Category Archives: Embryonic Stem Cells

DiNAQOR Acquires EHT Technologies GmbH to Advance Engineered Heart Tissue R&D Capabilities – PRNewswire

PFFFIKON, Switzerland, Jan. 19, 2021 /PRNewswire/ -- DiNAQOR, a gene therapy platform company,today announcedthat it has acquired EHT Technologies GmbH, a Germany-based engineered heart tissue (EHT) technology platform company. Financial terms of the transaction were not disclosed.

EHT Technologies was founded in 2015 based upon research on human induced pluripotent stem cells (hiPSC) at the University Medical Center Hamburg-Eppendorf. Cardiomyocytes derived from hiPSC are an innovative research technology for cardiac drug development programs. Engineered heart tissues are three-dimensional, hydrogel-based muscle constructs that can be generated from isolated heart cells of chicken, rat, mouse, human embryonic stem cells and hiPSC. Proof-of-concept studies have shown that EHT can be transduced efficiently with adeno-associated virus (AAV) vectors, including AAV9, validating the use of this platform for gene therapy applications.

"EHT Technologies' proprietary hiPSC platform for disease modeling is a perfect complement to DiNAQOR's research and development efforts and leaps forward our ability to develop creative approaches for treating heart diseases in the future. EHT's intellectual property and know-how is industry-leading and we are excited to be able to harness its platform at DiNAQOR," commented Johannes Holzmeister, M.D., Chairman and CEO at DiNAQOR.

"After more than 25 years of development, I'm very excited that our engineered heart tissue technology is making the transition from an academic research model to a drug development tool. The combined application of human cardiomyocytes and a versatile, 3D in vitro assay will facilitate development and reduce reliance on animal studies. The hiPSC-derived EHT assay has great potential for the development of innovative cardiovascular therapeutics and DiNAQOR is the perfect fit for this enterprise," commented Professor Thomas Eschenhagen, M.D., co-founder of EHT Technologies. Professor Eschenhagen serves on DiNAQOR's Scientific Advisory Board.

"The EHT technology will accelerate the advancement of our discovery pipeline and bridge the translational gap between the animal model and human disease. We are proud that DiNAQOR is on the forefront of implementing this innovative technology to expedite new therapies into the clinic," said Valeria Ricotti, M.D., Chief Medical Officer at DiNAQOR.

About DiNAQORFounded in 2019,DiNAQOR is a global gene therapy platform company focused on advancing novel solutions for patients suffering from heart disease.The company is headquartered in Pfffikon, Switzerland, with additional presence in London, England and Hamburg, Germany. For more information visitwww.dinaqor.com.

ContactKWM Communications Kellie Walsh [emailprotected] or Stephanie Marks [emailprotected]

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DiNAQOR: A global gene therapy platform company

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DiNAQOR Acquires EHT Technologies GmbH to Advance Engineered Heart Tissue R&D Capabilities - PRNewswire

TBC1D3 promotes neural progenitor proliferation by suppressing the histone methyltransferase G9a – Science Advances

INTRODUCTION

The expansion of the cerebral cortex during primate evolution is assumed to be associated with the acquisition of higher intelligence especially in the human species (1, 2). This process involves increased proliferative ability of cortical neural progenitors (NPs), including the ventricular radial glial cells (vRGs), the intermediate progenitors, and basal or outer RGs (bRGs or oRGs) (3, 4), which give rise to neurons or glia cells directly or indirectly. By contrast, disruption of the proliferative capacity of RGs has been shown to cause malformations of the cortex, which usually leads to intellectual disability (5). It has been shown that the epigenetic mechanisms, especially modifications of chromatin, play a critical role in regulating transcriptional programs that govern the stemness of NPs (6). However, the role of epigenetic factors in the process of cortical expansion during human evolution remains to be explored.

The epigenetic regulation at the level of chromatin is tightly controlled by posttranslational histone modification (7). The mono- or dimethylation of lysine 9 (K9) site at histone 3 (H3) (H3K9me1 and H3K9me2) mediated by histone methyltransferases G9a [also known as EHMT2 (euchromatic histone-lysine N-methyltransferase 2)] and G9a-like protein (GLP, also known as EHMT1) marks transcriptionally repressive genomic loci, which symbolize gene silencing in mammals (8). Notably, genetic ablation of G9a or GLP in the forebrain of adult mice has been shown to reactivate NP genes, leading to defects in cognitive and adaptive behaviors (9). Deletions or mutations of G9a/GLP genes are a cause of Kleefstra syndrome, a rare genetic disorder characterized by intellectual disability, autistic-like features, childhood hypotonia, and distinctive facial features (10). In addition, the differentiation of retinal progenitor cells requires G9a-mediated silencing of genes that sustain a proliferative state (11). It would be of interest to determine how the G9a activity is tightly controlled in neural stem cells during proliferation, in particular in human cortical NPs, which exhibit increased numbers of mitotic cell division compared with mice.

The cross-species analyses of epigenetic modifications between primate species and rodents have revealed major phenotypic changes during mammalian evolution (12, 13). Notably, comparative epigenetic analysis of human, rhesus macaque, and mouse genomes has identified the gained activity of promoters and enhancers in humans, which are substantially enriched in modules crucial for neural proliferation (14). Nevertheless, the factors responsible for these epigenetic differences underlying human brain development remain unclear.

Recently, we have found that the expression of the hominoid-specific gene TBC1D3 promotes production of cortical NPs, leading to expansion and folding of the cortex in mice (15). Here, we report a regulatory role of TBC1D3 for G9a-mediated H3K9me2 modification in human cortical NPs. We found that TBC1D3 physically interacted with G9a and inhibited G9a activity. Down-regulation of G9a promoted the proliferation of human NPs, resulting in expansion of human cerebral organoids. Disruption of TBC1D3/G9a interaction up-regulated the level of H3K9me2 and suppressed the expansion of human cerebral organoid. These results indicate that the inhibition of G9a by TBC1D3 ensures enhanced proliferation of human NPs and the expansion of human cerebral cortex.

In line with the hypothesis that duplication of specific genes in human might contribute to brain evolution, we found that expression of TBC1D3 gene, which is duplicated to form multiple paralogs in human genome and present in the chimpanzee genome as a single copy (16, 17), promoted cortex expansion and folding in mice (15). To assess whether the copy number correlated with the expression level of TBC1D3 in the chimpanzee and human, we analyzed accessible online datasets of RNA-sequencing (RNA-seq) information in NPs and differentiated neurons derived from induced pluripotent stem cells (18). Notably, the expression of TBC1D3 in chimpanzee cells was barely detectable, while it exhibited a substantial level in human cells, both in NPs and neurons (fig. S1A). To further determine the role of TBC1D3 in human cellular contexts, we generated human cerebral organoids by using guided differentiation of the H9 human embryonic stem cells (hESCs) into neuroectoderm with the addition of inhibitors for transforming growth factor (TGF-) or bone morphogenetic protein (BMP) pathway (19), as well as matrix embedding to promote neuroepithelium formation and organoid assembling (20). The quantitative polymerase chain reaction (PCR) analysis of organoids at different cultured stages revealed the expression of TBC1D3 since cultured day 16 (D16) as well as the appearance of forebrain marker FOXG1 (fig. S1, B and C). Then, we analyzed the effect of TBC1D3 up-regulation on organoid development by transducing hESCs with lentivirus encoding TBC1D3 (rLV-TBC1D3) or vehicle alone (rLV-Ctrl) (Fig. 1A). As shown in fig. S1D, the level of TBC1D3 mRNA in hESCs transduced with rLV-TBC1D3 was significantly higher than that in control cells. Intriguingly, the organoids with TBC1D3 up-regulation were markedly larger in size compared with control organoids at either the neuroectodermal stage (D12) or 6 days after induction for neuronal differentiation (D18) (Fig. 1B). Further analysis showed that the percentage of cells positive for KI67, a marker for proliferative cells, or phospho-vimentin (P-VIM), which marks mitotic radial glia cells, as well as PAX6, a typical marker for cortical NPs, among total DAPI+ (4,6-diamidino-2-phenylindolepositive) cells markedly increased in TBC1D3 organoids (Fig. 1C and fig. S1E). Thus, TBC1D3 up-regulation promotes the proliferation of NPs in human cerebral organoids.

(A) Schematic diagram for human cerebral organoid culture. (B) Analysis for the size of organoids (Ctrl, 6 organoids in D12 and 15 in D18; TBC1D3, 11 organoids in D12 and 17 in D18). Scale bars, 100 m. (C) Analysis for the percentage of KI67+ or P-VIM+ cells in D18 organoids (14 neuroepithelial rosettes from 6 control organoids; 16 rosettes from 8 TBC1D3 organoids). Scale bar, 20 m. (D) Analysis for the percentage of PAX6+ (white arrowheads) or DCX+ (yellow arrowheads) cells among GFP+ cells in D40 organoids infected with adenovirus (AV) expressing shTBC1D3 or shCtrl. Scale bar, 20 m. shCtrl, 23 rosettes from 15 organoids; shTBC1D3, 19 rosettes from 13 organoids. (E) TBC1D3 distribution in ReN cells. Scale bar, 10 m. (F) Immunostaining for PAX6, TBR2, and TBC1D3 in GW15.5 fetal human cortex. Scale bars, 200 m (left) and 5 m (magnified). Histograms show the percentage of cells with TBC1D3 enriched in cytoplasm/membrane (Cyt/Mem; gray arrows) or nucleus (Nuc; red arrows). Five regions for cortical plate (CP) or OSVZ; six regions for VZ/ISVZ from three slices. Data are presented as means SD, unpaired Students t test. **P < 0.01; ***P < 0.001.

To determine TBC1D3s loss-of-function effects, organoids at D37 were infected by adenovirus encoding small hairpin RNA sequence against TBC1D3 (AV-shTBC1D3) or scrambled control sequence (AV-shCtrl) (15), with vector-encoded green fluorescent protein (GFP) marking infected cells. As shown in Fig. 1D, the shTBC1D3 organoids at D40 exhibited marked decrease in the percentage of PAX6+ cells among GFP-marked infected cells, whereas the percentage of newborn differentiated neurons positively labeled by doublecortin (DCX) increased significantly (Fig. 1D). Furthermore, the shTBC1D3 organoids at D60 also exhibited a marked reduction in the percentage of cells labeled by HOPX (fig. S1F), a marker for oRGs (21). Thus, down-regulation of TBC1D3 impeded NP proliferation and caused precocious neuronal differentiation in human cerebral organoids.

