Alternative sources of pluripotency: science, ethics, and stem cells

Despite many advances in human embryonic stem cell (hESC) technology the ethical dilemma involving the destruction of a human embryo is one factor that has limited the development of hESC based clinical therapies. Two recent reports describing the production of pluripotent stem cells following the in vitro reprogramming of human somatic cells with certain defined factors illustrate one potential method of bypassing the ethical debate surrounding hESCs (Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007 Dec;318(5858):1917-1920; Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov;131(5): 861-872.). Other alternative methods include nuclear transfer, altered nuclear transfer, and parthenogenesis; each with its own set of advantages and disadvantages. This review discusses recent advances in these technologies with specific focus on the issues of embryo destruction, oocyte recovery, and the potential of each technology to produce large scale, patient specific cell transplantation therapies that would require little or no immunosuppression.     

Derivation of functional dopamine neurons from embryonic stem cells

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective degeneration of dopaminergic (DA) neurons in the substantia nigra of the midbrain. Pharmacological treatment of PD has been a prevailing strategy. However, it has some limitations because its effectiveness gradually decreases and side effects develop. As an alternative, cell transplantation therapy has been tried. Although transplantation of fetal ventral mesencephalic cells looks promising for the treatment of PD in some cases, ethical and technical problems in obtaining large numbers of human fetal brain tissues also lead to difficulty in its clinical application. Our recent studies showed that a high yield of DA neurons could be derived from embryonic stem (ES) cells and they efficiently induced behavioral recovery in a PD animal model. Here we summarize methods for generation of functional DA neurons from ES cells for application to PD models.     

Myogenic properties of human mesenchymal stem cells derived from three different sources

Mesenchymal stem cells (MSCs) of mammals have been isolated from many tissues and are characterized by their aptitude to differentiate into bone, cartilage, and fat. Differentiation into cells of other lineages like skeletal muscle, tendon/ligament, nervous tissue, and epithelium has been attained with MSCs derived from some tissues. Whether such abilities are shared by MSCs of all tissues is unknown. We therefore compared for three human donors the myogenic properties of MSCs from adipose tissue (AT), bone marrow (BM), and synovial membrane (SM). Our data show that human MSCs derived from the three tissues differ in phenotype, proliferation capacity, and differentiation potential. The division rate of AT-derived MSCs (AT-MSCs) was distinctly higher than that of MSCs from the other two tissue sources. In addition, clear donor-specific differences in the long-term maintenance of MSC proliferation ability were observed. Although similar in their in vitro fusogenic capacity with murine myoblasts, MSCs of the three sources contributed to a different extent to skeletal muscle regeneration in vivo. Transplanting human AT-, BM-, or SM-MSCs previously transduced with a lentiviral vector encoding β-galactosidase into cardiotoxin-damaged tibialis anterior muscles (TAMs) of immunodeficient mice revealed that at 30 days after treatment the frequency of hybrid myofibers was highest in the TAMs treated with AT-MSCs. Our finding of human-specific β-spectrin and dystrophin in hybrid myofibers containing human nuclei argues for myogenic programming of MSCs in regenerating murine skeletal muscle. For the further development of MSC-based treatments of myopathies, AT-MSCs appear to be the best choice in view of their efficient contribution to myoregeneration, their high ex vivo expansion potential, and because their harvesting is less demanding than that of BM- or SM-MSCs.     

Adult (somatic) stem cells and the musculoskeletal system

Stem cells offer a glimpse into a future that might unravel the promising ability of wide-ranging biological reconstructions.     

不同来源成体骨髓间充质干细胞调控免疫功能的比较研究

目的 比较霍奇金淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)患者与正常人骨髓间充质干细胞(MSC)的免疫调节能力.方法 获取正常人、HL和NHL患者的骨髓MSC,用低血清培养液进行培养.采用流式细胞仪检测骨髓MSC的免疫表型;应用酶联免疫吸附试验检测骨髓MSC培养上清液中转化生长因子β1(TGF-β1)的水平;采用Transwell培养体系检测骨髓MSC抑制T淋巴细胞增殖的能力;应用混合淋巴细胞反应检测骨髓MSC抑制异体T淋巴细胞增殖的能力.结果 正常人、HL和NHL患者骨髓间充质干细胞具有相似的细胞形态和免疫表型,均具有分泌TGF-β1的能力,均不表达HLA-DR和共刺激分子CD80、CD86和CD40.HL和NHL患者骨髓MSC具有抑制异体T淋巴细胞增殖的能力,这种抑制能力随着MSC细胞数量的增加而增强,并可以被抗TGF-β1抗体所逆转.体外诱导分化后的骨髓MSC仍然具有抑制异体T淋巴细胞增殖的能力.结论 HL和NHL患者骨髓MSC具有和正常成人骨髓MSC相同的免疫调控能力,这种抑制T淋巴细胞增殖的能力不随着其体外诱导分化而改变.     

