Archive for the ‘Induced Pluripotent Stem Cells’ Category

Regenerative Potential Of Induced Pluripotent Stem Cells …

Induced Pluripotent Stem Cells | Posted by admin
Mar 28 2019

1020 0 Posted on Mar 26, 2019, 9 p.m.

After the discovery of induced pluripotent stem cells in the Nobel Prize winning lab of researcher Shinya Yamanaka at Kyoto University in 2006, the thick morality lined clouds hovering over embryonic stem cell research began to fade away making room for happier times.

Stem cells harvested from adults cells rather than embryos are less controversial and hold some promise for practical benefits as well such as lower chance of rejection by the immune system as they can be created from the patients very own cells. iPSCs are a versatile tool which is proving to be useful in modeling disease, screening drugs, and holds massive promise in the field of regenerative medicine.

Osaka and Cardiff Universities explored how iPSCs may be able to restore vision in humans. Using stem cells they were able to generate multiple cells lineages that resulted in tissues being implanted into the eyes of rabbits with induced corneal blindness which repaired the organ and restored their vision. This demonstrates various types of human stem cells are able to take on characteristics of the cornea, lens, and retinas which paves the way for trials to explore the technology in humans. according to Professor Andrew Quantock.

Kyoto University researchers explored the idea of using iPSCs to arrest the slide in decline of dopamine which hampers motor skills in those with Parkinsons disease, using diseased monkey brains with cells converted into dopaminergic progenitors responsible for generating dopamine neurotransmitters. The research has gone so well they are now conducting human trials using the same technology; 7 Parkinsons disease patients were given 5 million iPSC derived dopaminergic progenitors which were transplanted directly into their brains with a special device in hopes that it will curtail effects of the disease. The patients are being closely monitored and observed over the next two years.

University of Minnesota researchers have created a cutting edge technology wherein cells can be converted into neural stem cells, which can be mixed and matched with alternating layers of silicon scaffold to be used to grow new connections in the spine between the nerves that remain in spinal cord injury. In lab testing this technology was found to grow nerves and connect undamaged separated cells. Testing showed new neurons could be grown in an injury site, however the work is still a way off in doing so in numbers that would allow a paralyzed human to walk again. Even with that being said partial repairing of the spinal cord could still improve functions such as bladder control, avoid involuntary movement of limbs, and improve quality of life which is still plenty reason to be excited.

Washington University research has made a breakthrough with a method of converting iPSCs into beta cells which were tested by implanting into diabetic mice incapable of producing insulin on their own. The animals began secreting insulin on their own within days in quantities sufficient to control their own blood sugar levels which functionally cured their condition.

Following the successful trials on pigs in 2017 the Japanese government has recently approved the first ever trial on humans wherein iPSCs are being used to create sheets bearing millions of heart muscle cells which are then grafted onto the heart of human patients with heart disease. It is hoped with the help of growth factors these sheets will promote regeneration of damaged muscles and improve the function of the heart. This first of its kind trial involves 3 patients, if all goes well the team hopes to have a larger trial with 10 patients, followed by commercial availability of the technique if all goes according to plan.

There is no shortage of research into male pattern baldness, recently we are being shown how stem cells may play a role in hair revival, such as wherein scientists had converted iPSCs into epithelial stem cells which gave rise to hair follicle on the skin of immunodeficient mice. Another technique coaxed iPSCs into the form of dermal papilla cells which were transplanted into mice which triggered new hair growth. Research from the University of Southern California harvested skin cells from adults, molecular events behind their growth was examined and then replicated in iPSCs to grow new hair follicles in mice.

iPSCs are opening up a diverse array of exciting new medical possibilities. At only 13 years of discovery this very well is likely just the tip of the iceberg of many wonderful things to come. What a great time this could be for the future of the entire medical field and the possibility of regenerative medicine.

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Regenerative Potential Of Induced Pluripotent Stem Cells ...

Induced Pluripotent Stem Cell (iPSC) Media and Reagents for …

Induced Pluripotent Stem Cells | Posted by admin
Mar 21 2019

Advancing your induced pluripotent stem cells or human embryonic stem cell therapy research to clinical applications requires careful material selection because the quality of starting materials significantly impact the properties of your final stem cell therapy product. Gibco CTS products have been developed to ease the transition from stem cell therapy research to clinical applications by providing high quality GMP manufactured, commercial scale ancillary materials with a high degree of qualification, traceability and regulatory documentation. In an effort to help you maximize the potential of your stem cell research and therapy, and simplify the transition to clinic-ready processes, we offer an extensive selection of research use stem cell research products with complementary CTS formulations. Our CTS products are used in commercially approved cell therapies as well as over 100 clinical trials and are backed by our professional regulatory support and over 30 years of GMP manufacturing experience.

Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) , sometimes collectively referred to as pluripotent stem cells (PSCs), are cells that have the ability to renew themselves indefinitely and differentiate into almost any cell type when exposed to the right microenvironment. These unique properties enable the application of induced pluripotent stem cells and embryonic stem cells in disease modeling, drug discovery, drug toxicity testing, and cell therapy. Strikingly, most embryonic stem cell and induced pluripotent stem cell applications have the potential to improve human health, none more directly so than ESC or iPSC therapy. The most intuitive approach for ES or iPS cell therapy is to transplant PSC-derived cells for the direct replacement of damaged or degenerated cells or tissue. However, there are many other approaches to ES or iPS stem cell therapy such as transplanting PSC-derived cells that then release signals triggering endogenous repair mechanisms.

At Thermo Fisher Scientific, we support the development of your human embryonic stem cell therapy or induced pluripotent stem cell therapy from the earliest stages of research and all the way to the clinic. We offer high-quality products across the iPS cell therapy workflow from reprogramming to differentiation. Most Gibco media and supplements for culture and differentiation are manufactured under GMP conditions at sites that use methods and controls that conform to current Good Manufacturing Practices (cGMP) for medical devices. These FDA-registered manufacturing sites are ISO 13485 and ISO 9001certified, and the rigorous practices we adhere to at these sites help ensure the consistency, reliability, and high quality of a wide variety of iPSC therapy workflow reagents.

To further help you maximize the potential of your research and streamline your transition to the clinic, we offer Gibco Cell Therapy Systems (CTS) equivalents for many of our research-use products. In addition to GMP manufacturing, Gibco CTS products undergo extensive safety testing and are accompanied by appropriate documentation so you can transition your cell therapy to the clinic with confidence.

*Adherence to supplier related responsibilities of USP

First off-the-shelf reprogramming system manufactured in accordance with GMP requirements. CTS CytoTune 2.1 kit offers high-efficiency Sendai delivery of reprogramming factors.

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Efficient reprogramming from adult human dermal fibroblasts, T cells, and CD34+ cells. These data demonstrate that the CytoTune-iPS 2.1 kit can be used to successfully reprogram human dermal fibroblasts (HDFa), T cells, and CD34+ cells.

Based on the widely cited Gibco Essential 8 Medium, Gibco CTS Essential 8 Medium is the first globally available human- and animal originfree culture medium for human pluripotent stem cells (hPSCs) and is designed to meet international regulatory requirements for cell therapy.

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Using Applied Biosystems TaqMan hPSC Scorecard Panel analysis, Gibco CTS Essential 8 Medium and research-use-only Essential 8 Medium were shown to support comparable expression of PSC markers and lineage markers in undifferentiated PSCs and PSC-derived embryoid bodies.

CTS Vitronectin (VTN-N) Recombinant Human Protein is a defined matrix for feeder-free culture of iPSCs. Designed in the laboratory of James Thomson, this recombinant protein is intended for use with the CTS Essential 8 culture system.

CTS RevitaCell Supplement (100X) is an animal-origin-free, chemically defined supplement used with PSCs for post-thaw recovery or in combination with CTS Essential 8 Medium for single cell passaging. To minimize both the loss of cell viability and differentiation of PSCs, use the CTS PSC Cryopreservation Kit.

CTS Versene is a gentle non-enzymatic cell dissociation reagent for use in routine clump passaging of PSCs while maintaining viability over multiple passages.

For the cryopreservation and recovery of PSCs, the CTS PSC Cryopreservation Medium and CTS RevitaCell Supplement minimize the loss of cell viability and maximize post-thaw recovery when used in combination. Both reagents are included in the CTS PSC Cryopreservation Kit.

The CTS PSC Cryopreservation Medium is a xeno-free solution for the cryopreservation of pluripotent stem cells (PSCs). Both CTS PSC Cryopreservation Medium and CTS RevitaCell supplement are included in the CTS PSC Cryopreservation Kit that helps minimize loss of cell viability and maximize post-thaw recovery.

CTS KnockOut SR XenoFree Medium is a defined, xeno-free serum replacement based on the traditional Gibco KnockOut Serum Replacement, which has been cited in more than 2,000 publications and trusted for over 20 years.

Maintenance of pluripotency using CTS KNOCKOUT SR XenoFree Medium. Following 10 passages in either KSR (left lane) or KSR XenoFree CTS (right lane) on HFF attached with CELLstart substrate, BG01v gene expression was examined (top). Gene expression of embryoid bodies generated from the same P10 BG01v/HFF cultures (bottom).