Previous studies have shown the cytoplasmic or membrane localization of TBC1D3 in several non-neuronal cell types (2224). To reveal the molecular mechanism of TBC1D3 in human cortical development, we determined the subcellular localization of TBC1D3 in human neural cells. First, immunostaining of cultured human neural stem cell ReN showed that albeit TBC1D3 was widely distributed in the whole cell, it was enriched in the nuclei, which were marked by DAPI and devoid of -tubulin (Fig. 1E). Next, we determined the expression of TBC1D3 in human fetal brain at gestation week 15.5 (GW15.5) and found that TBC1D3 was expressed widely in PAX6- or TBR2-labeled NPs in ventricular zone (VZ)/inner subventricular zone (ISVZ) or outer subventricular zone (OSVZ) regions (fig. S1G). Intriguingly, while the signal of TBC1D3 was distributed dominantly in cytoplasmic and membrane regions in cells located in the cortical plate, it was enriched in the nuclei of a majority of TBR2+ cells in the OSVZ and a fraction of PAX6+ cells in VZ/ISVZ (Fig. 1F). The nuclear localization of TBC1D3 was also confirmed by immunoblot analysis for the biochemical fractions of human fetal brain tissues and ReN cells (fig. S1H). Substantial TBC1D3 signals were also observed in the nuclei of cells in cultured human cerebral organoids at D16 or D24, and the cells in organoids at D40 exhibited much less TBC1D3 signals in the nuclei (fig. S1I). The immunofluorescence signals for TBC1D3 were specific because cells in organoids infected by AV-shTBC1D3 exhibited markedly decreased TBC1D3 signals compared with uninfected cells, while AV-shCtrl had no effect (fig. S1J). These results suggest that the subcellular localization of TBC1D3 is dynamic, and its nuclear distribution in human NPs suggests a mechanism underlying its role in regulating NP proliferation.

To gain further insights into molecular mechanisms by which TBC1D3 executes its functions in human cortex development, we searched for proteins that directly interact with TBC1D3 using the yeast two-hybrid (Y2H) system (Fig. 2A). A screen of human fetal brain complementary DNA (cDNA) library using as bait the full length of TBC1D3 led to the identification of around 20 hits. Among them, G9a was the only candidate that has been shown to control transcriptional regulation in nervous system (9). Three cDNA clones in Y2H encoded fragments of G9a, with the sequences covering a part of ankyrin repeats (ANK) and the entire SET domain, which has the methyltransferase activity (Fig. 2B) (8, 25). To determine whether TBC1D3 and G9a interact in mammalian cells, hemagglutinin (HA)tagged G9a and Myc-TBC1D3 were cotransfected into human embryonic kidney (HEK) 293 cells, and cell lysates were subjected to immunoprecipitation (IP). We found that IP of HA-G9a caused co-IP of Myc-TBC1D3, and vice versa (Fig. 2C). Furthermore, we observed the interaction between endogenous TBC1D3 and G9a, as IP with G9a antibody caused co-IP of TBC1D3 in homogenates of the GW15 human cortical tissue (Fig. 2D). In the human fetal brain, the TBC1D3 signals were colocalized with that of G9a in the nucleus of cells in the OSVZ (fig. S2A). The direct interaction between TBC1D3 and G9a was further verified using a pull-down assay. HEK293T cells were transfected with a construct encoding Myc-tagged TBC1D3 (Myc-TBC1D3), and then the cell lysates were incubated with beads containing glutathione S-transferase (GST) protein or GST-tagged recombinant fragment of 649 to 1210 amino acids of G9a, which contained C-terminal ANK and SET domains and thus was shortened as G9a-CF (CF represents C-terminal fragment). As shown in Fig. 2E, G9a-CF, but not GST alone, interacted with TBC1D3. The truncated CF containing the catalytic SET domain (879 to 1210 amino acids), but not the ANK domain (649 to 879 amino acids), was able to bind TBC1D3 directly (fig. S2B). These results indicate that TBC1D3 directly interacts with G9a in the developing human cortex.

(A) Schematic diagram for Y2H screening assay. (B) Domain structure of human G9a protein and sequences of positive clones. (C) IB analysis of reciprocal co-immunoprecipitation (co-IP) results in HEK293T cells transfected with HA-G9a or HA-G9a plus Myc-TBC1D3. (D) Homogenates of GW15 human fetal cortical tissues were subjected to IP with anti-G9a antibody with immunoglobulin G (IgG) as a control and IB with antibodies against TBC1D3 or G9a. Shown is an example of two independent experiments with similar results. (E) Homogenates of HEK293cells transfected with Myc-TBC1D3 were subjected to pull-down with beads coupled with GST or GST-G9a-CF [649 to 1210 amino acids (aa)], followed by IB with anti-Myc antibody. (F) Addition of 6xHis-TBC1D3 attenuates the Histone3 methylation activity of G9a. Relative levels of H3K9me2 with respect to that of Histone3 from three independent experiments were quantified. (G) Levels of H3K9me2 in ReN cells transfected with control (Myc-Ctrl) or Myc-TBC1D3 plasmid (six independent experiments). (H) Levels of H3K9me2 in human cerebral organoids infected with AV-shCtrl or AV-shTBC1D3 (three independent experiments). The quantified data are presented as means SD by unpaired Students t test. *P < 0.05; **P < 0.01. bp, base pair.

Having shown the interaction between TBC1D3 and G9a, we next investigated whether this interaction regulates the methyltransferase activity of G9a. To this end, we used the in vitro histone methylation system. Because the full length of G9a was difficult to be purified, we generated G9a-CF as the catalytic enzyme instead. As shown in fig. S2C, in the presence of methyl group donor SAM (S-adenosyl-l-methionine), purified G9a-CF was capable of mediating H3K9me2 modification. Addition of TBC1D3 to the histone methylation system significantly decreased the level of H3K9me2 (Fig. 2F, lanes 3 and 4), suggesting the inhibitory effect of TBC1D3 on G9a activity. TBC1D3 itself had no effect on H3K9me2 modification (Fig. 2F, lanes 1 and 2).

We then determined whether TBC1D3 regulates histone methylation in human neural stem cells. We found that transfection with Myc-TBC1D3 in ReN cells caused a marked decrease in the level of H3K9me2 but had no effect on some other histone modifications such as H3K9me3, H3K27me2, and H3K36me2, compared with the vehicle control group (Fig. 2G and fig. S2D). In addition, H9 hESCs transduced with rLV-TBC1D3 as well as later induced cerebral organoids also showed markedly decreased H3K9me2 compared with vehicle control group (fig. S2, E and F). By contrast, down-regulation of TBC1D3 by small interference RNA in D40 human cerebral organoids caused a marked increase in the level of H3K9me2 (Fig. 2H). Thus, the level of TBC1D3 is reversely correlated with that of H3K9me2, supporting the hypothesis that TBC1D3 suppresses the activity of G9a.

Our previous study has demonstrated that the TBC1D3 transgenic (TG) mice show increased cortical expansion and folding (15). We determined levels of H3K9me2 in wild-type and TG mice and found that TG mice exhibited a decreased level of H3K9me2 at embryonic days 14.5 (E14.5) and 17.5 (E17.5) (fig. S2, G and H). These results suggest a correlation between the states of H3K9me2 modification and cortex expansion during evolution.

Next, we determined whether inhibition of G9a had any effect on cortex development by using compound UNC0638, a specific and competitive inhibitor of G9a with high efficiency and low cytotoxicity (26). First, we treated ReN cells with different concentrations of UNC0638 for 24 hours and found that these treatments reduced the level of H3K9me2 in a dose-dependent manner (fig. S3A). Then, an optimized concentration of UNC0638 (1 M) was added into the medium during organoid induction (D12) with dimethyl sulfoxide (DMSO) as control, with drug-containing medium renewed every other day. Again, UNC0638 treatment resulted in notable reduction in the level of H3K9me2, as measured by either immunoblotting (IB) (Fig. 3A) or immunostaining (fig. S3B). UNC0638-treated organoids exhibited a marked increase in size at different culture stages as exampled in D30 and D40 (Fig. 3B). Then, different molecular markers were used for detailed immunochemistry analysis for NPs and differentiated neurons. We found that the percentage of cells labeled by KI67 or the expression of PAX6 increased significantly in UNC0638-treated organoids at D18 (fig. S3, C and D). The augmentation of NPs in UNC0638-treated organoids persisted until later stages at D30, as reflected from increased percentage of cells labeled by PAX6, P-VIM, or KI67 (Fig. 3, C, D, and E to G). The cortical identity of cultured organoids was further confirmed by the appearance of TBR2-marked intermediate progenitors and DCX-labeled differentiated neurons, which were also increased in UNC0638-treated samples (Fig. 3, C, D, F, and I). We believe that G9a inhibition might have promoted replenishment of NPs, subsequently leading to enhanced neurogenesis.

(A) Levels of H3K9me2 in D16 cerebral organoids treated with G9a inhibitor UNC0638 or DMSO control, determined by IB. (B) Representative images of D30 and D40 cerebral organoids treated with UNC0638 or vehicle control and quantification for the average diameter. Numbers of organoids analyzed: D30, 19 organoids for control and 20 organoids for UNC0638 group; D40, 11 organoids for control and 12 organoids for UNC0638 group. Scale bar, 1000 m. (C) Immunostaining for signals of PAX6, TBR2, and DAPI in D30 organoids treated with UNC0638 or DMSO. Scale bar, 50 m. (D) Immunostaining for signals of KI67, P-VIM, and DCX in D30 organoids treated with UNC0638 or DMSO control, with DAPI marking cell nucleus. Scale bar, 50 m. (E and F) Quantification for the percentage of PAX6+ cells (E) or TBR2+ cells (F) among DAPI+ cells (20 rosettes from 14 control organoids; 31 rosettes from 17 UNC0638-treated organoids). (G to I) Quantification for the percentage of KI67+ (G), P-VIM+ (H), or DCX+ cells (I). Twenty-three rosettes from 15 organoids were analyzed in each group. Data are presented as means SD, unpaired Students t test. ***P < 0.001.

