一种获得猪诱导性多能干细胞的新方法

诱导性多能干细胞技术表明,通过过表达4个重编程因子可使体细胞逆转到多能性的状态,为建立更多家畜动物的多能性干细胞系提供了新的方法,如猪、牛、羊等农业动物.莫洛尼氏鼠白血病逆转录病毒载体被广泛应用于小鼠iPS细胞的建系和机制研究上,然而这种病毒只能感染小鼠和大鼠细胞,这限制了它在其他哺乳动物iPS细胞系建立上的应用.本实验采用一种新的逆转录病毒系统,可以高效便捷地从猪成纤维细胞中获得iPS细胞.通过在GP2-293细胞中包装VSV-G蛋白包被的病毒,仅一步感染猪成纤维细胞即可转入4个人源重编程因子（Oct4,Sox2,Klf4和c-Myc）.在添加有碱性成骨因子bFGF的人类胚胎干细胞培养体系中,成功建立6株和人类胚胎干细胞形态极其相似的猪iPS细胞系.这些猪iPS克隆具有较大的细胞核/细胞质比例、边界清晰、细胞呈扁平状等特征.在体外可以分化成拟胚体,注射入免疫缺陷性小鼠体内可以形成畸胎瘤,含有3种胚层类型的组织.     

无饲养层和动物源蛋白的β-地中海贫血诱导多能干细胞系的建立

β-地中海贫血患者因无合适的造血干细胞供体来源从而不得不靠输血维持生命。诱导多能干细胞(iPS)技术为获得患者自身遗传背景的干细胞进行临床治疗开拓了新途径。目前,建立iPS细胞系的过程需要使用小鼠胚胎成纤维细胞作为饲养层和动物源的蛋白成分,因此建立的iPS细胞系存在病原体和动物源蛋白污染的可能性,不能应用于临床。采用目前商品化的TeSRTM2和StemAdhereTMDefined Matrix限定培养体系,利用Oct4、Sox2、Klf4、c-Myc 4个转录因子组装在同一表达载体的可切除的慢病毒感染人β-地中海贫血成纤维细胞,建立了5株无饲养层和动物源蛋白的β-地中海贫血iPS细胞系,这些iPS细胞系具有人胚胎干细胞典型的特征,表达人胚胎干细胞的多能性分子标记,如Oct4、Nanog、Tra-1-60等。在体外分化能够形成拟胚体,在体内分化能够形成含有3个胚层类型细胞的畸胎瘤。     

体细胞重编程为诱导多能干细胞

诱导多功能性干细胞（induced pluripotent stem cells,iPS细胞）是通过导入特定的转录因子（如Oct3/4、Sox2、c-Myc和Klf4等）将体细胞诱导重编程为多能性干细胞,其功能与胚胎干细胞相似.iPS细胞的建立,在生命科学领域引起了新的轰动.目前,iPS细胞的研究领域在转录因子的优化、iPS细胞的筛选、载体的运用、体细胞种类的选择和iPS细胞的应用等方面取得突破进展,但仍然存在致癌性、效率低等一系列急需解决的问题.     

利用piggyBac转座子制备牛成体多能干细胞诱导技术研究

通过逆转录病毒等媒介表达核转录因子Oct4、Sox2、Klf4、c-Myc可将体细胞重编程为诱导多能干细胞(induced pluripotent stem cells, iPSc)。时至今日,已经报道了小鼠、人、大鼠、猪、羊、马、牛的iPS细胞,但大动物iPS的多能性特别是嵌合体形成和生殖细胞传代还没有得到确认。与逆转录病毒等不同的是,piggyBac转座子转染效率高且无病毒源性、操作简单,可以在转座酶的存在下被安全切除。首次尝试了采用piggyBac转座子携带鼠源Oct4、Sox2、Klf4、c-Myc、Rarg和Lrh16个核转录因子诱导胎牛成纤维细胞,成功获得牛类iPS细胞,其形态与小鼠胚胎干细胞相似,克隆边界清晰、呈丘状、克隆内细胞致密、核大。RT-PCR与免疫组织化学染色分析均显示牛类iPS细胞表达多能性基因。该类细胞体外诱导分化可形成类胚体EB,且表达3个胚层的基因;体内诱导分化可形成畸胎瘤,苏木精、伊红染色显示瘤体有三胚层的分化。上述结果显示利用piggyBac转座子制备牛多潜能干细胞诱导技术可行,产生的牛类iPS细胞具有潜在多能性。     

体细胞重编程为诱导多能干细胞的研究

通过逆转录病毒将4个基因(Oct4 、 Sox2、c-Myc和Klf4)导入小鼠胚胎成纤维细胞 (mouse embryonic fibroblast, MEF)中,能诱导形成胚胎干细胞样特性的诱导多能干(induced pluripotent stem, iPS)细胞.人类iPS细胞的成功构建开拓了广泛的应用前景.本文简要综述了 iPS细胞的基因筛选,转导基因的选择以及iPS细胞的表观遗传特性等.     

