诱导多能干细胞体外向生精细胞自发分化潜能的研究

目的:探讨诱导多能干细胞(iPS)体外培养自发分化过程中生精细胞相关基因的表达,评估iPS体外向生精细胞自发分化的潜能。方法:经类胚体(EB)形成,体外诱导iPS向生精细胞分化,实时定量PCR和PCR检测生精细胞相关基因的表达。结果:实时定量PCR和PCR结果显示iPS经EB形成诱导分化后生精细胞不同时期的相关基因均有不同程度表达。iPS体外培养自发分化后生精细胞相关基因出现4种时间表达特征:Oct4基因表达量呈波浪状上升;Dppa3和Stra8基因表达量随诱导时间延长而下降;Dazl基因表达量呈波浪状下降;减数分裂前期基因Tex14、Msy2,减数分裂期基因Scp1、Scp3以及单倍体基因Akap3随着诱导时间延长先表达增加,而后表达下降。结论:iPS在经EB自发分化过程中表达生精细胞不同时期的相关基因,并且表达雄性配子单倍体基因,具有向雄性配子的分化潜能。     

以PECAM-1为标志去除残留未分化胚胎干细胞的研究

目的以血小板内皮细胞黏附分子-1(Platelet endothelial cell adhesion molecule-1,PECAM-1)为标志,去除小鼠胚胎干细胞(Embryonic stem cells,ESCs)中残留未分化的细胞,以去除其致瘤性,为ESCs在研究中的安全应用提供思路。方法将小鼠R1胚胎干细胞株在撤去白血病抑制因子的培养基中悬浮培养6 d,体外自发分化形成类胚体,消化打散后,以PECAM-1为标志进行磁珠分选,得到阳性与阴性细胞群体,分别以2×106个/点注射入裸鼠背部皮下,6~8周后观察畸胎瘤形成情况,组织学分析瘤体构成。结果裸鼠背部成瘤结果显示,PECAM-1+细胞群注射8个点中7个成瘤,成瘤率87.5%;而PECAM-1-细胞群注射8个点中1个成瘤,成瘤率12.5%。PECAM-1+细胞群与PECAM-1-细胞群成瘤率具有统计学差异(P=0.01)。结论应用PECAM-1可去除体外分化过程中的残留未分化ESCs,并去除其致瘤性。     

microRNAs对调控胚胎干细胞自我更新与分化的影响

胚胎干细胞（embryonic stem cell,ESCs）是指存在于胚泡内细胞群中、具有高度自我更新和多向分化潜能的细胞。ESCs可以在体外无限扩增并保持未分化状态,具有分化为胚胎或成体的各种细胞类型的潜能。因此,研究ESCs增殖与分化机制,可以更好地了解其生物学特性,对实现ESCs的定向分化和组织工程产品的研制具有推动作用。尽管目前ESCs自我更新与分化过程中的分子机制是研究热点,但距离全面厘清此问题仍相去甚远。     

去分化关节软骨细胞生物反应器培养反分化的实验研究

目的 观察体外经传代培养去分化的成人关节软骨细胞，在生物反应器培养后生物学性状的变化，探索去分化软骨细胞反分化的手段，为软骨细胞移植修复关节软骨缺损建立合适的体外培养方法。方法 无菌条件下取成人关节软骨组织，Ⅱ型胶原酶消化法（0．2％，37C，3h）分离软骨细胞，分成两组：一组常规单层传代培养，另一组添加重组人的生长因子（1ng／ml转化生长因子β1＋5ng／ml成纤维细胞生长因子2）体外培养传代大量扩增后，无微载体生物反应器内培养3周。血小板计数器行细胞计数，计算各代细胞倍增时间；细胞爬片和石蜡、冰冻切片进行HE、蕃红O、阿利新蓝染色，Ⅰ、Ⅱ型胶原和aggrecan免疫组织化学检测，观察细胞表型变化。结果 成人关节软骨细胞体外培养3代后迅速去分化，增殖缓慢。添加生长因子培养细胞去分化速度减缓；传10代，细胞扩增2000倍以上，部分去分化，但细胞扩增增殖能力仍很强；传20代软骨细胞表型基本丢失，但仍有增殖能力；置于生物反应器继续培养3周，细胞番红O染色强阳性、aggrecan和Ⅱ型胶原阳性，Ⅰ型胶原阴性，表型恢复良好。结论 软骨细胞在体外大量扩增后，在生物反应器培养，可恢复其表型，可望用于在体外培养时去分化软骨细胞的再分化。     

