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Objective:To investigate the role of nicotinamide (NIC) in the differentiation of neural crest cells from human embryonic stem cells (hESCs), and lay the foundation for the induction of hESC-derived corneal endothelial cells.Methods:hESCs line H1 cultured for 5-7 days was used for induction.According to the different components of the neural crest induction medium, cells were assigned into different groups for 7-days induction, including group treated without NIC cultured in induction medium only, group treated with NIC cultured in induction medium containing 10 mmol/L NIC, NIC+ resveratrol (Res) group cultured in induction medium containing 10 mmol/L NIC and 10 μmol/L Res and Sirtinol group cultured in induction medium containing 10 μmol/L Sirtinol.Res and Sirtinol were used as SIRT1 activity agonist and inhibitor, respectively.The relative mRNA expression levels of hESCs and neural crest cell markers were detected by real-time fluorescence quantitative PCR at 1, 3, 5 and 7 days during the induction.The expression of neural crest cells markers after 7 days of induction was assayed by immunofluorescence staining.The induction efficiency of NIC and the effect of SIRT1 regulation on human natural killer 1 (HNK-1) positive cells expression were evaluated through flow cytometry analysis of percentages of nerve growth factor receptor (P75) and HNK-1 + cells. Results:Compared with the group treated without NIC, the mRNA expressions of totipotent genes octamer transcription factor 4 (OCT4) and homeodomain proteins (NANOG) were significantly decreased, and the mRNA expression levels of neural crest cell markers P75, HNK-1, SRY-related HMG box (SOX) 9 and SOX10 were significantly increased in the group treated with NIC after 5 days of induction (all at P<0.05). In the group treated without NIC, P75 was weakly expressed, and HNK-1 was sporadically expressed, and transcription factor AP-2β (AP-2β) and paired-like homeodomain transcription factor 2 (PITX2) were not detected.In the group treated with NIC, P75, HNK-1, AP-2β and PITX2 were strongly expressed.The proportion of P75 + HNK-1 + cells and P75 + cells were both significantly higher in the group treated with NIC than without NIC ( t=8.481, P=0.001; t=2.987, P=0.041). The percentage of HNK-1 + cells in groups treated without and with NIC, NIC+ Res group and Sirtinol group were (34.267±12.522)%, (89.633±1.358)%, (64.667±6.429)% and (86.300±3.460)%, respectively, with a statistically significant overall difference ( F=36.799, P<0.001). The proportion of HNK-1 + cells in NIC+ Res group was significantly lower than that in the groups treated with NIC and Sirtinol (all at P<0.05). Conclusions:NIC promotes the differentiation of hESCs-derived neural crest cells by inhibiting the activity of SIRT1 to enhance the expression of HNK-1.NIC treatment may provide a new strategy for source of seed cells in the treatment of neural crest cell-related diseases, such as corneal endothelial transplantation.
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Objective To construct human embryonic stem cell (hESC) line with forkhead box G1 (FOXG1) gene knockout by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology, and to investigate the role of FOXG1 gene in the early neural induction of hESCs. Methods Two guide RNAs (gRNAs) were transfected to induce FOXG1 gene large fragment knockout in hESCs by CRISPR/Cas9 gene editing technology. FOXG1 gene knockout hESCs were confirmed by monoclonal screening, sequencing and Western blotting analysis. The expression of the key markers including paired box 6 (PAX6), sex-determining region Y-box 2 (SOX2) and orthodenticle homeobox 2 (OTX2) was detected by immunofluorescence staining and qRT-PCR in the early process of neural induction before and after FOXG1 gene knockout. Results hESCs with FOXG1 gene large fragment knockout were successfully obtained by CRISPR/Cas9 gene editing technology. The results of immunofluorescence staining and qRTPCR suggested that FOXG1 deletion did not significantly influence the expression of PAX6, SOX2 and OTX2 during neural induction. Conclusion FOXG1 gene large fragment knockout in hESCs can be quickly induced by a pair of gRNAs cotransfection. FOXG1 deletion has no significant impacts on neural induction of hESCs.
