Forced expression of theOct-4 gene influences differentiation of embryonic stem cells

By transfecting an Oct-4 expression plasmid into embryonic stem cells (ES cells), the ES-O cell line was constructed, which sustained the expression of Oct-4 gene when induced by retinoic acid. Forced expression of Oct-4 gene could not sustain the stem property of ES-O cells without the differentiation inhibiting factor LIF, but if LIF exists, forced expression of Oct-4 gene could enhance the ability to sustain the undifferentiation state and inhibit cell differentiation induced by retinoic acid. It was indicated that Oct-4 must cooperate with LIF to sustain the undifferentiation state of ES cells. During the cell differentiation, ES-O cells tend to differentiate into neural cells, suggesting that forced expression of Oct-4 gene may be in relation with the differentiation of neuroderm.     

胚胎干细胞向心肌细胞分化研究进展(综述)

胚胎干细胞(embryonic stem cells,ES)在体外分化培养条件下可以分化出各种组织细胞，其中包括心肌细胞。ES细胞在体外向心肌细胞分化与体内完整胚胎心肌发育过程相符合。该细胞在体外分化过程中顺序表达心肌细胞特有结构蛋白和离子通道，如肌球蛋白轻链和重链、特异性肌动蛋白、电压依赖性Ca^2 通道、K^ 通道等。ES细胞分化来源的心肌细胞具有体内心肌细胞的生理学特点，如产生的动作电位、表现自发性收缩等。因此，ES细胞是研究心肌细胞发育分化机制及鉴定其关键基因的理想模型。     

Properties and applications of embryonic stem cells

Mouse embryonic stem (ES) cells are pluripotent cells derived from the early embryo and can be propagated stably in undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in the embryonic and adult body in vivo, and can be induced to differentiate into many cell types under appropriate culture conditions in vitro. Using these properties, people have set up various differentiated systems of many cell types and tissues in vitro. Through analysis of these systems, one can identify novel bioactive factors and reveal mechanisms of cell differentiation and organogenesis. ES cell-derived differentiated cells can also be applied to cell transplantation therapy. In addition, we summarized the features and potential applications of human ES cells.     

Differentiation of human embryonic stem cells along a hepatocyte lineage and its application in liver regeneration

Hepatocyte transplantation and bioarUficial liver （BAL） as alternatives to liver transplantation offer the possibility of effective treatment for many inherited and acquired hepatic disorders. Unfortunately, the limited availability of donated livers and the variability of their derived hepatocytes make it difficult to obtain enough viable human hepatocytes for the hepatocyte-based therapies. Embryonic stem cells （ESCs）, which could be isolated directly from the blastocyst inner cell mass, have permanent self-renewal capability and developmental pluripotency and therefore might be an ideal cell source in the treatment of hepatic discords. However, differentiation of hESCs into hepatocytes with significant numbers remains a challenge. This review updates our current understanding of differentiation of ESCs into hepatic lineage cells, their future therapeutic uses and problems in liver regeneration.     

Induction of embryonic stem cells to hematopoietic cells in vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright's staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34+ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34-CD38+. Wright's staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Induction of embryonic stem cells to hematopoietic cellsin vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright’s staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34⁺ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34^－CD38⁺. Wright’s staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Effect of kinase-negativeEGFR gene on differentiation of embryonic germ cell line EG4

EG4 cells derived from primordial germ cells (PGCs) of 10.5 d post coitum 129/svJ mouse embryos can be used as a model system for in vitro differentiation study due to their pluripotential development ability. EG4 cell lines with stable expression of kinase-negative EGFR cDNA, designated EG4-EGFR^d, were generated by gene transfection. We found that: (ⅰ) EG4-EGFR^d cells share the similar morphology and growing character with wildtype cells that can maintain undifferentiated state in long term culture. (ⅱ) Treatment of EG4 cells with RA resulted in differentiation of adipocyte, while in mutant clones of EG4-EGFR^d, adipocytes were sparse or absent under the same condition, indicating the role of EGFR expressed during adipocyte development. (ⅲ) Histological analysis showed that predominant tissues in teratocarcinomas derived from EG4-EGFR^d cells and wildtype cells are different. A large amount of undifferentiated cells was present in those coming from mutant cell clones. In addition some cardiac and skeletal muscles are prominently differentiated cell types. EG4 wildtype cells produced multiple differentiated cell types of three primary germ layers such as cartilage, epithelia and neural tube. These studies suggested that EGFR-dependent differentiation was inhibited in kinase-negative EG4 cells.     