To investigate the proliferation and neuronal competency of NPs, D30 organoids were infected with GFP-expressing retrovirus, followed by culture for an additional 3 days in virus-free medium, to label dividing cells and the daughter progeny (fig. S3E). We found that the percentage of GFP+ cells among DAPI+ cells increased in UNC0638-treated organoids (fig. S3F), while the percentage of KI67+ GFP+ or DCX+ GFP+ among total GFP+ cells had no difference (fig. S3, G and H). These results suggest that G9a inhibition expanded the pool of dividing NPs without altering cellular lineage composition and neuronal competency. The effect induced by UNC0638 is reminiscent of the expansion of the human cerebral organoids with overexpression of TBC1D3 as well as a mutation of PTEN (27).

To precisely manipulate the interaction between TBC1D3 and G9a and seek the functional relevance, we mapped the TBC1D3 region that is essential for the interaction. We generated a battery of truncated forms of TBC1D3 tagged with Myc at the N terminus (Fig. 4A) and cotransfected each of them with HA-G9a into HEK293 cells. Then, the cell lysates were subjected to IP with anti-HA antibody. In the first round of domain mapping, we generated mutated forms of TBC1D3 with sequential deletion of a quarter of full length and found that the mutant with the deletion of C terminus (TBC1D3413549) failed to be associated with G9a, whereas other mutants maintained the binding activity (Fig. 4B). This result suggests that the C terminus of TBC1D3 is essential for the interaction with G9a. Based on this, we did other rounds of narrowing down to pinpoint the regions essential for the interaction (Fig. 4, C to E). Last, the minimal region was mapped to 465 to 481 amino acids, because TBC1D3465481 did not interact with G9a (Fig. 4E), but further partitioning had no effect (Fig. 4F).

(A) Schematic representation for full length of TBC1D3 protein and mutated forms with indicated fragment deletions. (B to F) HEK293 cells were cotransfected with constructs encoding HA-tagged G9a and Myc-tagged full-length or mutated forms of TBC1D3. Cell homogenates were subjected to IP with antibody against HA, followed by IB with antibody against Myc or HA. Data shown are blots of representative experiments performed for at least three times with similar results.

Because the 465 to 481amino acid segment of TBC1D3 was essential for the interaction with G9a, we asked whether the peptide covering this sequence is able to interfere with TBC1D3-G9a interaction. We synthesized the peptide composed of the cell-penetrating TAT sequence derived from the trans-activator of transcription of human immunodeficiency virus (28) and the 465 to 481 amino acids of TBC1D3 (shortened as T-T) or scrambled sequence (shortened as T-S) (Fig. 5A) and tested their effect on TBC1D3/G9a interaction. As shown in fig. S4A, the association between Myc-TBC1D3 and HA-G9a was attenuated in transfected HEK293 cells treated with T-T, but not T-S. Furthermore, the efficiency of GST-G9a-CF in pulling down Myc-TBC1D3 was markedly decreased in reactions with the presence of T-T, but not T-S (fig. S4B). Next, we asked whether the blockade of TBC1D3/G9a interaction changed the level of H3K9me2 or proliferation of NPs in human cerebral organoids. For this purpose, the organoids at D12 were treated with T-T or T-S for 4 days, followed by immunostaining with various antibodies. Notably, we found that the volume of human cerebral organoids reduced significantly in the T-T group (Fig. 5A). Moreover, the level of H3K9me2 was markedly increased, whereas the percentage of KI67+ cells or the intensity of PAX6+ signals was markedly decreased in T-Ttreated organoids (Fig. 5, B to D). These results were unlikely caused by a direct effect of peptides on G9a, because addition of T-T to the in vitro histone methylation assay did not change the level of H3K9me2 (fig. S4C). In addition, treatment of mouse neural stem cell N2A, which does not harbor TBC1D3, with T-T had no effect on H3K9me2 modification (fig. S4D). These results were in line with the idea that TBC1D3 interaction with G9a represses its histone dimethylation activity and thus maintains H3K9me2 at a low level, which may ensure high proliferative potency of human cortical NPs.

(A) Blockade of TBC1D3/G9a interaction with peptides suppresses organoid growth. At least 14 organoids were analyzed in each group. Scale bar, 200 m. (B) H3K9me2 signals in D16 organoids. Data are represented as means SD of 25 rosettes from 9 T-S organoids and 33 rosettes from 12 T-T organoids, with average value of T-S group normalized as 1. Scale bar, 20 m. (C) Analysis for the percentage of KI67+ in D16 organoids (29 rosettes from 7 T-S organoids; 16 rosettes from 6 T-T organoids). Scale bar, 40 m. (D) PAX6 signals in D16 organoids. Normalized intensity of PAX6 was quantified with the value of T-S group set as 1 (79 rosettes from 6 T-S organoids; 49 rosettes from 6 T-T organoids). Scale bar, 40 m. (E) Proliferative cells marked by EdU in mouse cortex transfected with indicated plasmids and yellow fluorescent protein (YFP). Histograms indicate percentage of EdU+ cells among YFP+ cells (9 embryos in the Myc-Ctrl or Myc-TBC1D3465481 group; 10 embryos in the Myc-TBC1D3 group). Scale bar, 20 m. Data are presented as means SD, unpaired Students t test. **P < 0.01; ***P < 0.001; ns, no significant difference.

We have shown previously that TBC1D3 expression promoted the generation and proliferation of basal cortical progenitors, leading to cortex expansion in mice (15). We wondered whether these effects were attributable to the TBC1D3 regulation of G9a. The fetal mice at E13.5 were subjected to in utero electroporation (IUE) to introduce various constructs into NPs in the VZ, followed by analysis of cell proliferation at E15.5. We first examined the effects of full-length (Myc-TBC1D3) and the mutated form of TBC1D3 with the deletion of 465 to 481 amino acids (Myc-TBC1D3465481) on NP proliferation by calculating the proportion of cells in the S phase determined by incorporation of pyrimidine analog 5-ethynyl-2-deoxyuridine (EdU) (Fig. 5E). Consistent with previous observation (15), we found that the Myc-TBC1D3 IUE mice exhibited an increase in EdU+ proliferating cells, as compared with control mice subjected to IUE with vesicle plasmid, while IUE with Myc-TBC1D3465481 had no effect on NP proliferation (Fig. 5E). These effects were evident in both apical and basal regions (Fig. 5E, right). Unlike the full length of TBC1D3, forced expression of TBC1D3465481 in ReN cells did not reduce the level of H3K9me2 (fig. S4E). These results suggest that the suppression of G9a-mediated histone dimethylation by TBC1D3 underlies its role in promoting NP proliferation.

As H3K9me2 modification is considered to be a typical repressive transcription mark (7), we analyzed the gene expression networks regulated by TBC1D3/G9a interaction. First, we compared global transcriptome profiles between human cerebral organoids (D16) treated with T-S or T-T using RNA-seq information. We conducted two replicates in each group to validate experimental consistency in terms of peptide treatment and sequencing process. The hierarchical clustering and Pearson correlation analysis revealed similar patterns in duplicates of either T-S (T-S-1 and T-S-2) or T-T (T-T-1 and T-T-2) duplicates (fig. S5, A and B). Among the differentially expressed genes (DEGs), the significantly changed ones were selected (fig. S5C) for further analysis. Gene ontology (GO) analysis showed that the down-regulated genes in T-T organoids were enriched in functional forebrain development or neuronal differentiation, while the up-regulated genes were enriched in apoptotic signaling pathways (fig. S5D). Further analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that DEGs were enriched in pathways involved in cell proliferation, such as AKT, WNT, or MAPK (mitogen-activated protein kinase) signaling, as well as pluripotency of stem cells (Fig. 6A). Among down-regulated genes, we found several genes that encode WNT ligands and receptor FZD10, FGF receptors, or PAX6 (Fig. 6B). Quantitative gene expression analysis using real-time reverse transcription PCR (RT-PCR) also validated the down-regulation of these representative genes in T-Ttreated organoids (Fig. 6C). These results support the conclusion that TBC1D3 interaction with G9a down-regulates the level of H3K9me2 and, hence, promotes the expression of genes involved in the proliferation of NPs.

(A) Top six enriched KEGG pathways by clustering analysis of down-regulated genes in T-T groups compared with T-S groups. (B) Heatmap showing representative down-regulated genes in T-Ttreated organoids. (C) Relative mRNA levels of indicated genes to GAPDH measured by quantitative PCR analysis for D16 organoids treated with T-T or T-S peptides. Data are presented as means SD of at least five experiments in each group with values from T-S group normalized as 1. Unpaired Students t test. **P < 0.01; ***P < 0.001. (D) ChIP-seq showing the count and distribution of peaks around TSSs in T-S or T-Ttreated organoids at D16. (E) H3K9me2-binding peaks increase in T-Ttreated organoids. (F) Genomic tracks showing differential H3K9me2 enrichment regions near TSS of FZD10 and WNT4. (G and H) Venn diagrams (G) and GO biological enrichment analysis (H) of genes corresponding to ChIP-seq peaks and DEGs identified by RNA-seq. (I) A proposed model showing a role of TBC1D3 in promoting NP proliferation and cortex expansion through down-regulating the level of H3K9me2. PI3K, phosphatidylinositol 3-kinase.

To identify the genomic locus associated with H3K9me2, we performed the genome-wide H3K9me2 chromatin immunoprecipitation sequencing (ChIP-seq) in human cerebral organoids treated with T-S or T-T peptides. The peak reads of H3K9me2 binding on transcriptional start sites (TSSs) were markedly increased in D16 organoids treated with T-T (Fig. 6, D and E, and fig. S5E). For example, many differential peaks appeared to be enriched in promoter regions of regulated genes, such as FZD10 or WNT4 (Fig. 6F, see the red peaks). Moreover, the comprehensive analysis showed that 253 genes were overlapped between the DEGs of RNA-seq and differential peaks observed in ChIP-seq (Fig. 6G). Further GO analysis indicated that the overlapped genes were mainly enriched in proliferative pathways, such as PI3K-Akt and MAPK signaling (Fig. 6H). These results support the conclusion that blockade of the TBC1D3/G9a interaction reactivates G9a activity, leading to increased level of H3K9me2, which marks suppressive gene expression. Together, the inhibitory role of TBC1D3 in G9a activity through direct interaction may maintain H3K9me2 at a low level, which allows expression of genes involved in NP proliferation and hominoid cortical expansion (Fig. 6I).