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BACKGROUND: In the research of human embryonic stem cells, introducing exogenous molecules such as DNA into cells is a common research method, but the transfection efficiency is relatively low. It is crucial to answer the question of how to optimize the existing conditions to improve the transfection efficiency. OBJECTIVE: To compare the effects of two different passaging methods on H9 transfection efficiency, in order to optimize the conditions required for embryonic stem cell transfection. METHODS: Human embryonic stem cell lines H9 were cultured for 48 hours after small clone passaging or single-cell passaging. Lipofectamine 3000 was used to transfect pAdTrack-AKT1 fluorescent plasmid into human embryonic stem cells. After 2 days of transfection, the expression of fluorescent plasmids was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry. RT-qPCR and western blot were used to detect the mRNA and protein expression levels of AKT1 respectively. RESULTS AND CONCLUSION: Under the fluorescence microscopy, the number of cells expressing fluorescent plasmids in the single-cell passaging group was more than that in the small clone passaging group, and the flow cytometry analysis showed that the transfection efficiency of cells in the single-cell passaging group was (47.18±2.00)%, which was significantly higher than (19.52±0.86)% in the small clone passaging group (P < 0.01). RT-qPCR and western blot analysis showed that the expression levels of AKT1 mRNA and protein in the single-cell passaging group were significantly higher than those in the small clone passaging group (P < 0.01). These findings indicate that single-cell passaging can increase the contact area between cells and transfection reagent liposomes, and improve the transfection efficiency of human embryonic stem cells.
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Dysregulation of circadian rhythms associates with cardiovascular disorders. It is known that deletion of the core circadian gene Bmal1 in mice causes dilated cardiomyopathy. However, the biological rhythm regulation system in mouse is very different from that of humans. Whether BMAL1 plays a role in regulating human heart function remains unclear. Here we generated a BMAL1 knockout human embryonic stem cell (hESC) model and further derived human BMAL1 deficient cardiomyocytes. We show that BMAL1 deficient hESC-derived cardiomyocytes exhibited typical phenotypes of dilated cardiomyopathy including attenuated contractility, calcium dysregulation, and disorganized myofilaments. In addition, mitochondrial fission and mitophagy were suppressed in BMAL1 deficient hESC-cardiomyocytes, which resulted in significantly attenuated mitochondrial oxidative phosphorylation and compromised cardiomyocyte function. We also found that BMAL1 binds to the E-box element in the promoter region of BNIP3 gene and specifically controls BNIP3 protein expression. BMAL1 knockout directly reduced BNIP3 protein level, causing compromised mitophagy and mitochondria dysfunction and thereby leading to compromised cardiomyocyte function. Our data indicated that the core circadian gene BMAL1 is critical for normal mitochondria activities and cardiac function. Circadian rhythm disruption may directly link to compromised heart function and dilated cardiomyopathy in humans.
ABSTRACT
Dysregulation of circadian rhythms associates with cardiovascular disorders. It is known that deletion of the core circadian gene Bmal1 in mice causes dilated cardiomyopathy. However, the biological rhythm regulation system in mouse is very different from that of humans. Whether BMAL1 plays a role in regulating human heart function remains unclear. Here we generated a BMAL1 knockout human embryonic stem cell (hESC) model and further derived human BMAL1 deficient cardiomyocytes. We show that BMAL1 deficient hESC-derived cardiomyocytes exhibited typical phenotypes of dilated cardiomyopathy including attenuated contractility, calcium dysregulation, and disorganized myofilaments. In addition, mitochondrial fission and mitophagy were suppressed in BMAL1 deficient hESC-cardiomyocytes, which resulted in significantly attenuated mitochondrial oxidative phosphorylation and compromised cardiomyocyte function. We also found that BMAL1 binds to the E-box element in the promoter region of BNIP3 gene and specifically controls BNIP3 protein expression. BMAL1 knockout directly reduced BNIP3 protein level, causing compromised mitophagy and mitochondria dysfunction and thereby leading to compromised cardiomyocyte function. Our data indicated that the core circadian gene BMAL1 is critical for normal mitochondria activities and cardiac function. Circadian rhythm disruption may directly link to compromised heart function and dilated cardiomyopathy in humans.
