The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

This study aimed to compare epithelial cells derived from human embryonic stem cells(hESCs) to human ameloblast-lineage cells (ALCs),as a way to determine their potential use as a cell source for ameloblast regeneration.Induced by various concentrations of bone morphogenetic protein 4(BMP4),retinoic acid(RA) and lithium chloride(LiCI) for 7 days,hESCs adopted cobble-stone epithelial phenotype(hESC-derived epithelial cells(ES-ECs)) and expressed cytokeratin 14.Compared with ALCs and oral epithelial cells(OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs.ES-ECs were compared with human fetal skin epithelium,human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well.ALCs had relatively high expression levels of cytokeratin 76,which was also found to be upregulated in ES-ECs.Based on the present study,with the similarity of gene expression with ALCs,ES-ECs are a promising potential cell source for regeneration,which are not available in erupted human teeth for regeneration of enamel.

Xeno-free culture of human spermatogonial stem cells supported by human embryonic stem cell-derived fibroblast-like cells

Spermatogonial stem cells （SSCs） divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells （hdFs）. Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen （SSEA）-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction （RT-PCR） analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the ‘niche＇ that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.

Noggin versus basic fibroblast growth factor on the differentiation of human embryonic stem cells

The difference between Noggin and basic fibroblast growth factor for the neural precursor differen- tiation from human embryonic stem cells has not been studied. In this study, 100 tJg/L Noggin or 20 IJg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen- tiate human embryonic stem cells H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro- scope. Immunofluorescence staining revealed expression levels of Nestin, [3-111 Tubulin and Sox-1 were higher in the induced cells and reverse-transcription PCR showed induced cells expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cell differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in- creases the differentiation of neural precursors.

Connexin mutant embryonic stem cells and human diseases

Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin(Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells(ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

Human embryonic stem cell-derived mesenchymal stem cells improved premature ovarian failure

BACKGROUND Premature ovarian failure(POF)affects many adult women less than 40 years of age and leads to infertility.According to previous reports,various tissue-specific stem cells can restore ovarian function and folliculogenesis in mice with chemotherapy-induced POF.Human embryonic stem cells(ES)provide an alternative source for mesenchymal stem cells(MSCs)because of their similarities in phenotype and immunomodulatory and anti-inflammatory characteristics.Embryonic stem cell-derived mesenchymal stem cells(ES-MSCs)are attractive candidates for regenerative medicine because of their high proliferation and lack of barriers for harvesting tissue-specific MSCs.However,possible therapeutic effects and underlying mechanisms of transplanted ES-MSCs on cyclophosphamide and busulfan-induced mouse ovarian damage have not been evaluated.AIM To evaluate ES-MSCs vs bone marrow-derived mesenchymal stem cells(BMMSCs)in restoring ovarian function in a mouse model of chemotherapy-induced premature ovarian failure.METHODS Female mice received intraperitoneal injections of different doses of cyclophosphamide and busulfan to induce POF.Either human ES-MSCs or BMMSCs were transplanted into these mice.Ten days after the mice were injected with cyclophosphamide and busulfan and 4 wk after transplantation of the ESMSCs and/or BM-MSCs,we evaluated body weight,estrous cyclicity,folliclestimulating hormone and estradiol hormone concentrations and follicle count were used to evaluate the POF model and cell transplantation.Moreover,terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling,real-time PCR,Western blot analysis and immunohistochemistry and mating was used to evaluate cell transplantation.Enzyme-linked immunosorbent assay was used to analyze vascular endothelial growth factor,insulin-like growth factor 2 and hepatocyte growth factor levels in ES-MSC condition medium in order to investigate the mechanisms that underlie their function.RESULTS The human ES-MSCs significantly restored hormone secretion,survival rate and reproductive function in POF mice,which was similar to the results obtained with BM-MSCs.Gene expression analysis and the terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling assay results indicated that the ES-MSCs and/or BM-MSCs reduced apoptosis in the follicles.Notably,the transplanted mice generated new offspring.The results of different analyses showed increases in antiapoptotic and trophic proteins and genes.CONCLUSION These results suggested that transplantation of human ES-MSCs were similar to BM-MSCs in that they could restore the structure of the injured ovarian tissue and its function in chemotherapy-induced damaged POF mice and rescue fertility.The possible mechanisms of human ES-MSC were related to promotion of follicular development,ovarian secretion,fertility via a paracrine effect and ovarian cell survival.

