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.

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid(LPA)is a small molecule glycerophospholipid,which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.In this study,sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.At first,the maturation medium containing estrus female sheep serum and synthetic oviduct fluid(SOF)were optimized for sheep IVF,and then the effects of LPA were investigated.From 0.1 to 10μmol L^(–1),LPA had no significant effect on the cleavage rate(P>0.05),but the maturation rate and blastocyst rate increased dependently with LPA concentration(P<0.05),and the blastocyst morphology was normal.When the LPA concentration was 15μmol L^(–1),the maturation rate,cleavage rate and blastocyst rate decreased significantly(P<0.05),and the blastocyst exhibited abnormal morphology and could not develop into highquality blastocyst.Besides,the exogenous LPA increases the expression of LPAR2,LPAR4,TE-related gene CDX-2and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10μmol L^(–1).The expression of LPAR2,LPAR4,CDX-2 and OCT-4 from the LPA-0.1μmol L^(–1)to LPA-10μmol L^(–1)groups in early embryos were extremely significant(P<0.05),while the expression of these genes significantly decreased in 15μmol L^(–1)LPA-treated embryos compared with LPA-10μmol L^(–1)group(P<0.05).The inner cell mass in 15μmol L^(–1)LPA-treated embryos was also disturbed,and the blastocysts formation was abnormal.Secondly,the sheep IVF blastocysts were applied to establish embryonic stem cells.The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10μmol L^(–1),while 15μmol L^(–1)LPA decreased OCT-4 and CDX-2 expression in the derived cells.The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1μmol L^(–1)group and LPA-10μmol L^(–1)group extremely significantly increased(P<0.05),but there was significant decrease in LPA-15μmol L^(–1)group compared with LPA-10μmol L^(–1)group(P<0.05).Meanwhile,the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10μmol L^(–1)concentration.This study references the IVF embryo production and embryonic stem cell research of domestic animals.

The combined application of stem cells and three-dimensional bioprinting scaffolds for the repair of spinal cord injury

Spinal cord injury is considered one of the most difficult injuries to repair and has one of the worst prognoses for injuries to the nervous system.Following surgery,the poor regenerative capacity of nerve cells and the generation of new scars can make it very difficult for the impaired nervous system to restore its neural functionality.Traditional treatments can only alleviate secondary injuries but cannot fundamentally repair the spinal cord.Consequently,there is a critical need to develop new treatments to promote functional repair after spinal cord injury.Over recent years,there have been seve ral developments in the use of stem cell therapy for the treatment of spinal cord injury.Alongside significant developments in the field of tissue engineering,three-dimensional bioprinting technology has become a hot research topic due to its ability to accurately print complex structures.This led to the loading of three-dimensional bioprinting scaffolds which provided precise cell localization.These three-dimensional bioprinting scaffolds co uld repair damaged neural circuits and had the potential to repair the damaged spinal cord.In this review,we discuss the mechanisms underlying simple stem cell therapy,the application of different types of stem cells for the treatment of spinal cord injury,and the different manufa cturing methods for three-dimensional bioprinting scaffolds.In particular,we focus on the development of three-dimensional bioprinting scaffolds for the treatment of spinal cord injury.

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.

Common stemness regulators of embryonic and cancer stem cells

Pluripotency of embryonic stem cells(ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal trans-ducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors(cancer stem cells), provides a common conceptual and research frame-work for basic and applied stem cell biology. In this review, we highlight current results on biomarkers, gene signatures, signaling pathways and epigenetic regulators that are common in embryonic and cancer stem cells. We discuss their role in determining the cell phenotype and finally, their potential use to design next generation biological and pharmaceutical approaches for regenerative medicine and cancer therapies.

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PGR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Effects of resveratrol on hydrogen peroxide-induced oxidative stress in embryonic neural stem cells

Resveratrol, a natural phenolic compound, has been shown to prevent cardiovascular diseases and cancer and exhibit neuroprotective effects. In this study, we examined the neuroprotective and antJoxJdant effects of resveratrol against hydrogen peroxide in embryonic neural stem cells. Hydrogen peroxide treatment alone increased catalase and glutathione peroxidase activities but did not change superoxide dismutase levels compared with hydrogen peroxide ＋ resveratrol treatment. Nitric oxide synthase activity and concomitant nitric oxide levels increased in response to hydrogen peroxide treatment. Conversely, resveratrol treatment decreased nitric oxide synthase activity and nitric oxide levels. Resveratrol also attenuated hydrogen peroxide-induced nuclear or mitochondrial DNA damage. We propose that resveratrol may be a promising agent for protecting embryonic neural stem cells because of its potential to decrease oxidative stress by inducing higher activity of antioxidant enzymes, decreasing nitric oxide production and nitric oxide synthase activity, and alleviating both nuclear and mitochondrial DNA damage.

