Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

我们以前证明 Wnt3a 能刺激人的胚胎的茎(hES ) 细胞增殖并且影响房间命运决心。当 feeder cell-derivedfactors 不在时，在一个喂入器饲料分送器免费的条件下面有教养的 hES 房间糟糕幸存并且增殖。当 feeder 房间不在时的 Addingrecombinant Wnt3a 导出因素的刺激 hES 细胞增殖而且区别。在现在的学习，我们进一步扩大了我们的分析到象 Wnt1 和 Wnt5a 那样的另外的 Wntligands。当 Wnt1 作为 Wnt3a 在 hES 房间上显示了类似的效果时，在这个系统的 Wnt5ahad 小效果。当喂入器饲料分送器导出自强因素和 bFGF 也是现在时， Wnt3a 和 Wnt1 提高了无差别的 hES 房间的增长。探索可能性由激活 Wnt 发信号支持无差别的 hES 细胞的增长，在使不朽的人的成年成纤维细胞(HAFi ) 的 weoverexpressed Wnt3a 或 Wnt1 基因在比主要老鼠支持无差别的 hES 细胞的长期的生长是优异的细胞胚胎的成纤维细胞。HAFi 房间与或没有 Wnt transgene 能被宣传 Wnt3a 基因的 indefinitely.Over 表示显著地提高了 HAFi 喂入器饲料分送器房间的能力支持我们测试了的 3 根不同 hES 房间线的无差别的生长。在 Wnt3a-overpressing HAFi 房间的 threecommonly-used 药选择基因的合作表示进一步使我们能在稳定的 transfection 或转导以后选择稀罕 hES 克隆。这些使不朽的设计喂入器饲料分送器房间(W3R ) 那列合作快车象 Wnt3a 那样的支持生长的基因和三药选择基因应该授权我们高效地为基本、翻译的研究使修改 hES 房间线基因。

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.

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.

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

为人的胚胎的茎(ES ) 的自强和区别的能力细胞为对待类型 Idiabetes mellitus 为胰腺的贝它细胞的产生使他们成为潜在的来源。这里，我们报导一最新发展了并且有效方法，在aserum免费的系统执行了，区分进生产胰岛素的 cells.Activin A 的导致的人的 ES 房间它在起始的阶段被使用从人的 EScells 导致权威的内胚叶区别，是由权威的内胚叶标记 Sox17 和 Brachyury.Further 的表示检测了， all-trans retinoic 酸( RA )被用来支持胰腺的区别，由早胰腺的抄写因素 pdx1 和 hlxb9 的表示显示了。在成熟 inDMEM/F12 以后有 bFGF 和菸碱的没有浆液的媒介，区分的房间表示了小岛特定的标记象 C 肽，胰岛素，胰高血糖素和 glut2 那样。百分比 ofC-peptide-positive 房间超过了 15% 。由这些房间的胰岛素和 C 肽的分泌物在葡萄糖层次对应于变化。当移植了进肾的囊时， ofStreptozotocin (STZ ) 对待裸体老鼠，这些区分的人的 ES 房间熬过并且维持贝它房间标记基因的表示包括 C 肽， pdx1， glucokinase， nkx6.1， IAPP， pax6and Tcf1。百分之三十只移植裸体老鼠展出了 stableeuglycemia 的明显的恢复；并且改正的显型被支撑超过六个星期。我们的新方法为学习人的胰开发的机制提供一个有希望的试管内区别模特儿并且说明为类型 Idiabetes mellitus 的处理使用人的 ES 房间的潜力。

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.

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.

Enhancement of cardiomyocyte differentiation from human embryonic stem cells

Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells.However,the differentiation efficiency is low,and appropriate culture protocols are needed to produce adequate numbers of cardiomyocytes for therapeutic cell transplantation.This study investigated the effects of serum on cardiomyocyte differentiation in suspension culture medium during embryoid body(EB) formation by human embryonic stem cells.The addition of ascorbic acid,dimethylsulfoxide and 5-aza-2'-deoxycytidine during days 5-7 at the EB-forming stage resulted in an increase in the numbers of rhythmically contracting clusters of derived cardiomyocytes.Treatment with 0.1 mmol L-1 ascorbic acid alone,or more notably in combination with 10 μmol L-1 5-aza-2'-deoxycytidine,induced the formation of beating cells within EBs.Most of the beating clusters had spontaneous contraction rates similar to those found in human adults,and their contractile ac-tivity lasted for up to 194 days.