Therapeutic potential of human embryonic stem cells in type 2 diabetes mellitus

AIM:To evaluate the safety and efficacy of human embryonic stem cells(h ESCs)for the management of type 2 diabetes mellitus(T2DM).METHODS:Patients with a previous history of diabetes and its associated complications were enrolled and injected with hE SC lines as per the defined protocol.The patients were assessed using Nutech functional score(NFS),a numeric scoring scale to evaluate the patients for 11 diagnostic parameters.Patients were evaluated at baseline and at the end of treatment period 1(T1).All the parameters were graded on the NFS scale from 1to 5.Highest possible grade(HPG)of 5 was considered as the grade of best improvement.RESULTS:Overall,94.8%of the patients showed improvement by at least one grade of NFS at the end of T1.For all the 11 parameters evaluated,54%of patients achieved HPG after treatment.The four essential parameters(improvement in glycated hemoglobin(HbA 1c)and insulin level,and fall in number of other oral hypoglycemic drugs with and without insulin)are presented in detail.For Hb A1c,72.6%of patients at the end of T1 met the World Health Organization cut off value,i.e.,6.5%of HbA 1c.For insulin level,65.9%of patients at the end of T1 were able to achieve HPG.After treatment,the improvement was seen in 16.3%of patients who required no more than two medications along with insulin.Similarly,21.5%of patients were improved as their dosage regimen for using oral drugs was reduced to 1-2 from 5.CONCLUSION:hE SC therapy is beneficial in patients with diabetes and helps in reducing their dependence on insulin and other medicines.

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells

Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

NEOPLASTIC TRANSFORMATION OF HUMAN EMBRYONIC NASOPHARYNGEAL EPITHELIAL CELLS INDUCED BY N,N'-DINITROSOPIPERAZINE (DNP) IN VITRO

This experiment is the first report on N, N'-dini-trosopiperazine (DNF)-induced neoplastic transformation of human embryonic nasopharyngeal (HENPE) cells. The transformed cells showed a prolonged life span, anchorage independent growth, chromosome aberration, tumorigenicity and an altered cell morphological appearance. The results demonstrated that DNP was able to induce not only nasopharyngeal carcinoma (NPC) of rats in vivo, but also neoplastic transformation of HENPE cells in vitro.

Mesenchymal Stromal Cells Derived from Human Embryonic Stem Cells, Fetal Limb and Bone Marrow Share a Common Phenotype but Are Transcriptionally and Biologically Different

Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.

Morphogenesis of human embryonic stem cells into mature neurons under in vitro culture conditions

AIM To describe the morphogenesis of different neuronal cells from the human embryonic stem cell(h ESC) line,SCT-N,under in vitro culture conditions.METHODS The directed neuronal cell line was produced from a single,spare,pre-implantation stage fertilized ovum that was obtained during a natural in vitro fertilization process. The h ESCs were cultured and maintained as per our proprietary in-house technology in a Good Manufacturing Practice,Good Laboratory Practice and Good Tissue Practice compliant laboratory. The cell line was derived and incubated in aerobic conditions. The cells were examined daily under a phase contrast microscope for their growth and differentiation. RESULTS Different neural progenitor cells(NPCs) and differentiating neurons were observed under the culture conditions. Multipotent NPCs differentiated into all three types of nervous system cells,i.e.,neurons,oligodendrocytes and astrocytes. Small projections resembling neurites or dendrites,and protrusion coming out of the cells,were observed. Differentiating cells were observed at day 18 to 20. The differentiating neurons,neuronal bodies,axons,and neuronal tissue were observed on day 21 and day 30 of the culture. On day 25 and day 30,prominent neurons,axons and neuronal tissue were observed under phase contrast microscopy. 4',6-diamidino-2-phenylindole staining also indicated the pattern of differentiating neurons,axonal structure and neuronal tissue. CONCLUSION This study describes the generation of different neuronal cells from an h ESC line derived from biopsy of blastomeres at the two-cell cleavage stage from a discarded embryo.