多顺反子质粒重编程人脂肪干细胞为诱导多能干细胞

为建立多顺反子质粒载体转染技术获得人脂肪干细胞(adipose stem cells,ASCs)来源的诱导多能干细胞(induced pluripotency stem cells,iPSCs),应用2A元件连接Oct4/Sox2/KLF4/c-Myc四因子基因,构建为单一开放阅读框的多顺反子质粒载体．使用该质粒对ASCs进行转染及重编程为iPSC．采用形态学观察、特异性抗体免疫荧光鉴定、体外拟胚体诱导分化和体内畸胎瘤形成等方法进行鉴定．结果显示,ASCs成功重编程为iPSCs,具有与人胚胎干细胞相似的形态学及多向分化潜能;通过拟胚体和畸胎瘤实验证实iPSCs能在体内外分化成三胚层细胞;DNA印迹实验显示质粒载体序列未整合至iPSCs基因组中．因此,通过多顺反子质粒载体重编程技术成功建立的人iPSCs具有多向分化潜能,可减免发生插入突变和免疫排斥问题,为iPSCs在遗传性或退行性疾病的治疗奠定了实验基础．     

转录因子与诱导型多能干细胞

小鼠的成纤维细胞通过转染四种转录因子（Oct3／4、Sox2、c-Myc和K1F4）可以被诱导转变成类似胚胎干细胞的多能性干细胞,称之为诱导型多能干细胞（induced pluripotent stem cell,iPS）,这种多能干细胞在细胞形态、增殖速率、致瘤性、基因表达以及形成嵌合小鼠的能力上与胚胎干细胞有许多相似之处,将来可能成为胚胎干细胞在临床应用中的替代。本文综述了iPS相关的几种转录因子,及其在重编程过程中的作用以及iPS的发展前景。     

诱导多能干细胞研究进展

成熟的体细胞过表达转录激活因子Oct4、Sox2、Klf4和c-Myc能够转化为具有多能性的干细胞,称为诱导多能干细胞。类似于胚胎干细胞,诱导多能性干细胞具有自我更新和多向分化潜能性两个主要特征。同胚胎干细胞相比,诱导多能干细胞不仅能够为以细胞替代治疗为核心的再生医学提供无限的细胞来源,而且有望解决胚胎干细胞临床开发面临的伦理道德及免疫排斥问题。从诱导多能干细胞技术的建立、重编程的机理及其在临床中的应用几方面作简要综述。     

特定因子诱导多能干细胞

胚胎干细胞由于具有发育上的全能性,被认为是用于移植治疗的最佳来源。然而,由于人的胚胎干细胞直接运用引发免疫排斥以及触及伦理矛盾,人们一直在研发多能干细胞。2006年,多能干细胞的研究有了重大进展。首先,Yamanaka实验室构建用逆转录载体将候选因子导入成纤维细胞,而后检测多能性标志基因的表达。结果发现,四种因子Oct3/4、Sox2、c-Myc以及Klf4的组合产生了表达多能性标志基因才有的抗药性的克隆,意味着细胞获得了多能性。用这种方法筛选的细胞无论在形态和增殖分化能力方面均类似于干细胞,而且表达干细胞标志基因以及在体内外能向三个胚层的细胞类型分化,这种细胞被命名为诱导性多能干细胞(iPS细胞)。进一步,用更严格的筛选基因nanog得到的iPS能够嵌合到生殖系中。而后,运用改进的方法从人的成体成纤维细胞也可以得到iPS细胞。然而,这种方法得到的嵌合体小鼠存在肿瘤形成现象,可能是由于c-Myc逆转录病毒整合到了基因组。通过替代的方法,去掉c-Myc的iPS也能够获得。为了进一步降低肿瘤形成的几率,近来发展了一种不依赖于病毒的方法,用质粒载体作为介质。iPS进一步的研究热点在于安全性以及从更严格的医学角度提高诱导iPS的效率,其分子机理和相关的技术问题也有待解决和克服。     