昆明鼠胚胎干细胞的分离培养与鉴定

目的：从昆明系小鼠的早期胚胎分离和培养胚胎干细胞(ES细胞)．方法：收集小鼠3．5d胚龄的囊胚，将其培养在小鼠胚胎成纤维细胞饲养层上，5—6d后取隆起生长的内细胞团块分离后再培养，观察集落的生长情况并通过碱性磷酸酶染色、原位杂交、细胞核型分析等对细胞集落进行鉴定．结果：KS细胞集落性生长，符合小鼠胚胎干细胞的一系列特性．结论：昆明系小鼠囊胚在胚胎成纤维细胞饲养层上可以发育成ES细胞，并能进行传代培养．     

胚胎干细胞及其在人类疾病治疗中的应用

胚胎干细胞是一类多能性干细胞,近年来已成为生命科学研究领域的热点之一．尤其在人类疾病治疗方面有着诱人的应用前景．本文主要介绍了胚胎干细胞在几种疑难疾病治疗上的应用及其前景,胚胎干细胞与异种器官移植。以及目前存在的一些问题．     

成纤维细胞和ES细胞在小鼠和兔去核卵母细胞内发育

以昆明白小鼠成纤维细胞和胚胎干（ES）细胞作为供核细胞,以昆明白小鼠和日本大耳白兔的MⅡ期去核卵母细胞作为受体,采用核移植方法,构楚了克隆胚胎．在同种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率明显低于以成纤维细胞为供核细胞的克隆胚胎卵裂率（24．4％相对于56．9％,P〈0．05）,1．8％的ES细胞克隆胚胎发育到囊胚阶段,而成纤维细胞克隆胚胎没能发育到囊胚阶段;在异种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率（89．6％）和囊胚发育率（18．8％）明显高于以成纤维细胞为供核细胞的克隆胚胎卵裂率（54．2％）和囊胚发育率（4．2％）．     

Maintenance of human embryonic stem cells on gelatin

Matrigel is routinely used as a coating material in the feeder-free culture system of human embryonic stem cells （hESCs）. However, matrigel is costive and inconvenient to use. In this study, the possibility of using gelatin as an alternative coating material was investigated. The results showed that, after trypsinization, hESCs were maintained undifferentiated on gelatin. These hESCs expressed pluripotent markers, formed teratoma and maintained a normal karyotype. As measured at passage 10, the hESCs expressed a high level of Oct4 on both gelatin and Matrigeh hESCs growing on gelatin formed AP-positive colonies in similar size and number to those growing on Matrigel （P〉 0.05）. Moreover, hESCs growing on gelatin contained a comparable percentage of SSEA-4-positive cells to those growing on Matrigel （95.1% vs.94.3%, P〉 0.05）. H-1 hESCs were maintained undifferentiated on gelatin for 20 passages and remained the stable normal karyotype. This gelatin-based culture protocol may allow us to propagate hESCs in large scale, with less cost.     

小鼠胚胎干细胞对病毒性心肌炎的影响

目的使用小鼠验证这样一个假设:外界病毒浸入诱发心肌炎时,机体的干细胞将进入心脏提高心肌的抗病毒能力。方法雄性BALB/c小鼠分为三组:小鼠胚胎干细胞对照组(ES),心肌炎病毒组(EM CV)及EM-CV加ES治疗组。通过尾静脉注射,令小鼠立即感染病毒。小鼠死亡率,炎性细胞浸润及心肌坏死等为观察指征。干细胞的游走及分化等通过免疫荧光法来验证。结果给予干细胞后的小鼠的存活率明显高于生理盐水对照组,炎性细胞侵润及心肌坏死亦明显低于生理盐水对照组。免疫荧光法表明,干细胞进入心肌并分化成新的心肌细胞。结论干细胞能明显提高心肌炎小鼠的存活率,减少心肌组织的坏死。同时,亦证明当心脏遭受病毒的侵入后,干细胞通过某种机理修复或再生心肌细胞,从而提高组织的抗病毒能力。     

Induction-dependent neural marker expression and electrophysiological characteristics of bone marrow mesenchymal stem cells that naturally express high levels of nestin