Differentiation of green fluorescent protein-labeled embryonic stem cell-derived neural precursor cells into Thy-1-positive neurons and glia after transplantation into adult rat striatum

OBJECT: The aim of this investigation was to assess new information concerning the capacity of transplanted embryonic stem cell (ESC)-derived neuronal cells to migrate into host brain and to evaluate these cells as a possible source for cell replacement therapy in neurodegenerative disorders such as Parkinson's disease (PD). METHODS: The authors investigated the ability of ESC-derived neural precursor cells to migrate and differentiate in a host striatum by using a D3-derived ESC clone that was transfected stably with a chicken beta-actin cytomegalovirus enhancer-driven green fluorescent protein (GFP)-labeled construct. This procedure allowed easy monitoring of all transplanted cells because of the green fluorescent labeling of donor cells. This approach also afforded easy estimation of cell integration and simultaneous observation of the entire transplanted cell population in relation to immunocytochemically identified neuronal and glial differentiation. After selection of nestin-positive neural precursor cells in a synthetic medium, they were implanted into the striatum of male adult Wistar rats. Their integration was analyzed on morphological studies performed 3 days to 4 weeks posttransplantation. CONCLUSIONS: The investigators found that after transplantation, a subpopulation of GFP-labeled cells differentiated into various neural morphological types that were positive for the mouse-specific Thy-1 antigen, which is known be expressed on neurons, as well as being positive for the astroglial marker glial fibrillary acidic protein. Moreover, GFP-expressing cells that were negative for either of these markers remained close to the injection site, presumably representing other derivatives of the neural lineage. Together, these findings contribute to basic research regarding future transplantation strategies in neurodegenerative diseases such as PD.     

成体干细胞在骨组织工程中的应用

种子细胞的来源、分离纯化、规模化扩增和保持体内成骨活性等是制约骨组织工程研究与应用发展的重要瓶颈问题之一.干细胞研究的进展和突破给骨组织工程种子细胞的选择带来了新希望.该文在论述骨组织工程对种子细胞要求的基础之上,对成体干细胞与胚胎干细胞进行比较,并对研究较多的骨髓间充质干细胞、脂肪干细胞、外周血干细胞及新近提取的胎盘来源干细胞的生物学特性、成骨能力、临床应用及其在骨组织工程中的应用前景进行阐述与讨论,以最终实现根据临床实际需要而选择合适的种子细胞的目标.     

Human umbilical cord blood cells can be induced to express markers for neurons and glia

Rare cells are present in human umbilical cord blood that do not express the hematopoietic marker CD45 and in culture do not produce cells of hematopoietic lineage. These umbilical cord multipotent stem cells (UC-MC) behave as multilineage progenitor cells (stem cells) and can be expanded in tissue culture. Exposure to basic fibroblast growth factor (bFGF) and human epidermal growth factor (hEGF) for a minimum of 7 days in culture induces expression of neural and glial markers. Western immunoblots demonstrate expression of both beta-tubulin III and glial fibrillary acidic protein (GFAP). Immunocytochemistry of the cells showed intense labeling to both compounds on the intracellular cytoskeleton. The oligodendrocyte cell surface marker galactocerebroside (Gal-C) was present on most cells. Many cells show dual labeling, expressing both neuronal and glial markers.     

骨折大鼠血清对不同来源间充质干细胞1的趋化作用

目的 对比不同来源间充质干细胞(MSC)形态的差异,观察骨折大鼠血清对不同来源MSC的趋化作用.方法 将36只SD大鼠完全随机地分为骨折组(24只,其中12只培养外周血MSC,12只分离血清)和对照组(12只),从两组大鼠骨髓和外周血中分离培养MSC,比较生长速度和细胞形态.观察两组血清对MSC的趋化作用及不同来源MS...     

