that Cxxc1-deficient TH17 cells transdifferentiated into Treg cells mainly due to IL-6/STAT3 signaling defects.

To further confirm IL-6R defects in Cxxc1-deficient TH17 cells, we overexpressed IL-6R in Cxxc1-deficient TH17 cells and carried out assays. We infected Cxxc1-deficient TH17 cells with retroviruses IL-6R or Cxxc1 complementary DNA as a positive control. The results showed that the overexpression of IL-6R potently increased the production of IL-17A and IL-17F relative to that in mock-transfected control Cxxc1-deficient TH17 cells (Fig. 6A). In addition, the overexpression of IL-6R could also efficiently reduce Foxp3 expression in Cxxc1-deficient TH17 cells (fig. S9F). Moreover, we found that the overexpression of Cxxc1 and the C-terminal domain of Cxxc1 (residues 361 to 656, C169A) increased the expression of IL-6R compared to that in mock-transfected Cxxc1-deficient TH17 cells, further confirming the regulation of IL-6R by Cxxc1 (Fig. 6B). We also overexpressed IL-6ST in Cxxc1-deficient TH17 cells and did not find the restoration of IL-17A and IL-17F production or the inhibition of Foxp3 expression compared with controls (Fig. 6A and fig. S9F).

(A) Naive CD4+ T cells from WT and RORtcreCxxc1fl/fl mice were differentiated into TH17 cells in the presence of TGF-1 and IL-6, and 20 to 24 hours, later the cells were transfected with the indicated retrovirus (Mock, Cxxc1, IL-6R, and IL-6ST). IL-17A and IL-17F levels were then measured by gated CD4+GFP+ cells after retrovirus infection for 72 hours. One of six experiments is shown. (B) Sorted naive CD4+ T cells were differentiated into TH17 cells in the presence of TGF-1 and IL-6, and 20 to 24 hours later, the cells were transfected with the indicated retrovirus. IL-6R levels were then measured by gated CD4+GFP+ cells after retrovirus infection for 72 hours. One of five experiments is shown. (C) Naive CD4+ T cells from WT and RORtcreCxxc1fl/fl mice were differentiated into TH17 cells in the presence of TGF-1 and IL-6, and 20 to 24 hours later, the cells were transfected with the indicated retrovirus [Mock, STAT3 (WT), STAT3 (A662C, N664C), and STAT3 (Y705A)]. IL-17A and IL-17F levels were then measured by gated CD4+GFP+ cells after retrovirus infection for 72 hours. One of five experiments is shown. (D) Naive CD4+ T cells from WT and RORtcreCxxc1fl/fl mice were differentiated into TH17 cells in the presence of TGF-1 and IL-6, and 20 to 24 hours later, the cells were transfected with the indicated retrovirus. CD4+GFP+ cells were then sorted after retrovirus infection for 72 hours and transferred into RAG1/ hosts. Two days later, the recipient mice were immunized with MOG3555 and FCA plus pertussis toxin to induce EAE. Clinical scores were recorded and calculated each day for the indicated times. Data are summed from three independent experiments. (E) IL-17A and IFN- production by CD4+ T cells isolated from CNS, draining lymph nodes, and spleens of Rag1/ mice at the peak of disease. One representative of three experiments is depicted. Error bars show the means SD. *P 0.05, **P 0.01, ***P 0.001 using the Students t test.

To further determine defects in the activation of STAT3, which is downstream of IL-6, we overexpressed STAT3 in Cxxc1-deficient TH17 cells. Our results showed that the overexpression of the active form of STAT3 (A662C, N664C) (30) strongly increased the production of IL-17A and IL-17F and inhibited Foxp3 expression relative to that in mock-transfected control Cxxc1-deficient TH17 cells, while the overexpression of WT STAT3 and the inactive form of STAT3 had almost no apparent effect (Fig. 6C and fig. S9G). However, we did not detect a change in IL-6R expression when the active form of STAT3 was overexpressed, indicating that IL-6R is upstream of STAT3 (fig. S9H). We also detected the reduced expression of RORt in Cxxc1-deficient TH17 cells, and RORt overexpression in Cxxc1-deficient TH17 cells partially rescued the TH17 differentiation defect under TH0 conditions and TH17-polarizing conditions (fig. S9I). Therefore, defects in STAT3 activation impaired RORt function at the Il17-Il17f locus.

Last, to better understand whether the overexpression of IL-6R in Cxxc1-deficient TH17 cells would affect the development of EAE in vivo, we sorted CD4+GFP+ T cells, transferred them into RAG1/ hosts, and then induced EAE with MOG3555. Mice that received WT TH17 cells developed the most severe disease, and mice that received Cxxc1-deficient TH17 cells overexpressing Cxxc1 or IL-6R developed more severe disease than those that received control Cxxc1-deficient TH17 cells (Fig. 6D). In addition, both the percentage and the number of TH17 cells in the CNS/draining lymph nodes/spleens of Rag1/ mice that received IL-6Roverexpressing TH17 cells were notably higher than those in the control mice (Fig. 6E). These data demonstrated that IL-6R could potently rescue the production of IL-17A in Cxxc1-deficient TH17 cells in vitro and in vivo and that Cxxc1 could restore IL-6R expression in Cxxc1-deficient TH17 cells.

Epigenetic regulation is an essential mechanism to coordinate T cell differentiation. Here, we report a crucial role of Cxxc1, which directly regulates promoter-associated H3K4me3 modification and the expression of genes such as Il6r that are essential for TH17 cell lineage specification. The transition of naive CD4 cells to TH17 cells in Cxxc1-deficient T cells was almost completely blocked, and the cells instead developed a Treg celllike transcriptional profile.

TH17 and Treg lineage differentiation is not only controlled by a combination of their specific cytokine milieus and TFs but also subjected to epigenetic control through various mechanisms. Previous work has shown that histones maintain TH17 cell differentiation and function by inducing histone modifications at the Il17a locus (31). The H3K4me3 methyltransferase MLL/menin/TrxG complex plays a critical role in the regulation of the TH2 cell program in murine and human systems (32). In addition, deletion of the H3K9me2 methyltransferase G9a also produces both TH17 and TH2 cell response defects (33). The conditional deletion of the H3K27me3 demethylases Jmjd3 and tripartite motif-containing 28 has been reported to have an inconsistent effect on the TH17 program, possibly due to different cellular environments (3437). However, our data showed that the loss of Cxxc1 led to a marked CD4+ T cell lineage switch from TH17 cells to Treg cells without affecting TH1 and TH2 cell differentiation. These data suggest that Cxxc1 is essential for TH17 cell differentiation and stability.

The functional receptor for IL-6 is composed of an IL-6R subunit that binds IL-6 and a gp130 subunit involved in signal transduction (38). The binding of IL-6 family of cytokines to their receptors activates STAT3, which is required for TH17 cell differentiation (26, 38). IL-6R is highly expressed in naive T cells and in the early phase of T cell activation, while IL-6R expression decreases in activated T cells. IL-6/gp130/STAT3 signaling is dominant in inhibiting the conversion of conventional T cells into Foxp3+ Treg cells in vivo, and in the absence of IL-6 signaling, almost no other cytokine can inhibit the conversion of T cells to Treg cells effectively (26). Our results showed a significant reduction in the IL-6R protein level in Cxxc1-deficient cells under TH17 cellpolarizing conditions (TGF-1 and IL-6) at different time points. Meanwhile, Cxxc1-deficient TH17 cells exhibited significantly defective TH17 differentiation and the strong expression of Foxp3 both in vitro and in vivo. ChIP-seq analysis revealed that Cxxc1 bound to and enhanced IL-6R by maintaining the appropriate H3K4me3 modification of its promoter regions. The cofactor that is bound by Cxxc1 and specifically enhances IL-6R expression in the early phase of TH17 cell differentiation remains to be elucidated in future studies.

Both the DNA methylation and H3K4me3 domains of Cxxc1 function in different cell types and tissues (1618). However, our overexpression assay indicated that the histone modification of Cxxc1 mostly functions in TH17 cell differentiation and stability. Our ChIP-seq data showed that Cxxc1 bound to TSS or gene body of several key genes involved in TH17 cell differentiation, including the Runx1, Satb1, IL21, Irf4, Rorc, and Rora gene loci, was associated with a significant decrease in the H3K4me3 modification of the promoter regions of these genes in Cxxc1-deficient TH17 cells. Although we suggested that IL-6R was the main target of Cxxc1 in TH17 cell differentiation, there are still some other target genes regulated by Cxxc1 that provide assistance in the TH17 cell differentiation process. In addition, our TH17 data illustrate that the cell typespecific binding profile of Cxxc1 may determine its primary function, which is consistent with our former analysis in thymocyte development and macrophage function.

In summary, we identified Cxxc1 to be a critical positive regulator of TH17 development at the early stage of differentiation, in which it positively regulates autoimmune disease and bactericidal activity mainly through promoting IL-6R expression and the subsequent activation of downstream pathways. Our findings provide insight into the association of epigenetic regulators with TH17 development and supply important clues for therapeutic approaches for the treatment of TH17-related inflammatory and autoimmune diseases.

The Cxxc1fl/fl mouse strain has been described previously (19). The ERT2cre mice were gifts from Y. W. He (Duke University Medical Center). The dLCKcre mice (JAX:012837) and IL-17AeGFP mice (018472 C57BL/6-IL-17atm1Bcgen/J) were from The Jackson Laboratories. The RORtcre mice (JAX: 022791) were gifts from J. Qiu (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). Rag1/ mice were purchased from The Jackson Laboratories. All experiments were performed with 6- to 10-week-old mice unless specified. All mice were kept in the Zhejiang University Laboratory Animal Center, and all animal experimental procedures were approved by the Animal Review Committee at Zhejiang University School of Medicine.

To induce EAE in Rag1/ mice, naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from WT and RORtcreCxxc1fl/fl or dLckcreCxxc1fl/fl mice were sorted by the Mouse CD4 Nave T cell Enrichment Kit (no. 8804-6824-74, Invitrogen) and intravenously transferred into Rag1/ mice at 2 106 cells per mouse. Two days later, the recipient mice were subjected to EAE induction.

Female age-matched Rag1/ mice (8 to 10 weeks old) were immunized with an emulsion containing the MOG peptide MOG3555 (200 g per mouse; MEVGWYRSPFSRVVHLYRNGK; Sangon) in an equal amount of complete Freunds adjuvant (200 l per mouse; no. 7027, Chondrex Inc.). Pertussis toxin (200 ng per mouse; no. 181, List Biological Laboratories) was administered intravenously 0 and 2 days after induction. Clinical evaluation was performed daily using a five-point scale: 0, no clinical signs; 1, limp tail; 2, paraparesis (weakness, incomplete paralysis of one or two hind limbs); 3, paraplegia (complete paralysis of two hind limbs); 4, hind limb and fore limb paralysis; and 5, moribund or death.

Mice were intracardially perfused with 50 ml of phosphate-buffered saline (PBS). The forebrain and cerebellum were dissected, and spinal cords were collected from the spinal canal. CNS tissue was cut into pieces and digested with collagenase D (2 g/ml; Roche Diagnostics) and deoxyribonuclease I (DNase I; 1 g/ml; Sigma-Aldrich) at 37C for 20 to 30 min while rotating. Mononuclear cells were isolated by passing the tissue through a 200-mesh cell filter membrane, followed by 80%/40% Percoll gradient centrifugation. Mononuclear cells were carefully removed from the interface, washed with PBS, and resuspended in culture medium for further analysis. For cytokine analysis, mononuclear cells were stimulated for 5 hours with phorbol 12-myristate 13-acetate and ionomycin (both from Sigma-Aldrich) in the presence of brefeldin A (eBioscience) and then subjected to flow cytometry analysis to detect intracellular IL-17A, IFN-, and Foxp3.

RAG/ mice were provided with autoclaved water supplemented with antibiotics [ampicillin (1 g/liter), metronidazole (1 g/liter), neomycin (1 g/liter), and vancomycin (0.5 g/liter)] for 6 days and then provided with autoclaved water for 1 day. Then, naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from WT and RORtcreCxxc1fl/fl mice were sorted and intravenously transferred into Rag1/ mice at 2 106 cells per mouse. Two days later, the recipient mice were subjected to C. rodentium infection as described (39). Briefly, mice were gavaged with 5 108 C. rodentium cells in 250 l of PBS per mouse. Bacteria were prepared by shaking at 37C overnight in LB broth, and then, the cultures were serially diluted and plated to measure the colony-forming units. Body weight was measured daily. Fecal pellets were collected, weighed, and then homogenized in sterile PBS, and C. rodentium colonies were identified on the basis of morphology after 18 to 24 hours of incubation at 37C on MacConkey agar plates.

To analyze CNS histology, mice were euthanized 22 days after EAE induction, and spinal cords were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were cut and stained with Luxol fast blue and hematoxylin and eosin (H&E). To analyze colon histology, the colons from Rag1/ hosts 7 days after inoculation with C. rodentium were collected, treated as described above, and stained with H&E.

Mouse small intestines were dissected, and fat tissues and Peyers patches were removed. The intestines were cut open longitudinally and washed with Dulbeccos modified Eagles medium (DMEM) until no fecal pellets were observed. The intestines were then cut into approximately 5-mm-long pieces. The intestinal pieces were incubated in 37C prewarmed DMEM containing 3% fetal bovine serum (FBS), 20 mM Hepes, 5 mM EDTA, and dithiothreitol (0.15 mg/ml) for 10 min with constant agitation by droppers in a 37C water bath. The digested cells that were collected were intraepithelial lymphocytes. Then, the left small intestine was incubated in a solution of 3% FBS, 20 mM Hepes, DNase I (0.125 mg/ml), and collagenase II (0.5 mg/ml) in 37C prewarmed DMEM for 5 min with constant agitation by droppers in a 37C water bath, and the dissociated cells that were collected were LP lymphocytes. Last, the collected cells were isolated by passing the tissue through a 200-mesh cell filter membrane, followed by 80%/40% Percoll (GE Healthcare) gradient centrifugation. Cells were carefully removed from the interface, washed with PBS, and resuspended in culture medium for further analysis.