The expansion in human cerebral cortex is believed to facilitate emergence of higher cognitive skills (1). Prolonged duration of cortical neurogenesis may contribute to cortical expansion and folding, and this process involves markedly increased proliferation capacity of cortical NPs. Recently, several human-specific genes have been shown to promote cortical progenitor proliferation and expansion, and the underlying mechanisms varied from cell cycle transition (29) to glutaminolysis regulation (30). In this study, we found that the level of H3K9me2 modification is reversely correlated with the proliferation capacity of cortical NPs. The hominoid-specific protein TBC1D3 inhibits G9a-mediated H3K9me2 modification, and this regulation underlies TBC1Ds role in promoting the proliferation of cortical NPs. The down-regulation of H3K9me2 caused by TBC1D3 interaction with G9a may derepress the expression of genes involved in the proliferation of NPs, which, in turn, resulted in cortical expansion. This study shows an epigenetic mechanism underlying enhanced stemness of NPs during the evolution of neocortex.

Compared with rodents, the human cortex exhibits increased radial and tangential expansion and more abundant cortical progenitors, which have sustained capability of multiple rounds of division and prolonged neurogenic period (4, 31). Interspecies comparisons have led to identification of specific genomic changes on the human linage, including individual nucleotide variation, insertion-deletions, gene duplications, and a few purely de novo human-specific genes (32). Besides coding regions, many forms of variants in regulatory regions or epigenetic elements, such as human-specific microRNAs (33) and differential histone methylation compared with other nonhuman primates, have been identified (34). Nevertheless, connecting these changes to functions in human brain development has been challenging due to the limitation of ethical issues and the lack of appropriate experimental approaches. In this study, we have established a link between TBC1D3 and H3K9me2 modification in cultured human cerebral organoid system.

Previous studies have shown that the level of H3K9me2 is dynamically regulated in the contexts of memory formation, addiction, and stress (3537). G9a-deficient mice display severe growth retardation and early lethality, and H3-K9 methylation is decreased markedly in G9a-deficient embryos (38). In humans, haploinsufficiency or disruption of the GLP gene has been shown to be associated with congenital intellectual disability, including Kleefstra syndrome and autism spectrum disorder (10). In mice, heterozygous ablation of GLP gene caused developmental delay and abnormal behavior (39). It would be of interest to determine whether TBC1D3 is involved in any intellectual disability or neural developmental disorders. Given that many mammals without TBC1D3 also have cortical expansion compared with rodents, this study does not preclude other mechanisms governed by multiple genetic elements underlying cortex expansion during evolution.

TBC1D3 has been shown to be involved in RAB guanosine triphosphatase signaling, vesicle trafficking, and tissue repair (16, 2224). All these functions seem to rely on its cytosolic and/or membrane localization. Notably, TBC1D3 can also shuttle between cytoplasm and nucleus, and its cytoplasmic retention needs microtubule network (40). In this study, we unraveled a role of TBC1D3 in the nucleus, especially in human NPs. Intriguingly, TBC1D3 was expressed in almost all PAX6+ or TBR2+ cells, and notably, most of those in the OSVZ and a fraction of vRGs had TBC1D3 enriched in the nucleus. This heterogeneous subcellular localization may reflect different states of vRGs. As shown in our previous study, the expression of TBC1D3 in vRGs caused destabilization of Cdh2 mRNA, leading to down-regulation of N-cadherin and delamination of vRGs in mice (15). How the dynamic spatial localization of TBC1D3 is determined in NPs at various states and/or positions warrants further study. Furthermore, its dominant distribution in the cytosol of differentiated neurons suggests multifaceted functions.

C57BL6/J mice and TBC1D3-TG mouse line maintained in C57BL6/J background were used for the IUE experiments. All animal manipulations including mouse housing, breeding, and surgical procedures were executed in compliance with the ethical guidelines of the Institutional Animal Care and Use Committee of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and ShanghaiTech University. All mice were housed under a 12-hour light-dark cycle in the institutional animal care facility. The TBC1D3-TG mice were generated as described previously (15). Mice at E13 to E17 were used for experimental processing without discrimination of the sex of embryos.

The human fetal cortical tissue samples were obtained from medical pregnancy termination. The collection and usage of the human fetuses were conducted in strict observance of the ethical guidelines approved by the ethics committee in Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (approval identifier number: ER-SIBS-221506). After release from clinical autopsy procedure, the brain tissues were transported in ice-cold Leibowitz-15 medium (Gibco, 21083027) and stored in liquid nitrogen for protein extraction or embedded in optimal cutting temperature (OCT) compound (Tissue-Tek Sakura, catalog no. 4583) for further frozen sectioning and immunostaining.

N2A and HEK293T cells were cultured in Dulbeccos modified Eagles medium (DMEM) (Gibco, 11966-025) supplemented with 10% fetal bovine serum (FBS; Gibco, 10099-141) in a 37C incubator with 5% CO2. HEK293T cells were transfected with plasmids using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019). ReN cells derived from human mesencephalon were grown in DMEM/F12 medium (Gibco, H330057) supplemented with B27 (Gibco, 17504044), heparin (10 U/ml; Sigma-Aldrich, H3393), epidermal growth factor (20 ng/ml; Stem cell, 78073), and fibroblast growth factor (10 ng/ml; Stem cell, 78003). The cultured dishes for ReN cells were pretreated with 0.5% laminin (Sigma-Aldrich, L2020) dissolved in DMEM medium for at least 4 hours at 37C. ReN cells were transfected with plasmids using Nucleofector (Lonza Nucleofector II, 2B). H9 hESCs (NEST) were grown on Matrigel (BD Biosciences, 354277)pretreated dishes and cultured in mTeSR medium (STEMCELL Technologies, 5850). Clones of H9 hESCs were passaged using the ReLeSR kit (STEMCELL Technologies, 5872) according to the protocol when the size of clones reached 1 to 2 mm in diameter.

Constructs encoding mutated forms of TBC1D3 with various deletions were generated using site-directed mutagenesis with a PrimeSTAR GXL DNA polymerase kit (Takara, R050A) according to standard protocol with Myc-TBC1D3 plasmid (15) as template and primers listed in table S1. TBC1D3 was also subcloned into PGB plasmid as bait for Y2H screening, into lentiviral vector rLV-EF1a-2A-EGFP-T2A-puro-WPRE for transduction of ES clones, and into PET-28a plasmid to produce 6xHis-tagged recombinant proteins. The full-length coding sequence of G9a or 649 to 1210amino acid fragment was amplified by PCR and subcloned into pGEX-2T-GST or pKH3-HA vector (see table S1 for the list of plasmids and sequences of primers used in PCR amplification).

Y190 yeast cells were cotransfected with PGB-TBC1D3 plasmid and the human fetal brain cDNA library (Clontech, catalog no. HL4028AH). The hits in the positive yeast clones were amplified and sequenced to obtain the gene information. False-positive clones were excluded from the following analysis.

The plasmids encoding GST-tagged G9a truncated fragments or 6xHis-tagged TBC1D3 were transformed into Rosetta Escherichia coli BL21 strain. After 0.5 mM isopropyl--d-thiogalactopyranoside (IPTG) induction (16C for 20 hours) for protein expression, cells were collected and lysed in phosphate-buffered saline (PBS) buffer supplemented with dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) using ultrasonication. For GST-fusion protein, the precleared supernatants were collected and incubated with Glutathione Sepharose 4B (GE Healthcare, 17-0756-01) beads, followed by washes in PBS and elution with glutathione (5 mg/ml). For 6xHis-tagged proteins, the lysis buffer was changed to phosphate buffer containing 10 mM imidazole, 300 mM sodium chloride, 50 mM sodium phosphate buffer, 10% glycerol, and 0.5% Tween, and recombinant proteins were purified with Ni column using elution buffer (30 mM sodium phosphate buffer, 300 mM imidazole, 300 mM sodium chloride, and 10% glycerol).

The cell lysates (1 mg/ml protein) of HEK293T cells transfected with Myc-TBC1D3 or various mutants were incubated with GST-tagged G9a fragments coupled with glutathione agarose beads at 4C with gentle rotation. For TAT blockade experiments, the peptide of T-S or T-T (100 M) was added into the mixture of Myc-TBC1D3 and GST-G9a-CF before following incubation. The beads were then washed in cell lysis buffer and subjected to IB analysis with corresponding antibody.

HEK293T cells transfected with various plasmids were lysed in modified radioimmunoprecipitation assay (RIPA) buffer containing 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA, and protease inhibitor cocktail. After centrifugation (12,000 rpm for 15 min), supernatants were collected and incubated with primary antibodies at 4C overnight and then incubated with Protein-G or Protein-A beads at 4C for 4 hours. After five washes with lysis buffer, the beads were boiled in 30 to 50 l of 1 SDS loading buffer and subjected to IB analysis. Nuclear and cytoplasmic fractions were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, 78833) following the manufacturers instructions. For IB analysis, protein samples were loaded and separated in SDSpolyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membranes. After blocking in 5% milk in TBS-T (tris buffered saline-Tween) for 1 hour at room temperature, the membranes were probed with primary antibodies and visualized with horseradish peroxidase (HRP)conjugated secondary antibodies. Antibodies for IB analysis were as follows: TBC1D3 (rabbit, Abcam, Ab139034), H3K9me2 (Cell Signaling Technology, 4658s), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Proteintech, 60004-1), Histone3 (Cell Signaling Technology, 4499s), Myc (rabbit, Sigma-Aldrich, C3956; mouse, Millipore, 05-419), HA (rabbit, Cell Signaling Technology, 3724s; mouse, Sigma-Aldrich, H3663), H3K9me3 (Abcam, Ab8898), H3K27me2 (Cell Signaling Technology, 9728T), H3K36me2 (Cell Signaling Technology, 2901T), HRP anti-mouse (Abcam, Ab64259), and HRP anti-rabbit (Abcam, Ab64261).