烧伤大鼠血清对不同来源间质干细胞的趋化作用

目的了解间质干细胞(MSC)的来源,观察烧伤大鼠血清对不同来源 MSC 的趋化作用。方法将72只大鼠采用完全随机法分成烧伤组(36只,制作背部30%TBSAⅢ度烧伤模型)、假伤组(36只,37℃模拟致伤)。从两组大鼠骨髓、外周血中分离并培养 MSC,在倒置显微镜下观察各组 MSC 的阳性率,比较其生长速度和细胞形态。同时观察不同血清对 MSC 的趋化作用及不同来源 MSC 的迁移能力。结果两组大鼠的骨髓内均培养出贴壁生长的 MSC。烧伤组12只大鼠外周血中有7只培养出 MSC,其阳性率(58%)明显低于骨髓培养(100%,P<0.05)。假伤组大鼠的外周血内未见 MSC(P<0.05)。倒置显微镜下可见原代接种后24h 有少量贴壁细胞,2～3d 后见其生长,散在贴壁,大多呈梭形。各组 MSC 形态无明显差异,均呈纺锤形生长。烧伤大鼠血清处理的假伤组骨髓 MSC 的迁移数[(94±11)个/高倍视野]明显多于正常大鼠血清和胎牛血清处理者[(37±6)、(38±11)个/高倍视野,P<0.01],后两者处理的 MSC 迁移数相近(P>0.05)。假伤组大鼠骨髓MSC 被不同血清趋化时迁移的细胞数明显少于烧伤组大鼠骨髓和外周血 MSC(P<0.05或0.01)。烧伤组大鼠骨髓 MSC 与外周血 MSC 的迁移能力虽有差异,但无统计学意义(P>0.05)。结论烧伤大鼠骨髓及外周血均能分离到 MSC,而正常大鼠仅骨髓能分离到 MSC。烧伤大鼠血清对 MSC 有较强的趋化作用。烧伤大鼠来源的 MSC 比正常大鼠来源的 MSC 具有更强的迁移能力。     

肝干细胞来源、定向分化及其转化机制

本文综述了近年来胚胎、造血、肝内祖细胞和小肝细胞样祖细胞、骨髓、间充质来源干细胞定向向肝细胞分化的研究进展,并阐述了干细胞转化为肝细胞的机制是融合还是转化.对此深入研究可望为肝干细胞移植和基因治疗带来希望,进而为临床肝移植面临的供肝短缺提供可选择的补充治疗方法.     

肝干细胞来源、定向分化及其转化机制

本文综述了近年来胚胎、造血、肝内祖细胞和小肝细胞样祖细胞、骨髓、间充质来源干细胞定向向肝细胞分化的研究进展,并阐述了干细胞转化为肝细胞的机制是融合还是转化.对此深入研究可望为肝干细胞移植和基因治疗带来希望,进而为临床肝移植面临的供肝短缺提供可选择的补充治疗方法.     

不同来源的间充质干细胞治疗骨与软骨组织疾病的研究进展

近年来,随着细胞和组织工程技术的发展,间充质干细胞广泛受到关注和研究,具有易分离获取、培养过程相对简单等优点,并且能够自我更新并分化成多种细胞类型,包括成骨细胞、软骨细胞、脂肪细胞等,是较为理想的种子细胞。在骨髓间充质干细胞大量的研究基础上,脂肪、骨骼肌、滑膜等多种不同来源的间充质干细胞也广泛应用在骨及软骨组织的体内研究和体外研究中。虽然间充质干细胞在基础性研究方面取得了飞跃进展,但在临床推广应用干细胞治疗上还面临着诸多问题,如对间充质干细胞的分化机理尚不明确,对其定向分化无法进行精确调控,且存在诸多限制骨和软骨再生的几个因素,很大程度上影响治疗的效果,故仍需进一步深入研究。     

放射性损伤对骨髓来源干细胞分化为实体细胞的影响

目的 观察骨髓来源干细胞(BMDSCs)能否在体分化为其他实体组织细胞.方法 野生型C57BL/6J雌性小鼠作为受体接受10 Gy的射线照射后,经尾静脉植入同等背景的转增强型绿色荧光蛋白(EGFP)基因的C57BL/6J雄性小鼠(绿鼠)骨髓细胞1×107个/只.移植受体稳定1年后检测各组织中EGFP的表达.结果 野生型小鼠各组织中EGFP的表达为0%,绿鼠各组织中EGFP的表达为100%.受体鼠各组织均有EGFP阳性细胞分布,表达率为100%,与野生型比较差异有统计学意义(P<0.01),但是表达强度均低于绿鼠组.EGFP阳性细胞主要存在于皮肤组织角质形成细胞、毛囊上皮,以及肺间质、支气管上皮、肺泡上皮中.结论 BMDSCs能在体内分化为其他实体细胞,并有组织特异性.     