牙髓干细胞研究进展

干细胞是一类具有自我更新、高度增生能力和多相分化潜能的细胞,能够产生高度分化的功能细胞,包括胚胎干细胞和成体干细胞。目前,已在体外成功地扩增出人体多种组织的成体干细胞,自从2000年Gronthos等[1]发现人牙髓组织中存在成体干细胞即牙髓干细胞（dental pulp stemcells,DPSCs）后,近十年来国内外学者对牙髓干细胞的培养、生物学特性、细胞表型、分化能力及重组为诱导性多潜能干细胞（induced pluripotent stem cells,iPSCs）等方面已进行了大量研究,本文就牙髓干细胞的研究现状综述如下。     

人牙髓干细胞的高效扩增及其对分化潜能的影响

目的检测不同接种密度对人牙髓干细胞（Dental pulp stem cells，DPSCs）增殖能力和分化潜能的影响．建立其高效扩增方法。方法不同密度接种培养DPSCs，计算细胞产量、倍增次数．观察细胞形态、检查克隆形成率和钙结节形成能力。结果低密度培养的乳牙牙髓干细胞（Stem cells from human exfoliated deciduous teeth，SHED）始终保持较高增殖、克隆形成率；而低密度DPSCs只在前3代保持与常规密度相似的增殖、克隆形成效率，3代以后其增殖和分化能力明显下降。低密度培养条件下DPSCs的矿化能力与常规密度的没有明显差别。结论1．5～3cells／cm^2低密度接种培养DPSCs有利于细胞快速扩增，扩增后的细胞保持较高的增殖和分化潜能。SHED的增殖能力、克隆形成效率和钙结节形成能力均优于DPSCs。     

非病毒载体诱导多能干细胞的研究进展

诱导多能干细胞（induced pluripotent stem cells,iPS细胞）是通过外源导入与多能性相关的转录因子来诱导体细胞发生重编程从而获得的一类具有多向分化潜能的细胞。iPS细胞在疾病的模型建立与机制研究、细胞治疗、药物的发现与评价等方面有着巨大的潜在应用价值。     

诱导人孤雌胚胎干细胞向类间充质干细胞分化的实验研究

目的探索人孤雌胚胎干细胞在体外向类间充质干细胞诱导分化的方法 ,并鉴定所得细胞的生物学特性。方法人孤雌胚胎干细胞在无血清条件下悬浮培养,形成拟胚体,10d后在含血清条件下使拟胚体贴壁生长,7d后胰酶消化,所得细胞在含血清的培养液中传代、扩增。观察传代、扩增后细胞的形态学变化;用免疫荧光染色和流式细胞技术进行细胞表型分析;取第9代细胞进行成脂、成骨和成软骨诱导,9～28d后行特殊染色及RT-PCR分析。结果人孤雌胚胎干细胞在诱导分化后,形态与骨髓间充质干细胞相似,多次扩增传代后仍保持细胞形态和扩增能力。免疫荧光染色发现,细胞表达中胚层标志波形蛋白(Vimentin)。流式细胞分析显示,细胞表达CD29、CD105、CD166、CD44等间充质干细胞表面标志。特殊染色及RT-PCR分析显示:成骨诱导后,细胞碱性磷酸酶和茜素红染色阳性,碱性磷酸酶和Cbfa-1表达增加;成软骨诱导后,细胞Ⅱ型胶原染色阳性,Ⅱ型胶原和软骨寡聚基质蛋白(COMP)表达增强;成脂诱导后,细胞油红染色阴性,脂蛋白酶和Leptin无表达。结论人孤雌胚胎干细胞可以诱导、分化为间充质干细胞,并具有成骨、成软骨分化潜能。     

间充质干细胞及创面修复的研究进展

干细胞是具有自我更新能力,且在适宜微环境下具有多分化潜能的原始细胞。近年研究发现,机体内除胚胎干细胞(embryonic stem cell,ES)外还存在自我更新和分化能力的成体干细胞,其中间充质干细胞(mesenchymal stem cells,MSCs)是目前备受关注的一类具有多向分化潜能的成体干细胞。MSCs可在体外分化培养扩增,遗传背景稳定,可分化为成骨细胞、成软骨细胞、脂肪细胞、神经细胞、肌肉细胞等     

成年山羊皮肤干细胞体外克隆与诱导分化

目的 探索山羊皮肤干细胞的分离方法与培养体系，为进一步研究皮肤干细胞增殖分化的分子机制订下基础。方法 从成年关中奶山羊耳尖获取皮肤，用组织块培养法分离皮肤干细胞，碱性磷酸酶(ALP)染色及体外分化实验鉴定，用条件培养基与细胞因子相结合行皮肤干细胞的体外诱导分化。结果 山羊耳尖皮肤分离得到类干细胞集落并传5代，细胞集落具有典型鸟巢状结构，ALP染色阳性，体外分化能形成类胚体。在细胞因子(IGF—1)及条件培养基的诱导下，所分离获取的皮肤干细胞能分化形成类毛囊，类星形胶质细胞及类成骨细胞。结论 用组织块培养法可以获得多能性皮肤干细胞；在体外培养条件下不仅能进行定向分化，还能进行横向分化。     