All flow cytometric data were collected on a FACS Calibur or FACS LSR II system (both from BD Biosciences) and analyzed using FlowJo analysis software v7.6.1. For intracellular cytokine staining, cells were stimulated for 5 hours at 37C with phorbol 12-myristate 13-acetate (50 ng/ml; Sigma), ionomycin (1 mg/ml; Sigma-Aldrich), and brefeldin A (eBioscience). After staining for surface markers, cells were fixed and permeabilized according to the manufacturers instructions (eBioscience). Intracellular staining was processed using intracellular fixation buffer (eBioscience), and a TF staining buffer set (eBioscience) was used for RORt and Foxp3 staining. For the detection of phosphorylated STAT3 by flow cytometry, BD Phosflow Fix Buffer I and Perm/Wash Buffer I were used.

The following antibodies (clone names are in parentheses) with different fluorochrome labels were purchased from eBioscience: CD4 (RM4-5), CD8a (53-6.7), TCR (H57-597), CD44 (IM7), CD62L (MEL-14), IFN- (XMG1.2), IL-17A (TC11-18H10.1), IL-4 (11B11), and RORt (B2D). The following reagents were purchased from BioLegend: IL-23R (12B2B64), IL-21R (4A9), CD126 (D7715A7), Foxp3 (MF-14), and IL-17F (9D3.1C8).

For Western blot and ChIP, anti-Cxxc1 (1:1000 dilution for Western blot; 6 g for each immunoprecipitation and ChIP reaction; ab56035) was purchased from Abcam. H3K4me3 (4 g for each ChIP reaction; 39915) was purchased from Active Motif. AntipC-SMAD2 (3101), anti-SMAD2 (3103), anti-SMAD3 (9523), antipC-SMAD3 (9520), anti-STAT3 (Tyr705) (9131), anti-STAT3 (9132), anti-Erk (Thr202/Tyr204) (4370), anti-Erk (4695), anti-JNK (T183/Y185) (9251), and anti-JNK (9258) were obtained from Cell Signaling Technology.

Naive CD4+ T cells (CD4+CD25CD62LhiCD44lo) were purified by a FACS Aria II flow cytometer or sorted by the Mouse CD4 Nave T cell Enrichment Kit (no. 8804-6824-74, Invitrogen). Naive CD4+T cells were cultured with irradiated (30 Gy) anaphase-promoting complex sorted from spleen at a ratio of 1:3 and were activated with anti-CD3 (2 g/ml) and anti-CD28 (3 g/ml) in a 48-well plate (5 105 T cells per well). T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, sodium pyruvate, penicillin-streptomycin, and 2-mercaptoethanol.

For nonpathogenic TH17 cell differentiation, culture medium was supplemented with IL-6 (20 ng/ml), TGF-1 (5 ng/ml), antiIL-4 (10 ng/ml), antiIL-12 (10 ng/ml), and antiIFN- (10 ng/ml). For pathogenic TH17 cells differentiation, culture medium was supplemented with IL-1 (20 ng/ml), IL-6 (20 ng/ml), and IL-23 (20 ng/ml), antiIL-4 (10 ng/ml), antiIL-12 (10 ng/ml), and antiIFN- (10 ng/ml). Other T cell differentiation were performed: TH1, IL-12 (20 ng/ml) and antiIL-4 (10 mg/ml); TH2, IL-4 (50 ng/ml), antiIFN- (10 ng/ml), and antiIL-12 (10 mg/ml); iTreg cells, TGF-1 (5 ng/ml), antiIL-4 (10 ng/ml), antiIL-12 (10 ng/ml), and antiIFN- (10 ng/ml). Neutralizing antiIFN- (XMG1.2), antiIL-4 (11B11), and antiIL-12 (C17.8) were from BioLegend.

Retroviruses were produced in Plat-E cells. Plat-E cells were transfected with pMX-IRES-GFP plasmids containing the indicated genes, and the medium was replaced twice with 3 ml of fresh medium every 10 hours after transfection. The retrovirus-containing supernatant was collected 72 hours after the medium was replaced for the second time and used to infect T cells.

Sorted naive CD4+ T cells were differentiated into TH17 cells in the presence of TGF-1 and IL-6 (48-well plate, 0.5 106 cells per well); 20 to 24 hours later, the cells were transfected with 1 ml of the indicated retrovirus in the presence of polybrene (10 g/ml) and 10 mM Hepes and infected for 2 hours at 1500g at 32C. After transfection, the cells were resuspended in TH17 differentiation medium and cultured for 3 days. The indicated cytokines (e.g., IL-17A and IL-17F) and other TFs (e.g., Foxp3 and RORt) were measured by gated CD4+GFP+ cells after retrovirus infection for 72 hours.

For RNA-seq, total RNA was extracted from naive CD4+ T cells differentiated in the presence of TGF-1 (5 ng/ml) and IL-6 (20 ng/ml) for 24 or 72 hours using the RNeasy kit (Qiagen). Library construction and sequencing were performed on a BGISEQ-500 platform by the Wuhan Genomic Institution (www.genomics.org.cn; BGI, Shenzhen, China). All reads were mapped to the mm10 mouse genome, and the uniquely mapped reads were subjected to RNA-seq data analysis using the Hierarchical Indexing for Spliced Alignment of Transcripts system (40).

ChIP assays were performed according to the manufacturers instructions with modifications using the ChIP-IT kit (Active Motif, USA). Briefly, the TH17 cells were fixed with 1% formaldehyde, and then, the cross-linked chromatin was sonicated in a 4C water bath using a Bioruptor Pico sonicator (Diagenode) to obtain DNA fragments between 150 and 500 base pairs (bp) in size. For Cxxc1 ChIP-seq, 5 106 TH17 of cells and 6 g of Cxxc1 antibody were used for each sample. For H3K4me3 ChIP-seq, 3 106 of TH17 cells and 4 g of H3K4me3 antibody were used for each sample.

The immunoprecipitated DNA was purified and subjected to sequencing library preparation using a VAHTSTM Universal DNA Library Prep Kit for Illumina V2 (Vazyme Biotech Co. Ltd.) according to the manufacturers protocol. The DNA libraries were then sequenced with an Illumina HiSeq X Ten system at Veritas Genetics in Hangzhou.

Sequenced reads of 150 bp were obtained using the CASAVA 1.8.2 package (Illumina). All reads were mapped to the mm10 mouse genome, and uniquely mapped reads were subjected to a further peak identification process. MACS2_V2.1.1 was used to identify significant peaks (q = 0.05) with both input DNA and ChIP DNA in Cxxc1-deficient cells as controls. The output of the peak files was converted by IGV browser. To calculate the tag density for Cxxc1-binding sites or H3K4me3 modifications around the TSS or at the centers of CGIs, uniquely mapped tags were summarized in 100-bp windows, and all window tag counts were normalized by the total number of bases in the windows and the total read number for the given sample.

Statistical analyses were performed using GraphPad Prism (GraphPad Software). The statistical significance was determined by Students t test. All error bars shown in this article represent SDs. Significance levels (P values) are presented in the figures.

Acknowledgments: We thank X. L. Liu (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) for gifts of cell lines; J. Qiu (Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences) for providing C. rodentium and mice; L. Shen (Zhejiang University) for helping with ChIP-seq data analysis; H. Y. Fang (Zhejiang University) for providing reagents; and L. R. Lu and D. Wang for their helpful discussion. We thank Y. Y. Huang, Y. W. Li, and J. J. Wang (Zhejiang University) for helping with the cell sorting; Y. Zhang and R. Ma (Zhejiang University) for feeding the mice. Funding: This work was supported, in part, by grants from the National Basic Research Program of China 973 Program (2015CB943301), the National Natural Science Foundation of China (81830006, 31670887, 31870874, and 31800734), Zhejiang Provincial Key Project of Research and Development (2019C03043), the Zhejiang Natural Science Foundation (LQ16H030003), and the Zhejiang Science and Technology Program(2017C37117 and 2017C37170). Author contributions: L.Wa. and F.L. designed the research. F.L., X.M., Y.G., W.C., Q.X., Z.H., W.L., J.C., S.H., and X.Z. performed the experiments and data analysis. L.Wa. and F.L. wrote the manuscript. L.L., C.W., J.W., W.Q., L.We., and D.W. provided expertise and advice. L.We. and L.Wa. supervised the project. Competing interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The ChIP-seq and RNA-seq datasets were deposited in the GSE accession:GSE132208 and the SRA accession: PRJNA545626. Additional data related to this paper may be requested from the authors.

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Epigenetic initiation of the TH17 differentiation program is promoted by Cxxc finger protein 1 - Science Advances

Interview with Tamer Mohamed of Aspect Biosystems on Advancing Tissue Therapeutics – 3DPrint.com

While attending The University of British Columbia (UBC), Tamer Mohamed, along with fellow graduate student Simon Beyer, began working at the Walus Laboratory on the development of a novel microfluidics-based bioprinting platform that could be used to fabricate human tissue constructs. One of the main reasons for their innovation was to potentially replace animal models in drug testing, which are costly, time-consuming and can have poor predictive accuracy. A few years went by and the two went on to win a MEMSCAP Design Award for their pioneering creation (the Lab-on-a-Printer Bioprinter) which would later become the basis for their startup, Aspect Biosystems. The UBC spinoff company was founded by Mohamed, Beyer, Konrad Walus (associate professor at UBC and head of the Walus Lab), and Sam Wadsworth, to turn their idea into a commercial product. The company quickly began providing pharmaceutical companies with high-efficacy tissue models that better mimicin vivoconditions, looking to improve the predictive accuracy of the front end drug discovery process. 3DPrint.com spoke to Mohamed to learn about his successful transition from graduate student to CEO of Aspect Biosystems.

Cofounders of Aspect Biosystems Tamer Mohamed and Simon Beyer at the Walus Lab when they were grad students

What was the inspiration behind Aspect Biosystems?

Aspect Biosystems was established with the vision of leveraging advancements in biology, microfluidics, and 3D printing to create technology-enabled therapeutics that will ultimately have a meaningful impact on patients. We are marrying our deep knowledge of human biology with cutting-edge 3D printing technology to create. Our story started almost a decade ago so weve spent years developing our foundational microfluidic bioprinting technology and are now applying our platform technology to create functional tissues, both internally through our proprietary programs, and with our partners around the world.

Can you tell me about the companys growth model?

Platform technologies often have the advantage of flexibility, as they could allow you to pursue multiple applications. This also presents a challenge though, in that it is easy to become unfocused. At Aspect, weve built a strategy that allows us to both focus and diversify. Internally, we are advancing proprietary tissue programs in regenerative medicine. But we also recognize that to achieve our vision of enabling human tissues on demand, we cant work alone. By providing access to our technology to partners around the world, we are able to create a network effect and tap into specific domain expertise. This allows our technology to be applied to a wide range of research purposes externally, without detracting resources or focus from our specific tissue programs internally. We collaborate with academia and industry on specific applications that allow us to fuel our growth and help generate revenue and a robust innovation pipeline.

How much has Aspect grown?

Aspect is the first and only company to leverage microfluidics to create functional tissue, and we are proud to pioneer this approach. Academically, we were one of the first groups in the world to print cells while at the UBC, so we see ourselves as pioneers in both bioprinting and platforms for creating tissue therapeutics. Five years ago, we had four full-time employees. Today we have a team of over 40 people focused on our mission and over 20 collaborations globally. We have attracted smart venture capital, partnered with some of the biggest names in our industry, and made major breakthroughs in applying our technology to create functional tissues. It is a great sign that, year-after-year, we continue to raise the bar. It is an even better sign that I believe the best is yet to come.

The Aspect Biosystem team celebrating Canada Day

What will the applications of this technology be in pharmaceutical research and drug trials?

I believe the opportunity with the highest value and best poised to make a significant impact on the pharmaceutical space is disease modeling. Using 3D bioprinting technology allows us to model diseases in a human-relevant system that would otherwise be difficult to study in animals or less sophisticated in vitro models. For example, working with GSK and Merck, we are leveraging our microfluidic 3D bioprinting platform to create physiologically-relevant 3D tissues containing patient-derived cells to assess the efficacy of anti-cancer drugs and to predict a patients response to treatment. This partnered program could unlock the discovery of novel therapeutic targets and the development of immuno-oncology therapeutics.

Would you tell us more about Aspects current and future work?

Our current internal programs are focused on orthopedic and metabolic diseases. On the orthopedic side, we are leveraging our deep knowledge of musculoskeletal biology and biomaterials to create knee meniscal replacements. On the metabolic side, we are focused on liver tissue and creating a therapeutic tissue for Type 1 diabetes. Externally, our partners around the world are using our 3D bioprinting technology to advance research in the brain, lungs, heart, pancreas, and kidneys, just to name a few. By being both focused internally and diversified externally, we are building a robust pipeline for the future. Our end goal is to enable the creation of human tissues on demand, and we know that we cant do it alone. Our network of academic researchers and industry partners are key to making our vision a reality.

How fast is the technology moving towards a future with lab-made functional organs?