Under certain experimental conditions, bone marrow mesenchymal stem cells (MSCs) express neuronal phenotypes and neuronal markers, which suggests that they could be used to treat various neurological diseases. In the present study, MSCs were isolated from adult rat bone marrow, cultivated, and evaluated for neurotrophin expression profiles, as well as the potential to differentiate into functional neuronal-like cells in vitro. MSCs from passage 5 were pre-induced with DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 10 ng/mL bFGF (fibroblast growth factor-2). Subsequently, a chemical inductor containing Dimethyl Sulphoxide (DMSO), Butylated Hydroxyanisole (BHA) and forskolin were used to induce neural expression of MSCs. Expression patterns of nestin, NF-200, and GFAP at time points before and after induction were detected by immunofluorescence. Nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF) expressions in MSCs were evaluated by RT-PCR. The whole-cell patch clamp technique was utilized to elucidate the electrical behavior of MSC before and after 24-h differentiation induction. Immunofluorescence analysis revealed that MSCs expressed nestin (57.1% ± 6.9%), but not NF-200 or GFAP. Following neural induction, the cells exhibited a neuronal-like appearance. Nestin and NF-200 expression was positive in the neuronal-like cells, but GFAP expression was negative. After 6-, 12- and 24-h induction, the ratio of nestin-positive cells was 96.5% ± 1.9%, 88.1% ± 5.4%, and 33.5% ± 5.4%. NF-200 positive cells were 90.1% ± 2.9%, 97.5% ± 1.3%, and 98.1% ± 1.6%, respectively. However, prior to induction, MSCs already expressed NGF and BDNF. With a stimulus impulse of 40 mV, the density of the transient outward K current was (9.95 ± 4.85) pA/pF (n = 9) and (328.50 ± 30.62) pA/pF (n = 9) before and after induction, and the density of transient calcium ion currents was (−0.059 ± 0.027) pA/pF (n = 7) and (−6.66 ± 0.50) pA/pF (n = 7), respectively. Transient outward potassium currents and calcium ions currents gradually increased following induction. In addition, MSCs isolated from bone marrow exhibited characteristics of neuronal progenitor cells and expressed neurotrophins. These cells exhibited the capacity to differentiate into functional neuronal-like cells in vitro. These results suggested that MSCs express high levels of nestin and could be utilized for therapeutic strategies to treat nervous system diseases.     

Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application ,we need to replace mouse embryonic fibroblasts with human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state,We Success-fully use human fibroblasts derved from aborted fetus and adult prepuces as feeder layer to maintain human embryonic stem cells growth ,During the passage and growth on this feeder layer,the human embryonic stem cells can keep their undifferentiated state.     

Advances in the study on induced pluripotent stem cells

Recently, the study on ＂induced pluripotent stem cells＂ （iPS cells） has made a great breakthrough, and it is considered as a new milestone in the history of life science. This progress has updated our traditional concepts about pluripotency control, and provided people with a brand-new strategy for somatic cell nuclear reprogramming. In virtue of its availability and stability, this method holds great potential in both biological and clinical research. In order to introduce this rising field of study, this paper starts with an overview of the development of iPS cell establishment, describes the key steps in generating iPS cells, elaborates several relevant scientific issues, and evaluates its current restrictions and promises in future research.     

哺乳动物胚胎干细胞的特性及利用

哺乳动物胚胎干细胞（ES细胞）是由动物早期胚胎发育的内细胞团（ICM）或原始生殖细胞（PGC）分离得到的。人们利用ES细胞所具有的全能性、体外分化以及稳定的遗传性能等特点，展示了ES细胞在建立哺乳动物的早期胚胎体外分化模型、转基因动物模型、器官和组织的修复和移植治疗、克隆动物的生产、发育生物学的研究等方面广阔的应用前景。但是，由于哺乳动物错综复杂的基因调控和环境因素的影响，对于胚胎干细胞的研究还存在诸多问题，还需作更深入细致的研究。     

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer （SCNT）, holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells （hESCs） have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmis- sion, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC （ntESC） lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results in- dicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.     

人胚胎脑神经干细胞分离纯化及体外培养的研究

为了探讨人胚神经干细胞体外培养条件下的生物学特性,为其应用于临床治疗奠定基础.取胎龄16周的人流产胚胎,胰酶消化结合机械法分离成单细胞悬液,以2×106个细胞/mL接种到含hEGF和h-bFGF的DMEM/F12、N2培养基进行体外培养;观察细胞生长情况,用10% FBS诱导神经干细胞球分化,免疫细胞化学鉴定. 结果显示从人胚大脑分离出的细胞经悬浮培养可以形成细胞球,表达Nestin蛋白.经诱导分化后具有表达神经元,神经胶质细胞的特异性抗原. 说明人胚神经干细胞在体外可以稳定生长,并能分化成为神经原及胶质细胞.     

微重力环境下Notch信号对神经干细胞分化的影响

基于微重力条件下神经干细胞的分化情况,结合Notch信号通路对神经干细胞分化的影响,论述模拟微重力对神经干细胞分化产生影响的可能分子机制。