Human embryonic stem cells as an in vitro model for studying developmental origins of type 2 diabetes

The developmental origins of health and diseases(DOHaD)is a concept stating that adverse intrauterine environments contribute to the health risks of offspring.Since the theory emerged more than 30 years ago,many epidemiological and animal studies have confirmed that in utero exposure to environmental insults,including hyperglycemia and chemicals,increased the risk of developing noncommunicable diseases(NCDs).These NCDs include metabolic syndrome,type 2 diabetes,and complications such as diabetic cardiomyopathy.Studying the effects of different environmental insults on early embryo development would aid in understanding the underlying mechanisms by which these insults promote NCD development.Embryonic stem cells(ESCs)have also been utilized by researchers to study the DOHaD.ESCs have pluripotent characteristics and can be differentiated into almost every cell lineage;therefore,they are excellent in vitro models for studying early developmental events.More importantly,human ESCs(hESCs)are the best alternative to human embryos for research because of ethical concerns.In this review,we will discuss different maternal conditions associated with DOHaD,focusing on the complications of maternal diabetes.Next,we will review the differentiation protocols developed to generate different cell lineages from hESCs.Additionally,we will review how hESCs are utilized as a model for research into the DOHaD.The effects of environmental insults on hESC differentiation and the possible involvement of epigenetic regulation will be discussed.

Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast lineage-associated gene expression in human embryonic stem cells

Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.

Preliminary Functional Study on Wnt9a Cloning from Human Embryonic Stem Cells

Wnts are secreted lipid-modified signaling proteins that influence multiple processes ranging from cell proliferation and differentiation, fate decisions, apoptosis, axial polarity and axonal guidance to stem cell loss, kidney and reproductive tract defects. Activation of Wnt signalling in many tissues has also been associated with cancer. In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a small double-stranded RNA corresponding to exon sequences in the mRNA. In this study, human Wnt9a was cloned from undifferentiated hES cells. The results of immunohistochemistry showed that Wnt9a protein was expressed in undifferentiated hES cells, pAVU6＋27 vectors were used to construct the siRNA expression vectors for human Wnt9a One kind of small interfering RNA inserts was designed, synthesized and tested for human Wnt9a. The results of in situ hybridization demonstrated that Wntga signal was dramatically reduced in the cells transfected with U6＋27/siWnt9a compared to the untransfected cells. The results of flow cytometry analysis showed that the human breast cancer MCF-7 cells proliferation was promoted after lowering the expression of human Wnt9a by RNAi, but inhibited after over-expression of human Wnt9a. All those suggest the expression level of human Wnt9a may play a role in adjusting the rate of cell proliferation ofhES and MCF-7 cells.

Malignant Transformation of Human Embryonic Liver Cells Induced by Hepatitis B Virus and Aflatoxin B_1

In order to investigate the effect of hepatitis B virus (HBV) and aflatoxin B 1 (AFB 1) on hepatocarcinogenesis, the human embryonic liver cells infected with HBV were transplanted to nude mice by subcutaneous route and the transplanted mice were divided into 4 groups for study, in which the group A of mice was injected with HBV-infected human embryonic liver cells and followed by injections of AFB 1 once a week (HBV+AFB 1); the group B was treated with HBV as group A, but no AFB 1 was given (HBV +); the group C was injected with normal human embryonic liver cells and AFB 1 was used as group (AFB 1 +) and the group D or control group was injected with normal embryonic liver cells without addition of AFB 1. The experimental results showed that the incidences of tumor formation in different groups were 27.3% (6/22) in group A; 0% (0/13) in group B; 13.3% (2/15) in group C and 0% (0/14) in group D respectively. All the tumors formed were proved to be human hepatocellular carcinoma (HCC) by pathological examinations and the tumor tissues were anthrogenetic as demonstrated by EMA monoclonal antibody. The HBV-X and HBV-S genes could be detected in the tumor tissues by means of slot hybridization and PCR amplification, indicating that the HBV-DNA genes had integrated into DNA of host cells. Thus, we have successfully induced the human HCC through HBV infection and introduction of AFB 1 with a synergistic effect between HBV and AFB 1 in hepatocarcinogenesis.

Search for naive human pluripotent stem cells

Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.

In vitro derivation of functional insulin-producing cells from human embryonic stem cells

The capacity for self-renewal and differentiation of human embryonic stem （ES） cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid （RA） was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdxl and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin （STZ）-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdxl, glucokinase, nkx6.1, lAPP, pax6 and Tcfl. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.

