Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells

BACKGROUND Embryonic stem cells(ESCs)serve as a crucial ex vivo model,representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos.ESCs exhibit a unique combination of self-renewal potency,unlimited proliferation,and pluripotency.The latter is evident by the ability of the isolated cells to differ-entiate spontaneously into multiple cell lineages,representing the three primary embryonic germ layers.Multiple regulatory networks guide ESCs,directing their self-renewal and lineage-specific differentiation.Apoptosis,or programmed cell death,emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development.How-ever,the molecular mechanisms underlying the dynamic interplay between diffe-rentiation and apoptosis remain poorly understood.AIM To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells,using mouse ESC(mESC)models-mESC-B-cell lym-phoma 2(BCL-2),mESC-PIM-2,and mESC-metallothionein-1(MET-1)-which overexpress the anti-apoptotic genes Bcl-2,Pim-2,and Met-1,respectively.METHODS mESC-T2(wild-type),mESC-BCL-2,mESC-PIM-2,and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation.The hanging drop method was adopted to generate embryoid bodies(EBs)and induce terminal differentiation of mESCs.The size of the generated EBs was measured in each condition compared to the wild type.At the functional level,the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control.At the molecular level,quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers:Troponin T,GATA4,and NKX2.5.Additionally,troponin T protein expression was evaluated through immunofluorescence and western blot assays.RESULTS Our findings showed that the upregulation of Bcl-2,Pim-2,and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs,in comparison with their wild-type counterpart.Additionally,a decrease in the count of beating cardiomyocytes among differentiated cells was observed.Furthermore,the mRNA expression of three cardiac markers-troponin T,GATA4,and NKX2.5-was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line.Moreover,the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression.CONCLUSION Our findings revealed that the upregulation of Bcl-2,Pim-2,and Met-1 genes altered cardiac differentiation,providing insight into the intricate interplay between apoptosis and ESC fate determination.

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells

BACKGROUND： Midbrain-derived neural stem cells （mNSCs） can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson＇s disease. OBJECTIVE： To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING： An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital （China） from March to November 2007. MATERIALS： Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein （GFAP） antibody and cyclic nucleotide 3＇-phosphohydrolase （CNPase） antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor （EGF） and fibroblast growth factor-2 （FGF2） were provided by R＆D Systems. METHODS： The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N： supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES： The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS： Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION： Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N： additive.

Undifferentiated liver embryonal sarcoma in adults: A report of four cases and literature review

AIM: To evaluate the undifferentiated embryonal sarcoma of liver (UESL) in adults in order to improve its diagnosis and treatment. METHODS: Four primary and one recurrent cases of UESL were clinicopathologically evaluated and immunohistochemically investigated with a panel of antibodies using the EnVision+ system. Relevant literature about UESL in adults was reviewed. RESULTS: Three males and one female were enrolled in this study. Their chief complaints were abdominal pain, weight loss, or fever. Laboratory tests, imaging and pathological features of UESL in adults were similar to those in children. Immunohistochemistry showed evidence of widely divergent differentiation into mesenchymal and epithelial phenotypes. The survival time of patients who underwent complete tumor resection followed by adjuvant transcatheter arterial chemoembolization (TACE) was significantly longer than that of those who underwent surgical treatment alone.CONCLUSION: UESL in adults may undergo pluripotential differentiation and its diagnosis should be made based on its morphological and immunohistochemical features. Complete tumor resection after adjuvant TACE may improve the survival time of such patients.

Neural differentiation from embryonic stem cells in vitro : An overview of the signaling pathways

Neurons derived from embryonic stem cells(ESCs) have gained great merit in both basic research and regenerative medicine. Here we review and summarize the signaling pathways that have been reported to be involved in the neuronal differentiation of ESCs,particularly those associated with in vitro differentiation. The inducers and pathways explored include retinoic acid, Wnt/b-catenin, transforming growth factor/bone morphogenetic protein, Notch, fibroblast growth factor, cytokine, Hedgehog, c-Jun N-terminal kinase/mitogen-activated protein kinase and others. Some other miscellaneous molecular factors that have been reported in the literature are also summarized and discussed. These include calcium, calcium receptor, calcineurin, estrogen receptor, Hox protein, ceramide, glycosaminioglycan, ginsenoside Rg1, opioids, two pore channel 2, nitric oxide, chemically defined medium, cellcell interactions, and physical stimuli. The interaction or crosstalk between these signaling pathways and factors will be explored. Elucidating these signals in detail should make a significant contribution to future progress in stem cell biology and allow, for example, better comparisons to be made between differentiation in vivo and in vitro. Of equal importance, a comprehensive understanding of the pathways that are involved in the development of neurons from ESCs in vitro will also accelerate their application as part of translational medicine.

Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stern cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, ~lll-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neuregenesis related genes （Sox-1, Nestin, GABA, Pax6, Zic5 and Pitxl） in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

MiR-301a promotes embryonic stem cell differentiation to cardiomyocytes

BACKGROUND Cardiovascular disease is the leading cause of death worldwide.Tissue repair after pathological injury in the heart remains a major challenge due to the limited regenerative ability of cardiomyocytes in adults.Stem cell-derived cardiomyocytes provide a promising source for the cell transplantation-based treatment of injured hearts.AIM To explore the function and mechanisms of miR-301a in regulating cardiomyocyte differentiation of mouse embryonic stem(mES)cells,and provide experimental evidence for applying miR-301a to the cardiomyocyte differentiation induction from stem cells.METHODS mES cells with or without overexpression of miR-301a were applied for all functional assays.The hanging drop technique was applied to form embryoid bodies from mES cells.Cardiac markers including GATA-4,TBX5,MEF2C,andα-actinin were used to determine cardiomyocyte differentiation from mES cells.RESULTS High expression of miR-301a was detected in the heart from late embryonic to neonatal mice.Overexpression of miR-301a in mES cells significantly induced the expression of cardiac transcription factors,thereby promoting cardiomyocyte differentiation and beating cardiomyocyte clone formation.PTEN is a target gene of miR-301a in cardiomyocytes.PTEN-regulated PI3K-AKT-mTOR-Stat3 signaling showed involvement in regulating miR-301a-promoted cardiomyocyte differentiation from mES cells.CONCLUSION MiR-301a is capable of promoting embryonic stem cell differentiation to cardiomyocytes.

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.

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.

Histone acetylation and its role in embryonic stem cell differentiation

The understanding of mechanisms leading to cellular differentiation is the main aim of numerous studies.Accessibility of DNA to transcription factors depends on local chromatin structure and chromatin compaction inhibits gene transcription.Histone acetylation correlates with an open chromatin structure and increased gene expression.Gene transcription levels are changed in early embryonic stem cells differentiation in a tissuespecific manner and epigenetic marks are modified,including increased global acetylation levels.Manipulation of histone deacetylases activity might be an interesting tool to generate populations of specific cell types for transplantation purposes.Thus,this review aims to show recent findings on histone acetylation,a post translational modif ication and its manipulation in embryonic stem cells differentiation.

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass （inner cell mass, ICM） which were isolated by mechanical method on the mouse embryonic fibroblaste cell （MEF） feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 （ stage specific embryonic antigen 1 ）. Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.

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。

Cell cycle exit and neuronal differentiation 1-engineered embryonic neural stem cells enhance neuronal differentiation and neurobehavioral recovery after experimental traumatic brain injury

Our previous study showed that cell cycle exit and neuronal differentiation 1(CEND1)may participate in neural stem cell cycle exit and oriented differentiation.However,whether CEND1-transfected neural stem cells can improve the prognosis of traumatic brain injury remained unclear.In this study,we performed quantitative proteomic analysis and found that after traumatic brain injury,CEND1 expression was downregulated in mouse brain tissue.Three days after traumatic brain injury,we transplanted CEND1-transfected neural stem cells into the area surrounding the injury site.We found that at 5 weeks after traumatic brain injury,transplantation of CEND1-transfected neural stem cells markedly alleviated brain atrophy and greatly improved neurological function.In vivo and in vitro results indicate that CEND1 overexpression inhibited the proliferation of neural stem cells,but significantly promoted their neuronal differentiation.Additionally,CEND1 overexpression reduced protein levels of Notch1 and cyclin D1,but increased levels of p21 in CEND1-transfected neural stem cells.Treatment with CEND1-transfected neural stem cells was superior to similar treatment without CEND1 transfection.These findings suggest that transplantation of CEND1-transfected neural stem cells is a promising cell therapy for traumatic brain injury.This study was approved by the Animal Ethics Committee of the School of Biomedical Engineering of Shanghai Jiao Tong University,China(approval No.2016034)on November 25,2016.

The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

Purpose:To study preliminarily induced differentiation of embryonic stem cells intocorneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal cornealepithelial cells in Transwell system to induce differentiation. Mophological andimmunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance.The cells formed a network and were confluent into film gradually after beingco-cultured with rabbit limbal corneal epithelial cells for 24 ～ 96 hours. The cells rangedmosaic structure and localized together with clear rim. Most of the cells showedpolygonal appearance. Transmission electron microscope showed lots of microvilli on thesurface of induced cells and tight junctions between them. These epithelial-like cellsexpressed the corneal epithelial cell specific marker cytokeratin3/cytokeratinl2.Conclusion: The potential mechanism of the differentiation of murine embryonic stemcells into corneal epithelial cells induced by limbal corneal epithelial cell-derivedinducing activity is to be further verified.