Tamer Mohamed

We are focused on identifying specific diseases or biological malfunction inside the body and rationally designing advanced tissue therapeutics that address these areas of unmet medical need. So, while we may not actually be making something that looks exactly like an organ, we are recreating the biological function that has been lost or damaged to address the problem. For example, someone with Type 1 diabetes has a pancreas that is unable to perform the vital function of creating insulin. We dont necessarily need to engineer something for them that looks exactly like a pancreas instead, we are creating an implantable therapeutic tissue that replaces function that has been lost. In this case, that function is sensing glucose levels in the blood and biologically releasing insulin in response. This is an example of one of our internal programs a bioengineered pancreatic tissue therapeutic that restores a critical function that been lost due to an autoimmune disease.

Is Canada a great place to develop a bioprinting company?

Canada has a long and rich history in the field of regenerative medicine, going back to the discovery of stem cells in the 1960s. As a country, we have an opportunity to be a global leader in the field. At Aspect, we are proud to be part of these efforts. We are in ongoing discussions with different government groups as to how we can play a role in helping to lead the charge and the government has been embracing that. We have seen significant federal and provincial support for innovation and public/private partnerships, which definitely help stimulate growth in the field.

How disruptive is the technology you created?

By combining microfluidics with 3D printing, we are disrupting tissue engineering. We are able to programmatically process multiple cells and biologically-relevant materials in high-throughput to rationally design and produce functional tissues. We are constantly integrating new microfluidic processing units within our printhead technology and leveraging continuous advancements in the lab-on-a-chip space. With our microfluidic technology, we are generating a large amount of data. By using this data and machine learning, we are improving the quality and automation of the biomanufacturing process.

Ultimately, bioprinting is only as good as our understanding of biology and our understanding of biology is growing wider and deeper. We are combining state-of-the-art stem cell science with our microfluidic 3D printing technology to create tissue therapeutics. For example, we are combining insulin-secreting cells derived from human embryonic stem cells (hESCs) with our printing technology to create therapeutic tissues for patients with Type 1 diabetes.

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Interview with Tamer Mohamed of Aspect Biosystems on Advancing Tissue Therapeutics - 3DPrint.com

Yale researchers develop way to help brain organoids thrive – Yale News

Brain organoids created from human embryonic stem cells offer scientists a powerful way to study the developing brain in three dimensions. However, organoids need nutrients and oxygen carried in blood to thrive, just as a developing fetal brain does. Now Yale researchers have developed a method to induce growth of blood vessels in organoids and prevent the death of cells, which has hindered efforts of scientists studying brain development, they report Oct. 7 in the journal Nature Methods.

The introduction of vasculature will hopefully lead to our ability to create larger, healthier organoids, said In-Hyun Park, associate professor of genetics and associate professor in the Child Study Center and Yale Stem Cell Center.

The development of organoids, created by spurring development of human embryonic stem cells, has been a boon to neuroscientists studying the origins of neurodevelopmental diseases such as autism and schizophrenia, which can arise from abnormalities that form early in brain development. Although just a few millimeters in diameter, organoids offer a new window into how developing regions interact in real time.

However, organoids are also prone to widespread death of cells caused by a lack of blood vessels, particularly within the interior of the developing organ. Parks lab overcame that problem by inducing the expression of hETV2, a gene associated with the creation of vasculature in the developing embryo, within human embryonic stem cells. Its studies, led by Bilal Cakir and Yangfei Xiang, found that cell death in the organoids with engineered cells dramatically decreased, but that new blood vessels were created when the organoids were transplanted into a mouse.

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Yale researchers develop way to help brain organoids thrive - Yale News

Catholics more motivated to donate if ethical investing is assured – The Catholic Spirit

An usher hands the collection basket to a Massgoer Oct. 28, 2018, at Jesus the Divine Word Church in Huntingtown, Md. Nearly nine in 10 Catholics surveyed said they want their donations to church institutions to be ethically invested and a majority of those responding voiced particular opposition to investments in companies that produce pornography, tobacco products and e-cigarettes, according to a new survey. CNS photo/Bob Roller

The survey for Boston-based Catholic Investment Services found that about 87% of respondents would be more likely to donate to Catholic institutions if they knew the money would be invested in ways consistent with Church teaching and values.

In contrast, about 14% of respondents said ethical investing was not a consideration when donating to Catholic institutions.

The results parallel those of a CIS survey released in April. Peter Jeton, the firms outgoing CEO, said the new study sought to more specifically identify what investments motivates or deters Catholics to give to Church entities.

People want their donations to go where its good and avoid evil, Jeton said.

Ethical investing can be a motivating factor in how much people give, he told Catholic News Service. And if I am the CFO (chief financial officer) of a diocese or if Im the bishop of a diocese, then I should be explaining very clearly and with frequency what the policy is regarding how peoples money is being invested.

The survey involved 500 Catholic adults who answered a series of online questions Sept. 7-9. It has a margin of error of plus or minus 4.5 percentage points.

It covered more than a dozen types of businesses from manufacturers of medications used during an abortion procedure to oil and natural gas mining to which respondents voiced objections.

In the case of the pornography industry, 60% of respondents said Church money should not be invested in such endeavors. Opposition also was raised to investing in tobacco products and e-cigarettes (57%), alcoholic beverages (52%), medications used during an abortion (52%), casinos and gaming (51%), medical facilities where abortions occur (50%), gun manufacturing (47%), for-profit prisons/detention centers (45%), security, defense and large-scale weapons (44%), research on embryonic stem cells (37%), manufacturers of birth control (31%) and oil and natural gas extraction (30%).

Jeton suggested that institutional financial officers and bishops be fully transparent about their investment decisions. The better the engagement, the better for the Church, he said.

They should emphasize the link with Catholic social teaching, he added. Because if our data is correct, then making people in the pews aware of that should, by all rights, increase the comfort level with the Church and a greater willingness to share more of their financial resources with the Church.

The survey also asked participants what investments Catholic institutions should be making. Among those cited by respondents were clean air and water (67%), workforce education/job training (66%), education technology (65%), agriculture and food (65%), health care providers (62%), affordable housing (60%), recycling (57%), renewable energy (53%), land conservation (50%), fitness/nutrition (49%) and banking and financial services to low-income people and small businesses (46%).

Catholic Investment Services was founded in 2013 to serve Catholic entities, including dioceses, colleges and universities, hospital systems and social service agencies. Jeton said the firm follows the socially responsible investment guidelines adopted by the U.S. Conference of Catholic Bishops.

Those guidelines, in place since 2003, are divided into six broad categories: protecting human life, promoting human dignity, reducing arms production, pursuing economic justice, protecting the environment and encouraging corporate responsibility. Each category lists specific areas of concern, such as abortion, racial discrimination, labor standards and other social needs in which the Church has had a voice.

Jeton said he hoped the data developed would influence how diocesan bishops in particular discuss finances with the folks in the pew.

They would do well to pay attention to mechanisms that would get more people engaged, Jeton said. Hopefully with greater engagement there is growing willingness (among donors) to go into their back pocket to help the Church.

Tags: Catholic Investment Services, Ethical investing

Category: U.S. & World News

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Catholics more motivated to donate if ethical investing is assured - The Catholic Spirit

Stem Cell Market: By Key Players, Market Competitive Landscape, Trends and Forecasts to 2024 – Joliet Observer

Global Stem Cell Market 2019 Analysis to serve a detailed research of primary, of this world, in-between and long-term trends to support the growth of already established challengers, emerging new companies and the detailed growth rate. This report also withgrowth trends,numerousstakeholders like investors, CEOs, traders, suppliers,analysis& media,internationalManager, Director, President, SWOT analysis i.e. Strength, Weakness, Opportunities and Threat to the organizationand others.

Top most List manufacturers/ Key player/ Economy by Business Leaders Leading Players of Stem Cell Market Are: CCBC, Vcanbio, Boyalife, Beikebiotech, . And More

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Overview of the Stem Cell Market:

Stem cells are a class of undifferentiated cells that are able to differentiate into specialized cell types. Commonly, stem cells come from two main sources: Embryos formed during the blastocyst phase of embryological development (embryonic stem cells) and Adult tissue (adult stem cells).,

Stem Cell Market Segment by Type covers:

Stem Cell Market Segment by Applications can be divided into:

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Stem Cell Market: By Key Players, Market Competitive Landscape, Trends and Forecasts to 2024 - Joliet Observer

Stem Cell Therapy Market to Witness a Pronounce Growth During 2020 – Gem Newz

Stem cells are most vital cells found in both humans and non-human animals. Stem cells are also known as centerpiece of regenerative medicine. Regenerative medicines have capability to grow new cells and replace damaged and dead cells. Stem cell is the precursors of all cells in the human body. It has the ability to replicate itself and repair and replace other damaged tissues in the human body. In addition, stem cell based therapies are used in the treatment of several chronic diseases such as cancer and blood disorders.

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The global stem cell therapy market is categorized based on various modes of treatment and by therapeutic applications. The treatment segment is further sub-segmented into autologous stem cell therapy and allogeneic stem cell therapy. The application segment includes metabolic diseases, eye diseases, immune system diseases, musculoskeletal disorders, central nervous system disorders, cardiovascular diseases and wounds and injuries.

In terms of geographic, North America dominates the global stem cell therapy market due to increased research activities on stem cells. The U.S. represents the largest market for stem cell therapy followed by Canada in North America. However, Asia is expected to show high growth rates in the next five years in global stem cell therapy market due to increasing population. In addition, increasing government support by providing funds is also supporting in growth of the stem cell therapy market in Asia. China and India are expected to be the fastest growing stem cell therapy markets in Asia.

In recent time, increasing prevalence of chronic diseases and increasing funds from government organizations are some of the major drivers for global stem cell therapy market. In addition, rising awareness about stem cell therapies and increasing focus on stem cell research are also supporting in growth of global stem cell therapy market. However, less developed research infrastructure for stem cell therapies and ethical issues related to embryonic stem cells are some of the major restraints for global stem cell therapy market. In addition, complexity related with the preservation of stem cell also obstructs the growth of global stem cell therapy market.

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Some of the major companies operating in the global stem cell therapy market are Mesoblast Ltd., Celgene Corporation, Aastrom Biosciences, Inc. and StemCells, Inc.

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Stem Cell Therapy Market to Witness a Pronounce Growth During 2020 - Gem Newz

On creativity, plasticity and repentance – Arutz Sheva

The young Israeli patient I visited in the isolation room at the Dana Farber Cancer Institute in Boston in the late 1970s was very ill. Ravages of the disease and its harsh treatment were clearly evident.

Diagnosed with a uniformly terminal disease, he had traveled to this renowned center in the Longwood Medical Area to be treated with a newly developed regimen of aggressive therapy. This world-famous academic hub is the medical campus where Harvard Medical School and many of its affiliated hospitals are all located. Major breakthroughs in medicine were developed here, including the first curative treatment of leukemia, the first kidney transplant, the first use of an electrical current to restore heart rhythm. It was here that creative man leaped forward with innovative advances that saved lives.

At the time I was a post-doctoral research fellow at Harvard Medical School. Our family resided several houses from the Ravs daughters home where the Rav was living, and our Maimonides community was fortunate to be able to spend eight to ten hours with the Rav every weekend in prayer and in learning. The creative gesture so central to the Ravs philosophy of halakhic man is a prime characteristic of biomedical research, and my oscillation between the universe of cutting edge biomedical research and the world of Torah learning with the Rav, while worlds apart, was harmonious. With the Rav often expressing curiosity about aspects of my research, I began to intuit that the work might not be merely creative but in fact a religious gesture.

A central theme in the Ravs weltanschauung is his emphasis on halakhic mans creative gesture. Ish haHalakhahs mission and continuous challenge are to heal and repair a purposely flawed world, for in the creation process a modicum of chaos was formed prior to the worlds creation and deliberately injected into both organic and inorganic matter, including into man himself.[1]

Man himself symbolizes, on the one hand, the most perfect and complete type of existence, the image of God, and, on the other hand, the most terrible chaos and void to reign over creation.[2] In order to enable man to cope with his inexorable chaos and sin, the tool of repentance was also created prior to the worlds formation (Pesahim 54a).

In a reference to creative introspection in Eight Chapters, Maimonides states: The perfect man needs to inspect his moral habits, weigh his actions, and reflect upon the state of his soul every single day. Whenever he sees his soul inkling toward one of the extremes, he should rush to cure it and not let the evil state become established (chap. 4).

Max Scheler, an early proponent of positive creative repentance, points out that modern philosophy, on the other hand, sees in retrospection and repentance mostly a negative, superfluous, uneconomical act due to disharmony of the mind and ascribed to lack of thought, sickness or various illusions..[3]

Emulating Maimonides, the Rav emphasizes an essential continuous remodeling, a re-creation of the sinners self as being a healthy, critically indispensable creative process. Halakhic man is engaged in self-creation, in creating a new I. He does not regret an irretrievably lost past but a past still in existence, one that stretches into and interpenetrates with the present and the future.[4]

The Ravs perspective on repentance is related to Schelers definition of creative repentance and to Henri Bergsons distinction between subjective, qualitative time-perception versus chronos, quantitative objective time. Both Scheler and Bergson ascribe to the principle of memory and experiential plasticity. The concept of plasticity, the property of being easily molded and remolded, has received intense scientific attention in the last decade, especially as related to the field of memory and neuroscience.