ABSTRACT
Dysregulation of circadian rhythms associates with cardiovascular disorders. It is known that deletion of the core circadian gene Bmal1 in mice causes dilated cardiomyopathy. However, the biological rhythm regulation system in mouse is very different from that of humans. Whether BMAL1 plays a role in regulating human heart function remains unclear. Here we generated a BMAL1 knockout human embryonic stem cell (hESC) model and further derived human BMAL1 deficient cardiomyocytes. We show that BMAL1 deficient hESC-derived cardiomyocytes exhibited typical phenotypes of dilated cardiomyopathy including attenuated contractility, calcium dysregulation, and disorganized myofilaments. In addition, mitochondrial fission and mitophagy were suppressed in BMAL1 deficient hESC-cardiomyocytes, which resulted in significantly attenuated mitochondrial oxidative phosphorylation and compromised cardiomyocyte function. We also found that BMAL1 binds to the E-box element in the promoter region of BNIP3 gene and specifically controls BNIP3 protein expression. BMAL1 knockout directly reduced BNIP3 protein level, causing compromised mitophagy and mitochondria dysfunction and thereby leading to compromised cardiomyocyte function. Our data indicated that the core circadian gene BMAL1 is critical for normal mitochondria activities and cardiac function. Circadian rhythm disruption may directly link to compromised heart function and dilated cardiomyopathy in humans.
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@#【Objective】 A human embryonic stem cell line derived from Preimplantation genetic testing （PGT） embryos was established in a xeno- free stem cell culture system to provide disease models for medical research. 【Methods】The xeno-free culture system using xeno-free human foreskin fibroblast feeder layers（XF-HFF）mixed with commercially available chemically-defined medium（CDM）was assessed. In the culture system，a new hESC cell line was established using discarded embryos derived from PGT in patients with chromosomal balance translocation.【Results】The new availabled stem cell line was successfully cultured in the xeno-free culture system for a long time（> 45 passages）. The karyotype analysis revealed that the new line kept the same karyotype over 45 passages. Moreover，the expression of pluripotent markers was detected by fluorescent immunostaining including SSEA- 3，SSEA- 4，SSEA- 1，TRA- 1- 60， and TRA-1-81. RT-PCR analysis showed that the stem cell markers were present in hESC grown on XF-HFF-CDM. In addition，the teratoma formation analysis demonstrated that the cells cultured in XF-HFF/CDM maintained their pluripotency in vivo.【Conclusions】Our study may provide the possibility to establish embryonic stem cells with certain pathogenic genes，which could be applied for clinical research and treatment.
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Objective • To generate a doxycycline (Dox)-inducible multiplexed CRISPR interference (CRISPRi) system for multiple gene inhibition in human embryonic stem cells (hESCs) to explore the function of gene families and model multigene diseases. Methods • A Dox-inducible multiplexed CRISPRi system was developed by Golden Gate assembly in hESCs. This system consisted of two plasmids, one expressing modified repressive nuclease-deactivated CRISPR-associated protein 9 (dCas9) and Krüppel-associated box (KRAB) transcriptional repressor domain under the control of Dox, the other carrying eight independent guide RNA (gRNA) expression cassettes. PCR was conducted using total genomic DNA as a template to confirm whether these two plasmids were integrated into genome. Western blotting was performed to confirm whether the expression of dCas9-KRAB could be induced by Dox treatment. Results • Using this tunable CRISPRi system, multiple genes were successfully silenced simultaneously in hESCs. The silence of genes and related to hESC self-renewal caused obvious cell differentiation in terms of changed cell morphology, decreased activity of alkaline phosphatase, and reduced expression of stage-specific embryonic antigen 4 (SSEA4), a marker of undifferentiated hESCs. Conclusion • This Dox-inducible multiplexed CRISPRi system can be used for quick and efficient silence of multiple genes in hESCs in a highly controlled manner.
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BACKGROUND: Human embryonic stem cells (hESCs) have the potential to treat various human disorders currently labeled as incurable and/or terminal illness. However, the fear that the patients' immune system would recognize them as non self and lead to an immune rejection has hampered their use. The main cause for immune rejection is usually the incompatibility of both donor and recipient's major histocompatibility complex (MHC). METHODS: We describe a hESC line developed through a patented technology that does not lead to immune reaction upon transplantation. We have transplanted these cells in >1,400 patients with chronic/terminal conditions and did not observe any immune reaction. No immunosuppressant were administered to these patients. We analyzed the expression levels of MHC-I and MHC-II on the surface of these hESCs using microarray technology. The gene targets for miRNA were analyzed using Gene ontology and DAVID database and pathways for these genes were determined using Reactome and Panther databases. RESULTS: Our results showed that the levels of expression of MHC-I and MHC-II on hESCs is almost negligible and thus the hESCs are less susceptible to an immune rejection. CONCLUSIONS: The hESCs cultured at our facility expresses low levels of MHC-I and do not produce an immune reaction. These can be administered universally and need no cross matching before transplantation.
