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

Endothelial cells (TEC_3 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 × 10~6 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 TEC_3 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.

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, PCR 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.

Applications of stem cells and bioprinting for potential treatment of diabetes

Currently, there does not exist a strategy that can reduce diabetes and scientists are working towards a cure and innovative approaches by employing stem cellbased therapies. On the other hand, bioprinting technology is a novel therapeutic approach that aims to replace the diseased or lost β-cells, insulin-secreting cells in the pancreas, which can potentially regenerate damaged organs such as the pancreas. Stem cells have the ability to differentiate into various cell lines including insulinproducing cells. However, there are still barriers that hamper the successful differentiation of stem cells into β-cells. In this review, we focus on the potential applications of stem cell research and bioprinting that may be targeted towards replacing the β-cells in the pancreas and may offer approaches towards treatment of diabetes. This review emphasizes on the applicability of employing both stem cells and other cells in 3 D bioprinting to generate substitutes for diseased β-cells and recover lost pancreatic functions. The article then proceeds to discuss the overall research done in the field of stem cell-based bioprinting and provides future directions for improving the same for potential applications in diabetic research.

Human parthenogenetic embryonic stem cells:one potential resource for cell therapy

Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem(iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies.However,the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells.Embryonic stem cells(ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy.Recent studies on human parthenogenetic embryonic stem cells(hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics,but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions.To generate various pluripotent stem cells,diverse reprogramming techniques and approaches will be developed and integrated.This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology,and ultimately benefit cell therapy and regenerative medicine.

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 transmission, 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 indicate 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.

Establishment of customized mouse stem cell lines by sequential nuclear transfer

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.

Stem cell therapy for idiopathic pulmonary fibrosis: How far are we from the bench to the bedside?

Idiopathic pulmonary fibrosis (IPF) is characterized by exuberant apoptosis and inadequate regeneration of lung parenchyma cells. Intratracheal alveolar type II epithelial cell instillation alleviates lung inflammation and fibrosis. Resident lung epithelial stem cells, as well as exogenous mesenchymal stem cells, are capable of differentiating into lung epithelial cells and repair the injured lung. It is thus supposed that, either engraftment of exogenous stem cells, or methods facilitating endogenous lung stem cell proliferation, are promising treatments for IPF, a devastating disease. Arrays of cellular and animal studies have shown the potential of stem cells in alleviating experimental lung fibrosis. Moreover, clinical trials have been launched to investigate the potentials of cell-based therapy in IPF patients. We intend to discuss the newest advances on stem cell therapy in pulmonary fibrosis, particularly the advantages, promises, and possible hurdles to pass from the successes in laboratory experiments to the eventual clinical applications.

巨噬细胞迁移抑制因子对干细胞的作用

背景:干细胞是一种可以自我更新并且具有分化潜能的细胞,干细胞治疗是一种很有前景的治疗方法。巨噬细胞迁移抑制因子几乎在所有哺乳动物细胞中都有表达,对许多生理过程至关重要。现有研究证明,巨噬细胞迁移抑制因子在多种干细胞上表达并且调节多种干细胞的增殖、分化和迁移。目的:描述巨噬细胞迁移抑制因子的基本特征并着重阐述巨噬细胞迁移抑制因子对于干细胞的作用及机制。方法:在PubMed和中国知网数据库中检索近10年文献,英文检索词为“macrophage migration inhibitory factor,cancer stem cell,embryonic stem cell,neural stem cell,mesenchymal stem cell,endothelial progenitor cell,stem cell therapy,tissue engineering”,中文检索词为“巨噬细胞移动抑制因子、肿瘤干细胞、胚胎干细胞、神经干细胞、间充质干细胞、内皮祖细胞、干细胞治疗、组织工程”,经过文题、摘要的筛选,排除相关性差及陈旧和重复的文献,对最终符合标准的85篇文献进行综述。结果与结论:(1)巨噬细胞迁移抑制因子蛋白由3个亚基构成,在细胞表面通过与受体CD74,CD44,CXCR2,CXCR4和CXCR7产生作用。巨噬细胞迁移抑制因子储存在细胞内,通过非经典途径分泌,多种因素能影响其表达以及分泌。(2)巨噬细胞迁移抑制因子通过不同的信号通路促进肿瘤干细胞的迁移、侵袭、转移、逃逸和致瘤性。(3)当前研究中,比较明确的是巨噬细胞迁移抑制因子促进小鼠胚胎干细胞的神经分化。(4)巨噬细胞迁移抑制因子通过多个信号通路促进神经干细胞的增殖和存活。(5)巨噬细胞迁移抑制因子作用于间充质干细胞能够抑制凋亡、促进存活、延缓衰老并增加间充质干细胞的旁分泌作用,除此之外,巨噬细胞迁移抑制因子也证明可以通过结合受体CD74抑制间充质干细胞的归巢。(6)多项研究证明巨噬细胞迁移抑制因子通过CXCR4受体激活下游信号通路促进内皮细胞的迁移以及血管重建。(7)巨噬细胞迁移抑制因子作用于肿瘤干细胞、胚胎干细胞、神经干细胞、间充质干细胞、内皮祖细胞,对它们的增殖、分化、迁移和凋亡等过程产生影响,但其中部分作用还存在争议,确切机制需要更进一步验证。