The presumed inability of the brain to generate new cells or to establish new neural networks is currently vigorously challenged and has indeed been proven incorrect. The process by which man can modify imprinted memories to affect his present and future behavior pattern is currently under scientific investigation. Epigenetic biochemical modifications of DNA and changes in neural networks triggered by ongoing experiences have been documented to alter both content and intensity of memories. The association between past triggering stimuli and the resurfacing of memories and behavior patterns has been shown to be moldable utilizing imaging and histological techniques. Previous memories can be reinforced, intensified, modified, or completely erased.

We no longer look at our genetic makeup and the mature brain as a fixed template that predicts our phenotype, and no longer are our memories an unalterable code. Rather, increasingly, biochemical data support the idea that they are templates upon which environmental and emotional stimuli can impact. Biochemical changes in the brain triggered by environmental and behavioral patterns were identified in identical twins raised in different environments. Scientists have defined conditions in which terminally differentiated cells, such as mature skin cells, which we assumed could never return to their embryonic pluripotent stem-cell status, have in fact definitively reverted and reprogrammed to evolve into new cell types. Recent reports have described the astonishing generation of live mice from skin cells reengineered to be ova.

If cells can revert to their embryonic state, if gene expression can be reprogrammed, if the brain can generate new nerve cells and establish new neural networks, the view of repentant man as a biologically defined new self is viable.

David Anderson from the California Institute of Technology describes a fascinating neuro-anatomical observation. The center in the brain that orchestrates emotion is the amygdala. It communicates with the hypothalamus, which houses the cells that control instinctive behavior like parenting, feeding, mating, fear, and fighting. Anderson found that a nucleus of cells within the hypothalamus contain two distinct populations of neurons: one that regulates aggression and one that regulates mating. About 20 percent of the cells in this nucleus are active both during mating activity and during aggressive behavior, which suggests that these two circuits are linked. How does the brain regulate these mutually exclusive behavior patterns? Anderson found that depending on the specific stimuli applied to this area it can trigger either mating activity or aggression.

Perhaps creating a new self through repentance from love (On Repentance, pp. 163) is associated with using mechanisms previously utilized for aggression and fear for productive activity such as love and fertility. A similar idea is found in the Babylonian Talmud (Shabbat 156a): He who is born under Mars will be a shedder of blood. Rabbi Ashi said: Either a surgeon, a thief, a slaughterer, or a circumciser. Through biochemical processes induced by the intense experiences of the teshuvah process confession, sacrifice, remorse, shame and a commitment to a new I a new self can emerge.

* * *

A decade after I visited the seriously ill young man at the Dana Farber Cancer Institute, the facility where chaos reigns and where creative man is faced with overwhelming challenges, I attended a scientific conference in Tiberias, burial place of Maimonides. I learned that a daily minyan was available at a nearby archeological site of an ancient synagogue on the shores of the magnificent Sea of Galilee. I was welcomed by a group of yeshiva students from Bnei Brak who had been coming weekly to maintain a minyan at this historical site.

Following services I was approached by a bearded man, who inquired:Are you Dr. Goldberg?

Since I had never practiced medicine in Israel I was surprised to be addressed as a physician.

Do you remember me? he asked. I am that patient you visited at the Dana Farber so many years ago. I am healthy, married and have several children.

The Prophets and the Torah as well recognized a strong connection between sin and illness on the one hand and between repentance and healing on the other (On Repentance, p. 80).

Through the creative gesture both the body and the spirit can be remodeled and healed.

Notes:

1. See Rabbi Joseph B. Soloveitchik, Halakhic Man (Philadelphia, 1983), p. 102.

2. Halakhic Man, p. 109.

3. Max Scheler, On the Eternal in Man (New Brunswick, NJ, 2010), p. 36.

4. Halakhic Man, p. 113.

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On creativity, plasticity and repentance - Arutz Sheva

What’s in the cards for this year’s Nobel Prizes? – STAT

Were not saying that discovering molecular drivers of cancer or cancer-causing genes doesnt deserve the Nobel Prize in medicine or physiology. But for Dr. Brian Druker (whose work led to the targeted leukemia drug Gleevec), Dr. Dennis Slamon (Herceptin), and Mary-Claire King (the BRCA breast- and ovarian-cancer gene), 2019 is probably not their year to be summoned to Stockholm: The 2018 medicine Nobel honored immuno-oncology, and according to STATs Nobel crystal ball, cancer wont win two years in a row.

With the naming of the science Nobels fast approaching the medicine prize will be announced on Oct. 7, physics on Oct. 8, chemistry on Oct. 9 polls, betting pools, and number crunching are in full swing, using approaches from toting up how many predictor prizes a scientist has won to calculating the periodicity of awards, meaning how many years pass before a specific subfield is honored again.

Past laureates, who get to submit nominations every year after their own anointing, have their favorites and, sometimes, their hunches, wrong though they usually are. As 2018 chemistry winner Frances Arnold of the California Institute of Technology said, Its not helpful to second-guess these things!

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Indeed, the Nobels are known for some head-scratching choices over the decades, though less in the science prizes than in peace and literature. Nevertheless, some experts have developed systems that do pretty well. Since 2002, David Pendlebury of Clarivate Analytics has made 50 correct predictions (though usually not in the right year) by analyzing how often a scientists key papers are cited by peers.

Using that strategy, Pendlebury thinks the chemistry Nobel could go toinventors of DNA sequencing techniques: Marvin Caruthers of the University of Colorado, Leroy Hood of the Institute for Systems Biology, and Michael Hunkapiller, CEO of DNA sequencing goliath Pacific Biosciences.Without their [1980s] inventions, Pendlebury said, there would be no map of the human genome.More on that below.

In medicine, Pendlebury likes the chances of Hans Clevers of the Netherlands Utrecht University for research on the Wnt signaling pathway. Wnt controls how stem cells differentiate and how some cancers develop. If Wnt wins, then the private biotech Samumed (valuation: $12 billion) can say its investigational Wnt-targeting drugs are based on Nobel-winning research.

Pendlebury also has his eyes on John Kappler and Philippa Marrack of National Jewish Health in Denver for discovering T-cell tolerance, a mechanism by which the thymus eliminates T cells that would attack the self. That advanced understanding of autoimmune diseases such as rheumatoid arthritis and lupus, and is so basic and important one wonders, Why no Nobel yet?

In 2019, the answer might be, because the 2018 prize honored the hybrid of immunology and cancer, so immunology might have to wait another decade for its next turn. Thats sad news for Jacques Miller, who has never received a Nobel for discovering the function of the thymus and the fact that immune cells include T and B cells in the 1960s. Look how many Nobels were given for immunology based on Millers work, said MITs Phillip Sharp, who shared the 1993 medicine Nobel. There is just a lot of good science that will never get recognized.

If immunology and cancer are off the table, it opens a lane for optogenetics, the revolutionary mashup of genetic engineering and neuroscience. Here, Pendlebury likes Ernst Bamberg of the Max Planck Institute of Biophysics in Germany, Karl Deisseroth of Stanford University, and Gero Miesenbck of the University of Oxford in the U.K. Honoring optogenetics would be wonderful on many levels, starting with the power of the technique to identify neural circuits involved in virtually any function and disease. (The Nobel committees like techniques that enable others to make cool discoveries, Sharp said.)

Even more fun, a prize for optogenetics could open the floodgates of controversy. The key discoveries have so many fathers (alas, optogenetics has no mothers) that picking any three, the maximum for a Nobel, would likely trigger weeks (maybe years!) of carping about who got left out. As it happens, on Thursday the $500,000 Warren Alpert Prize will honor optogenetics, but the winners are Deisseroth, Miesenbck, MITs Edward Boyden (Deisseroths former postdoc), and Peter Hegemann of Germanys Humboldt University. If science had cage matches, we could root for one between Bamberg, Boyden, and Hegemann for the third slot in an optogenetics Nobel.

Records of Nobel committee deliberations remain secret for 50 years, so its anyones guess if theyre influenced by other big prizes. But the latter do seem to have predictive value. Thats one reason David Allis of Rockefeller University and Michael Grunstein of Hebrew University, who in the 1990s discovered one way genes are activated and quieted (through proteins called histones), are favorites (again) for a medicine Nobel. Theyve shared a slew of awards, including a 2018 Lasker and a 2016 Gruber Prize in Genetics, so 2019 could (finally) be their year, especially since there hasnt been a Nobel for gene expression since 2006, and the Allis/Grunstein discovery basically launched the hot field of epigenetics.

Sharp points to another scientist who made seminal contributions to the understanding of genes off/on switches: Yale Universitys Joan Steitz, who in 1979 discovered small noncoding RNAs that control gene expression.

For his crystal ball, biologist Jason Sheltzer of Cold Spring Harbor Laboratory looks at periodicity, a strategy that helped him correctly predict last years medicine win for immune-oncology pioneer James Allison. Fields like infectious disease, immunology, and cancer win every 10 to 20 years, and so are probably off the table for 2019. But the last Nobel for DNA sequencing was way back in 1980, Sheltzer points out, and since then we have seen the complete sequencing of the human genome, one of humanitys towering achievements.

The problem is, hundreds of scientists worked on the Human Genome Project. Last year the Nobels honored the organizers of the project that discovered gravitational waves, so with similar reasoning the medicine or chemistry prize might go to Dr. Francis Collins, now director of the National Institutes of Health and the guy who herded all those cats to get the genome project done, and/or to the Broad Institutes Eric Lander, whose lab churned out much of the sequence. A third might be Craig Venter, the outsider whose private sequencing efforts raced the Collins/Lander government project to a bitter draw.

But if this trio wins, Pendleburys DNA-sequencing three (Caruthers, Hood, and Hunkapiller) wont. To complicate things even further, among the leading vote getters in an online poll for the chemistry Nobel is Shankar Balasubramanian of Cambridge University, who helped developed next-generation DNA sequencing. The Nobel committees grapple with questions of credit all the time: who did it, who did it first, who had the greatest impact, said chemist Peter Dorhout, past president of the American Chemical Society. I wouldnt want to be them.

For that and other messy reasons, when asked if sequencing has a shot at a Nobel, Sharp didnt hesitate: No, he told STAT.

Gene editing, on the other hand, last won in 1993, so it could be time for a new editing prize, Sheltzer tweeted. CRISPR in particular, [Jennifer] Doudna [of the University of California, Berkeley] will win for either chemistry or medicine.

The wisdom of the crowd agrees with him. Sigma Xi, the scientific research honor society, has been asking members to vote for most likely laureates in bracketology-style matchups. Doudna made the final four in chemistry. (Sigma Xi will announce the winner of its contest Thursday.) The other three finalists: Harvards Stuart Schreiber for research on signal transduction and master regulators of cell function such as the gene mTOR, now a hot target for cancer and other drugs; John Goodenough of the University of Texas for inventing lithium-ion batteries, and Stanfords Carolyn Bertozzi for basically developing bio-orthogonal chemistry, in which reporter molecules label biomolecules within cells.

Since STAT covers only life sciences, we have nothing to say about Goodenoughs chances (but thank you for making smartphones, digital cameras, and Teslas possible!), and agree that Schreiber and Bertozzi are stars. But the Doudna pick is a minefield. If she wins it alone, there will be hurt egos galore, starting with her collaborator Emmanuelle Charpentier of the Max Planck Institute for Infection Biology and including Virginijus iknys of Vilnius University. iknys is often called the forgotten man of CRISPR because Doudna and Charpentier had been running the table of prizes for their work turning a bacterial immune system into a DNA editor until he shared last years $1 million Kavli Prize in nanoscience.

If the Nobel committees prefer to keep firestorms of controversy for the peace prize (Greta Thunberg or Donald Trump?), they have many safe but stellar choices. Pierre Chambon and Ronald Evans have never won for discovering nuclear hormone receptors, where molecules as different as steroids and vitamins dock to make a whole suite of physiological reactions happen, Sharp points out. He also thinks Victor Ambros of the University of Massachusetts and Harvards Gary Ruvkun could bounce up this year for discovering microRNAs DNA-regulating molecules that turn out to control embryonic development, cancer, cell differentiation, and more. The pair has also snared a pile of predictor awards, including a Lasker, a Gairdner, a Breakthrough, and a Gruber Genetics Prize.

To all the many scientists whose work deserves a Nobel but who will not get an early-morning call from Stockholm next week, Sharp offers this quasi-consolation: Life is not fair.

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What's in the cards for this year's Nobel Prizes? - STAT

Moving beyond hype: Could one-two treatment restore damaged heart muscle? – University of Wisconsin-Madison

Heart attacks can cause immediate death. But in survivors, the blockage of blood flow can kill so many heart muscle cells that heart failure can follow months or years afterwards. Heart disease is the leading cause of hospital admission and death in the United States.

A heart attack causes a loss of muscle and leaves the heart with a scar that does not contract and so impairs the hearts pumping function, says Tim Kamp, a professor of medicine who is co-leader of a new grant designed to attack two roadblocks that have stymied efforts to restore heart muscle with muscle cells grown from stem cells.

Kamp, who directs the Stem Cell and Regenerative Medicine Center at the University of WisconsinMadison, says, Everybody involved in treating these patients knows that this scarring often leads to a continual decline in heart function with heart failure and even death.

The UWMadison researchers used approved surgical devices to locate the damaged heart muscle, and then injected the supportive matrix and committed cardiac muscle cells. The circle outlines target zone established before surgery; black dots show the sites that were injected in this mouse study. Amish Raval, work performed at UWMadison in collaboration with Biologics Delivery Systems.

Sixteen percent of men, and 22 percent of women, develop heart failure after myocardial infarction heart attack. Coronary artery disease the category that includes stoppage of blood flow causes one in seven deaths in the United States.

Adult stem cell injections seemed a logical way to form new heart muscle cells and repair the damaged muscle. But in dozens of experiments, the cells either washed out of the heart or failed to develop into the specialized muscle cells the cardiomyocytes that power cardiac contractions. The benefits were mixed, modest at best, says Kamp.