Epigenetic states and expression of imprinted genes in human embryonic stem cells

AIM: To investigate the epigenetic states and expres- sion of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan. METHODS: The heterozygous alleles of single nucleo- tide polymorphisms (SNPs) at imprinted genes were analyzed by sequencing genomic DNAs of hESC lines and the monoallelic expression of the imprinted genes were confirmed by sequencing the cDNAs. The expres- sion profiles of 32 known imprinted genes of five hESC lines were determined using Affymetrix human genome U133 plus 2.0 DNA microarray. RESULTS: The heterozygous alleles of SNPs at seven imprinted genes, IPW , PEG10 , NESP55 , KCNQ1 , ATP10A ,TCEB3C and IGF2 , were identified and the monoallelic expression of these imprinted genes except IGF2 were confirmed. The IGF2 gene was found to be imprinted in hESC line T2 but partially imprinted in line T3 and not imprinted in line T4 embryoid bodies. Ten imprinted genes, namely GRB10 , PEG10 , SGCE, MEST , SDHD , SN- RPN , SNURF , NDN , IPW and NESP55 , were found to be highly expressed in the undifferentiated hESC lines and down-regulated in differentiated derivatives. The UBE3A gene abundantly expressed in undifferentiated hESC lines and further up-regulated in differentiated tissues. The expression levels of other 21 imprinted genes were relatively low in undifferentiated hESC lines and five of these genes (TP73 , COPG2 , OSBPL5 , IGF2 and ATP10A ) were found to be up-regulated in differentiated tissues. CONCLUSION: The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.

Effect of VEGF on Neural Differentiation of Human Embryonic Stem Cells in vitro

The effects of vascular endothelial growth factor （VEGF） on neural differentiation of human embryonic stem cells （hESCs） in vitro and the possible mechanism were observed. The hESCs lines, TJMU1 and TJMU2, were established and stored by our laboratory, hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups： group A, routine induction; group B, routine induction＋10 ng/mL VEGF; group C, routine in- duction＋10 ng/mL VEGF＋10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were de- tected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence. The percentage of Nestin positive cells in group B was significantly higher than in groups A and C, while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C （P〈0.01）. There was no significant difference between groups A and C （P〉0.05）. It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.

Tyrosine hydroxylase-positive cells and dopaminergic neuronal function in human embryonic stem cells: An electrophysiological validation

BACKGROUND： Induced differentiation strategies and cytochemical properties of human embryonic stem ceils （hESCs） have been investigated. However, the electrophysiological functions of tyrosine hydroxylase （TH）-positive cells dedved from hESCs remain unclear. OBJECTIVE： To investigate the differentiation efficiency of TH-positive cells from hESCs in vitro using modified four-step culture methods, including embryoid body formation, and to examine the functional characteristics of the differentiated TH-positive cells using electrophysiological techniques. DESIGN, TIME AND SETTING： Neuroelectrophysiology was performed at the Reproductive Medicine Center and Stem Cell Research Center, Peking University Third Hospital, and the Neuroscience Research Institute and Department of Neurobiology, Peking University, from September 2004 to August 2008. MATERIALS： The hESC line, PKU-1.1, a monoclonal cell line derived from a pre-implantation human blastocyst in the Reproductive Medical Center of Peking University Third Hospital. The patch clamp recording system was provided by the Neuroscience Research Institute and Department of Neurobiology, Peking University. METHODS： The hESC line was induced to differentiate into TH-positive cells in vitro using a modified four-step culture method, including the formation of embryoid body, as well as the presence of sonic hedgehog and fibroblast growth factor 8. The cell karyotype was assessed by G-banding karyotype analysis techniques and specific markers were detected immunocytochemically. Whole-cell configuration was obtained after obtaining a tight seal of over 1 GΩ. Ionic currents were detected by holding the cells at -70 mV and stepping to test voltages between -80 and 40 mV in 10-mV increments in voltage-clamp configuration. MAIN OUTCOME MEASURES： We measured the cell karyotype, specific cell markers, and the electrophysiological properties of the voltage-gated ion channels on the cell membrane of TH-positive dopaminergic cells differentiated from our hESCs line in vitro. RESULTS： The differentiated cells had a consistent appearance, and the majority of cells （〉 90%） expressed TH and β-tubulion, as well as the neural progenitor marker, nestino Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype （46, XX） after differentiation. In addition, patch clamp recording showed that the 10 recorded TH-positive cells exhibited a fast inward current when the test voltage depolarized to -30 mV, and a delayed outward current when the test voltage depolarized to -10 mV. The peak of inward current was obtained at voltage between 10 mV and 0 mV, while the peak of outward current was obtained at 40 mV. The average peak of inward current density was （ -50.05 ± 15.50） pA/pF, and the average peak of outward current density was （41.98 ± 13.55） pA/pE CONCLUSION： More than 90% of the differentiated hESC-derived cells induced by the modified four-step culture method exhibit dopaminergic neuronal properties, including general electrophysiological functional properties, such as functional potassium and sodium channels.

Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells

Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem （ES） cells and induced pluripo- tent stem （iPS） cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDXl-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insuIin-producing ceils. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.