Your choice of chemically defined human- and animal origin-free basal media for pluripotent stem cell culture. Based on traditional DMEM and DMEM/F12 formulations, these basal media are:

Gibco CTS growth factors help enable you to easily qualify reagents during your transition from research applications to clinical applications. CTS products are supplied with harmonized documentation such as Certificates of Analysis and Certificates of Origin.

CTS offers high-quality growth factors and cytokines for T cell, stem cell and dendritic cell applications.


We offer full customization options to help meet your unique specifications for any project. Flexibility is yours in creating your own Gibco custom cell culture medium

Intended use of the products mentioned on this page vary. For specific intended use statements please refer to the product label.

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Embryonic or Induced Pluripotent Stem Cell Markers: R&D Systems

Induced Pluripotent Stem Cells | Posted by admin
Mar 11 2019


Embryonic stem cells (ESCs) have the exceptional ability to both self-renew and differentiate into nearly every cell of the human body. Induced pluripotent stem cells (iPSCs) are somatic cells that have been reprogrammed back into an ESC-like phenotype. Both ESCs and iPSCs have numerous roles to play in drug discovery studies, understanding mechanisms of disease, cell therapies, and developmental biology.

Expression of Pluripotency Markers in Human Embryonic Stem Cells. Pluripotency marker expression was detected in immersion-fixed BG01V human embryonic stem cells using antibodies supplied in the Human Pluripotent Stem Cell Markers Antibody Panel (R&D Systems, Catalog # SC008). Pluripotency marker expression was analyzed by dual immunofluorescence with the indicated primary antibodies supplied in the panel. The cells were stained using NorthernLights (NL) 493- and NL557-conjugated Secondary Antibodies (green and red, respectively). Where indicated, the nuclei were counterstained with DAPI (blue).

Verification of Pluripotency in Human Induced Pluripotent Stem Cells. iPS2 human induced pluripotent stem cells were grown on irradiated mouse embryonic fibroblasts (R&D Systems, Catalog # PSC001) and stained using antibodies included in the GloLIVE Human Pluripotent Stem Cell Live Cell Imaging Kit (R&D Systems, Catalog # SC023B). A. iPS2 cells stained with the NL493-conjugated SSEA-4 (green) and the NL557-conjugated SSEA-1 (red) antibodies. B. iPS2 cells stained with the NL493-conjugated SSEA-4 (green) and the N557-conjugated TRA-1-60(R) (red) antibodies. The cells were counterstained with Hoechst 33342 (blue). The colonies are positive for the stem cell markers SSEA-4 and TRA-1-60(R) and are negative for SSEA-1, suggesting that these colonies primarily contain undifferentiated human stem cells.

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Embryonic or Induced Pluripotent Stem Cell Markers: R&D Systems

Differentiation of human induced pluripotent stem cells into …

Induced Pluripotent Stem Cells | Posted by admin
Mar 08 2019

Culture of human iPSCs

The commercial human iPSCs used in this study were purchased from Saibei Biotechnology (Beijing, China). iPSCs (HiPSC-U1) were reprogrammed from human urine-derived cells of a 37-year-old male by the integration-free CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, MA, USA). iPSCs were cultured in 1% Matrigel-coated (BD Biosciences Co., Ltd., NM, USA) Petri dishes with E8 medium (Saibei Biotechnology) at 37C and 5% CO2. The medium was refreshed daily. iPSCs were passaged once every 6 days with 0.25% ethylenediaminetetraacetic acid (EDTA, Saibei Biotechnology). 1mL of 0.25% EDTA was added and the cells were placed at 37C and 5% CO2 for 5min. When iPSC colonies appeared white, the solution was gently removed, and the iPSCs were washed with Mg2+ and Ca2+-free Dulbeccos PBS (Sigma, MO, USA). iPSCs were then harvested by gently pipetting 710 times with 1mL E8 medium and seeded onto fresh six-well culture plates that were coated with 1% Matrigel at a ratio of 1:6. 10m Y-27632 (Sigma) was supplemented in the medium on the first day of passage.

Human LCs were obtained from nine male donors with a mean age of 45 years old through testes excision within 20h. Informed consent was obtained from each donor, and this study was approved by Human Research and Ethical Committee of Wenzhou Medical University. The testes were used to isolate ILCs. ILCs express all androgen biosynthetic enzymes56, and are capable of proliferation and differentiation57. The isolation of ILCs was performed as previously described56. In brief, the testes were perfused with collagenase (Sigma) via the testicular artery, and digested with M-199 buffer (Gibco, NY, USA) containing collagenase (0.25mg/ml) and DNase (0.25mg/mL, Sigma) for 15min. Then, the cell suspension was filtered through 100m nylon mesh and the cells were separated by a Percoll gradient (Sigma). The cells with the density of 1.071.088g/ml were collected. The purity of ILCs was evaluated by immunohistochemical staining HSD3B1, the biomarker of ILCs, as previously described58. The HSD3B1 staining solution contained with 0.4mm etiocholanolone (Sigma) as the steroid substrate and NAD+ as a cofactor58. The purity of ILCs was >95%.

The isolated ILCs were directly seeded into wells in the 24-well culture plates with the density of 2104 cells/well and incubated at a 37C, 5% CO2 incubator. The culture medium (LC-Medium) contains DMEM/F12 (Gibco), 5% fatal bovine serum (FBS, Gibco), 2.5% horse serum (HS, Gibco), and 1% penicillin/streptomycin (P/S, Gibco). In order to get ALCs, the culture medium were changed into differentiation-induced medium (DIM) contains DMEM/F12, 5mm ITS (insulin, transferrin, and selenium, Sigma), 5ng/ml luteinizing hormone (LH, PeproTech, NJ, USA), and 5mm lithium chloride (Li, Sigma) as our team previous report35.

The Sprague-Dawley rats (at 5 weeks of age) and immune deficiency (SCID) mice (at 5 weeks of age) were obtained from the laboratory animal center of Wenzhou Medical University, Wenzhou, China. All animals were kept under conditions with controlled temperature (232C), a 12h dark/light cycle, and relative humidity of 4555%. The standard drinking water and rodent diet were accessed ad libitum. All surgical procedures and postoperative care were approved by the Wenzhou Medical Universitys Animal Care and Use Committee, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

The point at which iPSCs were expanded to ~70% confluency in the E8 medium was defined as day 2, and at this point when iPSCs were changed into E7 medium (no FGF2) for 2 days to prepare differentiation. From day 5 to 0, the medium was refreshed daily. Prior to the beginning of differentiation, iPSCs were cultured in a differentiation-inducing medium composed of DMEM/F12, 1% bovine serum albumin (BSA) (Sigma), 5mm ITS, 5ng/mL LH. From 07 days, 0.2m SAG (DHH agonist, Sigma), 5m 22R-OHC (Steraloids, RI, USA), and 5mm Li were added into iPSC-DIM. From 710 days, 5ng/mL PDGF-AA (Sigma) and 5ng/mL FGF2 (Sigma) were added into iPSC-DIM. From 1017 days, 5ng/mL PDGF-AA, 5nM IGF1 (Sigma), and 10m Androgen (Sigma) were added into iPSC-DIM. From 1720 days, 10ng/mL PDGF-AA and 10ng/mL FGF2 were added into iPSC-DIM. From 2025 days, 5ng/mL LH, 0.5mm retinoic acid (RA, Sigma) and 1mm 8-Br-cAMP (Sigma) were added into iPSC-DIM. From day 0 to 25, the medium was changed every 2 days by fresh iPSC-DIM. From 2530 days, the cells were mechanically enriched by scraping away clonal iPSC-like cells. The remaining Leydig-like cells were kept in Enrichment Medium contained DMEM/F12, 5% FBS, 2.5% HS, 1sodiumpyruvate (Invitrogen), 1GlutaMAX (Invitrogen), and 1% P/S for the subsequent assays. The medium was changed every 2 days by fresh Enrichment Medium.

For TEM, the cells in different groups were prefixed with 2.5% glutaraldehyde in 0.1m PBS for 24h at 4C. Then, they were washed with PBS, and post-fixed with 1% osmium tetroxide. After gradient dehydration of acetone, they were embedded in Araldite M (Sigma Aldrich). Ultrathin sections (1m) were subsequently cut with an ultramicrotome, mounted on nickel grids, and stained with uranyl acetate and lead citrate. At last, the samples were sent to the electron microscope room at Wenzhou Medical University for subsequent processing and testing using a transmission electron microscope (H-600A-2; Hitachi, Tokyo, Japan).