胚胎干细胞生物学特性及其应用前景

胚胎干细胞是来源于着床前的囊胚内细胞团或早期胎儿的原始生殖细胞的一类未分化的全能性多能性干细胞，具有无限增殖和全能分化的潜力。胚胎干细胞在发育生物学基础研究、动物胚胎工程研究生产和临床医学上具有诱人的应用前景。

体外定向诱导小鼠胚胎干细胞向内皮细胞分化的研究

目的探讨体外定向诱导胚胎干细胞（embryonic stemcell，ESC）向内皮细胞分化的条件，为组织工程血管提供种子细胞。方法取怀孕12．5d的昆明小白鼠1只，断颈处死，取其胚胎，培养制备小鼠胚胎成纤维细胞（mouse embryonic fibroblast，MEF）。常规复苏ESC后进行体外培养，采用悬滴-悬浮法制备拟胚体（embryoid body，EB）。将EB分两组：实验组加入含有3ng／ml转化生长因子β1、50ng／ml血管内皮细胞生长因子及1μmol／L激活素受体样激酶选择性抑制剂的EB培养基；对照组只加入EB培养基。倒置显微镜下观察细胞生长情况。采用RT—PCR及免疫组织化学染色检测诱导细胞vWF和CD34的表达，验证细胞性质。结果原代MEF生长迅速，第3天细胞融合达90％左右，细胞呈长梭形，有少量侧支，细胞核饱满，有2～3个核仁，细胞排列紧密。传至3～5代，细胞呈多角形，胞浆饱满，有3～4个核仁，细胞表面分泌较多细胞颗粒。ESC在饲养层上保持未分化状态，细胞团形态呈鸟巢样，边缘平整，单个细胞体积小，折光性强，核质比高，增长速度快。悬滴培养3d的EB肉眼可见，再经悬浮培养3d后，形成较大的透亮EB；EB贴壁后第2天，球体略摊开。实验组第4～7天，EB周围有许多圆形细胞产生，第10b14天，可见由大量圆形细胞组成的血管样结构自EB周围生长；对照组EB周围未见血管样结构生长。免疫组织化学染色见EB周围大量vWF染色阳性细胞，RT—PCR检测到vWF和CD34的表达。结论ESC在特定的条件下可以分化为内皮细胞，有可能为组织工程血管提供大量的种子细胞。

内皮素1调控P19细胞向心脏传导细胞分化的机制

背景:内皮素1作为一种来源于心内膜和动脉血管内皮的旁分泌信号,能够诱导心脏传导细胞的分化成熟。目的:探讨内皮素1在体外诱导P19细胞向心脏传导细胞分化的作用。方法:P19细胞分别与1%二甲基亚砜和100 nmol/L浓度内皮素1孵育,通过Western blot、免疫荧光和qR T-PCR鉴定早期心肌细胞特征性转录因子GATA4、MEF2C和心肌细胞分化标志物MHC-α、cTnT,以及重要的传导细胞转录因子Nkx2.5、Tbx5和分化标志物Cx40、ANP的变化。qRT-PCR鉴定内皮素1特异性受体阻断剂和阻断PI3K-AKT-mTOR信号通路对内皮素1诱导P19细胞分化的影响。结果与结论:(1)内皮素1能够上调P19细胞早期心肌转录因子、心肌结构基因和传导系统特征表型表达;(2)与二甲基亚砜相比,内皮素1能够确实诱导P19细胞向心脏传导细胞分化;(3)内皮素1主要通过ETA通路上调Nkx2.5和Cx40表达水平从而诱导P19细胞向传导细胞分化,ETB通路可能还参与了一条负反馈调节通路;(4)内皮素1能够通过PI3K-AKT-mT OR信号通路调控Nkx2.5表达进而影响传导细胞分化。