Subject(s)
Humans , Cell Line , Gene Ontology , Human Embryonic Stem Cells , Immune System , Major Histocompatibility Complex , MicroRNAs , Neurons , Tissue Donors , ZygoteABSTRACT
Background Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Methods Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F +T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. Results The experiments indicated...
Subject(s)
Humans , Fibrin Tissue Adhesive , Bioengineering , Stem Cells , Sciatic Nerve , Nerve Regeneration , Peripheral Nerve InjuriesABSTRACT
Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Methods Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F +T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. Results The experiments indicated that sensory function improved when transgenic hESCs were used. The regeneration of sensory fibers indeed led to increased reflexes, upon stimulation of the paw ipsilateral to the lesion, as seen by von-Frey evaluation, which was supported by immunohistochemistry. Conclusions Overall, the present data demonstrated that transgenic embryonic stem cells, engineered to overexpress FGF-2 in an inducible fashion, could be employed to support regeneration aiming at the recovery of both motor and sensory functions.(AU)
Subject(s)
Animals , Male , Rats , Sciatic Nerve/transplantation , Transplantation, Heterologous/rehabilitation , Fibrin Tissue Adhesive , Embryonic Stem Cells , Nerve Regeneration , Mice, Inbred C57BLABSTRACT
Objective@#To compare the expressions of ALX gene family members (ALX1, ALX3, ALX4) in human embryonic stem cells (hESCs) and their differentiated neural crest cells (NCCs), as well as human mesenchymal stem cells (MSCs) and peripheral neurons derived from NCCs. The result provided a research foundation for understanding the expressions of ALX gene family in the process of NCCs differentiation.@*Methods@#The characteristics of hESCs, NCCs, MSCs and peripheral neurons were identified by RT-PCR, immunofluorescence staining and flow cytometry analysis. The expressions of ALX1, ALX3 and ALX4 were quantified by Real-time PCR.@*Results@#The expressions of ALX1 were significantly lower in MSCs than in NCCs (P < 0.05). However, the expression of ALX3 was significantly higher in MSCs than in hESCs, NCCs, peripheral neurons and osteoblasts (P < 0.05). The expressions of ALX4 were higher in MSCs, peripheral neurons and osteoblasts.@*Conclusions@#The expression profiles of different ALX genes in stem cells at different development stages are different, suggesting the regulation of ALX genes expressions is different in diverse cells.
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Human embryonic stem cell (hESC) culture system has been changing culture conditions from conventional to xeno-free for therapeutic cell applications, and N-glycolylneuraminic acid (Neu5Gc) could be a useful indicator of xenogeneic contaminations in hESCs because human cells can no longer produce it genetically. We set up the humanized culture condition using commercially available humanized materials and two different adaptation methods: sequential or direct. SNUhES4 and H1 hESC lines, previously established in conventional culture conditions, were maintained using the humanized culture condition and were examined for the presence of Neu5Gc. The hESCs showed the same morphology and character as those of the conventional culture condition. Moreover, they were negative for Neu5Gc within two passages without loss of pluripotency. This study suggested that this method can effectively cleanse previously established hESC lines, bringing them one step closer to being clinical-grade hESCs.
Subject(s)
Humans , Human Embryonic Stem Cells , MethodsABSTRACT
Although microRNAs have emerged as key regulators in diverse cellular processes, the roles of microRNAs are poorly understood in human embryonic stem cells (hESCs) during differentiation into specialized cell types. In this study, we used a microRNA array with 799 human microRNA probes to examine the expression profiles of microRNAs in hESCs during differentiation into endodermal and mesodermal lineages in vitro. Among the microRNAs analyzed, 7 and 20 microRNAs were enriched in the developmental process of hESCs into mesodermal and endodermal lineages, respectively. In particular, the expression levels of miR-200 family, which is known to regulate the epithelial to mesenchymal transition (EMT), gradually increased in hESCs during differentiation into hepatocytes while they gradually decreased during differentiation into vascular endothelial cells. Downregulation of ZEB1, a direct target of miR-200 family, and E-CADHERIN, a target protein of ZEB1, was observed in hESCs during differentiation into endodermal and mesodermal lineages, respectively. These results indicate that miR-200 family has an important role in determining the cell fate between endodermal and mesodermal lineages from the pluripotent state.