2012年第3期《遗传》封面说明

2006年,日本京都大学的Yamanaka研究组在《Cell》上率先报道了利用逆转录病毒感染技术,把转录因子Oct3/4、Sox2、Klf4和cMyc导入胚胎或成年小鼠的成纤维细胞中,诱导出与胚胎干细胞极为相似的多能性干细胞,并将其命名为诱导多能性干细胞(Induced pluripotent stem cells,iPS细胞)。相较于胚胎干细胞,iPS细胞的生产不需要卵母细胞或早期胚胎细胞,因而不涉及胚胎毁损等伦理问题;此外,利用自体来源的iPS细胞进行移植治疗将可避免异体间的免疫排     

Reprogramming the pluripotent cell cycle: restoration of an abbreviated G1 phase in human induced pluripotent stem (iPS) cells

Induced pluripotent stem (iPS) cells derived from terminally differentiated human fibroblasts are reprogrammed to possess stem cell like properties. However, the extent to which iPS cells exhibit unique properties of the human embryonic stem (hES) cell cycle remains to be established. hES cells are characterized by an abbreviated G1 phase (～ 2.5 h) and accelerated organization of subnuclear domains that mediate the assembly of regulatory machinery for histone gene expression [i.e., histone locus bodies (HLBs)]. We therefore examined cell cycle parameters of iPS cells in comparison to hES cells. Analysis of DNA synthesis [5-bromo-2'-deoxy-uridine (BrdU) incorporation], cell cycle distribution (FACS analysis and Ki67 staining) and subnuclear organization of HLBs [immunofluorescence microscopy and fluorescence in situ hybridization (FISH)] revealed that human iPS cells have a short G1 phase (～ 2.5 h) and an abbreviated cell cycle (16-18 h). Furthermore, HLBs are formed and reorganized rapidly after mitosis (within 1.5-2 h). Thus, reprogrammed iPS cells have cell cycle kinetics and dynamic subnuclear organization of regulatory machinery that are principal properties of pluripotent hES cells. Our findings support the concept that the abbreviated cell cycle of hES and iPS cells is functionally linked to pluripotency.     

Regulation of somatic cell reprogramming through inducible mir-302 expression

Global demethylation is required for early zygote development to establish stem cell pluripotency, yet our findings reiterate this epigenetic reprogramming event in somatic cells through ectopic introduction of mir-302 function. Here, we report that induced mir-302 expression beyond 1.3-fold of the concentration in human embryonic stem (hES) H1 and H9 cells led to reprogramming of human hair follicle cells (hHFCs) to induced pluripotent stem (iPS) cells. This reprogramming mechanism functioned through mir-302-targeted co-suppression of four epigenetic regulators, AOF2 (also known as KDM1 or LSD1), AOF1, MECP1-p66 and MECP2. Silencing AOF2 also caused DNMT1 deficiency and further enhanced global demethylation during somatic cell reprogramming (SCR) of hHFCs. Re-supplementing AOF2 in iPS cells disrupted such global demethylation and induced cell differentiation. Given that both hES and iPS cells highly express mir-302, our findings suggest a novel link between zygotic reprogramming and SCR, providing a regulatory mechanism responsible for global demethylation in both events. As the mechanism of conventional iPS cell induction methods remains largely unknown, understanding this microRNA (miRNA)-mediated SCR mechanism may shed light on the improvements of iPS cell generation.     

ROCK inhibition enhances the recovery and growth of cryopreserved human embryonic stem cells and human induced pluripotent stem cells

Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study, we demonstrate that Y‐27632, a specific inhibitor of Rho kinase (ROCK) activity, significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore, treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably, hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y‐27632. Additionally, we determined that Y‐27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus, Y‐27632 provides a means to “kick‐start” slow‐growing human pluripotent stem cells, especially after being thawed from frozen stocks. Together, these results argue that Y‐27632 is a useful tool in overcoming obstacles to studies involving the cultivation of both hES cells and human iPS cells. Mol. Reprod. Dev. 76: 722–732, 2009. © 2009 Wiley‐Liss, Inc.     