ES细胞源性表皮干细胞与胶原海绵构建组织工程皮肤

【目的】以胚胎干细胞(ES细胞)源性表皮干细胞为种子细胞与胶原海绵构建组织工程皮肤,探讨其对皮肤缺损修复的作用。【方法】胎鼠皮肤成纤维细胞与胶原海绵构建类真皮,植入小鼠全层皮肤缺损创面,以生物膜为载体,把羊膜诱导后带有核标记的ES细胞源性表皮干细胞覆盖在类真皮上,术后1～8周连续取材,HE染色,β1整合素、CK15、CK19、CK10和CEA免疫荧光双标和免疫组化观察。【结果】植入后3周,创面完全长合,较厚新生皮完全覆盖创面,基底层细胞增生,形成许多大小不一的细胞柱伸向真皮层,新生表皮中可见核标记的细胞呈β1整合素、CK15、CK19阳性,真皮中的管腔样结构呈核荧光和β1整合素、CEA免疫组化双标阳性,4～8周新生表皮基底层细胞呈CK19、CK10阳性,新生表皮下可见毛囊样、皮脂腺样结构。【结论】ES细胞源性表皮干细胞为种子细胞与胶原海绵构建的组织工程皮肤可以修复缺损皮肤,在其下真皮层有分化为毛囊样、皮脂腺样和汗腺样的结构,但是是否来源于ES细胞源性表皮干细胞仍需进一步证实。

IGF2基因印记丢失对胚胎干细胞向胰岛样细胞诱导分化的影响

背景:IGF2是一种重要的胚胎有丝分裂生长促进因子,在维持细胞正常生长过程中起关键作用,其基因表达以基因组印迹方式受表观遗传调控。当IGF2发生印迹丢失时,与个体的非正常发育以及肿瘤发生存在一些联系。目的:探讨IGF2基因发生印记丢失对小鼠胚胎干细胞定向诱导分化为胰岛样细胞的影响。方法:体外诱导2种小鼠胚胎干细胞(SF1-G和SF1-1)向胰岛样细胞分化;Real-Time PCR和细胞免疫荧光检测Insulin基因的表达;聚合酶链式反应-限制性内切酶片段长度多态性(PCR-RFLP)检测印迹基因IGF2在诱导分化前后细胞中的亲本表达;体外胰岛素释放实验检测诱导终末细胞的胰岛素释放水平。结果与结论:(1)IGF2基因印记正常(SF1-G)和印记丢失(SF1-1)的2种小鼠胚胎干细胞在诱导分化前后印迹状态没有发生改变;(2)在诱导的终末细胞中,尽管2种细胞的胰岛素释放水平差异无显著性意义,但是Insulin基因在SF1-1组中的表达水平要高于其在SF1-G组的表达水平;(3)IGF2基因印记丢失可能与诱导分化来源的终末细胞中Insulin基因的表达上调相关。

ES细胞源性表皮干细胞在皮肤缺损创面的分化

目的探讨ES细胞源性表皮干细胞移植入小鼠全层皮肤缺损后对创面的修复作用,为组织工程皮肤的研究提供新的思路。方法将羊膜诱导后的带有核荧光标记的ES细胞源性表皮干细胞以生物膜为载体,覆盖小鼠全层皮肤缺损创面。术后1～8周连续取材,HE染色观察,苏丹Ⅲ染色,β1整合素、CK15、CK19、CEA免疫组化荧光双标观察。结果术后2周,创面完全长合,较厚的新生皮完全覆盖创面,基底层细胞增生,形成细胞柱伸向真皮层,真皮层中可见管腔样结构,4周后新生表皮开始变薄,新生表皮下可见毛囊样、汗腺样、皮脂腺样等结构,皮脂腺样结构苏丹Ⅲ染色阳性。免疫双标结果显示,1～4周新生表皮基底层细胞呈β1整合素、CK19阳性,真皮层中带有核标记的管状或泡状结构呈CK15、CEA阳性。结论ES细胞源性表皮干细胞在小鼠全层皮肤缺损创面可分化为表皮样、汗腺样、毛囊样和皮脂腺样等结构的潜能。

核移植胚胎干细胞的研究及其应用前景

随着核移植技术和干细胞技术的逐渐成熟,目前已获得牛、小鼠核移植胚胎干细胞,以及人-兔异种间核移植胚胎干细胞,这些细胞在体外可分化成多种细胞形态. 已经进行的实验性动物克隆性治疗,显示了诱人的潜力,但人核移植胚胎干细胞研究还面临着许多问题,如建系效率低、卵母细胞来源有限以及伦理学和安全性问题等. 长远地看,随着克隆效率的提高,在道德与法律之间达成共识,核移植胚胎干细胞必将造福人类.