Histone proteins were extracted and purified from cultured ReN cells following the protocol as described previously (41). Briefly, 5 106 cells were collected and lysed in hypotonic lysis buffer containing 10 mM tris-HCl (pH 8.0), 1 mM KCl, 1.5 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail (Selleck, B14001) to release the intact nuclei. The nuclei pellets were then resuspended in 0.4 N H2SO4 and incubated on a rotator for at least 30 min or overnight at 4C. Histone proteins were precipitated in 33% trichloroacetic acid (TCA) (Sigma-Aldrich, T4885), washed with ice-cold acetone, air dried for 20 min at room temperature, and, lastly, dissolved in appropriate volume of ddH2O.

The purified histone proteins (about 30 ng/l) were mixed with GST-G9a (649 to 1210 amino acids) (30 ng/l) in 30 l of reaction buffer containing 0.5 mM SAM (New England Biolabs) as the methyl group donor, 50 mM tris-HCl (pH 8.0), 2 mM MgCl2, 0.01% Trion X-100 (Takara), 1 mM tris (2-carboxyethyl) phosphine (TCEP) (Hampton Research), and protease inhibitor cocktail, and incubated for 12 hours at room temperature, without or with 6xHis-TBC1D3 (30 ng/l). The SDS-PAGE sample buffer was added to stop the reaction, and the products were subjected to IB analysis.

Human cerebral organoids were derived from H9 ES cells following the protocol introduced previously (20) with some modifications. First, after culturing for 48 hours, the ES clones were digested to single cells and passaged to new dishes. Then, the clones were digested into single cells using 1 ml of Accutase at 37C for 5 min, washed in mTeSR medium, centrifuged at 800 rpm for 90 s, and resuspended in 2 ml of mTeSR medium. The cell suspension was seeded into low-attachment V-bottom 96-well plates with about 7000 cells per well in 180 l of mTeSR medium supplemented with 10 M Y27632 (Stem cell, 72304). After 2 days, the medium was changed to the hES medium containing 80% DMEM/F12, 20% KSR (KnockOutTM Serum Replacement) (Gibco, 10828028), 1% GlutaMAX (Thermo Fisher Scientific, 35050061), 1% MEM_NEAA (Invitrogen, 11140050), 0.0004% 2-mercaptoethanol, 2.5 M dorsomorphine (Tocris, Oct-93), and 2 M A83-01 (Tocris, Oct-39) and cultured for 4 days. At D6 to D12, half of the hES medium was replaced with neural induction medium: DMEM/F12 supplemented with 1% N2 supplement (Thermo Fisher Scientific, 17502048), 1% GlutaMAX, 1% MEM_NEAA, heparin (1 g/ml; Sigma-Aldrich, H3393), 200 nM LDN-193189 (Selleck, S7507), and 2 M SB431542 (Selleck, S1067). At D12, the organoids were embedded in Matrigel and cultured in neural differentiation medium containing 50% DMEM/F12, 50% Neurobasal medium (Life Technology, 12348-017), 0.5% N2 supplement, 1% GlutaMAX, 1% MEM_NEAA, 1% B27, 0.0004% 2-mercaptoethanol, and 0.025% insulin (Sigma-Aldrich, I9278) for 4 days, without or with the addition of small molecular inhibitors UNC0638 (1 M; Selleck, S8071), DMSO vehicle control, or 30 M peptides (T-T: YGRKKRRQRRR-EGPWFRHYDFRQSCWVR; T-S: YGRKKRRQRRR-FRVRYWFQGCHSEDPWR). The drug-containing medium was renewed every other day. At D16, B27 supplemented with vitamin A was used in differentiation medium, and the culture condition was maintained for the following days. For lentivirus-transduced cerebral organoids, H9 ES clones (1 to 2 mm in diameter) were infected with control lentivirus (rLV-EF1a-2A-EGFP-T2A-puro-WPRE, shortened as rLV-Ctrl) or TBC1D3 expressing lentivirus (rLV-EF1a-TBC1D3-2A-EGFP-T2A-puro-WPRE, shortened as rLV-TBC1D3). Two days later, puromycin (1/1000; Sigma-Aldrich, P8833) was added to select transfected cells with GFP as selection marker. The clones grown from single GFP+ cells were subjected to quantitative real-time PCR to determine the RNA level of TBC1D3 and used in the following analyses. The nucleotide sequence of small interference RNA for TBC1D3 (target sequence: 5-GCCTCTATGAAGAAACTAA-3) or control (target sequence: 5-TTCTCCGAACGTGTCACGT-3) was inserted into adenovirus vector to generate the pDKD-CMV-eGFP-U6-shTBC1D3 construct. The packaged adenovirus was added into organoid culture medium at D37 and maintained for 3 days. For retrovirus infection, virus was added into organoid culture medium at D26 and maintained for 24 hours. Then, the medium with virus was removed, and organoids were washed immediately with fresh medium for at least three times. After another 3-day culture, organoids were fixed in 4% paraformaldehyde (PFA) and subjected to immunostaining.

The total RNA from brain tissue or human brain organoids was extracted with TRIzol reagent (Life Technology, 15596018) following the manufacturers instructions. RNA samples were subjected to reverse transcription and quantitative real-time PCR using SYBR Green (Selleck, B21702) on QuantStudio 7 Flex System (Life Technologies). The QuantStudio Real-Time PCR Software v1.3 was used for data analysis. The primers used were as follows: TBC1D3, 5-AGGTTCAGCAGAAGCGCCTCA-3 (forward), 5-GCCTGGATGCCGACGACCCTT-3 (reverse); human GAPDH, 5-GACCTGCCGTCTAGAAAAACCT-3 (forward), 5-CTGTTGCTGTAGCCAAATTCGT-3 (reverse); mouse GAPDH, 5-GGGTCATCATCTCCGCCCC-3 (forward), 5-TTGGCAGCACCAGTGGATGCA-3 (reverse); PAX6, 5-TGCATTTGCATGTTGCGGAG-3 (forward), 5-TTAGCGAAGCCTGACCTCTG-3 (reverse); FZD10, 5-CAAACCTCGAAACAGCTGCC-3 (forward), 5-AACAATACCGGGAAGCGAGG-3 (reverse); FGFR3, 5-AGGAGCTCTTCAAGCTGCTG-3 (forward), 5-ACAGGTCCAGGTACTCGTCG-3 (reverse); WNT1, 5-CAAGATCGTCAACCGAGGCT-3 (forward), 5-AAGGTTCATGAGGAAGCGCA-3 (reverse); WNT4, 5-CGTGCCTGCGTTCGCT-3 (forward), 5-GGCAAGGAGTCGAGTGTGG-3 (reverse); FOXG1, 5-CCCTCCCATTTCTGTACGTTT (forward), 5-CTGGCGGCTCTTAGAGAT (reverse).

High-throughput sequencing of total RNA isolated from human cerebral organoids was performed on Illumina NovaSeq 6000 system with average length of 150 nucleotides for every read of paired end. Raw data were filtered by FASTX-Toolkit to generate clean reads and then mapped to human GRCh38. The level of a specific transcript was expressed as FRKM (fragments per kilobase of transcript per million fragments mapped) measured using StringTie software with statistical criterion set as P < 0.05. GO analysis was performed using EdgeR software with false discovery rate <0.05 and log2 (fold change) >1 or <1.

The samples for ChIP analysis were extracted using SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, 91820s). Briefly, tissues were fixed in 1% formaldehyde to cross-link proteins to DNA for 10 to 20 min at room temperature, and the reaction was stopped by addition of glycine. Then, cells were lysed to release nucleus, and Micrococcal Nuclease was added to digest chromatin into protein-associated DNA fragments, followed by sonication to break nuclear membranes and generate chromatin fragments of appropriate size. The samples were incubated with antibody against H3K9me2 (Cell Signaling Technology, 4658s) at 4C overnight and then incubated with ChIP-grade Protein G magnetic beads at 4C for 2 hours, followed by DNA elution and purification. DNA samples in input and IP groups were pair-end sequenced on HiSeq 2500 (XTen) platform. The sequence reads were trimmed for adaptor sequence using FASTP software (version 0.19.11), and peak calling was conducted using MACS2 (version 2.1.0). Peaks were mapped to genome using BWA software (version 0.7.12-r1039), and GO analysis was conducted using Goseq and Bioconductor (version 4.10.2). The enrichGO function in the clusterProfiler (v3.13) R package was used for overrepresentation analysis of GO biological processes overlapped in ChIP-seq and RNA-seq. Venn diagram is plotted by VennDiagram R package.

Pregnant mice with embryos at D13.5 or D14.5 were anesthetized with a mixture of pentobarbital sodium (2.5 g/kg body weight) and ketamine (50 mg/ml of solution) and subjected to IUE. The uterus was exposed under sterile conditions, and plasmid solutions containing DNA (1 to 2 g/l) mixed with fast green (0.1 mg/ml; Sigma-Aldrich, F7252) were manually injected into the lateral ventricles with a beveled glass micropipette (VWR International, 53432-921). Two tweezer electrodes connected to an electroporator (BTX830) were used in the electroporation procedure to deliver five 50-ms pulses of 30-V voltage with 950-ms interval. The mice were surgically sutured and placed on warm electric blanket until recovery.

Cultured cells on coverslips were fixed in 4% PFA for 10 min at room temperature. Embryonic mouse brains were dissected out and postfixed in 4% PFA overnight at 4C. Cultured human brain organoids were soaked in 4% PFA for 2 to 4 hours at 4C. The fixed tissues were dehydrated in 20% sucrose in PBS at 4C and then sectioned at 30-m (mouse brain) or 20-m thickness (organoids) using a freezing microtome (Leica, CM1950). Sections of control and experimental groups were pasted on the same slide to maintain uniform conditions during staining and image collection processes. For immunohistochemistry, fixed cells were washed with PBS for three times and permeabilized in 0.1% Triton X-100 in PBS for 10 min. Tissue slices were subjected to antigen retrieval by citrate and then permeabilized in 0.3% Triton X-100 in PBS for 30 min. After blocking with 10% FBS for 50 min, the cells or slices were incubated with various primary antibodies at 4C overnight, washed with PBS for three times, incubated with secondary antibodies for 2 hours at room temperature in the dark, and mounted with mounting reagent (DAKO, S3023) for observation.