The cell culture supernatants and serum were collected at each experimental time point for the quantitative measurement of testosterone. For the cell culture supernatants, 10ng/mL LH was in advance at least 3h added into the medium (just having DMEM/F12) to stimulate the testosterone production of LCs or iPSC-LCs. Testosterone levels were measured with a tritium-based radioimmunoassay using anti-testosterone antibody as previously described59. Standards ranging between 10 and 2000pg/mL testosterone were prepared in triplicate. Standards and samples were incubated with tracer and antibody at 4C overnight and charcoal-dextran suspension was used to separate the bound and free steroids. The bound steroids were mixed with a scintillation buffer and counted in a scintillation counter (PE, CA, USA). The minimum detectable concentration for testosterone was 5pg/mL. Quality control samples contain 100pg/mL testosterone. The intra-assay and inter-assay coefficients of variation were within 10%.

Immunofluorescence was used to identify iPSC-LCs as a previous report60. In brief, after fixation with 4% paraformaldehyde (Sigma) for 15min, cells were washed three times with PBS. Then cells were permeabilized with 0.1% TritonX-100 in PBS for 15min at room temperature, and incubated with 3% (w/v) BSA in PBS for 1h at room temperature. The cells were then incubated with primary antibodies as TableS1 overnight at 4C, and then with fluorescein isothiocyanate (FITC)-conjugated anti-mouse, FITC-conjugated anti-rabbit, Cy3-conjugated anti-mouse, and Cy3-conjugated anti-rabbit IgG secondary antibodies (1:1000, Bioword, USA) for 60min at room temperature. Then the cells were rinsed three times with PBS thrice for 5min each and then incubated for 15min with DAPI (Sigma) for nuclear staining and washed three times with PBS before examination by an inverted fluorescence microscope (OLYMPUS, Japan).

Total RNA from the cells was extracted using Trizol reagent (Invitrogen, CA, USA) according to the manufacturers instruction. The RNA was reversely transcribed into cDNA using the Superscript II kit (Invitrogen). The cDNAs templates were diluted 1:10, which were used to perform RT-PCR and qPCR to analyze the gene expressions. RT-PCR was performed using an authorized thermal cycler (Eppendorf, Hamburg, GER). After amplification, 1L of 6Loading buffer and 5L of each PCR product were mixed and electrophoresed on a 2% agarose containing 0.5g/mL ethidium bromide. Gels were scanned for further analysis. qPCR was performed using the Thunderbird SYBR qPCR Mix (Takara, Tokyo, Japan) according to the manufacturers instructions. Signals were detected using a Light Cycler 480 Detection System (Roche, Basel, Switzerland). The relative expression of genes was normalized to GAPDH. The melting curve was examined for the quality of PCR amplification for each sample, and quantification was performed using the comparative 2-Ct method. The primer sequences were shown in TableS2.

Total RNA from each sample was extracted using Trizol reagent (Invitrogen, CA, USA). 12g total RNA was used to prepare the sequencing library. To sequence the libraries, the barcoded libraries were mixed, denatured to single stranded DNA, captured on Illumina flow cell, amplified in situ, and subsequently sequenced for 150 cycles for both ends on Illumina HiSeq 4000 instrument. Sequence quality was examined using the FastQC software. The transcript abundances for each sample were estimated with StringTie, and the FPKM value for gene and transcript level were calculated with R package Ballgown. The differentially expressed genes and transcripts were filtered using R package Ballgown. The correlation analysis was based on gene expression levels. Hierarchical Clustering, Gene Ontology, Pathway analysis, scatter plots and volcano plots were performed with the differentially expressed genes in R, Python for statistical computing and graphics61.

Cells were washed with cold PBS and were lysed in 1radioimmunoprecipitation assay lysis buffer in the presence of a protease inhibitor mixture/1% phosphatase inhibitor mixture (Roche). 50g of protein samples were applied to a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred into the polyvinylidene difluoride membranes (Sigma) by an electroblot apparatus. After being blocked with a blocking solution (5% fat-free milk) for 2h at 4C, the membranes were incubated with primary antibodies as TableS1 in the blocking solution at 4C overnight. The membranes were washed with tris-buffered saline with Tween 20 (TBST) five times (10min each), and incubated with horseradish peroxidase-conjugated secondary antibody (1:3000, Bioword) at room temperature for 2h. The membranes were then washed five times (10min each) with TBST. Bands were visualized with enhanced chemiluminescence (ECL, Pierce, USA). The protein expression was normalized to -actin.

The cell samples were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% TritonX-100 (Sigma). The samples were then labeled with primary or isotype control antibodies for 30min at 4C. Primary and isotype control antibodies that were not conjugated to fluorophores were labeled with fluorophore-conjugated secondary antibody for 30min at 4C. The labeled samples were detected by flow cytometry analyzer (BD, USA). Data analysis was performed on FCS Express 4 Flow Research Edition software.

The standard protocol was conducted as PKH26 Product Information Sheet (Sigma, MINI2). In brief, the suspension containing 2107 cells were centrifuged (400g, 5min) and were washed once using fresh medium without serum. After centrifuging, the supernatant was removed and no more than 25L of 2Cell Suspension was prepared by adding 1mL of Diluent C, the cells were resuspended with gentle pipetting to ensure complete dispersion. 2Dye Solution (4106m) was prepared by adding 4L of the PKH26 ethanolic dye solution to 1mL of Diluent C and mixed well. Then 1mL of 2Dye Solution was rapidly added into the 1mL of 2Cell Suspension. Final concentration after mixing was 2106m PKH26 with 1107 cells/well. The mixing suspension was incubated at room temperature for 5min with periodic mixing. The action of the staining was stopped by adding an equal volume (2mL) of serum. Then the suspension was centrifuged at 400g for 10min and washed three times. Finally, the cells tagged with PKH26 were seeded on fresh wells and used for injection.

For evaluating whether iPSC-LCs could facilitate the recovery of LC dysfunction of rats, iPSC-LC transplantation was performed as previously described with some modifications62. Sixty 49-days-old male Sprague-Dawley rats (n=5 for each group at each time point) were used in this study. Before transplantation, male rats were administered a single intraperitoneal injection of EDS (75mg/kg, Pterosaur Biotech Co., Ltd., Hangzhou, China), which was dissolved in dimethyl sulfoxide (Sigma): H2O (1: 3, v/v). This treatment resulted in the elimination of LCs in the adult testes of rats63. Then iPSC-LCs labeled with PKH26 (red) were resuspended manually, and harvested in a 15mL Falcon tube. Cells were rinsed twice with PBS following centrifugation at 200g for 5min. Finally each pellet was resuspended in PBS for transplantation. Cells were loaded into a 1mL syringe for injection into the testis of adult Sprague-Dawley male rats that had been treated with EDS. Approximately 2106 PKH26-labeled iPSC-LCs in 40mL of PBS were injected into the parenchyma of recipient testes 7 days after the rats received EDS. The control animals for the experimental group were EDS-treated rats that had received a testicular injection of the PBS vehicle. Testes from all animals were examined at 0, 7, 14, and 21 days after EDS treatment.

One testis from each rat was used for immunohistochemistry (Vector Laboratories, Inc., Burlingame, CA, USA) according to the manufacturers instructions. The rats were killed with an overdose of sodium pentobarbital (Sigma). Testes were removed and fixed in 4% paraformaldehyde overnight at 4C. Then testes were dehydrated with a graded series of ethanol and xylene and subsequently embedded in paraffin. Five micrometer-thick transverse sections (5m) were cut, de-waxed in water, and were mounted on glass slides. Antigen retrieval was performed by microwave irradiation for 10min in 10mm (pH 6.0) of citrate buffer, after which endogenous peroxidase was blocked with 0.5% of H2O2 in methanol for 30min. Some sections were fixed with 4% paraformaldehyde for 15min and washed 3 times with PBS. Then they were permeabilized with 0.1% TritonX-100 in PBS for 15min at room temperature, and incubated with 3% (w/v) BSA (Sigma) in PBS for 1h at room temperature. Then these sections were then incubated with an CYP11A1 polyclonal antibody diluted 1:1000 for 2h at room temperature, and then with FITC-conjugated IgG secondary antibodies (1:1000, Bioword) for 1h at room temperature. These sections were rinsed with PBS three times for 5min each time. Then the sections were incubated for 15min with DAPI (10g/mL, Sigma) for nuclear staining and washed three times with PBS. The sections were cover-slipped with resin (Thermo Fisher Scientific, Waltham, UK). At last, they were examined by an inverted fluorescence microscope (OLYMPUS). The cells with CYP11A1 staining in the interstitial area represent the LC64.

Other sections were directly incubated with CYP11A1 polyclonal antibody diluted 1:1000, for 2h at room temperature. Diaminobenzidine was used for visualizing the antibodyantigen complexes, positive labeling LCs by brown cytoplasmic staining. Mayer hematoxylin was applied in counterstaining. The sections were then dehydrated in graded concentrations of alcohol and cover-slipped with resin (Thermo Fisher Scientific, Waltham, UK). Lastly, they were examined by a fluorescence microscope (LEICA). The cells with CYP11A1 staining in the interstitial area represent the LCs.