After years of preliminary investigations, however, Kamp and Amish Raval, a professor of cardiology, researcher and entrepreneur, hope that a combination of two cutting-edge approaches would use a fabric-like material to prevent wash-out and successfully implant cardiomyocytes to damaged hearts.

Aided by a Regenerative Medicine Innovation Project grant from the National Heart, Lung, and Blood Institute, part of the National Institutes of Health, the two will lead a group to test that idea in pigs over two years.

Having committed cells could be a major advance, Raval says. The first stem-cells therapies started with cells that I call the model T. Now, we are moving to the Buick. The cells originate as induced pluripotent stem cells (iPSCs) a relative of embryonic stem cells that is based on reprogramming adult cells.

Two Madison-based businesses, and sources at the University of WisconsinMadison, also helped to fund the research. Fujifilm Cellular Dynamics Inc., one of the largest commercial sources of stem cell products, produces the committed cardiac progenitor cells that will be tested. These committed cells are ready to transform themselves into cardiomyocytes.

Fujifilm bought CDI, a company whose founders included Kamp and UWMadison stem cell pioneer James Thomson, but the operations remain in Madison. Kamp has no ownership position but is a consultant for the company.

Raval is a founder and board chair of the second commercial supporter, Cellular Logistics, Inc., which makes a freeze-dried matrix from the same proteins that naturally holds cardiomyocytes in place in the heart. The material is called extracellular matrix (ECM) because it scaffolds cells from the outside.

When the heart pumps, internal pressures often eject would-be replacement cells through lymph channels and blood vessels. Ravals group has already shown in mice that injecting extracellular matrix proteins along with new cells creates mechanical restraints that avoid the wash-out problem.

The extra-cellular matrix to be used in the NIH grant at UWMadison helped retain stem cells (yellow dots) in a pig heart. When similar cells (blue) were injected without the matrix, the cells spilled out of the heart muscle through the needle track and lymph channels.Eric Schmuck and Amish Raval, work performed at UWMadison. Eric Schmuck and Amish Raval, work performed at UWMadison

The injected scaffold may have another advantage for regenerating muscle after heart attack, Kamp notes. The ECM replenishes the scarred area to become more hospitable to the replacement cardiomyocytes. The effect may be based on chemical and mechanical signaling between the ECM and the regenerating cells.

Pigs hearts are quite close to human hearts in size and structure. The grant will cover tests on four groups of 12 pigs each following myocardial infarction:

If the combination is effective, Raval adds, We plan to proceed toward a Food and Drug Administration application for an investigational new drug, which would allow us to begin human trials.

With the passion and concern of a working cardiac surgeon, Raval says those trials would focus on patients who have not been helped by the best medical management we know today and they are not candidates for heart transplant or mechanical assist devices. The only other option is palliative or hospice care.

As Raval notes, More people are surviving heart attacks, and thats great. But many are left with a scar in the heart muscle a dead zone. That scar can enlarge, and the damage can spread. So we are seeing an increasing number of patients with heart failure. Thats why we are moving forward with this project.

This research is being funded by NIH grant 1U01HL148690-01.

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Moving beyond hype: Could one-two treatment restore damaged heart muscle? - University of Wisconsin-Madison

Conjugated polymers optically regulate the fate of endothelial colony-forming cells – Science Advances

Abstract

The control of stem and progenitor cell fate is emerging as a compelling urgency for regenerative medicine. Here, we propose a innovative strategy to gain optical control of endothelial colony-forming cell fate, which represents the only known truly endothelial precursor showing robust in vitro proliferation and overwhelming vessel formation in vivo. We combine conjugated polymers, used as photo-actuators, with the advantages offered by optical stimulation over current electromechanical and chemical stimulation approaches. Light modulation provides unprecedented spatial and temporal resolution, permitting at the same time lower invasiveness and higher selectivity. We demonstrate that polymer-mediated optical excitation induces a robust enhancement of proliferation and lumen formation in vitro. We identify the underlying biophysical pathway as due to light-induced activation of TRPV1 channel. Altogether, our results represent an effective way to induce angiogenesis in vitro, which represents the proof of principle to improve the outcome of autologous cell-based therapy in vivo.

In recent years, organic semiconductors have emerged as highly promising materials in biotechnology, thanks to several key-enabling features. Differently from silicon-based electronics, they support both electronic and ionic charge transport (1); they can be easily functionalized with specific excitation and sensing capabilities (24); and they are solution processable, soft, and conformable (5). They are highly biocompatible, being suitable for in vivo implantation and long-term operation, as recently reported for many different applications, including electrocorticography, precise delivery of neurotransmitters, electrocardiography, deep brain stimulation, and spinal cord injury (69). An important, distinctive feature of organic semiconductors is their sensitivity to the visible and near-infrared light. Recently, our and other groups have exploited it for optical modulation of cell electrophysiological activity, by using conjugated polymers and organic molecules as exogenous light-sensitive actuators (1012). Interesting applications have been reported in the field of artificial visual prosthesis (5), photothermal excitation or inhibition of cellular activity (13, 14), and modulation of animal behavior (15).

In this framework, the opportunity to use polymer-based phototransduction mechanisms to regulate the very early stages of living cell development has been very scarcely considered (16, 17). The possibility to selectively and precisely regulate a number of cell processes, such as adhesion, differentiation, proliferation, and migration, would be key to regenerative medicine and drug screening. The presently dominant approaches to reliably regulate stem and progenitor cell fate for regenerative purposes mainly rely on the use of chemical cues. However, irreversibility and lack of spatial selectivity represent important limitations of these methods. Whenever targeting in vivo applications, one must face the major, unsolved problem of diffusion of neurotrophic molecules by the conventional intravenous or oral routes. In addition, the therapeutic outcome of autologous cell-based therapy is often impaired by low engraftment, survival, and poor integration of stem cells within the environment of the targeted tissue. Other stimuli, mainly consisting of mechanical and electrical cues, were recently reported to have some notable effects, and recent advances in nanotechnology and material science enabled versatile, robust, and larger-scale modulation of the cell fate. In particular, carbon-based materials and conjugated polymers led to interesting results (18). However, their distinctive visible light absorption was never exploited in optically driven techniques.

Use of light actuation has been proposed either by viral transfer of light-sensitive proteins, by optogenetics tools, or by absorption of endogenously expressed light-sensitive moieties, based on low lightlevel therapies (1921). In the first case, interesting results were obtained (22); however, this approach bears all the drawbacks related to the need for viral gene transfer. Photobiomodulation led to interesting outputs as well, but overall efficiency is hampered by the limited absorption of light-responsive molecules endogenously expressed in living cells.

In this work, we propose to couple the use of conjugated polymers with visible light excitation to gain optical control of cell fate. We focus our attention on endothelial progenitor cells (EPCs) and, in particular, on endothelial colony-forming cells (ECFCs), which are currently considered the bona fide best surrogate of EPCs (23). ECFCs are mobilized from the bone marrow and vascular stem cell niche to reconstruct the vascular network destroyed by an ischemic insult and to restore local blood perfusion (24). ECFCs may be easily harvested from peripheral blood, display robust clonogenic potential, exhibit tube-forming capacity in vitro, and generate vessel-like structures in vivo (24, 25), thereby representing a promising candidate for autologous cell-based therapy of ischemic disorders (24). Manipulating the signaling pathways that drive ECFC proliferation, migration, differentiation, and tubulogenesis could represent a reliable strategy to improve the regenerative outcome of therapeutic angiogenesis in the harsh microenvironment of an ischemic tissue, such as the infarcted heart (24, 25). Intracellular Ca2+ signals play a crucial role in stimulating ECFC proliferation and tubulogenesis by promoting the nuclear translocation of the Ca2+-sensitive nuclear transcription factor B (NF-B) (2628). It has, therefore, been suggested that intracellular Ca2+ signaling could be targeted to boost the regenerative potential of autologous ECFCs for regenerative purposes (29). For the above-mentioned reasons, ECFCs represent a valuable test bed model for assessing the possibility to exploit the visible light sensitivity of conjugated polymers to gain touchless, optical modulation of cell proliferation and function.

In this framework, we demonstrate that polymer-mediated optical excitation during the first steps of ECFC growth leads to a robust enhancement of both proliferation and tubulogenesis through the optical modulation of the Ca2+-permeable transient receptor potential vanilloid 1 (TRPV1) channel and NF-Bmediated gene expression. Our results represent, to the best of our knowledge, the first report on the use of polymer photoexcitation for the in vitro modulation of ECFC fate and function, thereby representing the proof of principle to obtain direct control of progenitor cell fate.

Figure 1A shows a sketch of the bio/polymer interface developed for obtaining optical control of ECFC proliferation and network formation, together with the polymer chemical structure and the optical absorption spectrum. The material of choice for light absorption and phototransduction is a workhorse organic semiconductor, widely used in photovoltaic and photodetection applications, namely, regioregular poly(3-hexyl-thiophene) (P3HT) (6). It is characterized by a broad optical absorption spectrum, in the blue-green visible region, peaking at 520 nm. P3HT outstanding biocompatibility properties have been reported in a number of different systems, both in vitro and in vivo, including astrocytes (30), primary neurons and brain slices (14), and invertebrate models of Hydra vulgaris (15). Chronical implantation of P3HT-based devices in the rat subretinal space did not show substantial inflammatory reactions up to 6 months in vivo (10). Here, polymer thin films (approximate thickness, 150 nm) have been deposited by spin coating on top of polished glass substrates, as detailed in Materials and Methods. Both polymer-coated and glass substrates have been thermally sterilized (120C, 2 hours), coated with fibronectin, and, lastly, used as light-sensitive and control cell culturing substrates, respectively. ECFCs have been isolated from peripheral blood samples of human volunteers and seeded on top of polymer and glass substrates.

(A) P3HT polymer optical absorption spectrum. Insets show the chemical structure of the conjugated polymer and a sketch of the polymer device used for cell optical activation. ECFCs are cultured on top of P3HT thin films, deposited on glass substrates. (B) ECFC viability at fixed time points after plating (24, 48, and 72 hours). Cell cultures were kept in dark conditions at controlled temperature (37C) and fixed CO2 levels (5%). No statistically significant difference was observed between the glass and polymer substrates at any fixed time point (unpaired Students t test). (C) Experimental setup and optical excitation protocol for evaluation of polymer-mediated cell photoexcitation effects on cell fate. Polymer and control samples are positioned within a sterilized, home-designed petri holder. Light scattering effects are completely screened. The geometry and the photoexcitation protocol have been implemented to minimize overheating effects and to keep the overall extracellular bath temperature fairly unaltered. Thirty-millisecond-long green light pulses are followed by 70 ms in dark condition.

ECFC proliferation on polymer substrates has been primarily assessed in dark conditions at three different time points, namely, 24, 48, and 72 hours after plating (Fig. 1B). Polymer-coated samples, while showing from the very beginning a slightly lower number of cells as compared with control substrates, exhibit a proliferation rate fully similar to cells plated on glass substrates (slope of the linear fitting is 0.034 0.003, R2 = 0.99 and 0.034 0.005, R2 = 0.96 for control and P3HT polymer samples, respectively).

Once assessed that the P3HT polymer surface represents a nicely biocompatible substrate for ECFC seeding and proliferation in the dark, we moved to investigate the effect of polymer photoexcitation. In more detail, to evaluate the effect of optical stimulation on cell proliferation and network formation, we continuously shined light for the whole temporal window required for cell growth, and we realized an ad hoc system suitable for operation within the cell incubator. The experimental configuration and the excitation protocol are schematically represented in Fig. 1C. Optical excitation is provided by a light-emitting diode (LED) source, with maximum emission wavelength at 525 nm, incident from the substrate side. The choice of the protocol, continuously administered to the cell cultures during early seeding and proliferation stages, has been mainly dictated by the need to avoid overheating effects, with possible negative outcomes on the overall cell culture viability. On the basis of these considerations, we opted for a protocol based on 30-ms excitation pulses, followed by a 70-ms dark condition, at a photoexcitation density of 40 mW/cm2. The whole protocol is continuously repeated for a minimum of 4 up to 36 hours, depending on the type of functional assay, at controlled temperature (37C) and CO2 levels (5%).

The temporally precise and spatially localized measurement of the temperature variation upon polymer photoexcitation at the polymer/cell interface (i.e., within the cell cleft) is not straightforward because it requires the use of localized, submicrometer probes with a fast response time. However, according to the heat diffusion equation, we expect that dissipation occurs within a few milliseconds, following exponential decrease dynamics (14). Moreover, we used the well-known method of the calibrated pipette (31) to characterize the temperature variation dynamics within the extracellular bath volume, defined by the cylinder with the base area corresponding to the light spot size and the height of about 1 m. This choice is a good approximation of the overall volume occupied by a single ECFC cell; thus, it provides a realistic estimation of the average heating experienced by the cell (fig. S1A). We observe that temperature variation closely follows short optical pulse dynamics, reaching a maximum temperature at the end of the 30-ms illumination period, quickly followed by an almost complete thermal relaxation to the basal temperature during the 70-ms-long dark period. We conclude that our polymer-based system provides a highly spatially and temporally resolved method for optical excitation, making it possible, in perspective, to selectively target single cells and even cell subcompartments. Upon prolonged illumination (hours), one should also consider possible overheating effects of the whole extracellular medium volume. The average temperature of the bath for the entire duration of the long-term experiment was measured by a thermocouple immersed in the medium. Data show that an equilibrium situation is established after 5 hours and that the absolute temperature of the bath is increased by about 1.5 (fig. S1B). The adopted prolonged excitation protocol does not negatively affect overall cell culture viability (see below).