Antibody-directed lentiviral gene transduction for live-cell monitoring and selection of human iPS and hES cells

The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular, in the identification of induced pluripotent stem (iPS) cells during the reprogramming process. Based on the selective expression of stem cell surface markers, a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts, allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells, but that specific genes, including positive and negative selection markers, regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies.     

Molecular basis for an attenuated cytoplasmic dsRNA response in human embryonic stem cells

The introduction of double stranded RNA (dsRNA) into the cytoplasm of mammalian cells usually leads to a potent antiviral response resulting in the rapid induction of interferon beta (IFNβ). This response can be mediated by a number of dsRNA sensors, including TLR3, MDA5, RIG-I and PKR. We show here that pluripotent human cells (human embryonic stem (hES) cells and induced pluripotent (iPS) cells) do not induce interferon in response to cytoplasmic dsRNA, and we have used a variety of approaches to learn the underlying basis for this phenomenon. Two major cytoplasmic dsRNA sensors, TLR3 and MDA5, are not expressed in hES cells and iPS cells. PKR is expressed in hES cells, but is not activated by transfected dsRNA. In addition, RIG-I is expressed, but fails to respond to dsRNA because its signaling adapter, MITA/STING, is not expressed. Finally, the interferon-inducible RNAse L and oligoadenylate synthetase enzymes are also expressed at very low levels. Upon differentiation of hES cells into trophoblasts, cells acquire the ability to respond to dsRNA and this correlates with a significant induction of expression of TLR3 and its adaptor protein TICAM-1/TRIF. Taken together, our results reveal that the lack of an interferon response may be a general characteristic of pluripotency and that this results from the systematic downregulation of a number of genes involved in cytoplasmic dsRNA signaling.Key words: dsRNA, interferon, innate immunity, pluripotency, stem cells     

Making gametes from alternate sources of stem cells: past,present and future

Infertile couples including cancer survivors stand to benefit from gametes differentiated from embryonic or induced pluripotent stem (ES/iPS) cells. It remains challenging to convert human ES/iPS cells into primordial germ-like cells (PGCLCs) en route to obtaining gametes. Considerable success was achieved in 2016 to obtain fertile offspring starting with mouse ES/iPS cells, however the specification of human ES/iPS cells into PGCLCs in vitro is still not achieved. Human ES cells will not yield patient-specific gametes unless and until hES cells are derived by somatic cell nuclear transfer (therapeutic cloning) whereas iPS cells retain the residual epigenetic memory of the somatic cells from which they are derived and also harbor genomic and mitochondrial DNA mutations. Thus, they may not be ideal starting material to produce autologus gametes, especially for aged couples. Pluripotent, very small embryonic-like stem cells (VSELs) have been reported in adult tissues including gonads, are relatively quiescent in nature, survive oncotherapy and can be detected in aged, non-functional gonads. Being developmentally equivalent to PGCs (natural precursors to gametes), VSELs spontaneously differentiate into gametes in vitro. It is also being understood that gonadal stem cells niche is compromised by oncotherapy and with age. Improving the gonadal somatic niche could regenerate non-functional gonads from endogenous VSELs to restore fertility. Niche cells (Sertoli/mesenchymal cells) can be directly transplanted and restore gonadal function by providing paracrine support to endogenous VSELs. This strategy has been successful in several mice studies already and resulted in live birth in a woman with pre-mature ovarian failure.     

Trophoblast differentiation defect in human embryonic stem cells lacking PIG-A and GPI-anchored cell-surface proteins

Pluripotent human embryonic stem (hES) cells can differentiate into various cell types derived from the three embryonic germ layers and extraembryonic tissues such as trophoblasts. The mechanisms governing lineage choices of hES cells are largely unknown. Here, we report that we established two independent hES cell clones lacking a group of cell surface molecules, glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs). The GPI-AP deficiency in these two hES clones is due to the deficiency in the gene expression of PIG-A (phosphatidyl-inositol-glycan class A), which is required for the first step of GPI synthesis. GPI-AP-deficient hES cells were capable of forming embryoid bodies and initiating cell differentiation into the three embryonic germ layers. However, GPI-AP-deficient hES cells failed to form trophoblasts after differentiation induction by embryoid body formation or by adding exogenous BMP4. The defect in trophoblast formation was due to the lack of GPI-anchored BMP coreceptors, resulting in the impairment of full BMP4 signaling activation in the GPI-AP-deficient hES cells. These data reveal that GPI-AP-enhanced full activation of BMP signaling is required for human trophoblast formation.