For teratoma formation, iPSCs (5106 cells) were dissociated with 0.5mm EDTA, centrifuged, resuspended in 100L E8 with 1% Matrigel, and injected into the hind limbs of 6-week-old male SCID mice. Teratomas were collected after 6 weeks, and fixed in 4% paraformaldehyde for paraffin embedding and hematoxylin and eosin staining. Slides were imaged and analyzed by a qualified clinical pathologist.

The division of iPSCs was blocked with 50g/mL of colcemid solution (Invitrogen, USA). Cells were washed with PBS and harvested with trypsin at room temperature for 2min. Then cells were fixed in methanol/glacial acetic acid (3:1) for three times and dropped onto slides for chromosome spreads. At last, the slides were baked at 55C for overnight. Standard G-banded karyotypes were obtained using Giemsa solution staining (Giemsa, Japan).

To enumerate CYP11A1-positive Leydig cell numbers, sampling of the testis was performed according to a fractionator method as our previous report65. Identification of all Leydig cell lineages was done by the staining of CYP11A1. About 10 testis sections per rat were sampled from each testis. The total number of LCs was calculated by multiplying the number of LCs counted in a known fraction of the testis by the inverse of the sampling probability.

All experiments were performed at least thrice, and the data are presented as the meanstandard error of the mean. Statistical analyses were evaluated using an unpaired Students t test or one-way analysis of variance for more than two groups. P

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Differentiation of human induced pluripotent stem cells into ...

Do You Know the 5 Types of Stem Cells? | BioInformant

Induced Pluripotent Stem Cells | Posted by admin
Mar 04 2019

*Post also available in: Espaol Romn

As you start to learn about stem cells, one of the most common questions tohave is, What types of stem cells exist?There is not an agreed-upon number of stem cell types, because one can classify stem cells either by differentiation potential(what they can turn into) or by origin (from where they are sourced).This post is dedicated to explaining the five types of stem cells, based on differentiation potential.

The five different types of stem cells discussed in this article are:

All stem cells that exist can be classified into one of five groups based on their differentiation potential. Each of these stem cell types is explored in greater detail below.

The Rise of Direct Cell Reprogramming | BioInformant #totipotent #pluripotent #multipotent #autologous

Todd C Bertsch (@todd_bertsch) February 19, 2018

These stem cells are the most powerful that exist.

They can differentiate into embryonic, as well as extra-embryonic tissues, such as chorion, yolk sac, amnion, and the allantois. In humans and other placental animals, these tissues form the placenta.

The most important characteristic of a totipotent cell is that it can generate a fully-functional, living organism.

The best-known example of a totipotent cell is a fertilized egg (formed when a sperm and egg unite to form a zygote).

It is at or around four days post-fertilization that these cells begin to specialize into pluripotent cells, which as described below are flexible cell types, but cannot produce an entire organism.

Theyre aliveeee!! Turned our human pluripotent stem cells into beating cardio!!! ::happy tears:: Next up crispR KO fun #stemcellscientist #WomenInScience #futureBIOhacker

Kristin Pagel (@DeeDeeTroit84) March 31, 2018

The next most powerful type of stem cell is the pluripotent stem cell.

The importance of this cell type is that it can self-renew and differentiate into any of the three germ layers, which are: ectoderm, endoderm, and mesoderm. These three germ layers further differentiate to form all tissues and organs within a human being.

There are several known types of pluripotent stem cells.

Among the natural pluripotent stem cells, embryonic stem cells are the best example.However, a type of human-made pluripotent stem cell also exists, which is the induced pluripotent stem cell (iPS cell).

iPS cells were first produced from mouse cells in 2006 and human cells in 2007, and are tissue-specific cells that can be reprogrammed to become functionally similar to embryonic stem cells.

Because of their powerful ability to differentiate in a wide diversity of tissues and their non-controversial nature, induced pluripotent stem cells are well-suited for use in cellular therapy and regenerative medicine.

Did you know that bone marrow contains multipotent stem cells that give rise to all the cells of the blood?

caremotto (@caremotto) January 17, 2018

Multipotent stem cells are a middle-range type of stem cell, in that they can self-renew and differentiate into a specific range of cell types.

An excellent example of this cell type is the mesenchymal stem cell (MSC).

Mesenchymal stem cells can differentiate into osteoblasts (a type of bone cell), myocytes (muscle cells), adipocytes (fat cells), and chondrocytes (cartilage cells).

These cells types are fairly diverse in their characteristics, which is why mesenchymal stem cells are classified as multipotent stem cells.

The next type of stem cells, oligopotent cells, are similar to the prior category (multipotent stem cells), but they become further restricted in their capacity to differentiate.

While these cells can self-renew and differentiate, they can only do so to a limited extent. They can only do so into closely related cell types.

An excellent example of this cell type is the hematopoietic stem cell (HSC).

HSCs are cells derived from mesoderm that can differentiate into other blood cells. Specifically, HSCs are oligopotent stem cells that can differentiate into both myeloid and lymphoid cells.

Myeloid cells includebasophils, dendritic cells, eosinophils, erythrocytes, macrophages, megakaryocytes, monocytes, neutrophils, and platelets, while lymphoid cells include B cells, T cells, and natural kills cells.

Finally, we have the unipotent stem cells, which are the least potent and most limited type of stem cell.

An example of this stem cell type would be muscle stem cells.

While muscle stem cells can self-renew and differentiate, they can only do so into a single cell type. They are unidirectional in their differentiation capacity.

The purpose of these stem cellcategories is to assess thefunctional capacity of stem cells based on their differentiation potential.

Importantly, each category has different stem cell research applications, medical applications, and drug development applications.

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Do You Know The 5 Types Of Stem Cells?

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Global transcriptome analysis of pig induced pluripotent …

Induced Pluripotent Stem Cells | Posted by admin
Mar 04 2019

The progress of next-generation sequencing technology has caused a technological breakthrough at the whole-genome level in a large number of species1. Especially, RNA-sequencing (RNA-Seq) has enabled us to take a snapshot of global gene expression in various organs and cells, regardless of any information of a reference genome. RNA-Seq outputs are digital data that can be uploaded to the public database, and sequence information can be shared worldwide.

RNA-Seq analysis also allows us to compare the biological similarity of embryonic stem cells (ES cell) with induced pluripotent stem (iPS) cells. In general, stem cells can be classified into two major subtypes: nave and primed states2,3. ES/iPS cells at nave state of pluripotency, reflect the characteristics of pre-implantation embryos and are applicable in rodents, which contribute to chimeras and germ line4,5. The growth of nave stem cell depends on the activation of LIF (Leukemia Inhibitory Factor) signaling, whereby the cell forms colonies with three-dimensional shape.

On the other hand, primed cells have the characteristics of post-implantation embryos and rarely contribute to chimeras and germ line. In brief, primed cells are already at a more differentiated stage compared to nave cells. Primate ES/iPS cells were conventionally believed to be established in primed state6,7. However, recent publications have demonstrated a reliable method for transforming human ES cells from primed to nave state8,9. Transcriptome analysis using RNA-Seq played an important role in identifying the cellular characteristics reported in those articles.

In the case of pig iPS cells, the status of the cellsnave or primedremains inconclusive since the pluripotent genes have a wide variety of phenotypes. To understand the biological variety of pig iPS cells, multiple datasets of global gene expression profiling would be needed. Although a significant number of reports on the establishment of pig iPS cells have been published10,11,12,13,14,15,16,17,18,19,20, expression profiling data in Sequence Read Archive (SRA) database are quite limited. Therefore, detailed biological features of pig iPS cells need to be addressed with whole expression profiling.

In our previous publication, we had reported that pig iPS cell, derived from six reprogramming factors, has more advantageous than that derived from four factors. Especially, the expression of six reprogramming factors was suitable for X chromosome re-activation21, which is one of the mile-stone characteristics of nave-type stem cells. Our previous data using Ion Torrent sequencing also proved that the expression of six reprogramming factors was more advantageous to activate various pluripotent genes. Although the data obtained from Ion Torrent is suggestive, at least 20M reads would be necessary to obtain a quantitative evaluation of the relatively low-expressing genes. The data obtained in our previous publication seem insufficient in terms of the number of sequencing reads required to conclude. This situation led us to detect the global expression profile of pig iPS cells, derived from the expression of six reprogramming factors, using Illumina short-read sequencer, HiSeq 2500. Currently, there are no publicly available dataset of six factor-derived pig iPS cells using Illumina sequencing platform.

The aim of this study was to clarify the difference of mRNA expression profiles between pig iPS cells derived from six and four reprogramming factors. We found relevant submitted data from two research groups on pig iPS cells with four reprogramming factors, in SRA22,23. We could compare ours with these gene expressions since both datasets were obtained with Illumina sequencer. In this study, we describe the detailed expression profile of pig iPS cells derived from four and six reprogramming factors. Multiple analyses demonstrated that the pig iPS cells derived from six factors formed independent clusters compared to those derived from four factors, and were distant from fibroblasts. Furthermore, we detected that the expression levels of various nave-specific genes were relatively elevated in pig iPS cells derived from six factors. Our data set would contribute to the understanding of biological differences between the iPS cells derived from six and four reprogramming factors, and provide the scientific explanation of how diversity of pluripotency-related genes related to the process of animal evolution.