不同来源间充质干细胞抑制滤泡辅助性T细胞的作用及其机制研究

目的  探讨不同来源的间充质干细胞（MSC）对滤泡辅助性T细胞（Tfh细胞）的抑制作用及其机制。方法  采用脐带来源MSC（UC MSC）、骨髓来源MSC（BM MSC）、脂肪来源MSC（Fat MSC）分别与外周血单核细胞（PBMC）混合培养48 h,并设立对照组,采用流式细胞仪检测4组培养物中Tfh细胞占淋巴细胞比例,采用酶联免疫吸附试验（ELISA）法检测4组培养物上清中白细胞介素（IL）-21的含量。将BM MSC与PBMC共同培养,分别加入吲哚胺2, 3-双加氧酶（IDO）抑制剂1-甲基色氨酸（1-MT）、IL-10抗体、人类白细胞抗原（HLA）-G抗体,分别为1-MT组、IL-10抑制组、HLA-G抑制组,另设不加其他物质的BM MSC组,培养48 h后采用流式细胞仪检测Tfh细胞占淋巴细胞比例。结果  流式细胞术检测结果显示,与对照组比较,BM MSC组Tfh细胞的比例降低,差异有统计学意义（P < 0.05）;与BM MSC组比较,UC MSC组和Fat MSC组Tfh细胞的比例较高,差异均有统计学意义（均为P < 0.05）。ELISA结果显示,与对照组比较,BM-MSC组IL-21含量降低,差异有统计学意义（P < 0.05）;与BM MSC组比较,UC MSC组和Fat MSC组IL-21含量较高,差异均有统计学意义（均为P < 0.05）。机制研究结果显示1-MT组、IL-10抑制组和HLA-G抑制组Tfh细胞的比例分别为（1.75±0.07）%、（1.31±0.09）%和（1.50±0.03）%,与BM MSC组[（1.03±0.43）%]比较,差异均有统计学意义（均为P < 0.05）。结论  BM MSC对Tfh细胞分化的抑制效果最好,抑制IL-21的效果最好,其发挥抑制Tfh细胞分化的机制可能与促进IDO分泌有关。     

不同来源血清体外培养骨髓间充质干细胞的研究

目的 适当的血清浓度可维持骨髓问充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的体外培养,而不同来源的血清对BMSCs的培养效果是否小同.本研究探讨自体血清、脐带血清、AB血清、胎牛血清在体外培养人BMSCs的可行性.方法 Ficoll密度梯度离心法结合贴壁法分离纯化人BMSCs,在培养过程中分别加入白体血清、脐带血清、AB血清、胎牛血清,观察比较细胞形态、表面抗原、生长曲线及各组在诱导剂作用下表面抗原和诱导分化的相关性.结果 从BMSCs的形态学、生长曲线、表面抗原、诱导分化上看各组无明显差异,生长状况良好.结论 自体血清、脐带血清、AB血清、胎牛血清均可维持BMSCs的体外培养,而自体血清解决异种和异体血清带来的一些潜在的风险,更为安全.     

One-step induction of neurons from mouse embryonic stem cells in serum-free media containing vitamin B12 and heparin

We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B12 and heparin, but without any expensive cytokines. After 14 days in culture, beta-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B12/heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B12/heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5-fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B12/heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinson's disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.     

高效诱导人胚胎干细胞分化为表达生殖功能调控相关基因的下丘脑神经细胞

目的优化体外诱导人胚胎干细胞向类下丘脑弓状核神经细胞分化方案、提高分化效率;明确分化神经细胞中是否表达生殖功能调控相关基因。方法利用接受辅助生殖技术治疗患者捐赠的冷冻胚胎建立人胚胎干细胞系CCRM14;饲养层上培养CCRM14,经纯化后消化为单细胞、诱导其向下丘脑神经细胞分化。分化策略:神经分化早期抑制转化生长因子β和骨形态发生蛋白信号通路;接着激活sonic hedgehog信号通路,促使下丘脑神经前体细胞生成;随后抑制Notch信号通路,添加脑源性神经营养因子诱导类下丘脑弓状核神经细胞的分化与成熟。分化不同时期采样,利用RT-qPCR、细胞免疫荧光、流式细胞术方法鉴定分化细胞。结果依据本优化分化方案,CCRM14分化为下丘脑神经前体细胞的效率达95%以上,分化的细胞表达下丘脑弓状核神经细胞特异性标志物POMC和NPY/AGRP,同时表达参与生殖功能调控基因KISS1和AR。结论该类分化细胞有作为细胞模型用于研究下丘脑弓状核在生殖相关疾病发生过程中调控机制的潜力。