Generation of pancreatic islet cells from human embryonic stem cells

Efficiently obtaining functional pancreatic islet cells derived from human embryonic stem(hES) cells not only provides great potential to solve the shortage of islets sources for type I diabetes cell therapy,but also benefits the study of the development of the human pancreas and diabetes pathology.In 2001,hES cells were reported to have the capacity to generate insulin-producing cells by spontaneous differentiation in vitro.Since then,many strategies(such as overexpression of key transcription factors,delivery of key proteins for pancreatic development,co-transplantation of differentiated hES cells along with fetal pancreas,stepwise differentiation by mimicking in vivo pancreatic development) have been employed in order to induce the differentiation of pancreatic islet cells from hES cells.Moreover,patient-specific induced pluripotent stem(iPS) cells can be generated by reprogramming somatic cells.iPS cells have characteristics similar to those of ES cells and offer a new cell source for type I diabetes cell therapy that reduces the risk of immunologic rejection.In this review,we summarize the recent progress made in the differentiation of hES and iPS cells into functional pancreatic islet cells and discuss the challenges for their future study.

Cytotoxic effects of mono-（2-ethylhexyl） phthalate on human embryonic stem cells

Background Mono-（2-ethylhexyl） phthalate （MEHP）, the metabolite of di-（2-ethylhexyl） phthalate （DEHP）, was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by an embryonic stem cell test （EST） based on two human embryonic stem cell （hESCs） lines. Methods CH1 established in our own lab and H1, a federally registered cell line were two human embryonic stem cell lines used in this test. Four endpoint measurements were performed consisting of cell viability, proliferation ability, apoptosis as well as changes of gene expression patterns after spontaneous differentiation were determined. For measuring effects on the first three endpoints, the cells were treated with various concentrations of MEHP dissolved in dimethyl sulfoxide （DMSO） and only with DMSO which served as control and harvested after 5 days. For measuring effects during spontaneous differentiation, the RNA of embryoid bodies （EBs） formed after 8 days＇ MEHP exposure was collected and changes in differentiation specific gene expression patterns were analyzed by quantitative real time RT-PCR. Results As a result the viability and proliferation ability of both cell lines decreased significantly at 1000 μmol/L MEHP, while there was no effect on apoptosis or cell morphology. In addition MEHP also changed the gene expression pattern in the EBs of both cell lines. Conclusion MEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicitv in human embrvos.

Shp2-mediated molecular signaling in control of embryonic stem cell self-renewal and differentiation

在干细胞生物学要处理的一个关键问题是控制胚胎的茎(ES ) 的分子的发信号机制细胞 pluripotency。干细胞性质被象脱氧核糖核酸甲基化并且染色质改变那样的特定的抄写因素和 epigenetic 过程支配。几个 cytokines/growth 因素作为批评 ES 房间管理者被识别了。然而，在我们在 ES 房间连接细胞外的信号到 transcriptional 规定的细胞内部的发信号小径的知识有差距。这短评论讨论 Shp2 的生理的角色，细胞质的酷氨酸磷酸酶，在管理 EScell 自强对区别的分子的开关中。Shp2 支持 ES 细胞分化，英皇家空军之阶级最低之兵的主要 throughbi 方向性的调整和 Stat3 小径。在老鼠 ES 房间的 Shp2 的删除导致更有效的自强。这观察提供动力在文化为 ES 房间的维护和扩大开发 Shp2 禁止者。

Neural precursors derived from human embryonic stem cells

Human embryonic stem (hES) cells provide a promising supply of specific cell types for transplantation therapy. We presented here the method to induce differentiation of purified neural precursors from hES cells. hES cells (Line PKU-1 and Line PKU-2) were cultured in sus- pension in bacteriological Petri dishes, which differentiated into cystic embryoid bodies (EBs). The EBs were then cultured in N2 medium containing bFGF in poly-L-lysine-coated tissue culture dishes for two weeks. The central, small cells with 2―3 short processes of the spreading out- growth were isolated mechanically and replated. The resulting neurospheres were cultured in suspension for 10 days, then dissociated into single cell suspension with a Pasteur pipette and plated. Cells grew vigorously in an attached way and were passed every 4―5 days. Almost all the cells were proved nestin positive by immunostaining. Following withdrawal of bFGF, they differentiated into neurons expressing β-tubulin isotypeIII, GABA, serotonin and synaptophysin. Through induction of PDGF-AA, they differentiated into astrocytes expressing GFAP and oli- godendrocytes expressing O4. The results showed that hES cells can differentiate into typical neural precursors expressing the specific marker nestin and capable of generating all three cell types of the central nervous system (CNS) in vitro.