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Global transcriptome analysis of pig induced pluripotent ...

Hypoimmunogenic derivatives of induced pluripotent stem …

Induced Pluripotent Stem Cells | Posted by admin
Feb 20 2019

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Hypoimmunogenic derivatives of induced pluripotent stem ...

microRNA-690 regulates induced pluripotent stem cells (iPSCs …

Induced Pluripotent Stem Cells | Posted by admin
Feb 17 2019

Stem Cell Research & Therapy201910:59

The Author(s).2019

The regulatory mechanism of insulin-producing cells (IPCs) differentiation from induced pluripotent stem cells (iPSCs) in vitro is very important in the phylogenetics of pancreatic islets, the molecular pathogenesis of diabetes, and the acquisition of high-quality pancreatic -cells derived from stem cells for cell therapy.

miPSCs were induced for IPCs differentiation. miRNA microarray assays were performed by using total RNA from our iPCs-derived IPCs containing undifferentiated iPSCs and iPSCs-derived IPCSs at day 4, day 14, and day 21 during step 3 to screen the differentially expressed miRNAs (DEmiRNAs) related to IPCs differentiation, and putative target genes of DEmiRNAs were predicted by bioinformatics analysis. miR-690 was selected for further research, and MPCs were transfected by miR-690-agomir to confirm whether it was involved in the regulation of IPCs differentiation in iPSCs. Quantitative Real-Time PCR (qRT-PCR), Western blotting, and immunostaining assays were performed to examine the pancreatic function of IPCs at mRNA and protein level respectively. Flow cytometry and ELISA were performed to detect differentiation efficiency and insulin content and secretion from iPSCs-derived IPCs in response to stimulation at different concentration of glucose. The targeting of the 3-untranslated region of Sox9 by miR-690 was examined by luciferase assay.

We found that miR-690 was expressed dynamically during IPCs differentiation according to the miRNA array results and that overexpression of miR-690 significantly impaired the maturation and insulinogenesis of IPCs derived from iPSCs both in vitro and in vivo. Bioinformatic prediction and mechanistic analysis revealed that miR-690 plays a pivotal role during the differentiation of IPCs by directly targeting the transcription factor sex-determining region Y (SRY)-box9. Furthermore, downstream experiments indicated that miR-690 is likely to act as an inactivated regulator of the Wnt signaling pathway in this process.

We discovered a previously unknown interaction between miR-690 and sox9 but also revealed a new regulatory signaling pathway of the miR-690/Sox9 axis during iPSCs-induced IPCs differentiation.

Type 1 diabetes (T1D) is defined as dysregulation of homeostatic control of blood glucose due to an absolute insulin deficiency caused by autoimmune destruction of insulin-secreting pancreatic -cells [1]. The transplantation of -cells from a pancreatic donor or augmentation of endogenous -cells regeneration may lead to a cure for T1D. Unfortunately, these methods are restricted by donor tissue availability and tissue rejection and are thus far from being widely applied [2]. Insulin-producing cells (IPCs) derived from pluripotent stem cells in vitro may provide an alternative source of -cells [3]; however, the rate of development of functional and mature IPCs is very low according to the present protocols [4], which will be improved by a thorough understanding of pancreatic organogenesis, including proliferation, differentiation, migration, and maturation of pancreatic progenitor cells.

Considerable evidence has verified that microRNAs (miRNAs) in pancreatic cells regulate gene expression through post-transcriptional modulation [5, 6]. Recently, the global influence of miRNAs on pancreatic development has been assessed by Dicer-knockout mouse embryos. Dicer deficiency resulted in alterations of islet architecture and differentiation markers, accompanied by enhanced apoptosis and defects in all types of endocrine cell formation, particularly that of -cells [7]. Similarly, miR-375 is expressed specifically in pancreatic islets and regulates the proliferation and insulin secretion of -cells by targeting myotrophin (MTPN) and phosphoinositide-dependent protein kinase-1 [8]. Knockdown of miR-375 in ob/ob mice led to a disproportionate ratio of -cells to -cells, high plasma glucagon levels, or even diabetes [9]. In addition, other miRNAs, such as miR-7 and miR-199b-5p, have been studied functionally and reported to selectively affect the development of pancreatic islets, promoting the proliferation of -cells and miR-124a and regulating Foxa2 expression and intracellular signaling in -cells [1012]. These findings, as highlighted above, encouraged us to identify different layers of miRNA regulatory networks, which will provide greater insights into the roles of noncoding RNAs and help further elucidate -cell biology, pancreas formation, and the molecular mechanisms of diabetes etiopathogenesis.

During pancreatic development, the sex-determining region Y (SRY)-box9 (Sox9) factor, which is known to function in campomelic dysplasia, XY sex reversal, and skeletal malformations, has been linked to the proliferation and differentiation of endocrine progenitors [13, 14]. Analysis of cases with Sox9 loss in pancreatic progenitor cells demonstrated a proportional reduction in FoxA2 and Onecut1 expression, along with upregulation of Hnf1b (TCF2), which resulted in a dramatic decrease in endocrine cells without changes in exocrine compartments [15]. Despite a fair understanding of the molecular mechanism by which Sox9 controls pancreatic development, only a few pathways regulated by Sox9 are known. Wnt/-catenin signaling (WNT) has been demonstrated to participate broadly in the differentiation of stem cells, showing a negative regulatory relationship with Sox9 in various contexts [16, 17]. Furthermore, both CTNNB1 (-catenin) and pGSK3 act as downstream target genes, increasing transcriptional activity and decreasing degradation by overexpression of Sox9 [14].

In this study, we identified miR-690 as a differentially expressed transcript during induced pluripotent stem cell (iPSCs)-induced IPCs differentiation in vitro. Surprisingly, predicted mRNA targets, such as Sox9, CTNNB1 (-Catenin), and Stat3, were found to be crucial during the specification of pancreatic progenitor cells and terminal maturation of endocrine cells. Furthermore, the augmentation of miR-690 destabilized IPCs differentiation through direct binding to Sox9 and was likely to have a repressive effect on the Wnt pathway, suggesting an unreported role of miR-690 in modulating key transcription factors and signaling pathways.

C57BL/6J mice were from the animal center of Nantong University. All animal experiments were performed according to the Institutional Animal Care guidelines and were approved by the Animal Ethics Committee of the Medical School of Nantong University.

Mouse GFP-iPSCs were obtained from the Innovative Cellular Therapeutics, Ltd. (Shanghai, China), maintained on feeders in mESC culture conditions, and induced to differentiate into pancreatic IPCs via a three-step protocol as previously described.

Total RNA was isolated using RNAiso Plus (TaKaRa). The first-strand cDNA synthesis for miRNA was performed by using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) and following the manufacturers instructions. The relative expression levels of each miRNA and mRNA were calculated by the 2Ct method as previously described, and GAPDH and U6 were used as the internal normalization controls. Each experiment was performed independently and repeated three times. The qRT-PCR primer sequences were designed and synthesized by GenScript Biotech Corp. (Nanjing, China).

miRNA profiling of iPSC-derived IPCs was carried out by the Professional Oebiotech Corporation (Shanghai, China). In brief, total extracted RNA was labeled with the Agilent miRNA Complete Labeling and Hyb kit (Agilent, Santa Clara, CA, USA) and hybridized to an Agilent Mouse microRNA microarray V21.0. Then, a Gene Expression Wash Buffer kit (Agilent) was used to wash the microarray. Differentially expressed miRNAs (DEmiRNAs) were identified using GeneSpring software (version 13.1, Agilent Technologies, fold change 1.5, P value 0.05). TargetScan and were used to select target genes of DEmiRNAs (P0.05 for both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis). The feasible regulatory relationships between miRNAs and target genes were analyzed using Cytoscape software (

Cells were washed with PBS and lysed on ice for 30min with RIPA buffer (high) (Solarbio). Protein concentrations were detected using the BCA Protein Assay (Thermo Fisher Scientific). Total proteins were separated by SDS-PAGE, blotted on PVDF membranes (Millipore, Bedford, MA, USA), and probed with primary antibody in Antibody Dilution Buffer (Solarbio) at 4C overnight. After three washes in TBST, the membranes were incubated with HRP-conjugated secondary antibodies for visualization. Primary antibodies and HRP-conjugated secondary antibodies are listed: anti-Sox9 antibody (Abcam), anti-beta catenin antibody (Abcam), anti-beta actin antibody as a loading control (Abcam), anti-phospho-GSK-3 (Ser9) rabbit mAb (Cell Signaling Technology), anti-phospho-CyclinD1 (Ser90) antibody (affinity), and goat anti-rabbit HRP antibody (affinity).

iPSCs-derived IPCs were transferred into new 24-well plates for 12h. After preincubation in Krebs-Ringer bicarbonate buffer (KRB) without glucose for 120min, the cells were stimulated with KRB containing 0, 5, 15, 30, and 45mM glucose for 120min. The supernatant was collected. Insulin content and secretion from iPSC-derived IPCs were assessed by ELISAs, which were carried out using an ultrasensitive mouse insulin assay kit (Mercodia) following the manufacturers instructions.