Figure 2 reports specific effects mediated by P3HT substrates and visible light stimulation on ECFC proliferation. ECFCs were plated in the presence of EGM-2 medium to facilitate the adhesion to the substrate. After 12 hours, the medium was switched to EBM-2 supplemented with 2% fetal bovine serum, and the cells were subjected to the long-term lighting protocol for 36 hours at controlled temperature (37C) and CO2 levels (5%). Under these conditions, ECFCs seeded on P3HT and subjected to light stimulation undergo a significant increase in proliferation rate, as compared with the control condition, i.e., to cells also seeded on P3HT polymer substrates but kept in dark conditions for the whole duration of the experiment (+158% versus P3HT dark; P < 0.05). No statistically significant difference in proliferation was observed among cells seeded on glass, whether they were subjected to optical excitation or not (Fig. 2A).

(A) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT thin films, together with corresponding control samples kept in dark conditions. Cell proliferation was measured after 36 hours of culture in the presence of EBM-2 supplemented with 2% fetal calf serum. (B) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence (CTRL) and presence of 10 M capsazepine (CPZ), 10 M ruthenium red (RR), 20 M RN-1734 (RN-1734), and 30 M BAPTA-AM (BAPTA). The results are represented as the means standard error of the mean (SEM) of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way analysis of variance (ANOVA) coupled with Tukey (A) or Dunnetts (B) post hoc test. *P < 0.05.

Recent evidence demonstrated an interesting correlation between processes key to ECFC vascular regeneration, including proliferation and network formation, and activation of TRPV1 channels, which are expected to be endogenously expressed in ECFCs (32). In addition, we recently reported that polymer photoexcitation leads to selective TRPV1 activation in transfected human embryonic kidney (HEK) cell models (33). Therefore, we were prompted to evaluate whether the increase in cell proliferation is distinctively determined by a polymer-mediated photoactivation of the TRPV1 channel. To this goal, we preliminarily checked the actual expression of the TRPV1 channel in the ECFC models by carrying out electrophysiology experiments in patch-clamp configuration. Methods and results are extensively discussed in the Supplementary Materials (fig. S2 and related description). Briefly, the expression of the TRPV1 channel was confirmed, as well as the capability to selectively excite its activity through localized polymer excitation at high optical power density. To establish whether the TRPV1 channel also has a role in the observed increase in cell proliferation upon polymer excitation, we performed the experiments under light illumination upon administration of a highly specific TRPV1 antagonist [capsazepine (CPZ), 10 M], an aspecific TRPV channel inhibitor [ruthenium red (RR), 10 M], and a selective antagonist of a different temperature-sensitive channel, TRPV4, which is also endogenously expressed in ECFCs (RN-1734, 20 M) (34) (Fig. 2B). TRPV1 inactivation by CPZ and RR results in a relative, strong reduction in cell proliferation by 51 and 30%, respectively, as compared with untreated cells. Conversely, in the case of RN-1734 treatment, the proliferation increase due to polymer photoexcitation is completely unaltered.

As mentioned earlier, intracellular Ca2+ signaling has been reported to drive ECFC proliferation (26, 28). To further investigate whether TRPV1-mediated extracellular Ca2+ entry mediates the proangiogenic response to light illumination, we pretreated ECFCs with [1,2-Bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester) BAPTA-AM] (30 M), a membrane-permeable buffer of intracellular Ca2+ levels (26, 28). BAPTA-AM is widely used to prevent the increase in intracellular Ca2+ concentration ([Ca2+]i) induced by extracellular stimuli and inhibits the downstream Ca2+-dependent processes. For instance, BAPTA-AM represents the most suitable tool to prevent the activation of Ca2+-sensitive decoders residing within tens of nanometers from the inner pore of plasmalemmal Ca2+ channels (35). It was recently reported that, in the absence of Ca2+-mobilizing growth factors, it does not impair the low rate of ECFC growth (27). Here, however, BAPTA-AM clearly reduced the light-driven proliferation increase, thus confirming that TRPV1 stimulates ECFCs through an increase in [Ca2+]i (Fig. 2B).

We further examined the physiological outcome of chronic light stimulation by carrying out a tube formation assay within an extracellular matrix protein-based scaffold, which is a surrogate of the basement membrane extracellular matrix. This assay recapitulates many steps of the angiogenic process, including adhesion, migration, protease activity, and tubule formation (27, 28). ECFCs were plated in the presence of EBM-2 medium supplemented with 2% fetal calf serum and subjected to the long-term lighting protocol for 8 hours at controlled levels of temperature and CO2. Control experiments carried out in dark conditions, either onto glass (see Fig. 3A for a representative optical image) or onto polymer substrates (Fig. 3C), as well as control experiments carried out upon photoexcitation of cells seeded on glass substrates (Fig. 3B), do not show remarkable differences. Conversely, ECFC cultures subjected to polymer-mediated optical excitation clearly tend to assemble into an extended bidimensional capillary-like network (Fig. 3D). Cell cultures were monitored up to 24 hours after illumination onset, but results were comparable to observations reported here, after 8 hours of illumination. This qualitative observation is fully confirmed by quantitative morphological analysis (27). As depicted in the sketch of Fig. 3E, we quantitatively evaluated the main features typical of the capillary-like network formation and, in particular, the number of master segments (Fig. 3F), master junctions (Fig. 3G), and meshes (Fig. 3H). In all cases, a notable, statistically relevant difference is observed between cells subjected to polymer-mediated optical excitation and controls. Within the same considered temporal window, the combined use of polymer substrates and visible light stimuli does not lead to sizable toxicity effects or delays in cell proliferation. Conversely, it leads to enhanced cell proliferation (Fig. 2) and allows the achievement of the formation of a more extended and mature tubular network (Fig. 3).

(A to D) Representative images of in vitro tubular networks of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT, as well as on corresponding control samples in dark conditions. Cultures were observed up to 24 hours, but their appearance did not substantially change after pictures were taken after 8-hour culture. Scale bars, 250 m. (E) Sketch representing the main features typical of the capillary-like network that were considered for the topologic analysis. Number of master segments (F), master junctions (G), and meshes (H) analyzed in the different conditions. The results are represented as the means SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Tukey post hoc test. **P < 0.01 and ***P < 0.001.

As evidenced for the proliferation rate, the TRPV1 channel activation emerges to play also a fundamental role in tubulogenesis (Fig. 4). The TRPV1 pharmacological blockade with the specific inhibitor CPZ deterministically leads to a marked reduction in network formation (Fig. 4A). Upon CPZ administration, a statistically significant decrease in the relative variation of the number of master segments (Fig. 4E), master junctions (Fig. 4F), and meshes (Fig. 4G) is observed. In line with the results shown in Figs. 2 and 3, RR administration resulted in a less marked but still sizable reduction in the tubular network (Fig. 4, B and E to G), probably due to the minor specificity toward TRPV1, while the protubular effect of light remained fully unaltered in the presence of the TRPV4 inhibitor RN-1734 (Fig. 4, C and E to G). Notably, the treatment with BAPTA-AM (30 M), which affected ECFC proliferation, was able to prevent also in vitro tubulogenesis, thus corroborating the key role of intracellular Ca2+ signaling in the proangiogenic response to light illumination (Fig. 4, D and E to G). Control measurements carried out in dark conditions on polymer substrates upon the considered pharmacological treatments do not show any relevant effect (fig. S4, A to C). Overall, this evidence supports the notion that TRPV1 stimulates ECFC proliferation and network formation and demonstrates that optical excitation, properly mediated by biocompatible polymer substrates, positively affects ECFC fate by spatially and temporally selective activation of the TRPV1 channel.

(A to D) Representative optical images of in vitro tubular network of ECFCs subjected to long-term optical excitation seeded either on bare glass or on P3HT thin films and treated respectively with CPZ (A), RR (B), RN-1734 (C), and BAPTA-AM (D). Scale bars, 250 m. (E to G) Relative variation of number of master segments (E), master junctions (F), and meshes (G) of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence [control (CTRL)] and presence of 10 M CPZ, 10 M RR, 20 M RN-1734 (RN-1734), and 30 M BAPTA-AM (BAPTA). The results are represented as the means SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Dunnetts post hoc test. *P < 0.05 and **P < 0.01.

We now turn our attention to elucidating the possible mechanisms leading to optically enhanced tubulogenesis, through TRPV1 channel activation, upon prolonged polymer excitation.

Reliable optical modulation of the cell activity mediated by polymer photoexcitation has been reported in several, previous reports, both in vitro, at the level of single cells, and in vivo, at the level of the whole animal, as evidenced by behavioral studies on both invertebrate and vertebrate models. Three different photostimulation mechanisms, active at the polymer/cell interface, have been proposed so far. These include (i) the creation of an interface capacitance, i.e., of a localized electric field, possibly affecting the cell membrane potential (11); (ii) photothermal processes, establishing a localized temperature increase upon polymer photoexcitation (13, 36); and (iii) photoelectrochemical reactions, mainly oxygen reduction processes, leading to a local variation of extracellular and/or intracellular pH (33) and sizable production of reactive oxygen species (ROS), at a nontoxic concentration, and intracellular calcium modulation (37).

In electrophysiological experiments, carried out at a photoexcitation density higher than the one used in chronic stimulation by about two orders of magnitude, we clearly observe TRPV1 excitation, corresponding however to a small variation of the cell membrane potential, in the order of a few millivolts (Supplementary Materials). Thus, upon much lower light intensity, the effects of either direct photothermal channel activation and of photocapacitive charging are expected to be negligible. To further corroborate this hypothesis, we carry out control experiments aimed at disentangling photoelectrical from photothermal transduction processes.

First, we use a different material as a cell-seeding substrate, characterized by optical absorption and heat conductivity similar to the ones typical of P3HT (13) but fully electrically inert (i.e., unable to sustain electronic charge generation upon photoexcitation). The material of choice is a photoresist (MicroPosit S1813). S1813 thin films are realized by spin coating, and deposition parameters are optimized to obtain optical absorbance values similar to the semiconducting polymer samples at the considered excitation wavelength. The capability of photoresist substrates to sustain ECFC proliferation was successfully assessed in a control measurement, obtaining fully comparable results with respect to the P3HT substrates (Fig. 5A). The functional effect eventually driven by photoresist optical excitation on tubulogenesis was then investigated by using the same experimental conditions and analysis technique previously adopted for polymer and glass substrates (Fig. 5B). Data show that long-term photoresist excitation does not lead to sizable enhancement of the cellular network formation, thus pointing out that a purely photothermal effect does not play a major role in boosting the tubulogenesis process at variance with semiconducting polymer substrates. In a complementary experiment, we directly assessed the occurrence of photoelectrochemical reactions at the polymer/extracellular bath interface by measuring ROS production. We previously demonstrated that P3HT polymer thin films exposed to saline electrolytes sustain efficient light-triggered charge generation and charge transfer processes, giving rise to photoelectrochemical reactions (38, 39). Moreover, we also reported that P3HT nanoparticles are efficiently internalized within the cytosol of secondary line cell models (HEK-293) and that their photoexcitation leads to the production of ROS and subsequent intracellular calcium modulation (15, 37). However, the actual capability to sustain photoelectrochemical reactions in the specific experimental conditions used in this work (polymer film deposition conditions, sterilization process, prolonged exposure to specific cellular growth medium in an incubating environment, prolonged exposure to a light excitation protocol, light wavelength, pulses duty cycle, and power density) was never assessed. In particular, direct measurement of intracellular ROS was never carried out in the presence of polymer thin films. To this goal, we realized ECFC cultures on top of polymer and glass control substrates, and we exposed them to the same optical stimulation protocol previously used in the tubulogenesis assay. ROS production was then evaluated by means of a fluorescence experiment based on the use of the well-known ROS probe 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Fig. 5C). Results show that light induces an increase in ROS production both on glass and polymer substrates. Relative percentage variation amounts to +34 and +200%, respectively, thus pointing out that polymer surface photocatalytic activity plays a major role in the phototransduction phenomenon.

(A) An electrically insulating, thermally conducting material (photoresist) is successfully used as an ECFC seeding substrate. (B) Photoresist long-term photoexcitation does not lead to sizable enhancement in tubulogenesis parameters. (C) Evaluation of intracellular ROS production following long-term photoexcitation protocol of ECFC cultures on polymer and glass substrates (glass dark, n = 629; glass light, n = 656; P3HT dark, n = 686; and P3HT light, n = 583). For each panel, the results are represented as the means SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with unpaired Students t test (A and B) or one-way ANOVA coupled with Tukey post hoc test (C). ***P < 0.001.

Altogether, data in Fig. 5 indicate that photoelectrochemical reactions induced by light at the interface between the organic semiconducting polymer and the extracellular bath play a key role in triggering the observed enhancement in cell network formation through indirect activation of the TRPV1 channel. The occurrence of faradaic phenomena at the polymer/bath interface may give rise to material degradation effects. The photostability of the polymer substrates was carefully checked by optical absorption, photoluminescence, and Raman spectra measurements. By treating the samples with the same experimental protocol used for cell tubulogenesis assays (photoexcitation density, pulses frequency, overall exposure duration, temperature, and humidity levels), no sign of irreversible polymer degradation was observed, as compared with nonilluminated samples (fig. S5).