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.

Immunological properties of embryonic and adult stem cells

The possibility of treating degenerative diseases by stem cell-based approaches is a promising therapeutical option.Among major concerns for the clinical application of stem cells,some derive from the possibility that stem cells may be rejected by the immune system as a consequence of histoincompatibility and that stem cells themselves may interfere with the normal functions of host immune response.Therefore,the immunogenicity and the immunomodulatory properties of stem cells must be carefully addressed.Although these properties are common features of different stem cell types,some peculiarities can be recognized and characterized for their proper clinical use.

Intravenous transplantation of mouse embryonic stem cells attenuates demyelination in an ICR outbred mouse model of demyelinating diseases

Induction of demyelination in the central nervous system （CNS） of experimental mice using cuprizone is widely used as an animal model for studying the pathogenesis and treatment of demyelination. How- ever, different mouse strains used result in different pathological outcomes. Moreover, because current medicinal treatments are not always effective in multiple sclerosis patients, so the study of exogenous cell transplantation in an animal model is of great importance. The aims of the present study were to establish an alternative ICR outbred mouse model for studying demyelination and to evaluate the effects of intrave- nous cell transplantation in the present developed mouse model. Two sets of experiments were conducted. Firstly, ICR outbred and BALB/c inbred mice were fed with 0.2% cuprizone for 6 consecutive weeks; then demyelinating scores determined by luxol fast blue stain or immunolabeling with CNPase were evaluated. Secondly, attenuation of demyelination in ICR mice by intravenous injection of mES cells was studied. Scores for demyelination in the brains of ICR mice receiving cell injection （mES cells-injected group） and vehicle （sham-inoculated group） were assessed and compared. The results showed that cuprizone signifi- cantly induced demyelination in the cerebral cortex and corpus callosum of both ICR and BALB/c mice. Additionally, intravenous transplantation of mES cells potentially attenuated demyelination in ICR mice compared with sham-inoculated groups. The present study is among the earliest reports to describe the cuprizone-induced demyelination in ICR outbred mice. Although it remains unclear whether mES cells or trophic effects from mES cells are the cause of enhanced remyelination, the results of the present study may shed some light on exogenous cell therapy in central nervous system demyelinating diseases.

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.

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 × l06 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6～8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6～8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.

Tissue factor expression and methylation regulation in differentiation of embryonic stem cells into trophoblast

Objective:To explore tissue factor(TF)expression and methylation regulation in differentiation of human embryonic stem cells(hESCs)into trophoblast.Methods:Differentiation of hESCs into trophoblast was induced by bone morphogenetic protein 4(BMP4).Expression of gene,protein of TF and DNA methylation at different time points during induction process was detected by RTPCT,Western blot,flow cytometry and MSP-PCR method.Results:The expression of mRNA,protein level of TF could be detected during directional differentiation of hESCs to trophoblast cells,semi methylation-semi non methylation expression appeared at TF DNA promoter region,and it showed decreased methylation level and increased non methylation level with formation of trophoblast cell and increased expression of TF.Conclusions:It shows that during differentiation of hESCs into trophoblast,the differential expression of TF is related with DNA methylation level,and it is changed with the methylation or non methylated degree.It provids new platform to furtherly explore the regulation mechanisms of specific expression of tissue factor in the process of the embryonic stem cell development.

WWP2 promotes degradation of transcription factor OCT4 in human embryonic stem cells

POU 抄写因素 OCT4 不仅在维持 pluripotent 和房间而且幕作为通过基因剂量的一个房间命运决定因素完成的胚胎的茎(ES ) 的自我更新的状态起一个必要作用。然而，控制细胞内部的 OCT4 蛋白质水平的分子的机制留下逃犯。这里，我们报导那人的 WWP2， E3 ubiquitin (Ub ) 蛋白质 ligase，通过它的 WW 领域明确地与 OCT4 交往并且在 vitro 并且在 vivo 提高 OCT4 的 Ub 修正。我们首先证明在人的 ES 房间的内长的 OCT4 能被 Ub post-translationally 修改。而且，我们发现 WWP2 以一种剂量依赖者方式，和 WWP2 的活跃地点半胱氨酸残余通过 26S proteasome 支持了 OCT4 的降级在 OCT4 上为它的酶的活动和解朊的效果被要求。显著地，我们当 WWP2 表示是由特定的 RNA 干扰(RNAi ) 的 downregulated 时，内长的 OCT4 蛋白质水平显著地被提高的数据表演，建议那 WWP2 是为在人的 ES 房间维持合适的 OCT4 蛋白质水平的一个重要管理者。而且，北污点分析证明 WWP2 抄本在多样的人的织物 / 器官是广泛地在场的并且高度在无差别的人的 ES 房间表示了。然而，它的表示水平快速在区分的人的 ES 房间以后被减少，显示 WWP2 表示力量发展地被调整。我们的调查结果证明 WWP2 是在人的 ES 房间的 OCT4 蛋白质水平的一个重要管理者。