For EdU labeling, pregnant mice that recovered from the IUE surge were injected intraperitoneally with EdU (50 mg/kg body weight) (Thermo Fisher Scientific, C10640). EdU staining was performed immediately after the secondary antibody incubation using a Click-iT Plus EdU Imaging Kit (Thermo Fisher Scientific, C10640). Antibodies for immunostaining were as follows: DAPI (Beyotime, C1002), TBC1D3 (rabbit, Abcam, Ab139034), TBC1D3 (mouse, Santa Cruz, sc-376073), KI67 (Abcam, Ab66155), PAX6 (Covance, PRB-278P), H3K9me2 (Cell Signaling Technology, 4658s), G9a (Abcam, Ab185050), -tubulin (Cell Signaling Technology, 2128s), TBR2 (Invitrogen, 14-4877-82), DCX (Santa Cruz, sc-8066), HOPX (Sigma-Aldrich, HPA030180), Alexa Fluor 488 (Jackson, 703-546-155), Alexa Fluor 555 (Invitrogen, A31572), and Alexa Fluor 647 (Invitrogen, A31571).

Images of immunostaining were collected using confocal microscopy with Nikon TiE, Nikon A1R, Leica P8, or Olympus FV3000 and processed with ImageJ software. The shooting parameters were kept the same between each control and experimental group. Statistical tests were performed using GraphPad Prism software, and data were presented as means SD. Data satisfied to Gaussian distribution test were quantified with Students t test, while others were quantified with unpaired Students t test. The statistical significance was indicated by *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference.

Acknowledgments: We are grateful to A. L. Sheng for the assistance with brain organoid culture, L. Du for suggestions on ChIP-seq analysis, Y. Jin for sharing ReN cells, and J. P. Ding for the modified PET-28a vector. Funding: This study was partially supported by grants from the National Natural Science Foundation of China (31490591 to Z.-G.L. and 31871034 to X.-C.J.), the National Key R&D Program of China (2017YFA0700500), the Frontier Key Project of the Chinese Academy of Sciences (QYZDJ-SSW-SMC025), and Shanghai Municipal Science and Technology Projects (2018SHZDZX05 and 201409001700). Author contributions: Q.-Q.H. designed the experiments, conducted data collection and analysis, and wrote the original draft. Q.X. and X.-C.J. participated in RNA-seq and ChIP- seq data analysis. X.-Y.S. participated in human cerebral organoid culture. Z.-G.L. conceived the project and wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: RNA-seq data and ChIP-seq data were deposited in the Gene Expression Omnibus (GEO) with accession number GSE136283. The dataset for evaluating TBC1D3 expression in human and chimpanzee neural cells was deposited in the GEO with accession number GSE83638. 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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TBC1D3 promotes neural progenitor proliferation by suppressing the histone methyltransferase G9a - Science Advances

‘He was very honored in his work’ – Mercer Island Reporter

Dr. Sen-itiroh Hakomori was honored as a hometown hero at the Mercer Island Summer Celebration Parade in 2000. Courtesy photo

Pioneering scientist Dr. Sen-itiroh Hakomori dies at age 91.

Dr. Sen-itiroh Hakomori couldnt leave his lab work alone. He would often log 13-hour days, seven days a week, and even spend some time at his job on Christmas morning, intensely focused on his glycosphingolipid medical and biochemical research.

I realized fairly recently that all of the cells are just like a pet. You have to check up on them and make sure that things are going well because theyre alive, said his daughter Naoko Vaughn. As children, we did not understand why he went to work every single day. I realized that he had to.

Vaughns father, who she said had a heart of gold and would help anybody, died of natural causes at the age of 91 on Nov. 10 at his home on Mercer Island.

Roger Laine, a colleague and friend who was the last professor to visit Hakomori, said that he was truly a pioneering scientist in glycobiology, a field in which he spent seven decades participating in groundbreaking studies.

He worked most of his career showing differences between cancer cells and normal cells that could be targeted for therapy. If you asked him what was his goal in life, he would answer, cure cancer, said Laine, a professor, scientist and researcher at Louisiana State University.

Hakomori is survived by his wife, Mitsuko (they were married for 74 years); Vaughn; sons, Yoichiro and Kenjiro; four grandchildren and two great-grandchildren; two brothers and a sister.

A native of Japan, professor Hakomori made crucial contributions to new cancer-cell studies at the Cancer Research Institute at Tohoku Pharmaceutical University, and continued his vast research in the field when the family immigrated to the United States to the Boston area.

The family moved from Boston to Bellevue in the late 1960s and set up their new home on Mercer Island 46 years ago. Hakomori relocated his family to the Pacific Northwest to become involved with the Fred Hutchinson Cancer Research Center, and served as University of Washington professor of pathobiology and professor of microbiology. He was named a UW professor emeritus of pathobiology and global health in 2006.

Hakomori retired at the age of 88, finishing his career working at the Pacific Northwest Cancer Center in Seattle. During a celebration in Japan three years ago, Hakomori who was a member of the prestigious National Academy of Sciences spoke to the attendees and noted, We are all globally connected with research and science.

His two sons reflected on their fathers vital contribution to their lives.

Dad was passionate about his work and a great mentor to many of his younger colleagues. He has inspired me to try to emulate that passion in the work I do as an architect and professor, said Yoichiro.

Added Kenjiro: Dad taught me by example to work hard on trying to find out and work on resolving research questions it is a lifelong passion.

Vaughn added that her father instilled a high-level work ethic in his children, telling them, Whatever you do, whatever you choose to do, whatever your passion is, you do it 120 percent.

Hakomori made a worldwide impact with his research and was nominated five times for the Nobel Prize in chemistry. He received numerous awards, including the Philip Levine Immunohematology Award, the Karl Meyer Award from the Society of Glycobiology and the Rosalind Kornfield Award for Lifetime Achievement in Glycobiology.

He published 585 articles in peer-reviewed journals, and a pair of his many major scientific achievements were methylation analysis of glycoconjugates with mass spectrometry, and cell adhesion based on carbohydrate-carbohydrate interaction, particularly through GSL clusters at the embryonic stem cell surface.

He was very honored in his work, and he did not do it for money. He was very much just trying to help, which is rare these days, said Vaughn, adding that a host of his students and colleagues from around the world are assembling a memorial for her father to be published in a glycosphingolipid journal.

He was just a wonderful person. I think the most important thing is his colleagues really admired him. He made them successful in their lives, Vaughn added.

When Sarah Spiegel was a graduate student, she was drawn to Hakomoris papers and reviews on the role of glycoconjugates in cancer. Those documents sparked her imagination and inspired her to pursue a career in sphingolipids, she wrote on the Evergreen Washelli Funeral Home & Cemetery memorial page.

He was a champion of advancing careers of young female scientists and his generous spirit influenced my generation and generations to come, said Spiegel, Ph.D., professor and chair in the Department of Biochemistry and Molecular Biology at the Virginia Commonwealth University School of Medicine. His legacy will live on through the works of countless researchers who continue working in the field of sphingolipids and many colleagues throughout the world will miss him tremendously.

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'He was very honored in his work' - Mercer Island Reporter

Israeli biotech firm’s ALS treatment shows safety of use in trials – The Jerusalem Post

Ness Ziona-based biotech firm Kadimastem has shown encouraging results of Cohort B of its Phase 1/2a clinical trial for AstroRx, its Amyotrophic Lateral Sclerosis (ALS) treatment trial. The objective of this trial was to evaluate the safety of their treatment, with a secondary objective of the trial of estimating its preliminary efficacy. The treatment was developed by Kadimastem and contains functional, healthy astrocytes (nervous system support cells) derived from Human Embryonic Stem Cells (hESC) that aim to protect diseased motor neurons. The company's technology allows injecting AstroRx into the spine of the patient, to slow down the progression of the disease. The treatment has been granted orphan drug designation by the FDA for the treatment of ALS. The five patients included in this part of the trial showed no serious adverse effects during the half a year follow up after the treatment was given. The rate in which it slows down the disease was also tested for, using the ALS Functional Rating Scale-Revised (ALSFRS-R), which tracks ALS progression, and has shown that after the treatment was given, there was a 45% decline in the disease's progression rate. At the end of the 6-month post-treatment period, the rate of ALSFRS-R progression was similar to the rate that was measured before treatment. "The results after 6 months of follow up are encouraging, as they suggest a clinically meaningful signal of effect for 3 months by a single administration of AstroRx and confirm the safety of AstroRx," said Dr. Marc Gotkine, Head of the ALS Clinic at the Department of Neurology at Hadassah Medical Center in Jerusalem, and the Principal Investigator of the trial.

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Ca Bishops To Work w/ Govt on Vaccination Campaigns – Catholic Herald Online

The bishops of California have said that taking either the Moderna or Pfizer COVID-19 vaccine is not a sin, despite controversy that the vaccines are derived from embryonic stem cells.

In a December 3 statement, the California Catholic Conference of bishops stated that it, affirms that the imminent Pfizer and Moderna COVID-19 vaccines are morally acceptable and commits to working closely with Catholic health care ministries and Catholic Charities to promote and encourage COVID-19 vaccinations in collaboration with state and local governments and other entities, Catholic San Francisco reported.

The California bishops join many other bishops, including those of Alberta and Northwest Territories, who stated Dec. 2, The Catholic Church does certainly support and encourage ethical scientific research into the development of vaccines that will mitigate or even end the harm caused by this terrible disease.

However, the bishop of Fresno said a few weeks prior that there are concerns that the vaccine is derived from stem cells of an aborted baby.

Citing ethical concerns about the use of fetal cells in vaccine development, Bishop Joseph Brennan of Fresno has urged Catholics not to jump on the COVID-19 vaccine bandwagon, Los Angeles Times reported a few weeks ago.

Experts have explained that the vaccines by Moderna and Pfizer are made from proteins that do not come from embryonic or fetal tissues, ABC Action News stated.

Bishop Brennan explained, I wont be able to take a vaccine, I just wont, brothers and sisters, and I encourage you not to, if it was developed with material derived from stem cells of a baby who was aborted. Or material that was cast off from artificial insemination.

Kevin McCormack of the California Stem Cell Agency in Oakland explained that the Pfizer and the Moderna vaccines, use messenger RNA. So these are made from genetically tweaked proteins so they have nothing to do with embryonic tissue or fetal tissue, he said.

The AstraZeneca/Oxford vaccine has reportedly been developed from cell-lines originating from the cells of an aborted fetus in 1983, and the bishops of England and Wales have stated that the vaccine is still morally acceptable, according to The Catholic Universe.