Preliminary Study on in Vitro Induced Differentiation of Embryonic Stem Cells into Neurons

Purpose:To study preliminarily in vitro induced differentiation of embryonic stem cells into neurons for further investigation of an alternative for the treatment of glaumatous neuropathy.Materials and methods:Supernatant of cultured Buffalo rat liver cells(buffalorat liver cell-conditioned medium,BRL-CM)was used for culturing embryonic stem cells(ES-D3 cell line),Morphological features of undifferentiated EScells were studied by HE staining and electron microscopy.Based on the methods used by Bain et al,we modified the methods and used retinoic acid(RA)as an inducer to differentiate ES-D3 cells and cytosine arabinoside(Ara-C)as inhibitor of proliferative cells.The growth of the cells was observed under phase contrast microscope.Results:ES-D3cells cultured by BRL-CMgrew in aggregates and remained undifferentiated.Electromicroscopy showed large nucleus and a large amount of mitochondria in undifferentiated ES cells and many processes on the surfaces.In the first day after the adding of retinoic acid,some neuron-like cells with one,two or more processes were present.In the second day after adding RAand the first day after the plus of 10μm Ara-C,a large amount of neuron-like cells appeared,with the formation of neuron-like networks.Con clusions:Combined use of RA and Ara-Ccan induce ES cells to differentiate into neuron-like cells.Our present preliminary study might provide insights into an alternative for the treatment of glaucomatous neuropathy by the transplantation of embryonic stem cells.Eye Science2000;16:1-6.

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.

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。

Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes.METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression prof iles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were usedfor the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells.RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by signif icant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages.CONCLUSION: Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines.

Differentiation of mouse embryonic stem cells into insulin-secreting cells in vitro

包括房间代替策略，再生药可以在与减少的小岛房间质量被联系的类型 1 糖尿病的治疗有一个重要角色。迄今为止，重要进步在从 pluripotent 老鼠产生胰岛素秘密房间被成为了胚胎的干细胞(转换字符) 。这研究的目的探索调整转换字符的区别进胰腺的内分泌的房间的潜力能够综合包括在合适的条件下面的胰岛素， glucagon， somatostatin 和胰腺的多肽的胰腺的荷尔蒙。无差别的 ES 房间线稳定地，有在没有浆液的媒介的老鼠 RIP-YFP plasmid 建设的 transfected 使用 Lipofectamine？2000 试剂。我们测试了胰腺的特定的基因表示并且描绘了这些导出转换字符的胰腺的内分泌的房间。大多数这些胰岛素秘密房间共同表示许多象 insulin1 那样的房间的 phenotypic 标记特征， insulin2， Islet1， MafA，联系 insulinoma 的抗原 1 (IA1 ) 等等，在 vivo 显示一个类似的基因表示模式到成年小岛房间。这张人口的描述表明它由能在适当条件下面经历胰腺的说明的胰腺的内分泌的房间主要组成了。我们也表明了那锌补充有效 inducer 支持了导出转换字符的糖尿病的发展的胰岛素秘密房间的调停的起来规定治疗。在结论，这个工作不仅在 vitro 从转换字符建立了有效胰腺的区别策略到胰腺的内分泌的系，而且导致了新策略的开发为一个干细胞的未来应用程序从胚胎的干细胞导出可移植的小岛代替房间糖尿病的基于的治疗。

Preparation of Mouse Embryonic Stem Cells and Cardiomyocyte Differentiation Induced with Retinoic Acid and Ascorbic Acid

The experiment was designed to study effects of retinoic acid and ascorbic acid on differentiation of mouse embryonic stem cells to cardiomyocytes. Embryonic bodies （EB） were developed from mESC in suspension culture, different levels of concentration of retinoic acid and ascorbic acid were used to determine the optimal conditions for EB formation. The results showed that the optimal concentrations were 10.9 mol. L-1 and 0.1 mg. mL-1 for retinoic acid and ascorbic acids, respectively. 50% of EB which was significantly （p〈0.05） different from the control group developed to cardiomyocytes. In conclusion, rctinoic acid and ascorbic acid had strong ability to promote cardiomyocyte differentiation of mouse embryonic stem cells. 10-9 mol. L-1 retinoic acid and 0.10 mg. mL-1 ascorbic acids were recommended to induce differentiation of mouse ES ceUs toward cardiomyocytes.