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.

Smad2 mediates Activin/Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells

尽管发信号的 Activin/Nodal 调整人的胚胎的茎(ES ) 的 pluripotency 细胞，这个发信号怎么在老鼠 ES 细胞行动，仍然保持大部分不清楚。调查这，我们证实了房间拥有活跃调停 Smad2 的 Activin/Nodal 发信号的那老鼠 ES 并且发现调停 Smad2 的 Activin/Nodal 发信号为自强维护是非必需的，但是向 mesendoderm 系为合适的区别被要求。到进内在的机制的获得卓见，联系 Smad2 的基因被染色体宽的染色质 immunoprecipitation 薄片分析识别。结果证明在 Smad2 绑定和 Activin/Nodal 发信号调整之间有 transcriptional 关联，并且发展相关的基因在 Smad2 固定的目标之中被充实。我们进一步在 Activin/Nodal-Smad2 小径下游地行动的老鼠 ES 房间的 mesendoderm 区别作为一个关键播放器识别了 Tapbp。一起拿，我们的调查结果建议发信号的调停 Smad2 的 Activin/Nodal 通过相应发展管理者表示的直接调整安排鼠标 ES 房间的 mesendoderm 系承诺。

Skeletal myogenesis by human embryonic stem cells

我们检验了在一个 xeno 移植动物的细胞建模的人的胚胎的茎(hES ) 的 myogenic 潜力。这里，我们证明区分开来与 hES 房间的先锋能在成年环境经历 myogenesis 并且在 myogenic 系产生房间类型的一个范围。这研究提供 hES 房间能在 vivo 改革肌肉和卫星房间并且是为对待肌肉的另一种有希望的房间类型的直接证据除了另外的 myogenic 房间类型的退化混乱。房间研究(2006 ) 16：713-722。做 i：10.1038/sj .cr.7310080；出版联机 2006 年 6 月 20 日。

Embryonic stem cells develop into hepatocytes after intrasplenic transplantation in CCl_4-treated mice

AIM: To transplant undifferentiated embryonic stem (ES) cells into the spleens of carbon tetrachloride (CCl4)-treated mice to determine their ability to differentiate into hepatocytes in the liver. METHODS: CCl4, 0.5 mL/kg body weight, was injected into the peritoneum of C57BL/6 mice twice a week for 5 wk. In group 1 (n = 12), 1 x 105 undifferentiated ES cells (0.1 mL of 1 x 106/mL solution), genetically labeled with GFP, were transplanted into the spleens 1 d after the second injection. Group 2 mice (n = 12) were injected with 0.2 mL of saline twice a week, instead of CCl4, and the same amount of ES cells was transplanted into the spleens. Group 3 mice (n = 6) were treated with CCl4 and injected with 0.1 mL of saline into the spleen, instead of ES cells. Histochemical analyses of the livers were performed on post-transplantation d (PD) 10, 20, and 30. RESULTS: Considerable numbers of GFP-immunopositive cells were found in the periportal regions in group 1 mice (CCl4-treated) on PD 10, however, not in those untreated with CCl4 (group 2). The GFP-positive cells were also immunopositive for albumin (ALB), alpha-1 antitrypsin, cytokeratin 18, and hepatocyte nuclear factor 4 alpha on PD 20. Interestingly, most of the GFP-positive cells were immunopositive for DLK, a hepatoblast marker, on PD 10. Although very few ES-derived cells were demonstrated immunohistologically in the livers of group 1 mice on PD 30, improvements in liver fibrosis were observed. Unexpectedly, liver tumor formation was not observed in any of the mice that received ES cell transplantationduring the experimental period. CONCLUSION: Undifferentiated ES cells developed into hepatocyte-like cells with appropriate integration into tissue, without uncontrolled cell growth.

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.