Teratoma formation and hepatocyte differentiation in mouse liver transplanted with mouse embryonic stem cell-derived embryoid bodies

We previously reported that mouse embryonic stem (ES) cells are capable of differentiating into hepatocytes in cultured embryoid bodies (EBs) and that hepatocytes generate in the recipient liver injected with cultured day-9 EB cells via spleen without the formation of a teratoma. Because ES cells frequently form teratomas in recipient mice, we investigated incidence of teratoma formation when day-9 EBs derived from ES cells were transplanted directly into the subcapsule of mouse liver. In contrast to injection of day-9 EB cells through the portal vein via the spleen, direct subcapsular injection of cultured day-9 EB cells into liver, and even of cultured day-15 EBs, resulted in an high incidence of teratoma in the liver. In teratomas of livers injected directly with day-15 EBs, hepatocytes were detected singly and in clusters. These results imply that undifferentiated cells capable of developing into teratomas exist in cultured EBs, and even in cultured day-15 EBs containing differentiated hepatocytes.     

Generation of induced pluripotent stem cells from human kidney mesangial cells

Glomerular injury and podocyte loss leads to secondary tubulointerstitial damage and the development of fibrosis. The possibility of genetically reprogramming adult cells, termed induced pluripotent stem cells (iPS), may pave the way for patient-specific stem-cell-based therapies. Here, we reprogrammed normal human mesangial cells to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4 and c-Myc). The kidney iPS (kiPS) cells resembled human embryonic stem-cell-like colonies in morphology and gene expression: They were alkaline phosphatase-positive; expressed OCT3/4, TRA-1 to 60 and TRA-1 to 81 proteins; and showed downregulation of mesangial cell markers. Quantitative (qPCR) showed that kiPS cells expressed genes analogous to embryonic stem cells and exhibited silencing of the retroviral transgenes by the fourth passage of differentiation. Furthermore, kiPS cells formed embryoid bodies and expressed markers of all three germ layers. The injection of undifferentiated kiPS colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. These results suggest that reprogrammed kidney induced pluripotent stem cells may aid the study of genetic kidney diseases and lead to the development of novel therapies.     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

Evidence for hepatocyte differentiation from embryonic stem cells in vitro

We confirmed hepatocyte differentiation from embryonic stem (ES) cells in vitro. RT-PCR analysis revealed that a broad range of hepatic gene expression was observed in ES cells differentiated through formation of embryoid bodies (EBs) and its attachment culture. Quantitative PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of ES cells. The presence of albumin-producing cells in the peripheral region of attached EBs was confirmed by immunocytochemical analysis. Future experiments will reveal the molecules that induce hepatocyte differentiation from ES cells in vitro. This research will provide systems for the investigation of mechanisms in liver development and establish a method of ES cell-based therapy for liver diseases.     

Differentiation of murine embryonic stem cells in skeletal muscles of mice

Possible myogenic differentiation of SSEA-1- and OCT-4-positive murine embryonic stem cells (ESCs) and embryoid bodies (EBs) was studied in vitro and in vivo. In vitro, ESC- or EB-derived ESCs (EBs/ESCs) showed only traces of Pax 3 and 7 expression by immunocytochemistry and Pax 3 expression by immunoblot. By RT-PCR, myogenic determinant molecules (myf5, myoD, and myogenin) were expressed by EBs/ESCs but not by ESCs. However, in such cultures, very rare contracting myotubes were still present. Suspensions of LacZ-labeled ESCs or EBs were injected into anterior tibialis muscles (ATM) of different cohorts of mice for the study of their survival and possible myogenic differentiation. The different cohorts of mice included isogenic adult 129/Sv, nonisogenic CD1 and mdx, as well as mdx immunosuppressed with 2.5 mg/kg daily injections of tacrolimus. Ten to 90 days postinjections, the injected ATM of nonisogenic mice did not contain cells positive for LacZ, SSEA-1, OCT-4, or embryonic myosin heavy chain. The ATM of intact mdx mice contained very rare examples of muscle fibers positive for dystrophin and/or embryonic myosin heavy chain. In the ATM of the isogenic normal and the immunosuppressed mdx mice, as expected, large teratomas developed containing the usual diverse cell types. In some teratomas of immunosuppressed mdx mice, small pockets of muscle fibers expressed dystrophin and myosin heavy chain. Our studies indicated that in muscles of animals nonisogenic with the used ESCs, only very rare ESCs survived with myogenic differentiation. These studies also indicated that ESCs will not undergo significant, selective, and preferential myogenic differentiation in vitro or in vivo in any of the models studied. It is probable that this strain of murine ESC requires some experimentally induced alteration of its gene expression profile to secure significant myogenicity and suppress tumorogenicity.     