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Human Embryonic Stem Cells (HESC) Market 2019 | Analyzing The Impact Followed By Restraints, Opportunities And Projected Developments | DataIntelo -…

DataIntelo, one of the worlds prominent market research firms has released a new report on Global Human Embryonic Stem Cells (HESC) Market. The report contains crucial insights on the market which will support the clients to make the right business decisions. This research will help both existing and new aspirants for Human Embryonic Stem Cells (HESC) market to figure out and study market needs, market size, and competition. The report talks about the supply and demand situation, the competitive scenario, and the challenges for market growth, market opportunities, and the threats faced by key players.

The report also includes the impact of ongoing global crisis i.e. COVID-19 on the Human Embryonic Stem Cells (HESC) market and what the future holds for it. The published report is designed using a vigorous and thorough research methodology and DataIntelo is also known for its data accuracy and granular market reports.

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A complete analysis of the competitive scenario of the Human Embryonic Stem Cells (HESC) market is depicted by the report. The report has a vast amount of data about the recent product and technological developments in the markets. It has a wide spectrum of analysis regarding the impact of these advancements on the markets future growth, wide-range of analysis of these extensions on the markets future growth.

Human Embryonic Stem Cells (HESC) market report tracks the data since 2015 and is one of the most detailed reports. It also contains data varying according to region and country. The insights in the report are easy to understand and include pictorial representations. These insights are also applicable in real-time scenarios.

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Components such as market drivers, restraints, challenges, and opportunities for Human Embryonic Stem Cells (HESC) are explained in detail. Since the research team is tracking the data for the market from 2015, therefore any additional data requirement can be easily fulfilled.

Some of the prominent companies that are covered in this report:

ESI BIO Thermo Fisher BioTime MilliporeSigma BD Biosciences Astellas Institute of Regenerative Medicine Asterias Biotherapeutics Cell Cure Neurosciences PerkinElmer Takara Bio Cellular Dynamics International Reliance Life Sciences Research & Diagnostics Systems SABiosciences STEMCELL Technologies Stemina Biomarker Discovery Takara Bio TATAA Biocenter UK Stem Cell Bank ViaCyte Vitrolife

*Note: Additional companies can be included on request

The industry looks to be fairly competitive. To analyze any market with simplicity the market is fragmented into segments, such as its product type, application, technology, end-use industry, etc. Segmenting the market into smaller components helps in understanding the dynamics of the market with more clarity. Data is represented with the help of tables and figures that consist of a graphical representation of the numbers in the form of histograms, bar graphs, pie charts, etc. Another key component that is included in the report is the regional analysis to assess the global presence of the Human Embryonic Stem Cells (HESC) market.

Following is the gist of segmentation:

By Application:

Research Clinical Trials Others

By Type:

Totipotent Stem Cells Pluripotent Stem Cells Unipotent Stem Cells

By Geographical Regions

Asia Pacific: China, Japan, India, and Rest of Asia Pacific Europe: Germany, the UK, France, and Rest of Europe North America: The US, Mexico, and Canada Latin America: Brazil and Rest of Latin America Middle East & Africa: GCC Countries and Rest of Middle East & Africa

You can also go for a yearly subscription of all the updates on the Human Embryonic Stem Cells (HESC) market.

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Below is the TOC of the report:

Executive Summary

Assumptions and Acronyms Used

Research Methodology

Human Embryonic Stem Cells (HESC) Market Overview

Human Embryonic Stem Cells (HESC) Supply Chain Analysis

Human Embryonic Stem Cells (HESC) Pricing Analysis

Global Human Embryonic Stem Cells (HESC) Market Analysis and Forecast by Type

Global Human Embryonic Stem Cells (HESC) Market Analysis and Forecast by Application

Global Human Embryonic Stem Cells (HESC) Market Analysis and Forecast by Sales Channel

Global Human Embryonic Stem Cells (HESC) Market Analysis and Forecast by Region

North America Human Embryonic Stem Cells (HESC) Market Analysis and Forecast

Latin America Human Embryonic Stem Cells (HESC) Market Analysis and Forecast

Europe Human Embryonic Stem Cells (HESC) Market Analysis and Forecast

Asia Pacific Human Embryonic Stem Cells (HESC) Market Analysis and Forecast

Middle East & Africa Human Embryonic Stem Cells (HESC) Market Analysis and Forecast

Competition Landscape

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Stem Cell Market Technology 2021 and Application, Segmentation by Leading Global Players, Market Status by Share and Size Forecast to 2024 – The…

International Stem Cell Corporation

Key Market Trends:

Oncology Disorders Segment is Expected to Exhibit Fastest Growth Rate Over the Forecast Period

Cancer has a major impact on society in the United States and across the world. As per the estimation of National Cancer Institute, in 2018, 1,735,350 new cases of cancer were anticipated to get diagnosed in the United States, and 609,640 deaths were expected from the disease. This increasing medical burden is due to population growth. Bone marrow transplant or stem cell transplant is a treatment for some types of cancers, like leukemia, multiple myeloma, multiple myeloma, neuroblastoma, or some types of lymphoma.

Embryonic stem cells (ESC) are the major source of stem cells for therapeutic purposes, due to their higher totipotency and indefinite lifespan, as compared to adult stem cells with lower totipotency and restricted lifespan. However, the use of ESCs for research and therapeutic purposes is restricted and prohibited in many countries throughout the world, due to some ethical constraints. Scientists from the University of California, Irvine, created the stem cell-based approach to kill cancerous tissue while preventing some toxic side effects of chemotherapy by treating the disease in a more localized way.

Although the market shows positive growth, due to the growing focus of stem cell-based research that can further strengthen the clinical application, its expensive nature for stem cell therapy may still hamper its growth.

North America Captured The Largest Market Share and is Expected to Retain its Dominance

North America dominated the overall stem cell market with the United States contributing to the largest share in the market. In 2014, the Sanford Stem Cell Clinical Center at the University of California, San Diego (UCSD) Health System, announced the launch of a clinical trial, in order to assess the safety of neural stem cell-based therapy in patients with chronic spinal cord injury. Researchers hoped that the transplanted stem cells may develop into new neurons that could replace severed or lost nerve connections, and restore at least some motor and sensory functions. Such numerous stem cell studies across the United States have helped in the growth of the stem cell market.

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Market Overview:

Report Highlights:

Scope of the Report:

The scope of this market is limited to tracking the stem cell market. As per the scope of this report, stem cells are biological cells that can differentiate into other types of cells. Also, various types of stem cells are used for therapeutic purposes.

Competitive Landscape:

Most of the companies present in the market are efficient at the technological front, but require significant support for enhancing their services and expanding their businesses. Thus, mergers and acquisitions offer significant opportunities to gain the attention of a large number of providers across developed regions.

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Detailed TOC of Stem Cell Market Report 2024:

1 INTRODUCTION 1.1 Study Deliverables 1.2 Study Assumptions 1.3 Scope of the Study

2 RESEARCH METHODOLOGY

3 EXECUTIVE SUMMARY

4 MARKET DYNAMICS 4.1 Market Overview 4.2 Market Drivers 4.2.1 Increased Awareness about Umbilical Stem Cell 4.2.2 Increase in the Approval for Clinical Trials in Stem Cell Research 4.2.3 Growing Demand for Regenerative Treatment Option 4.2.4 Rising R&D Initiatives to Develop Therapeutic Options for Chronic Diseases 4.3 Market Restraints 4.3.1 Expensive Procedures 4.3.2 Regulatory Complications 4.3.3 Ethical and Moral Framework 4.4 Industry Attractiveness- Porters Five Forces Analysis 4.4.1 Threat of New Entrants 4.4.2 Bargaining Power of Buyers/Consumers 4.4.3 Bargaining Power of Suppliers 4.4.4 Threat of Substitute Products 4.4.5 Intensity of Competitive Rivalry

5 MARKET SEGMENTATION 5.1 By Product Type 5.1.1 Adult Stem Cell 5.1.2 Human Embryonic Cell 5.1.3 Pluripotent Stem Cell 5.1.4 Other Product Types 5.2 By Therapeutic Application 5.2.1 Neurological Disorders 5.2.2 Orthopedic Treatments 5.2.3 Oncology Disorders 5.2.4 Diabetes 5.2.5 Injuries and Wounds 5.2.6 Cardiovascular Disorders 5.2.7 Other Therapeutic Applications 5.3 By Treatment Type 5.3.1 Allogeneic Stem Cell Therapy 5.3.2 Auto logic Stem Cell Therapy 5.3.3 Syngeneic Stem Cell Therapy 5.4 By Banking Service and Technology 5.4.1 Stem Cell Acquisition and Testing 5.4.2 Cell Production 5.4.3 Expansion 5.4.4 Sub-culture 5.4.5 Cryopreservation 5.5 By Type of Banking 5.5.1 Public 5.5.2 Private 5.6 Geography 5.6.1 North America 5.6.1.1 US 5.6.1.2 Canada 5.6.1.3 Mexico 5.6.2 Europe 5.6.2.1 UK 5.6.2.2 Germany 5.6.2.3 France 5.6.2.4 Italy 5.6.2.5 Spain 5.6.2.6 Rest of Europe 5.6.3 Asia-Pacific 5.6.3.1 China 5.6.3.2 Japan 5.6.3.3 India 5.6.3.4 Australia 5.6.3.5 South Korea 5.6.3.6 Rest of Asia-Pacific 5.6.4 Middle East & Africa 5.6.4.1 GCC 5.6.4.2 South Africa 5.6.4.3 Rest of Middle East & Africa 5.6.5 South America 5.6.5.1 Brazil 5.6.5.2 Argentina 5.6.5.3 Rest of South America

6 COMPETITIVE LANDSCAPE 6.1 Company Profiles 6.1.1 Osiris Therapeutics Inc. 6.1.2 Pluristem Therapeutics Inc. 6.1.3 Thermo Fisher Scientific 6.1.4 Qiagen NV 6.1.5 Sigma Aldrich Corporation 6.1.6 Becton, Dickinson and Company 6.1.7 Stem Cell Technologies Inc. 6.1.8 AllCells LLC 6.1.9 Miltenyi Biotec 6.1.10 International Stem Cell Corporation

7 MARKET OPPORTUNITIES AND FUTURE TRENDS

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Stem Cell Market Technology 2021 and Application, Segmentation by Leading Global Players, Market Status by Share and Size Forecast to 2024 - The...