iPSCs-derived IPCs grown on glass coverslips were washed with PBS and fixed with 4% paraformaldehyde for 15min at room temperature. Then, these cells were washed thrice (10min every time) and permeabilized with 0.5% (v/v) Triton X-100 for 15min at room temperature. Next, 5% donkey serum was added for 60min, and the cells were stained with different primary antibodies at 4C overnight. Then, the cells were stained with fluorescence secondary antibodies for 1h and DAPI (Solarbio) for 15min. Images were acquired using a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Ltd.). Primary antibodies are listed as follows: anti-insulin antibody (Abcam), anti-C-peptide antibody (Abcam), anti-PDX1 antibody (Abcam), anti-SOX9 antibody (Abcam), antibody-beta catenin antibody (Abcam), anti-NKX6.1 (D804R) rabbit mAb (Cell Signaling Technology). Secondary antibodies included donkey anti-rabbit (Alexa Fluor 647, Abcam), donkey anti-rabbit (Alexa Fluor 555, Abcam), goat anti-guinea pig (Alexa Fluor 647, Abcam), donkey F(ab,)2 anti-goat (Alexa Fluor 594, Abcam), and donkey anti-goat (Alexa Fluor 647, Abcam) antibodies.

For identification of the insulin-positive population, 1106 iPSCs-derived IPCs were digested with trypsin, washed with PBS, and resuspended as single cells by incubation in Reagent 1: Fixation (Beckman Coulter) for 15min. Then, the cells were washed once in PBS, incubated in Reagent 2: Permeabilization (Beckman Coulter) for 20min, and washed once in PBS. Next, the cells were resuspended in PBS with primary antibody and incubated for 30min. The cells were then washed with PBS twice and analyzed with the BD FACSCalibur system (BD Biosciences). The results were analyzed using FlowJo software. All procedures were carried out at room temperature. The primary antibody was anti-h/b/m insulin APC-conjugated rat IgG2A (R&D Systems). The isotype antibody was rat IgG2A control APC-conjugated.

A luciferase reporter assay was performed to observe interactions between miR-690 and Sox9. Wild-type Sox9 and the mutant Sox9 were cloned into the Pezx-FR02 reporter vector for miR-690 targeting. Pezx-FR02 or Pezx-FR02-Sox9-MUT was co-transfected with miR-690 mimic or miRNA mimic control. Firefly and Renilla luciferase activities were assayed with a Dual-Luciferase Assay (Promega, Madison, USA) at 48h post-transfection according to the manufacturers instructions.

Data are presented as the meanstandard deviation (SD) from at least three independent experiments. Significant differences in the relative miRNA or mRNA levels between the experimental groups and their negative controls were determined via Students t test using GraphPad Prism 7.0 (GraphPad Software, Inc.). A P value

The differentiation protocol has been described by Huang et al. (Fig.

a, b) [


]. The iPSCs obtaining from the Innovative Cellular Therapeutics, Ltd., were identified (Additionalfile

: Figure S1). Importantly, pancreatic -cells are the only IPCs in humans and animals. C-peptide is the active form of insulin. We detected these two markers of mature -cells in iPSC-derived IPCs on day 21 of step 3 to evaluate the efficiency of these insulin-secreting cells. Immunofluorescence assays showed that the majorities of the cells were positive for insulin and C-peptide (Fig.

c). The flow cytometry results also showed that 41.3%0.35% of iPSCs-derived IPCs at the final stage were insulin


d). To determine whether the differentiated cells respond to glucose stimulation, we assessed insulin secretion by exposing IPCs to glucose at different concentrations (0, 5, 15, and 30mM). Treatment with glucose increased insulin secretion in these IPCs, with a peak at the 15mM glucose concentration. No more insulin was induced when the glucose concentration increased to 30mM, suggesting that these IPCs reached the upper limit of their insulin secretion capacity in response to glucose (Fig.


Overview of the differentiation protocol. a Summary of the three-step differentiation protocol. EBs embryoid bodies, MPs multilineage progenitor. b Morphologies of differentiating iPSCs into IPCs at different time points during differentiation. Scale bar: 20m. c Immunofluorescence assay of iPSCs at step 3 on day 21. Co-immunostaining of insulin (red) with C-peptide (green); nuclear DAPI staining is shown in blue. Scale bar: 75m. d Flow cytometry plots illustrating the protein expression of insulin in populations of iPSC-derived IPCs. Black text indicates the percentage of insulin. e Glucose-stimulated insulin secretion in vitro. iPSC-derived IPCs on day 21 of the three-step protocol were exposed to different glucose concentrations (0, 5, and 15mM). The insulin concentration levels were determined

To screen the differentially expressed miRNAs (DEmiRNAs) related to IPCs differentiation, we performed miRNA microarray assays by using total RNA from our iPSCs-derived IPCs containing undifferentiated iPSCs and iPSCs-derived IPCs at day 4 (early stage), day 14 (middle stage), and day 21 (late stage) during step 3. A Venn diagram was used to compare several miRNAs differentially expressed during the three-step induction. The results showed that there were 13 common miRNAs during the three-step induction (Fig.

a). The miRNA expression levels at different time points were clustered and are shown graphically (Fig.


Differentially expressed miRNA profiling and bioinformatic analysis. Differentially expressed miRNAs (P

To further understand the role of 13 common DEmiRNAs in iPSCs-derived IPCs, we performed the bioinformatics prediction analysis using two databases (TargetScan and miRanda) respectively to search for putative target genes. There were 332 common target genes after combining data predicted by two databases (miRanda threshold value: binding energy 16.0, align score 158, TargetScan threshold value: context score percentile 30, data not shown). We explored the connections between the DEmiRNAs and putative target genes by building a regulatory network for miRNA-target genes using Cytoscape software (Fig.2c). Then, we investigated the target genes in the KEGG pathways to further study the biological function of the DEmiRNAs (Fig.2d). Interestingly, the WNT signaling pathway was located at the top of the 20 most enriched pathways. Our pathway analysis partly revealed the function of the signature miRNAs, and signal-related function was highlighted among all the subsystems, which was consistent with GO function analyses of the target genes (Fig.2e). To verify the bioinformatic results, we performed qPCR, showing that miR-296, miR-331, miR-345, and miR-690 levels were consistent with the previous trends (Additionalfile2: Figure S2). Of the transcripts that we identified, miR-690, which was persistently highly expressed in the full step 3, drew our attention, as it was reported to regulate Runx2-induced osteogenic differentiation of myogenic progenitor cells; these findings suggest that it may mediate organism differentiation and development. Then, we concentrated on the miR-690 functions during IPCs differentiation.

To explore the specific function of miR-690 in the progression of the three-step induced differentiation, we constructed an agomir vector targeting miR-690 (miR-690-agomir), and miR-690 was overexpressed in MPCs on day 4. The overexpression efficiency of agomir-miR-690 was confirmed by qPCR analysis (Fig.

a). Upregulated miR-690 in MPCs reduced the mRNA expression of several key transcription factors critical for early pancreatic development such as Pdx1, Ngn3, Nkx6.1, Gata4, and Pax4, although the deletion of these nonspecific factors alone was enough to abrogate pancreatic lineage induction (Fig.

b). Immunostaining assays partially verified the results of quantitative RT-PCR analysis (Fig.

c). As expected, IPCs overexpressing miR-690 showed a weak response to glucose stimulation, and high expression of these markers was correlated with the maturation of -cells. Moreover, flow cytometry showed that the population of insulin

cells significantly decreased from 42.4%0.25% to 22.8%0.007% from cells with NC-agomir compared to cells with miR-690-agomir (Fig.

a). The ELISA results of mature IPCs (late stage/day 21) showed that insulin secretion decreased after glucose stimulation (Fig.

b), indicating that IPCs were unable to reduce their glucose concentrations compared with NC cells. Also, we found that IPCs generated after overexpression of miR-690 showed significantly lower mRNA levels of mature -cell and mature -cell markers, such as insulin 1, insulin 2,


, and

, than NC-agomir-transfected cells on day 21 of the late stage through quantitative RT-PCR analysis. Interestingly,

expression of -cells was opposite to that of mature -cells and mature -cells, and Mafa expression showed no significant difference between the two groups of cells (Fig.

c). In addition, immunostaining assays confirmed that the co-expression of insulin/C-peptide, insulin/Nkx6.1, and insulin/Pdx1 was consistent with the results from previous quantitative RT-PCR assays (Fig.

d). All these findings showed that miR-690 suppressed the maturation and endocrine functions of IPCs derived from iPSCs, indicating that miR-690 might be a critical regulator of -cells differentiation.