Differentiation of embryonic stem cells after transplantation into peritoneal cavity of irradiated mice and expression of specific germ cell genes in pluripotent cells

Permanent embryonic stem cell lines (ES cells) are considered as one of the most promising cellular sources for regenerative medicine. ES cells have a high proliferative potency and ability to differentiate into all kinds of somatic and germ cells. However, transplantation of undifferentiated ES cells into adult recipient tissue results in the formation of teratomas. To understand the mechanisms underlying self-renewal and determination of pluripotent cells, we investigated differentiation potencies of undifferentiated ES cells and differentiating embryoid bodies (EB). ES cells and EBs growing on acetate-cellulose membranes were transplanted into the peritoneal cavity of irradiated mice. Behavior and differentiation of transplanted cells were studied within 1, 2, 3, and 6 weeks after transplantation. No differences in the cell composition were found in the teratomas formed by ES cells and differentiating EBs. The pattern of expression of the genes specific for pluripotent and germ cells was studied in all types of experimental teratomas. The expression of oct4, stella, fragilis was detected in the teratomas, but nanog was not expressed. We conclude that pluripotent cells are retained in the experimental teratomas formed after transplantation of ES cells and EBs but the pattern of expression of the studied genes underwent changes.     

小鼠iPS细胞具备诱导性原始生殖细胞分化潜能的研究

目的:探讨小鼠诱导性多能干细胞IP14D-1是否具备诱导性原始生殖细胞(induced primordial germcells,iPGCs)分化潜能,以及特异基因表达变化及可能机制。方法:未分化IP14D-1培养扩增,分化形成诱导性拟胚体(induced embryoid bodies,iEBs)。RT-PCR和免疫荧光分别检测4、7、9 d的iEBs中Lin28、Blimp1、Stra8和Mvh的表达变化和蛋白定位情况。结果:未分化IP14D-1同小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)相同,Lin28表达较弱,Blimp1表达相对较强,Mvh和Stra8也在这两种细胞及其相应iEBs和EBs中表达,但均无明显差别。IP14D-1分化形成的iEBs从4 d生长到7 d时,Lin28表达逐渐增强,到9 d时表现为下降,Blimp1表达则随着iEB生长时间延长而逐渐降低。结论:建立了IP14D-1和相应拟胚体(iEBs)的完善、稳定的培养及分化体系;未分化IP14D-1与mESCs在Lin28、Blimp1、Mvh和Stra8表达方面无明显差别;iEB和EBs的Mvh和Stra8表达也无明显差别。IP14D-1及iEBs具有iPGCs分化潜能,且可能是7 d的iEBs内iPGCs分化数量较多,之后进入iPGCs分化的Lin28低表达时期。     

Transplantation of osteogenically differentiated mouse iPS cells for bone repair

Induced pluripotent stem (iPS) cells are a type of undifferentiated cell that can be obtained from differentiated cells and have the pluripotent potential to differentiate into the musculoskeletal system, the myocardium, vascular endothelial cells, neurons, and hepatocytes. We therefore cultured mouse iPS cells in a DMEM containing 15% FBS, 10(-7) M dexamethasone, 10 mM β-glycerophosphate, and 50 μg/ml ascorbic acid for 3 weeks, in order to induce bone differentiation, and studied the expression of the bone differentiation markers Runx2 and osteocalcin using RT-PCR in a time-dependent manner. Osteocalcin, a bone differentiation marker in bone formation, exhibited the highest expression in the third week. In addition, the deposition of calcium nodules was observed using Alizarin red S staining. iPS cells cultured for bone differentiation were transplanted into severe combined immunodeficiency (SCID) mice, and the osteogenic potential exhibited after 4 weeks was studied. When bone differentiation-induced iPS cells were transplanted into SCID mice, bone formation was confirmed in soft X-ray images and tissue specimens. However, teratoma formation was confirmed in 20% of the transplanted models. When mouse iPS cells were treated with irradiation of 2 Gray (Gy) prior to transplantation, teratoma formation was inhibited. When mouse iPS cells treated in a likewise manner were xenotransplanted into rats, bone formation was confirmed but teratoma formation was not observed. It is believed that irradiation before transplantation is an effective way to inhibit teratoma formation.