Subject(s)
Humans , Humans , Cadherins , Down-Regulation , Endoderm , Endothelial Cells , Hepatocytes , Human Embryonic Stem Cells , In Vitro Techniques , Mesoderm , MicroRNAsABSTRACT
Pluripotent stem cells can differentiate into many cell types including mature hepatocytes, and can be used in the development of new drugs, treatment of diseases, and in basic research. In this study, we established a protocol leading to efficient hepatic differentiation, and compared the capacity to differentiate into the hepatocyte lineage of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Optimal combinations of cytokines and growth factors were added to embryoid bodies produced by both types of cell. Differentiation of the cells was assessed with optical and electron microscopes, and hepatic-specific transcripts and proteins were detected by quantitative reverse transcription polymerase chain reaction and immunocytochemistry, respectively. Both types of embryoid body produced polygonal hepatocyte-like cells accompanied by time-dependent up regulation of genes for α-fetoprotein, albumin (ALB), asialoglycoprotein1, CK8, CK18, CK19, CYP1A2, and CYP3A4, which are expressed in fetal and adult hepatocytes. Both types of cell displayed functions characteristic of mature hepatocytes such as accumulation of glycogen, secretion of ALB, and uptake of indocyanine green. And these cells are transplanted into mouse model. Our findings indicate that hESCs and hiPSCs have similar abilities to differentiate into hepatocyte in vitro using the protocol developed here, and these cells are transplantable into damaged liver.
Subject(s)
Adult , Animals , Humans , Mice , Cytochrome P-450 CYP1A2 , Cytochrome P-450 CYP3A , Cytokines , Embryoid Bodies , Glycogen , Hepatocytes , Human Embryonic Stem Cells , Immunohistochemistry , In Vitro Techniques , Indocyanine Green , Induced Pluripotent Stem Cells , Intercellular Signaling Peptides and Proteins , Liver , Pluripotent Stem Cells , Polymerase Chain Reaction , Reverse Transcription , Up-RegulationABSTRACT
BACKGROUND: The normal cells derived from human embryonic stem cells (hESCs) are regarded as substitutes for damaged or dysfunctional adult cells. However, tumorigenicity of hESCs remains a major challenge in clinical application of hESC-derived cell transplantation. Previously, we generated monoclonal antibody (MAb) 57-C11 specific to the surface molecule on undifferentiated hESCs. The aim of this study is to prove whether 57-C11-positive hESCs are pluripotent and tumorigenic in immunodeficient mice. METHODS: Undifferentiated hESCs were mixed with retinoic acid (RA)-differentiated hESCs at different ratios prior to 57-C11-mediated separation. To isolate 57-C11-positive hESCs from the mixture, biotinylated 57-C11 and streptavidin-coated magnetic beads were added to the mixture. Unbound 57-C11-negative hESCs were first isolated after applying magnet to the cell mixture, and 57-C11-bound hESCs were then released from the magnetic beads. In order to measure the efficiency of separation, 57-C11-positive or -negative hESCs were counted after isolation. To evaluate the efficiency of teratoma formation in vivo, 57-C11-positive or negative cells were further injected into left and right, respectively, testes of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. RESULTS: Approximately 77~100% of undifferentiated hESCs were isolated after applying 57-C11-coated magnetic beads to the mixed cell populations. Importantly, teratomas were not observed in NOD/SCID mice after the injection of isolated 57-C11-negative hESCs, whereas teratomas were observed with 57-C11-positive hESCs. CONCLUSION: 57-C11-positive hESCs are pluripotent and tumorigenic. The combination of 57-C11 and magnetic beads will be useful to eliminate remaining undifferentiated hESCs for the safe cell transplantation.
Subject(s)
Adult , Animals , Humans , Mice , Cell Transplantation , Human Embryonic Stem Cells , Teratoma , Testis , Transplants , TretinoinABSTRACT
Objective To investigate the effect of phosphoinositide-3-kinase inhibitor LY294002 on the differentiation of human embryonic stem cells (HESC) into more mature insulin-producing cells. Methods HESCs were induced to differentiate into insulin-producing cells through five stages. Nicotinamide and B27 (group B27), nicotinamide and LY294002 (group LY) were used to induce the nesting positive cells into mature insulin-producing cells. The morphological change of each stage was observed under microscope , and expressions of insulin, c-peptide, somatostatin and glucagon were identified by immunofluorescence staining. Results After 14 days in stage 5 , there was no significant difference in rate of insulin positive cells between group LY and group B27 (P﹥0.05), but rates of somatostatin and glucagon positive cells in group LY were lower than those in group B27(P﹤0.05). Furthermore, the co-stained rate of somatostatin and insulin in group LY was also lower than that in group B27 (P﹤0.05). Conclusion HESCs can be induced to differentiate into more mature insulin-producing cells by phosphoinositide-3-kinase inhibitor LY294002 in serum-free culture medium.