Overexpression of miR-690 inhibits pancreatic differentiation potential. This group of experiments tested the functions of iPSCs-derived IPCs on day 4 of the second step. Quantitative RT-PCR analysis of the expression levels of a miR-690 and b several key transcription factors during the development of pancreatic -cells (Pdx1, NGN3, Nkx2.2, Nkx6.1, Gata4, Gata6, Pax4, Pax6). GAPDH was used as the internal control. Error bars show meanstandard deviation (SD) (n=3). *P

Overexpression of miR-690 impaired the functions of terminal iPSCs-derived IPCs. This group of experiments tested the functions of iPSCs-derived IPCs on day 21 of the second step. a Flow cytometry plots illustrating the protein expression of insulin in populations of iPSCs-derived IPCs. Black text indicates the percentage of insulin. b Glucose-stimulated insulin secretion in vitro. iPSCs-derived IPCs on day 21 of the third step were exposed to different glucose concentrations (0, 5, 15, and 45mM). The insulin concentration levels were determined. c Quantitative RT-PCR analysis of the mRNA expression levels of key endocrine markers (insulin1, insulin2, GCG, SST, GCK, Mafa, ISL, Glut2). GAPDH was used as the internal control. Error bars show meanstandard deviation (SD) (n=3). *P

To further dissect the molecular mechanism of the inhibitory effect of miR-690 on IPCs differentiation, we performed the bioinformatics prediction analysis by using TargetScan and miRanda and combined with the results from RNA-seq (Huang, et al.) to predict the target genes of DEmiRNAs. miR-690 has 15 putative target genes (Prkca, Nedd4l, Ulk2, Prkcz, Csnk1g1, Mllt3, Enah, Pcgf3, Impa1, Stat3, Grm5, Cnot6, Sox9, Wasl, and Ctnnb1). Then, we built a regulatory network to show the connections between DEmiRNAs and target genes (Fig.

c). Among these predicted genes,

, a marker of pancreatic progenitor cells, and the genes encoding key transcription factors for the development of -cells were notable. Next, we performed a dual-luciferase reporter assay to experimentally determine whether miR-690 targeted Sox9 directly. We transfected HEK293T cells with a luciferase plasmid containing the wild-type 3 UTRs of Sox9 or its mutant version downstream of the firefly luciferase cDNA in the pEZX-FR02 vector (Fig.

a). The results showed that the co-transfection of miR-690 mimics into 293T cells led to a decrease of up to 83% in luciferase activity by miR-690 but had nearly no effect on the mutant reporter activity (Fig.

b). Furthermore, knockdown of miR-690 reversed the repressive effects of siRNA-Sox9 on the mRNA and protein levels of Sox9 (Fig.

ce). These findings indicated that Sox9 was the authentic target of miR-690 in our induced IPCs.

miR-690 directly targeted Sox9 in iPSCs-derived IPCs. a Predicted miR-690 targeting sequence in the 3UTR of Sox9 (Sox9 WT-3UTR) and the mutant form of the Sox9 3UTR (Sox9 MUT-3UTR). b Dual-luciferase reporter assays to determine the influence of miR-690 on Sox9 3UTR activity in iPSCs-derived IPCs. Data are the meanSD of three independent assays. ce Quantitative RT-PCR and Western blotting analyses of the effects of miR-690 knockdown (miR-690 inhibitor) on the expression level of Sox9 and the effects of miR-690 knockdown (miR-690) on the repressive effects of Sox9 knockdown (Sox9 siRNA). -Actin was used as the loading control. GAPDH was used as the internal control for mRNA. Error bars show the SD (n=3)

Sox9 has been reported to play a role in regulating Wnt signaling, which influences pancreatic development and modulates mature -cell functions, such as insulin secretion, survival, and proliferation. Sox9 was chosen for further analysis in our study and validated by both qPCR analysis at the mRNA level and Western blot and immunostaining assays at the protein level (Fig.

ac and h). Because the phosphorylation and inactivation of GSK3- may lead to activation and nuclear translocation of -catenin, we detected the level of GSK3- phosphorylation when miR-690 was overexpressed. As expected, a more than 1.5-fold decrease in phosphorylated GSK3- and a more than 2-fold decrease in -catenin activity were observed (Fig.

dh). We speculated that in our induced models, miR-690 may inactivate the WNT signaling pathway through Sox9 which will be the focus of our future research (Fig.


miR-690 may affect the differentiation of IPCs by inactivating the expression of the Wnt signaling pathway. a Quantitative RT-PCR analysis of the expression levels Sox9 and -catenin. The scale bar represents 100m. Western blotting analysis of the effects of miR-690 overexpression on Sox9 (b, c), p-GSK3 (phosphorylated-GSK3) (d, e), and -catenin (f, g) (690-OE means 690 overexpression/miR-690 agomir). -Actin was used as the loading control. h Immunofluorescence assay (Sox9 and -catenin, red; nuclei, blue; scale bar 75m) of the protein level of Sox9 and -catenin. i Schematic diagram of the supposed role of miR-690 in iPSCs-derived IPCs differentiation.

We next sought to explore whether miR-690 could modulate glucose homeostasis by transplanting miR-690-overexpressing IPCs and negative control cells into anemic capsule kidneys of mice treated with streptozotocin (STZ), which specifically destroys mouse -cells (Fig.

a). After transplantation, populations from the NC group needed nearly 28days to reverse the hyperglycemia. Although the blood glucose level was decreased, mice transplanted with the miR-690 agomir still showed glycemia (Fig.

b). Not surprisingly, the body weight of transplanted mice in the miR-690 overexpression group was significantly lower than that of the control group and healthy mice (data has not shown). At 40days post-transplant, excised iPSCs-derived IPCs grafts were highly compact and homogenous and did not have regions of expanded ducts (Fig.

c). Immunofluorescence staining revealed insulin-positive clusters of cells in the graft surrounded by connective tissue producing endocrine hormones (Fig.


iPSCs-derived IPCs reverse diabetes in vivo. a Image of the entire kidney with iPSCs-derived IPCs engrafted under the kidney capsule and harvested at 25days post-transplant. (~1106 cells/mouse, n=6 /group). b Blood glucose levels were measured pre- and post-transplantation for over 30days. c Hematoxylin and eosin (H&E) staining image of iPSCs-derived IPCs grafts in the kidney capsule 25days after transplantation. Scale bar 200m. d Immunofluorescence staining of whole grafts for insulin (red); nuclear DAPI staining is shown in blue. Scale bar 75m

iPSCs, which are derived from somatic cells, allow for the patient-specific functional -cells in vitro that will free diabetic populations from daily insulin injections and prevent life-threatening complications, generate sufficient -cells for transplantation, and also avert immune suppression to repress auto- and allo-immunity [1, 19]. Although many attempts have been made to acquire mature, glucose-responsive IPCs entirely in vitro, the results of these studies lacked convincing evidence [19]. Multiple core transcription factors, signaling pathways, and noncoding RNAs have been confirmed to be required for pancreatic -cells differentiation potential in potent stem cells [10, 2024]. Increasing evidence shows that miRNAs, as important epigenetic factors that regulate gene expression and determine cell fate in pancreatic -cells, mediate -cells biological activities, including differentiation, proliferation, apoptosis, and insulin secretion [6, 25]. However, the mechanisms of miRNAs in -cells differentiation of iPSCs remain unknown.

This study adopted a three-step protocol mimicking normal pancreatic formation to screen for differentiation-associated miRNAs during iPSCs-induced IPCs differentiation in culture. According to the miRNA array analysis data, 13 miRNAs with markedly different expression levels were identified (Additionalfile1: Figure S1), and we found that miR-690 was significantly upregulated in step 3 compared to that in iPSCs. To explore the specific function of miR-690 in IPCs differentiation, we overexpressed miR-690 in progenitor cells on day 4 of step 3 and found that pancreatic progenitor markers, such as Pdx1 and Sox9, and the early endocrine progenitors NGN3, Nkx6.1, and Pax4 were downregulated after 48h. At the final stage of our protocol, miR-690 overexpression significantly impaired the maturation and endocrine function of IPCs (Fig.3). However, the mRNA level of SST increased unexpectedly after miR-690 overexpression, suggesting that this miRNA may promote the differentiation of -cells.