Predicting differentiation potential of human pluripotent stem cells:Possibilities and challenges

The capability of human pluripotent stem cell(hPSC)lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine.However,variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost.To screen the qualified cell lines and exclude problematic cell lines,their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by highthroughput gene expression profiling assay.Additionally,their differentiation potential,particularly the lineage-specific differentiation propensities of hPSC lines,should be predicted in an early stage.As a complement to the teratoma assay,RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs in vivo.Moreover,multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation.For clinical application of hPSCs,the malignant potential of the cells must also be evaluated.A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas,malignancy marker expression,and other parameters.Although various prediction methods are available,distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost,restricting their wide applications in routine studies.Therefore,simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.

Immunomodulative effects of mesenchymal stem cells derived from human embryonic stem cells in vivo and in vitro

Objective: Human embryonic stem cells (hESCs) have recently been reported as an unlimited source of mesenchymal stem cells (MSCs).The present study not only provides an identical and clinically compliant MSC source derived from hESCs (hESC-MSCs),but also describes the immunomodulative effects of hESC-MSCs in vitro and in vivo for a carbon tetrachloride (CCl4)-induced liver inflammation model.Methods: Undifferentiated hESCs were treated with Rho-associated kinase (ROCK) inhibitor and induced to fibroblast-looking cells.These cells were tested for their surface markers and multilineage differentiation capability.Further more,we analyzed their immune characteristics by mixed lymphocyte reactions (MLRs) and animal experiments.Results: hESC-MSCs show a homogenous fibroblastic morphology that resembles bone marrow-derived MSCs (BM-MSCs).The cell markers and differentiation potential of hESC-MSCs are also similar to those of BM-MSCs.Unlike their original cells,hESC-MSCs possess poor immunogenicity and can survive and be engrafted into a xenogenic immunocompetent environment.Conclusions: The hESC-MSCs demonstrate strong inhibitory effects on lymphocyte proliferation in vitro and anti-inflammatory infiltration properties in vivo.This study offers information essential to the applications of hESC-MSC-based therapies and evidence for the therapeutic mechanisms of action.

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.

Chemical approach to generating long-term self-renewing pMN progenitors from human embryonic stem cells

Spinal cord impairment involving motor neuron degeneration and demyelination can cause lifelong disabilities,but effective clinical interventions for restoring neurological functions have yet to be developed.In early spinal cord development,neural progenitors of the motor neuron(pMN)domain,defined by the expression of oligodendrocyte transcription factor 2(OLIG2),in the ventral spinal cord first generate motor neurons and then switch the fate to produce myelin-forming oligodendrocytes.Given their differentiation potential,pMN progenitors could be a valuable cell source for cell therapy in relevant neurological conditions such as spinal cord injury.However,fast generation and expansion of pMN progenitors in vitro while conserving their differentiation potential has so far been technically challenging.In this study,based on chemical screening,we have developed a new recipe for efficient induction of pMN progenitors from human embryonic stem cells.More importantly,these OLIG2+pMN progenitors can be stably maintained for multiple passages without losing their ability to produce spinal motor neurons and oligodendrocytes rapidly.Our results suggest that these self-renewing pMN progenitors could potentially be useful as a renewable source of cell transplants for spinal cord injury and demyelinating disorders.

ETV2 expression increases the efficiency of primitive endothelial cell derivation from human embryonic stem cells

Background:Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body.Ets variant 2(ETV2)is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification.Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells.Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.Findings:We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells(ESCs)to determine when the peak of ETV2 expression occurs.We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.Conclusions:Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification.This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

Prostaglandin E2 promotes hematopoietic development from human embryonic stem cells

Recent studies have suggested that prostaglan-din(PG)E2(PGE2)and the prostaglandin pathway are essential for hematopoietic stem cell growth and develop-ment.However,similar studies on hematopoietic commit-ment from human embryonic stem cells(hESCs)are still limited.Here we report that the addition of PGE2 promotes hematopoietic differentiation of hESCs.The induced cells from hESCs/OP9 co-culture and in the presence of PGE2 were characterized by reverse transcription-PCR(RT-PCR),flow cytometry,colony-forming assays and Wright-Giemsa staining.Our results demonstrated that PGE2 exposure could alter the gene expression pattern and morphology of co-cultured hESCs and resulted in a robust hematopoietic differentiation with higher frequencies of CD34+and CD45+cells.Furthermore,the Smad signaling pathway may be involved in PGE2 and OP9 induced hematopoietic differentiation of hESCs.This research may improve our knowledge of stem cell regulation and hopefully lead to better stem cell-based therapeutic options.