The Ca2+-sensitive transcription factor NF-B might provide the missing link between the influx of Ca2+ through TRPV1 and the increase in proliferation and tubulogenesis observed in ECFCs upon photostimulation (26). We therefore monitored the nuclear translocation of the cytoplasmic p65 NF-B subunit via immunofluorescence staining and mRNA levels of a number of genes induced during tubulogenesis in an NF-Bdependent manner (26, 40) (Fig. 6). Our data indicate that ECFCs seeded on polymer and subjected to light stimulation have a significantly enhanced p65 NF-B nuclear translocation compared with the control conditions consisting of cells also seeded on P3HT but kept in dark conditions (+35% versus P3HT dark; P < 0.05; Fig. 6, A and B), and seeded on bare glass (+28% versus glass dark; P < 0.05; Fig. 6B). No differences were observed between samples seeded on glass, whether they were subjected to optical excitation or not (fig. S6).

ECFCs seeded on P3HT samples and glass controls are subjected to long-term photostimulation protocol. Corresponding control samples are kept in dark conditions. After photostimulation, p65 NF-B nuclear translocation (A and B) and mRNA levels of tubulogenic/angiogenic genes that have been shown to be activated downstream of NF-B (C) are evaluated. (A) Representative images of immunofluorescence staining showing p65 NF-B (green) nuclear translocation. Cell nuclei are detected by 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 m. (B) Quantitative evaluation of p65 NF-B nuclear translocation, as evidenced by colocalization experiments. Results are expressed as means SEM of the relative percentage of p65 nucleipositively stained cells to the total number of cells (glass dark, n = 151; glass light, n = 125; P3HT dark, n = 147; and P3HT light, n = 159). Ten fields per condition are analyzed. Data are obtained from two different experiments conducted on cells harvested from two different donors. (C) mRNA levels of intercellular adhesion molecule 1 (ICAM1), selectin E (SELE), and matrix metalloproteinases (MMP1, MMP2, and MMP9) are quantified by real-time polymerase chain reaction (PCR). Data are expressed as means SEM of percentage variation with respect to cells grown in the dark (n = 6). The significance of differences was evaluated with unpaired Students t test (C) or one-way ANOVA coupled with Tukey post hoc test (B). *P < 0.05 and **P < 0.01.

In addition, we have checked the expression of nine genes whose expression is known to be induced in endothelial cells during tubulogenesis/angiogenesis in an NF-Bdependent manner. We considered intercellular adhesion molecule 1 (ICAM1); vascular adhesion molecule 1 (VCAM1); selectin E (SELE), matrix metalloproteinases (MMPs) 1, 2, and 9; vascular endothelial growth factor A (VEGFA); cyclooxygenase 2 (COX2, PTGS2); and cyclin D1 (CCND1) (40). Of these, five are significantly up-regulated by light exposure in cells grown on P3HT substrates, namely, ICAM1 (+90% versus P3HT dark; P < 0.05), SELE (+1119%; P < 0.01), MMP1 (+242%; P < 0.01), MMP2 (+467%; P < 0.05), and MMP9 (+458%; P < 0.05) (Fig. 6C). Conversely, VCAM1, VEGFA, PTGS2, and CCND1 do not show relevant variation upon light stimulation (fig. S7A). Light excitation on cells grown on bare glass substrates does not show any significant effect as compared with control samples in dark conditions (fig. S7B).

Therapeutic angiogenesis via autologous EPC transplantation represents a promising strategy to preserve or, at least, partially restore cardiac function after myocardial infarction (24, 41). Nevertheless, the regenerative outcome of EPC-based therapies in preclinical studies was rather disappointing and did not lead to sufficient neovascularization of the ischemic heart (41). This led to the proposal to boost their angiogenic activity by using emerging technologies, including tissue engineering of vascular niches, pharmacological preconditioning, or genetic and epigenetic reprogramming (42). ECFCs are regarded among the most suitable EPC subtypes to induce therapeutic angiogenesis and cardiac regeneration due to their high clonal proliferative potential and ability to assemble into capillary-like structures (23, 24). In addition, they can be easily isolated and expanded from the peripheral blood of patients and healthy donors. It has recently been suggested that their angiogenic activity could be boosted by targeting the intracellular Ca2+ toolkit (29). Here, we target ECFCs by adopting a fully different approach, i.e., by exploiting visible light as a modulation trigger and by the use of a thiophene-based conjugated polymer as the exogenous, light-responsive actuator. We demonstrate that photoexcitation of the organic material deterministically leads to robustly enhanced proliferation and tubulogenesis. Pharmacological assays, supported by electrophysiology experiments, allow the identification of TRPV1 selective excitation as a key player in the molecular pathway leading to macroscopic outcomes, as observed by quantitative analysis of the angiogenic response.

All data unambiguously show that polymer photoexcitation leads to selective activation of the TRPV1 channel, which has recently been shown to be expressed and drive angiogenesis in human ECFCs (32). TRPV1 is a polymodal Ca2+-permeable channel that integrates multiple chemical and physical cues to sense major changes in the local microenvironment of most mammalian cells (43). TRPV1 is activated by either noxious heat (>42C) and acidic solutions (pH < 6.5), whereas mild acidification (pH 6.3) of the extracellular milieus sensitizes TRPV1 to heat stimulation and results in channel activation at temperature thresholds (30 to 32C) well below the normal one (43). ROS production is also expected to further contribute to TRPV1 activation, as previously reported in mouse coronary endothelial cells (44), in which hydrogen peroxide elicits a depolarizing inward current at negative holding potentials. Likewise, ROS may stimulate TRPV1 to depolarize the membrane potential, thereby triggering trains of action potentials in airway C fibers (45, 46).

On the basis of measurements carried out in cells seeded on the photoresist substrate, as well as on direct evaluation of a limited, local temperature increase upon light stimuli during the long-term photoexcitation protocol, we infer that the excitation of the TRPV1 channel through direct photothermal transduction is not the predominant process leading to enhanced tubulogenesis.

We have previously demonstrated that polymer photoexcitation leads to generation of faradaic current, to electron transfer reactions at the polymer/electrolyte interface, and to sizable intracellular enhancement of ROS (37, 38). Briefly, optical excitation of P3HT polymer thin films leads to photoexcited species (Eq. 1), namely, singlets and charge states, which react with the oxygen dissolved in the cell medium, thus reducing oxygen (Eq. 2)P3HT+hP3HT*(1)P3HT*+O2P3HT++O2(2)

The superoxide further evolves, leading to the generation of different ROS and, lastly, ending up with hydrogen peroxide production. It has been reported that extracellular H2O2 can cross the plasma membrane through aquaporin AQP3, thereby triggering intracellular ROS signaling (47, 48). In line with our previous results, we have demonstrated here that intracellular ROS enhancement does occur in ECFCs upon photoexcitation of polymer thin films, thus contributing to TRPV1 activation.

Altogether, the evidence supports the hypothesis of a transduction mechanism mainly governed by photoelectrochemical reactions. Moreover, these same observations could explain why TRPV4, which is also expressed in ECFCs (34), is not sensitive to optical modulation. Although TRPV4 is activated by moderate heat (24 to 38C), it is supposed to be inhibited by local pH variation, although this is still a matter of debate (49, 50).

On the one hand, the role attributed in the phototransduction mechanism to the capability of the polymer to generate and transport electronic charges, as well as to its photocatalytic activity in an aqueous environment, clearly implies the need for a biocompatible, visible lightresponsive, semiconducting material. This excludes any possible implementation of the reported technique by using a thermally conducting, electrically insulating plastic substrate. Suitable cell-seeding materials have to be selected and developed within the wide arena of organic semiconducting polymers. On the other hand, the key role played by ROS raises additional issues about material photostability, cell viability, and overall safety and reliability of the technique. We extensively verified that the main polymer optoelectronic properties are not substantially altered by the exposure to light and to incubating conditions. From the biological point of view, it is very well known that high ROS levels can induce highly toxic effects and, finally, lead to cell death. We notice, however, that the established photoactivation protocol (illuminator geometry and air flow, light photoexcitation density, duty cycle, and repetition rate) has been implemented to avoid any detrimental effect. Accordingly, no toxicity effects were detected for the overall duration of the experiments, as proven by the robust increase in ECFC proliferation and tubulogenesis exposed to light. This observation is consistent with the emerging notion that appropriate ROS levels can exert a signaling role and control angiogenesis in endothelial cells (51).

The biophysical mechanisms whereby the photoactivation of TRPV1 stimulates in vitro angiogenesis in ECFCs deserve a more detailed discussion as well. Earlier work showed that TRPV1 stimulates proliferation and tube formation in vascular endothelial cells by mediating extracellular Ca2+ entry. The following increase in intracellular Ca2+ concentration ([Ca2+]i) leads to the recruitment of several downstream Ca2+-dependent decoders, such as endothelial nitric oxide synthase and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (52). Recently, TRPV1 was found to induce also proliferation and tube formation in ECFCs by mediating the uptake of the endocannabinoid anandamide (32). This study, however, did not investigate whether TRPV1 activation was per se able to stimulate ECFCs by engaging Ca2+-dependent pathways. Intracellular Ca2+ signaling is a crucial determinant of ECFC fate and behavior (2628). Accordingly, light-induced ECFC proliferation and tube formation were markedly reduced by the pharmacological blockade of TRPV1-mediated Ca2+ entry with CPZ and RR and by preventing the subsequent increase in [Ca2+]i with BAPTA-AM. This finding endorses the view that optical excitation stimulates ECFCs through TRPV1-mediated extracellular Ca2+ entry, and we suggest here that this occurs via downstream activation of transcriptional factor NF-B. NF-B has previously been shown to stimulate cell proliferation and tubulogenesis in endothelial cells (53, 54) and in hepatocytes (55). Our group has shown that NF-B triggers the transcriptional program underlying the angiogenic response to extracellular Ca2+ entry in ECFCs (26). Moreover, NF-B activation in response to extracellular stimulation and Ca2+ entry through TRPV1 has also been demonstrated (56, 57). Under resting conditions, NF-B is retained in the cytoplasm by the complex with the inhibitory protein IB. An increase in [Ca2+]i results in IB degradation by ubiquitination, which is triggered upon the Ca2+-dependent phosphorylation of IB. As a consequence, the p65 NF-B subunit is released from IB inhibition and translocates into the nucleus (58) where it induces the expression of multiple proangiogenic genes (40). Consistently, we found that optical excitation significantly boosted the nuclear translocation of p65 in ECFCs cultured on the conjugated polymer compared with those not exposed to light. Robust up-regulation of several angiogenic genes, such as ICAM, SELE, MMP1, MMP2, and MMP9, which are under NF-Bdependent transcriptional control, was also consequently observed. Intriguingly, NF-B also mediates VEGFA-induced gene expression and angiogenesis in vascular endothelial cells (59, 60) through an increase in [Ca2+]i (61). These observations strongly hint at NF-B as the Ca2+-sensitive decoder that translates optical excitation into an angiogenic response in human ECFCs interfaced with the light-sensitive conjugated polymer.

Overall, our findings represent the proof of principle that optical modulation may be successfully exploited to directly control the fate of a progenitor cell population, i.e., ECFCs, which has been shown to support revascularization of ischemic tissues. The in vitro activation of ECFC angiogenic activity is made possible by the use of a biocompatible, light-sensitive polymer as the phototransduction element.

The combined use of optical excitation and organic polymer technology can open interesting perspectives for several different reasons. First, the use of light modulation allows unprecedented spatial and temporal resolution to be achieved in a fully reversible way. Light temporal and spatial patterns can be specifically designed and adapted to different in vitro cell models, allowing ideally endless combinations of possibilities, to finely tune overall output in cell proliferation and network formation. The demonstrated technology is minimally invasive, allows for massive parallelization of experiments, and can be virtually implemented in any cell therapy model in a straightforward way. In addition, the use of different polymers, with lower energy gap and in the form of nanobeads, may pave the way to the optical enhancement of therapeutic angiogenesis in vivo. Further work is needed to understand whether the pattern and/or intensity of the illumination protocol may be adjusted to further boost the angiogenic response. For instance, the optical excitation protocol consisted of 30-ms-long light pulses that were delivered at 1 Hz for 4 (tubulogenesis) up to 36 (proliferation) hours. This is likely to result in oscillations in [Ca2+]i, which are known to deliver the most instructive signal for ECFCs to undergo angiogenesis by inducing the nuclear translocation of the p65 NF-B subunit (26). As the frequency of intracellular Ca2+ oscillations can be artificially manipulated to regulate NF-Bdependent gene expression in virtually any cell type (62), we envisage an additional layer of specificity and control that could be exploited to further improve the angiogenic response to optical excitation. Future work will also be devoted to assess the outcome of optical modulation on patient-derived ECFCs. One of the main hurdles associated to autologous cell-based therapy is the impairment of the angiogenic activity of EPCs, including ECFCs harvested from cardiovascular patients (29). The therapeutic translation of our findings will require the demonstration that light-induced TRPV1 activation boosts angiogenesis also in ECFCs derived from individuals affected by severe cardiovascular disorders, such as hypertension, atherosclerosis, and heart failure. In this view, the combination of organic semiconductors and genetic manipulation to increase endogenous TRPV1 expression could be sufficient to restore the reparative phenotype of autologous ECFCs from cardiovascular patients.

Regioregular P3HT (99.995% purity; Mn 54,000 to 75,000 molecular weight) was purchased from Sigma-Aldrich and used without any further purification. The samples for cell cultures were prepared by spin coating on a square 18 mm by 18 mm glass (VWR International) substrates carefully rinsed in subsequent ultrasonic baths of ultrapure water, acetone, and isopropanol. P3HT solution was prepared in chlorobenzene at a final P3HT concentration of 20 g/liter and spin coated on the cleaned substrates with a two-step recipe: (i) 3 s at 800 rpm and (ii) 60 s at 1600 rpm. Polymer film thickness is about 150 nm.