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BACKGROUND AND OBJECTIVES: Genomic imprinting is an inheritance phenomenon by which a subset of genes are expressed from one allele of two homologous chromosomes in a parent of origin-specific manner. Even though fine-tuned regulation of genomic imprinting process is essential for normal development, no other means are available to study genomic imprinting in human during embryonic development. In relation with this bottleneck, differentiation of human embryonic stem cells (hESCs) into specialized lineages may be considered as an alternative to mimic human development. METHODS AND RESULTS: In this study, hESCs were differentiated into three lineage cell types to analyze temporal and spatial expression of imprinted genes. Of 19 imprinted genes examined, 15 imprinted genes showed similar transcriptional level among two hESC lines and two human induced pluripotent stem cell (hiPSC) lines. Expressional patterns of most imprinted genes were varied in progenitors and fully differentiated cells which were derived from hESCs. Also, no consistence was observed in the expression pattern of imprinted genes within an imprinting domain during in vitro differentiation of hESCs into three lineage cell types. CONCLUSIONS: Transcriptional expression of imprinted genes is regulated in a cell type-specific manner in hESCs during in vitro differentiation.
Subject(s)
Female , Humans , Pregnancy , Alleles , Embryonic Development , Embryonic Stem Cells , Genomic Imprinting , Human Development , Parents , Pluripotent Stem Cells , WillsABSTRACT
Objective A lot of drugs have side effects on the central nerves system. Especially in children. In vivo neurotoxicity tests are time-consuming and expensive. The neural differentiation of human embryonic stem cells and amniotic fluid stem cells provides all ideal in vitro system that Can be applied to evaluate neurotoxicity of drugs. This study was to try to establish such a system. The kainie acid was selected to test the neurotoxicity. Methods The human embryonic stem cells and amniotic fluid stem cells were indueed to differentiate into neural cells by a chemically defined neural induction medium. The induced neural cells were propagated in the presence of basic fibroblast growth factor. Immunocytochemical staining Was applied to confirm these cells' neural identity. The induced cells were propagated under different concentration of kainic acid, then the gosh curve were made based on the cell numbers. Results Both of the human embryonic stem cells and amniotic fluid stem cells could be efficiently induced to be differentiated into neural cells. The neural differentiation efficiency of human embryonic stem cells is higher than that of human amniotic fluid stem cells. The kainic acid has neurotoxieity to the indueed neural cells. Conclusions The neural differentiation of human embryonic stem cells and amniotic fluid stem cells were proved to provide a rapid and convenient approach for estimating the neurotoxlcity of drugs.
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Recent advances in stem cell biology, including the development of optimized cell type-specific culture systems, and the broader understandings of biochemical and molecular signals involved in cell self-renewal and differentiation have brought cell-based therapy closer to practical application. As of now, at least 250 adult stem cell therapies are being used or tested in clinical situations. Stem cells have two important properties that distinguish them from other types of cells; they can both proliferate without changing their phenotypes indefinitely, and they also can differentiate into one or more new kinds of cells depending on their culture conditions. Thus, stem cell therapy could be most effective for treating the diseases that are marked by the loss of cells. The typical examples are Parkinson's disease, Alzheimer's disease, diabetes, heart failure, blindness, spinal cord injury, and stroke. Additionally, stem cell derivatives can be used in drug discovery as well. In the last decade, various types of stem cells have been identified from preimplantation stage embryos, fetuses, placentas, and adult tissues. Moreover, it is now almost a common practice to produce induced pluripotent stem (iPS) cells from various adult somatic cells using only a few defined factors. Thus, it is feasible that patient-specific stem cells will be generated with less controversy in the near future. However, human embryonic stem (ES) cells firmly remain "the gold standard" because of their greatest potential to become any type of cell in the body. The vast knowledge obtained from human ES cell research in the past decade has made cell-based therapy more promising than ever. Even the recent establishment of iPS cell technology is the culmination of human ES cells research. In our laboratory, interesting human cardiovascular cells including endothelial precursor cells and beating myocardiac cells, artificial blood cells, and retinal pigment epithelial cells were successfully differentiated and their therapeutic potential was confirmed after cell transplantation into animal models. Thus, here, the current research status of human embryonic stem cell-based therapy will be introduced and the future directions of stem cell applications in clinical trials will be discussed.