Stem Cell Therapy Market Size, Opportunities, Dynamic, Outlook and Forecast To 2027 – Cheshire Media

Allosource

Impact of Covid-19 on this Market:

Stem Cell Therapy Market report analyses the impact of Coronavirus (COVID-19) on the Stem Cell Therapy industry. Since the COVID-19 virus outbreak in December 2019, the disease has spread to almost 180+ countries around the globe with the World Health Organization declaring it a public health emergency. The global impacts of the coronavirus disease 2019 (COVID-19) are already starting to be felt, and will significantly affect the Stem Cell Therapy market in 2020.

The outbreak of COVID-19 has brought effects on many aspects, like flight cancellations; travel bans and quarantines; restaurants closed; all indoor events restricted; emergency declared in many countries; massive slowing of the supply chain; stock market unpredictability; falling business assurance, growing panic among the population, and uncertainty about future.

COVID-19 can affect the global economy in 3 main ways: by directly affecting production and demand, by creating supply chain and market disturbance, and by its financial impact on firms and financial markets.

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Market Segments and Sub-segments Covered in the Report are as per below:

1.Stem Cell Therapy Market, By Cell Source:

Adipose Tissue-Derived Mesenchymal Stem Cells Bone Marrow-Derived Mesenchymal Stem Cells Cord Blood/Embryonic Stem Cells Other Cell Sources

2.Stem Cell Therapy Market, By Therapeutic Application:

Musculoskeletal Disorders Wounds and Injuries Cardiovascular Diseases Surgeries Gastrointestinal Diseases Other Applications

3.Stem Cell Therapy Market, By Type:

Allogeneic Stem Cell Therapy Market, By Application Musculoskeletal Disorders Wounds and Injuries Surgeries Acute Graft-Versus-Host Disease (AGVHD) Other Applications Autologous Stem Cell Therapy Market, By Application Cardiovascular Diseases Wounds and Injuries Gastrointestinal Diseases Other Applications

This Market Study covers the Stem Cell Therapy Market Size across segments. It aims at estimating the market size and the growth potential of the market across segments by component, data type, deployment type, organization size, vertical, and region. This Stem Cell Therapy study also includes an in-depth competitive analysis of the key market players, along with their company profiles, key observations related to product and business offerings, recent developments, and key market strategies.

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Reason to purchase Stem Cell Therapy market report:

Finally, the Stem Cell Therapy Market Report is a credible source of market research that will accelerate your business exponentially. The report gives the most important regional framework conditions, economic situations with item value, advantage, limit, production, supply, demand, market development rate and number, etc. Stem Cell Therapy Industry Report Also includes a new SWOT review task, speculative test research, and corporate return on investment research.

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Stem Cell Therapy Market Size, Opportunities, Dynamic, Outlook and Forecast To 2027 - Cheshire Media

Stem Cells Market will grow at CAGR of 8.61% by 2027 Cheshire Media – Cheshire Media

According to Precedence Research, The Global Stem Cells Market size will reach USD 17.79 bn by 2027, growing at a CAGR of 8.61% during the forecast period 2020-2027.

Precedence Research started a new study on the global Stem Cells market, providing forecast for the period 2020-2027. The study provides an analysis of the global Stem Cells industry for the period 2016-2027, wherein 2020 to 2027 is the forecast period and 2019 is considered as the base year.

The study also includes key indicator assessment to define the potential growth prospects of the global market, along with forecast statistics regarding the progress of the market based on value (US$ million) and volume (tons). In the Stem Cells market study, lucrative opportunities are seen for Stem Cells. The report enumerates valuable insights to enable readers to make winning business decisions for the future growth of their businesses. The report sheds light on the significant factors that are constantly shaping the growth of the market, untapped opportunities for manufacturers, trends and developments, and other insights across various key segments. Macroeconomic factors that are directly or indirectly affecting the growth of the market are also incorporated in the report.

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Key Questions Answered

Key indicators associated with the Stem Cells market have been evaluated thoroughly in the report. The study highlights vital market dynamics such as key drivers, challenges, and trends, along with opportunities in the global market. A comprehensive study on the supply chain of the global market has also been encompassed in the report.

Other key aspects laid down in this market include pricing strategy of the leading market players. Furthermore, forecast factors and forecast scenario of the market have been encompassed in the report to understand future prospects of the market.

The report also renders imperative numbers such as historical and forecast size of various segments of this market.

Y-o-Y growth comparison, volume and revenue comparison, and market share comparison of various market segments have been delivered in the report. The Stem Cells market has been analyzed at both regional and country levels.

The research report provides an exhaustive evaluation on the structure of the market, in tandem with a dashboard view of all the leading companies profiled in the report. A company share analysis on this market players has also been presented in the report, apart from the footprint matrix on the profiled market players. The report depicts the presence of manufacturers by leveraging an intensity map. In this market report, readers can avail a detailed taxonomy along with a comprehensive analysis on the competitive landscape. The study profiles incumbent companies as well as new entrants in the market, wherein, new product innovations and strategic initiatives of these players have been detailed.

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Competition Landscape

The report has engulfed a chapter on the global Stem Cells markets competitive landscape, which provides detailed analysis and insights on companies offering. Profiles of key companies, along with a strategic overview of their M&A and expansion plans across geographies, have been delivered in this chapter. This chapter is priceless for report readers, as its enables them in gauging their growth potential in the market and implement key strategies for extending their market reach. This chapter offers key recommendations for both new and existing market participants, enabling them to emerge sustainably and profitably. Intelligence on the market players has been delivered on the basis of their product overview, SWOT analysis, key developments, key financials and company overview. Occupancy of these market participants has been tracked by the report and portrayed via an intensity map.

Key Companies:

Various players operating in the global Stem Cells markets are

Market Segmentation as below:

By Product

By Application

By Technology

By Therapy

By Regional Outlook

Research Methodology

A realistic methodology, along with a holistic approach makes the base for incisive insights provided in this market for the study evaluation period. The report comprises of detailed information on the growth prospects of this industry, along with riveting insights into the forecast assessment of the market.

Extensive primary and secondary research have been employed to garner incisive insights into the forecast study of this market. The research report has undergone through a cross-validation by in-house professionals to make the Stem Cells market report one-of-its-kind, with the highest credibility.

The Final Report will cover the impact analysis of COVID-19.

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Stem Cells Market will grow at CAGR of 8.61% by 2027 Cheshire Media - Cheshire Media

Stem Cell Medical Research to Expand in California Following Passage of Prop. 14 – Times of San Diego

Share This Article: A stem cell research center at UC Davis. Courtesy California Institute for Regenerative Medicine By Barbara Feder Ostrov | CalMatters

Californias stem cell research agency was supposed to be winding down its operations right about now, after a 16-year run and hundreds of millions in grants to scientists researching cutting-edge treatments for diabetes, cancer, Alzheimers and other diseases.

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Instead, the taxpayer-supported California Institute for Regenerative Medicine will get a $5.5 billion reboot after voters earlier this month narrowly passed the Proposition 14bond measure. The overall cost of the bonds with interest will total about $7.8 billion.

Were thrilled that California voters saw fit to continue the work weve done, said Jonathan Thomas, chair of the agencys governing board. California has always had a frontier mentality and a love for the cutting edge, and the work that CIRM has done has put it on the very forefront of regenerative medicine.

Even with Californias economy in a coronavirus-induced tailspin and somescientists arguingthat stem cell research no longer needs taxpayer support,Prop. 14passed with 51 percent of the vote after well-financed supporters pourednearly $21 millioninto the Yes on 14 campaign. The measure was essentially a rerun of Proposition 71, which California voters approved in 2004 after a since-revoked federal ban on embryonic stem cell research.

The cash infusion is expected to keep the institute running for another 10 to 15 years, although the agency will see some significant changes under Prop. 14.

The institute also must contend with longstanding concerns over conflicts of interest that have dogged it since its inception, observers say. About 80% of the money distributed has gone to universities and companies tied to agency board members, according to an analysisby longtime agency watchdog David Jensen, a former Sacramento Bee journalist who runs theCalifornia Stem Cell Reportblog and wrote abookon the institute.

Prop. 14 allows the agency to fund a wider array of research projects even some that dont involve stem cells, but instead are related to genetics, personalized medicine and aging.

Thats necessary because the field has evolved, said Paul Knoepfler, a UC Davis professor of cell biology who studies the role of stem cells in cancer and writes a stem cell blog. He received a 2009 grant from the institute.

Stem cells are interesting and important, but there are going to be a lot of new therapies in the next 10 years that are not stem-cell centric, Knoepfler said.

Other changes for the agency include:

Ysabel Duron, who joined the institutes board late last year, said she sees her role as promoting equity in opportunities for both researchers and patients and ensuring that treatments resulting from the research can benefit all Californians.

Researchers in particular need to boost the diversity of patients in their clinical trials and do a better job communicating the value of their work to the public, Duron said, noting that nearly 40% of Californians are Latino.

We need to keep researchers feet to the fire, said Duron, a former television journalist and founder of the Latino Cancer Institute. They need to show us a plan and we need to reward them.

To date, the agency has funded 64 clinical trials of treatments for many types of cancer, sickle cell disease, spinal cord injuries, diabetes, kidney disease and amyotrophic lateral sclerosis, commonlyknown as Lou Gehrigs disease.But the most advanced trials involve therapies for relatively rare conditions, such asSevere Combined Immunodeficiency known as the bubble baby disease, Jensen noted. That therapy is being reviewed by the FDA but has not yet been approved.

Cancer, heart disease these are the big killers. Thats what most people are interested in, Jensen said. You can fund something for a rare disease, but that doesnt affect the majority of Californians.

And, Jensen asks, what will happen after the agency runs out of money again? Will taxpayers once again be asked to refill its coffers? There was hope when the agency began that revenues from successful treatments would sustain its grant-making in the years to come, but the institute has only received a few hundred thousand dollars, not nearly enough to become self-sustaining without taxpayer support, according to theLegislative Analysts Office.

The sustainability issue is important and its hard to address, Jensen said. The money doesnt last forever.

Stem Cell Medical Research to Expand in California Following Passage of Prop. 14 was last modified: November 27th, 2020 by Editor

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Stem Cell Medical Research to Expand in California Following Passage of Prop. 14 - Times of San Diego