Low level of activin A secreted by fibroblast feeder cells accelerates early stage differentiation of retinal pigment epithelial cells from human pluripotent stem cells

Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hPSC-RPE is still poorly understood and current differentiation protocols rely on spontaneous differentiation on fibroblast feeder cells or as floating cell aggregates in suspension. The fibroblast feeder cells may have an inductive effect on the hPSC-RPE differentiation, providing variable signals mimicking the extraocular mesenchyme that directs the differentiation in vivo. The effect of the commonly used fibroblast feeder cells on the hPSCRPE differentiation was studied by comparing suspension differentiation in standard RPEbasic (no bFGF) medium to RPEbasic medium conditioned with mouse embryonic (mEF-CM) and human foreskin (hFF-CM) fibroblast feeder cells. The fibroblast secreted factors were found to enhance early hPSC-RPE differentiation. The onset of pigmentation was faster in the conditioned media (CM) compared to RPEbasic for both human embryonic (hESC) and induced pluripotent (iPSC) stem cells, with the first pigments appearing around two weeks of differentiation. After four weeks of differentiation, CM conditions consistently contained higher number of pigmented cell aggregates. The ratio of PAX6 and MITF positive cells was quantified to be clearly higher in the CM conditions, with mEFCM containing most positive cells. The mEF cells were found to secrete low levels of activin A growth factor that is known to regulate eye field differentiation. As RPEbasic was supplemented with corresponding, low level (10 ng/ml) of recombinant human activin A, a clear increase in the hPSC-RPE differentiation was achieved. Thus, inductive effect provided by feeder cells was at least partially driven by activin A and could be substituted with a low level of recombinant growth factor in contrasts to previously reported much higher concentrations.

Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

单性生殖是之一主要、很有用，方法到导出胚胎的干细胞(转换字符) ，它可以为房间治疗是 histocompatible 房间和纸巾的重要来源。这里，我们描述从在 vitro 的线(hPES-1 和 hPES-2 ) 开发了胚囊列在后面的二个转换字符的推导和描述人的卵母细胞的单性生殖的激活。典型转换字符形态学被看见，并且转换字符标记的表示是是为碱的磷酸酶期望， octamer 有约束力的抄写因素 4，阶段特定的胚胎的抗原 3，阶段特定的胚胎的抗原 4， TRA-1-60，和 TRA-1-81，并且有否定标记例如的表示的缺席阶段特定的胚胎的抗原 1。为不同胚胎的细菌层特定的基因的表示从两根 hESC 线的胚胎植物或动物身体(EB ) 被检测，建议他们在 vitro 的区别潜力。在 vivo，然而，仅仅 hPES-1 形成了由所有三胚胎的细菌层组成的 teratoma (hPES-2 ) 。在为超过 100 个段落的连续增长以后，有趣地， hPES-1 房间仍然维持了正常 46 XX karyotype；hPES-2 在长期的段落以后显示了象染色体 translocation 那样的畸形。短双人脚踏车重复(STR ) 结果证明 hPES 线是有鸡蛋施主的基因火柴，并且基因印数据证实了这些 ES 房间的单性生殖的起源。染色体宽的 SNP 分析显示出典型地代表单性生殖的一个模式。所有这些结果表明了可行性孤立并且建立人的单性生殖的转换字符线，它提供一个重要工具因为学习 epigenetic 在一个临床的背景在转换字符以及为未来完成治疗学的干预。

Nutrient supplemented serum-free medium increases cardiomyogenesis efficiency of human pluripotent stem cells

AIM: To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells. METHODS: Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcriptionpolymerase chain reaction. RESULTS: hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar. CONCLUSION: The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac marker expression during cardiomyocyte differentiation, translating to an overall increase in cardiomyocyte yield.