Microposit S1813 photoresist was purchased from Shipley and used without any further purification. Photoresist thin films were prepared by spin coating on cleaned substrates with a two-step recipe: (i) 3 min at 300 rpm and (ii) 30 s at 2600 rpm. Parameters were adjusted to obtain homogeneous films and similar optical absorbance to the one of the polymer thin films, at the same excitation wavelength used in the long-term stimulation protocol (see below). All films were thermally treated in an oven at 120C for 2 hours for annealing and sterilization. To promote adhesion, samples were coated with fibronectin (from bovine plasma; Sigma-Aldrich) at a concentration of 2 mg/ml in phosphate-buffered saline (PBS) for at least 30 min at 37C and then rinsed with PBS.

ECFCs were isolated from peripheral blood and expanded as shown elsewhere (26). Blood samples (40 ml) collected in EDTA-containing tubes were obtained from healthy male human volunteers aged from 28 to 38 years. The Institutional Review Board at Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo Foundation in Pavia approved all protocols and specifically approved this study. Informed written consent was obtained according to the Declaration of Helsinki of 1975 as revised in 2008. We focused on the so-called ECFCs, a subgroup of EPCs that are found in the CD34+ CD45 fraction of circulating mononuclear cells (MNCs), exhibit robust proliferative potential, and form capillary-like structures in vitro (23). To isolate ECFCs, MNCs were obtained from peripheral blood by density gradient centrifugation on lymphocyte separation medium for 30 min at 400g and washed twice in EBM-2 with 2% fetal calf serum. A median of 36 106 MNCs (range, 18 to 66) was plated on fibronectin-coated culture dishes (BD Biosciences) in the presence of the endothelial cell growth medium EGM-2 MV (Lonza) containing endothelial basal medium (EBM-2), 5% fetal bovine serum (FBS), recombinant human (rh) EGF, rhVEGF, recombinant human Fibroblast Growth Factor-Basic (rhFGF-B), recombinant human Insulin-like Growth Factor-1 (rhIGF-1), ascorbic acid, and heparin and maintained at 37C in 5% CO2 and humidified atmosphere. Nonadherent cells were discarded after 2 days, and thereafter, medium was changed three times a week. The outgrowth of ECFCs from adherent MNCs was characterized by the formation of a cluster of cobblestone-shaped cells. That ECFC-derived colonies belonged to the endothelial lineage was confirmed by staining with anti-CD31, anti-CD105, anti-CD144, anti-CD146, antivon Willebrand factor, anti-CD45, and anti-CD14 monoclonal antibodies and by assessment of capillary-like network formation in the in vitro tube formation assay.

For our experiments, we have mainly used endothelial cells obtained from early-passage ECFCs (P2-4, which roughly encompasses a 15- to 18-day period) with the purpose to avoid, or maximally reduce, any potential bias due to cell differentiation. However, to make sure that the phenotype of the cells did not change throughout the experiments, in the preliminary experiments, we tested the immunophenotype of ECFCs at different passages, and we found no differences. We also tested whether functional differences occurred when early (P2) and late (P6)passage ECFCs were used by testing the in vitro capacity of capillary network formation in a Cultrex assay and found no differences between early- and late-passage ECFC-derived cells (data not shown).

Electrophysiological recordings were performed using a patch-clamp setup (Axopatch 200B; Axon Instruments) coupled to an inverted microscope (Nikon Eclipse Ti). ECFCs were measured in whole-cell configuration with freshly pulled glass pipettes (3 to 6 M), filled with the following intracellular solution: 12 mM KCl, 125 mM K-gluconate, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM EGTA, 10 mM Hepes, and 10 mM ATP (adenosine 5-triphosphate)Na2. The extracellular solution contained the following: 135 mM NaCl, 5.4 mM KCl, 5 mM Hepes, 10 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2. Only single cells were selected for recordings. Acquisition was performed with the pCLAMP 10 software (Axon Instruments). Membrane currents were low pass filtered at 2 kHz and digitized with a sampling rate of 10 kHz (Digidata 1440 A; Molecular Devices). Data were analyzed with Clampfit (Axon Instruments) and Origin 8.0 (OriginLab Corporation).

For optical excitation of the polymer, a homemade petri cell culture illuminator, compatible with the use within the cell incubator, was designed and implemented. Its design included a black spacer made by fused filament fabrication, both to minimize overheating effects in the extracellular bath and to avoid unwanted light scattering/diffusion effects and cross-talk between different specimens. Optical excitation was provided by a green LED system, whose duty cycle, repetition rate, and intensity were set through a custom-made control circuit, comprising a microcontroller, a digital-to-analog converter, and an analog LED driver. The driver was connected to five green LEDs (SMB1N-525V-02; Roithner LaserTechnik GmbH, Vienna, Austria), with maximum emission wavelength at 525 nm, each carrying a collimator lens reducing the emission angle to 22. This way, up to five 3.5-cm petri dishes can be simultaneously treated with a homogeneous photoexcitation density of 40 mW/cm2. The long-term optical excitation protocol adopted for cell fate modulation consists of 30-ms-long pulses, followed by 70-ms-long dark conditions, continuously repeated for a minimum of 4 up to 36 hours in the case of tubulogenesis and proliferation assays, respectively.

Growth dynamics were evaluated by plating a total of 5 103 ECFC-derived cells into 10-mm fibronectin-treated cloning cylinders (5 104/cm2) in the presence of EGM-2 MV medium to facilitate the adhesion. After 12 hours, the medium was switched to EBM-2 supplemented with 2% fetal calf serum. For the pharmacological treatment, one of compounds was added to the medium: BAPTA (30 M), CPZ (10 M), RN-1734 (20 M), or RR (10 M). Cultures were incubated at 37C (in 5% CO2 and humidified atmosphere), and cell growth was assessed after 36 hours since the beginning of the long-term illumination protocol. At this point, cells were recovered by trypsinization from all the dishes, and the cell number was assessed by counting in a hemocytometer. Preliminary experiments showed no unspecific or toxic effect for each agent when used at these concentrations. Each assay was repeated in triplicate.

ECFC-derived cells from early-passage (P2 to P4) cultures were obtained by trypsinization and resuspended in EBM-2 supplemented with 2% FBS. EPC-derived cells (10 103) per well were plated in Cultrex basement membrane extract (Trevigen Inc., Gaithersburg, MD, USA) 10-mm fibronectin-treated cloning cylinders. Plates were then incubated at 37C, 5% CO2, and capillary network formation was assessed starting from 4 to 24 hours later. At least three different sets of cultures were performed every experimental point. Quantification of tubular structures was performed after 8 hours of incubation by measuring the total length of structures per field with the aid of the ImageJ software (National Institutes of Health, USA; http://rsbweb.nih.gov/ij/). To evaluate the role of TRPV1, the same protocol was repeated in the presence of the following drugs: BAPTA (30 M), CPZ (10 M), RN-1734 (20 M), or RR (10 M).

H2DCF-DA (Sigma-Aldrich) was used for the intracellular detection of ROS. ECFCs were seeded onto polymer and control substrates and subjected to the same photoexcitation protocol used for the in vitro tube formation assay. Immediately after the end of the protocol, cell cultures were incubated with the ROS probe for 30 min. After careful washout of the excess probe from the extracellular medium, the fluorescence of the probe was recorded (excitation/emission wavelengths, 490/520 nm; integration time, 70 ms for H2DCF-DA) with an inverted microscope (Nikon Eclipse Ti) equipped with an Analog-WDM Camera (CoolSNAP MYO, Teledyne Photometrics). To minimize the effects of the spectral overlap between the polymer absorption and emission spectra, and the probe emission, samples were turned upside down by using a homemade chamber with a 500-m-thick channel filled with extracellular medium. Variation of fluorescence intensity was evaluated over regions of interest covering single-cell areas, and reported values represent the average over multiple cells. See figure captions for additional details about statistical analysis. Image processing was carried out with ImageJ and subsequently analyzed with Origin 8.0.

Two sets of P3HT thin films (n = 12) were prepared as described above. The optical absorbance, the emission, and the Raman spectrum were measured immediately after fabrication. Then, all samples were exposed to ECFC growth medium (EBM-2 supplemented with 2% FBS) and incubated at 37C, 5% CO2 for 24 hours. The first set was taken in dark conditions (n = 6), and the second one was treated with the same optical excitation protocol used in the tubulogenesis assays (n = 6). After incubation, absorption, emission, and Raman spectrum were measured again in the same conditions as before. Absorption spectra were recorded by using a spectrophotometer (PerkinElmer Lambda 1040) in transmission mode. Photoluminescence spectra were acquired by using a Jobin-Yvon spectrofluorometer; the excitation wavelength was set at the polymer absorption peak wavelength (530 nm). Resonant Raman spectra were recorded by using visible light excitation at 532 nm (HORIBA Jobin-Yvon HR800 micro-Raman spectrometer system). Laser power intensity on the sample was kept at values lower than 0.03 mW to avoid laser-induced sample degradation. Spectra were typically recorded in the region 600 to 2000 cm1 and were calibrated against the 520.5 cm1 line of an internal silicon wafer. The signal-to-noise ratio was enhanced by repeated acquisitions (100). The measurements were conducted at room temperature (RT), and the resulting spectral resolution was 0.4 cm1.

To examine NF-B p65 subunit translocation into the nucleus in the individual ECFCs, the coverslips were fixed with 4% formaldehyde in PBS (20 min at RT) and permeabilized with 0.1% Triton X-100 in PBS (7 min at RT). Primary rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology, catalog no. Sc-372) was applied at a final dilution of 1:100 for 1 hour at 37C in a humidified chamber. After three washes with PBS, secondary chicken anti-rabbit Alexa(488)-conjugated antibody (1:200; Invitrogen, catalog no. A-21441) was applied for 1 hour at RT. After washing (three times in PBS), nuclei were counterstained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI; 1:5000 dilution in PBS; 20 min at RT; Invitrogen, catalog no. D1306). Last, the coverslips with cells were mounted on microscope glass slides using Fluoroshield mount medium (Sigma, catalog no. F6182). Fluorescence images were taken with the same fluorescence microscope used for the electrophysiology experiments, using standard DAPI and fluorescein isothiocyanate filters set for the acquisition of DAPI and Alexa(488) fluorescence emission, respectively.

Cells were lysed in 0.5 ml of TRI Reagent (Sigma, catalog no. T9424), and total RNA was extracted according to the manufacturers protocol. One microgram of total RNA was retrotranscribed using SensiFAST cDNA Synthesis Kit (Bioline, London, UK, catalog no. BIO-65054). Real-time polymerase chain reaction (PCR) was performed using iTaq qPCR master mix according to the manufacturers instructions (Bio-Rad, Segrate, Italy, catalog no. 1725124) on a SFX96 Real-Time System (Bio-Rad). As a control, S18 ribosomal subunit was used, whose expression did not change across the conditions. For each gene, Ct was calculated by using the formula Ct = 2^(Ct(gene) Ct(S18)). The data are expressed as a percentage variation between P3HT light and glass light conditions and P3HT dark and glass dark samples, respectively. Sequences of oligonucleotide primers are listed in table S1.

The significance of differences was evaluated with unpaired Students t test or one-way analysis of variance (ANOVA) coupled with Tukey or Dunnetts post hoc test, as appropriate. Data are represented as means standard error of the mean (SEM). P < 0.05 was considered statistically significant. Statistical analysis was performed using the GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA).

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaav4620/DC1

Fig. S1. Local and global evaluation of the extracellular bath temperature.

Fig. S2. TRPV1 is endogenously expressed in ECFCs, and it is efficiently activated by polymer photostimulation.

Fig. S3. Current clamp measurements in HEK-293 cells.

Fig. S4. Pharmacological study on ECFCs seeded on polymer substrates in the darkEvaluation of effect on tubulogenesis.

Fig. S5. Polymer photostability.

Fig. S6. p65 NF-B nuclear translocation is unaltered in ECFCs seeded on glass subjected to light-induced photostimulation.

Fig. S7. mRNA levels of proangiogenic genes downstream of NF-B signaling.

Table S1. List of oligonucleotide primers used for real-time PCR.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We gratefully thank I. Abdel Aziz for the characterization of the homemade petri cell culture illuminator used for long-term optical excitation and P. Falvo for the constructive criticism of the manuscript and the helpful scientific discussions. Funding: This work was jointly supported by the European Research Council (ERC) under the European Unions Horizon 2020 research and innovation program LINCE, grant agreement no. 803621 (M.R.A.), the EU Horizon 2020 FETOPEN-2018-2020 Programme LION-HEARTED, grant agreement no. 828984 (F.L., F.M., and M.R.A.), the Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (20182022)Department of Biology and Biotechnology L. Spallanzani, University of Pavia (F.M.), and Fondo Ricerca Giovani from the University of Pavia (F.M.). Author contributions: F.L., F.M., and M.R.A. planned the experiments. F.L. carried out the experimental measurements (electrophysiology, short- and long-term photoexcitation, evaluation of effects on proliferation, tubulogenesis, and ROS production). V.R. provided the ECFC models, took care of the cell cultures, and contributed to the tubulogenesis and proliferation experiments. G.T. prepared the polymer samples. A.D. designed, realized, and optimized the experimental setup for the long-term photoexcitation. L.T. and D.L. carried out the immunofluorescence and real-time PCR assays. P.C. contributed to the methodological discussion about gene expression. F.L. and M.R.A. wrote the main manuscript, with help from F.M. All authors contributed to the data interpretation and approved the final manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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