To elaborate on the mechanism by which miR-690 regulates IPCs formation, we used bioinformatic analysis. Combined with the RNA-seq data detected previously, these results identified Sox9 as an underlying target gene of miR-690. Sox9 is widely known as a pancreatic progenitor marker that influences endocrine pancreatic development and modulation of mature -cells functions [14]. The prevailing theory is that miRNAs regulate gene expression post-transcriptionally by binding to the 3 untranslated sequence of the targeted mRNA to silence its corresponding target genes [26, 27]. Then, we demonstrated that Sox9 was a direct target of miR-690 using a luciferase reporter assay (Fig.5). Furthermore, overexpression of miR-690 decreased the protein levels of Sox9 and -catenin (Fig.6), indicating that this noncoding RNA may regulate the Wnt signaling pathway, which has been thoroughly investigated and is necessary for controlling the development of -cells and their function [16, 28, 29]. These findings suggested that the important function of miR-690 during IPCs differentiation was predominantly regulated by the miR-690/Sox9 and -catenin axes, confirming that the interactions of miRNAs and transcription factors were involved in the differentiation of mouse iPSCs to IPCs. -catenin is an important effector of the Wnt pathway [30]. To date, the role of Wnt signaling in pancreatic development has been disputed. The majority of studies have noted the primary role of Wnt signaling in the development of the exocrine compartments of the pancreas and confirmed that abolishment of the signaling pathway resulted in an almost complete lack of exocrine cells; however, the influence of Wnt signaling on endocrine cells, especially on pancreatic -cell development, is still undefined [31]. Previous studies have reported that knockdown of the Sox9 gene in human islet epithelial cells significantly decreases the expression of phosphorylated GSK3- at the protein level, leading to a prominent decline in the expression of cyclin D1 and other target genes of the Wnt signaling pathway [14]. Therefore, we examined the Wnt signaling activity by detecting the expression of p-GSK3-. Interestingly, the results showed that miR-690 overexpression simultaneously decreased Sox9 and phosphorylated GSK3- at the protein level. We speculated that miR-690 may mediate the Wnt signaling pathway via binding to Sox9 and lead to a decline in phosphorylation of GSK3- and a decrease in -catenin, which are the effectors of this pathway. Furthermore, other researchers have shown that pancreatic -cells differentiation is complex and a result of the interaction of multiple signaling pathways, such as Notch, Fgf, Wnt, and others. Thus, the specific regulatory mechanism between miR-690 and the Wnt signaling pathway and whether other signaling pathways are regulated by miR-690 require further exploration.

Recently, miR-690 was reported to mediate osteogenic differentiation of human myogenic progenitor cells through its target NF-kappaB p65, indicating that miR-690 may play different roles in the development and differentiation of different organs and tissues [32]. Many studies have shown that Sox9 downregulation is important for early lineage bifurcation of endocrine progenitors and pancreatic -cells development [15, 3336]. In our study, the expression of miR-690 at an appropriate level is vital to the maturation and differentiation of IPCs. However, prematurely upregulated Sox9 resulted in deficient IPC differentiation in vitro, indicating that miR-690 activity may need to be within a narrow range to avoid detrimental consequences. Therefore, further exploration of the function of the miR-690/Sox9/Wnt signaling pathway in pancreatic -cells differentiation, development, and maturation may be required to systematically uncover the critical function and mechanism of miR-690 in vitro and in vivo.

We found that miR-690, a rarely studied noncoding RNA, played an important role in the differentiation of iPSCs-derived IPCs. MiR-690 regulates the expression of transcription factor Sox9 and may have an influence on Wnt signaling pathway in the differentiation process. These findings not only indicate that miR-690 mediates differentiation of iPSCs-derived IPCs through Sox9 and affects Wnt signaling pathway, but also provide novel evidence for the regulatory potential mechanisms of miRNAs in development associated with insulin-producing cells derived from induced pluripotent cells.

Yang Xu, Yan Huang and Yibing Guo contributed equally to this work.

Differentially expressed miRNAs

Embryoid Bodies

GATA binding protein 4

GATA binding protein 6



Facilitated glucose transporter, member 2

Gene ontology

Hematoxylin and eosin

Insulin-producing cell

Induced pluripotent stem cell

ISL LIM homeobox

Kyoto Encyclopedia of Genes and Genomes

v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A


Multilineage precursor stem cell

Negative control

Neurogenin 3

NK2 homeobox 2

NK6 homeobox 1

Paired box 4

Paired box 6

Pancreatic and duodenal homeobox 1

Phosphorylated glycogen synthase kinase-3

Real-time quantitative polymerase chain reaction

Original post:
microRNA-690 regulates induced pluripotent stem cells (iPSCs ...

Induced pluripotent stem cells have been generated for the …

Induced Pluripotent Stem Cells | Posted by admin
Feb 17 2019

Induced pluripotent stem cells have been generated for the first time from tumor cells in order to study therapies for tumors developed in patients with hereditary diseases with predisposition to cancer

The Hereditary Cancer Research Group at the Germans Trias i Pujol Research Institute (IGTP) on the Can Ruti Campus, Badalona has for the first time generated induced pluripotent stem cells (iPSCs) from tumors from people with the hereditary disease Neurofibromatosis type 1 (NF1). The work has been carried out in conjunction with Angel Raya of the Centre for Regenerative Medicine of Barcelona (CMRB) and published in Stem Cell Reports, the official journal for the International Society for Stem Cell Research (ISSCR).

iPSCs are stem cells capable of giving rise to most other types of cell in the body. It is quite normal to generate stem cells by reprogramming skin cells extracted from patients with hereditary diseases such as NF1 to study them as a model for the disease, but the IGTP researchers have now generated iPSCs for the first time as a valid model for NF1 from cells from tumors. Instead of using skin cells, we have reprogrammed cells from tumors from patients with NF1 in order to have a model of cells genetically identical to the tumor cells, explains Eduard Serra, leader of the work at the IGTP.

One of the difficulties of studying these pathologies with cells obtained directly from benign tumors is that they are finite, but now we have achieved a cellular model which will not run out because, due to their characteristics, we can culture these iPSCs indefinitely and then convert them into the same cells that make up a tumor, Serra adds. The work has invested most of the efforts in converting these iPSCs into Schwann cells, which are cells which make up plexiform neurofibromas, typical of NF1. The resulting cells have the same capacity to proliferate as the original tumor cells. As iPSCs are an endless source of cells, we have been able to generate the resource we needed. Now we can test drugs that inhibit proliferation, study the mechanisms by which these tumors develop and try to stop them developing into malignant tumors, explains Meritxell Carri, first author of the article.

New ways to study NF1

The Hereditary Cancer Research Group at the IGTP has been studying Neurofibromatoses for many years, this includes NF1, a minority disease that affects 1 in every 3,000 people in the world. It is a hereditary disease, which brings a high predisposition to develop tumors in the peripheral nervous system. One of these types of tumor is the plexiform neurofibromas. It is a tumor that appears at birth, or in the first years of life, and forms on major nerves in the body, disorganizing and thickening the tissue surrounding the nerve and forming a tumor mass that can reach great dimensions. These tumors can impede functionality and disfigure the part of the body where they appear, which can be on the extremities or even on the face. Additionally, there is a risk that these tumors progress to become malignant; a sarcoma of the peripheral nerve sheath.

Although they start as benign tumors, we do not have drugs to put the patient into remission. To develop effective drugs for this type of tumor we need cellular models that are faithful copies of the tumor and that do not run out, this is the model that now has been achieved.

Available to the scientific community

The cell lines generated have been deposited in the Carlos III Stem Cell Bank at the node kept at the CMRB in Barcelona. They are available to other researchers in the world who want to study this disease and these tumors.

This work was led by Eduard Serra at the IGTP and carried out in collaboration with Angel Raya at the CMRB alongside other participating institutions such as the Catalan Institute of Oncology (ICO), Sage Bionetworks, and the Germans Trias Hospital. This project has been financed by the Neurofibromatosis Therapuetic Acceleration Program (NTAP,, a program based at the Johns Hopkins University School of Medicine in Baltimore, Maryland in the United States, whose mission is to accelerate the development of therapies for Neurofibromatosis Type 1. The team has also had the support of the Spanish and Catalan Associations of Neurofibromatosis Patients.

The Johns Hopkins University, and the Germans Trias i Pujol Research Institute (IGTP) ( are academic institutions based respectively in Baltimore, Maryland, and Badalona (Barcelona), Spain.

The Center of Regenerative Medicine in Barcelona (CMR[B]) ( is a public research centre (non-profit foundation) based in Barcelona, Spain. The overall mission of the CMR[B] is to conduct fundamental research of excellence forthe advancement inthe clinical translation of regenerative medicine strategies basedin pluripotent stem cells, mainly in the context of heart failure, neurodegenerative diseases, non-malignant hematological diseases and age-related macular degeneration.

Sage Bionetworks is a nonprofitbiomedical research and technology organization. We develop and apply open practices to data-driven research for the advancement of human health. Our interdisciplinary team of scientists and engineers work together to provide researchers access to technology tools and scientific approaches to share data, benchmark methods, and explore collective insights, all backed by Sages gold-standard governance protocols and commitment to user-centered design.Sage, founded in Seattle in 2009, is supported through a portfolio of competitive research grants, commercial partnerships, and philanthropic contributions. Learn more

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Induced pluripotent stem cells have been generated for the ...

Induction of Human Embryonic and Induced Pluripotent Stem …

Induced Pluripotent Stem Cells | Posted by admin
Feb 17 2019

Public Summary:

The authors evaluated different methods to induce human embryonic stem cells and induced pluripotent stem cells into urothelial cells, which are specialized cells that line the inside of the bladder, ureters and kidneys. They also evaluated regulatory events that occur during the differentiation of these cells. This technology shows the feasibility of producing urologic tissue from stem cells for bioengineering purposes.

Scientific Abstract:

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.

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Induction of Human Embryonic and Induced Pluripotent Stem ...