人胚胎干细胞的研究

来自着床前的囊胚和早期人胚胎的人胚胎干细胞是未分化的多能干细胞 ,具有无限增殖和分化的潜力 ,这种特性使之在基础研究和移植治疗中具有广泛的应用。尤其是胚胎干细胞可以产生任何类型的可供临床使用的细胞、组织和器官的潜力 ,将会带来一场医学革命。

人胚胎干细胞向肌腱细胞分化方法的专家共识

胚胎干细胞具有无限增殖能力，可能为组织工程提供肌腱细胞或肌腱祖细胞的来源。为规范人胚胎干细胞来源的肌腱细胞的分化方法，提高人肌腱细胞的纯度，保证分化方法的可重复性，使其更系统、规范和有效地应用于临床和生物安全性评价体系中，科技部国际合作专项有关专家制定了《人胚胎干细胞向肌腱细胞分化方法共识》。共识建议通过两步法将人胚胎干细胞诱导为肌腱细胞：首先利用不同材料表面将人胚胎干细胞诱导为成体间充质干细胞，然后采用高密度种植形成无支架组织工程肌腱，并利用静态或动态力学刺激在体内外环境下将成体间充质干细胞诱导成肌腱细胞。由此建立的体外组织工程肌腱可作为检测评价模型。对小分子化合物、医用材料和药物进行肌腱相关的毒理学系统分析和安全性评价。

组织工程研究中的胚胎干细胞问题

胚胎干细胞研究是组织工程基本科学问题中一个重要的组成部分。本文回顾了近年来胚胎干细胞领域的研究进程 ,尤其是人体胚胎干细胞研究的最新成果 ;提出了组织工程中“治疗性克隆”的战略目标。并且确立了未来人体胚胎干细胞研究解决组织工程种子细胞问题的具体步骤 ;指出了每项具体研究中所要解决的关键问题。

组织、器官重建的基础:干细胞

种子细胞是应用组织工程技术重建组织、器官的前提和基础,干细胞已成为最佳种子细胞来源。成体干细胞在动物及临床试验中的成功,证实了组织工程技术的可行性,展示了其广阔的发展前景。而同种异体干细胞的研究以及通用型胚胎干细胞研究的拓展,将为实现组织工程产业化奠定基础。

Y-27632促进人胚胎干细胞向类神经元细胞的分化

背景:Y-27632是Rho相关卷曲螺旋形成的蛋白激酶抑制剂,已被证实具有调节干细胞的自我更新、促进克隆形成和存活的作用。另外,其对神经细胞生长和发育也具有调节和保护作用。而如何提高胚胎干细胞向类神经元细胞分化是神经损伤修复和神经再生领域热点问题。目的:探讨Y-27632对人胚胎干细胞向类神经元细胞分化效率的作用。方法:取16代人胚胎干细胞复苏后,进行形态观察和染色鉴定,采用悬浮培养法制备类胚体,培养8 d后,进行分组贴壁培养:对照组细胞为常规Sato培养基诱导分化;Y-27632组分别在Sato培养基加入5,10,20,40μmol/L Y-27632。培养10 d后进行染色鉴定;10-18 d后,进行免疫荧光染色检测胚胎干细胞分化效率和类神经元细胞突起生长情况。结果与结论:(1)人胚胎干细胞共表达Oct4和SSEA-3干细胞特异蛋白;(2)悬浮分化培养8 d后,进一步贴壁培养10 d,可观察到具有神经形态,且表达Tuj-1的类神经元细胞;(3)经不同浓度的Y-27632诱导后,免疫荧光染色发现贴壁细胞数和Tuj-1阳性细胞数显著增加,提示胚胎干细胞向类神经元细胞分化速率加快,且在浓度为10μmol/L时最为明显;(4)分化培养18 d后,免疫荧光染色显示,与对照组相比,10μmol/L Y-27632组的Tuj-1阳性细胞数及突起数和突起长度显著增加;(5)结果说明,Y-27632不仅能够促进人胚胎干细胞向类神经元细胞分化,同时促进类神经元细胞突起的生长。