Induced pluripotent stem cells from pigs and other ungulate species: an alternative to embryonic stem cells?

Robust embryonic stem cell (ESC) lines from livestock species have been difficult to derive and maintain, and unlike mouse ESC, have not contributed to our ability to understand directed differentiation in vitro. Nor have such cells yet provided a simpler means than pronuclear injection to manipulate the genomes of agriculturally important species, such as cattle, sheep and pigs. Induced pluripotent stem cells (iPSC) generated by reprogramming somatic cells, such as fibroblasts, with a set of stemness genes, most usually but not exclusively POU5F1, SOX2, KLF4 and c-MYC, offer an alternative to ESC in these regards, as they exhibit a pluripotent phenotype resembling that of ESC, yet are readily generated in the laboratory. Accordingly, such cells, in association with cloning technologies, may be useful for introducing complex genetic changes into livestock, although this potential has yet to be demonstrated. Porcine iPSC may be especially valuable because the pig is a prime biomedical model for tissue transplantation. In general, iPSC from livestock, like those from humans, are of the epiblast type and depend upon FGF2 and activin/nodal signalling systems to maintain their pluripotency and growth. Recent experiments, in which newly reprogrammed porcine and bovine cells were selected on a LIF-based medium in presence of specific protein kinase inhibitors, have allowed iPSC cells of the na?ve type, resembling the more amenable blastocyst-derived mouse ESC and iPSC to be isolated. However, hurdles still remain if such cells are to achieve their biotechnological promise.     

猪多潜能干细胞系的研究进展

猪多潜能干细胞是在体外建立起来的具有自我更新及三胚层分化潜能的一类干细胞,可应用于发育生物学研究、基因组编辑和疾病模型建立等各方面,在畜牧业生产及再生医学研究中具有重要的应用价值。培养体系对多潜能干细胞的成功构建具有重要意义,其包含多种成分,包括基础培养液、氨基酸等营养成分、小分子化合物(信号通路激活剂/抑制剂及细胞因子)共同维持细胞的多能性并抑制其分化。目前已有诸多关于猪多潜能干细胞的报道,但尚未获得高效的培养体系可以维持猪多潜能干细胞的长期传代并完成生殖嵌合。猪多潜能干细胞可分为原始态(Na6ve)、形成态(Formative)及始发态(Primed)3种不同多能性的状态特点,根据建系来源的不同分为猪扩展多潜能干细胞、猪胚胎干细胞、猪前原肠胚上胚层干细胞、猪诱导多能干细胞4种干细胞类型。作者综述了猪多潜能干细胞培养体系中常用的细胞因子和信号通路激活剂/抑制剂对猪多潜能干细胞的多能性和分化能力的影响和作用,为进一步建立具有真正生殖嵌合能力的Na6ve多能性的猪多潜能干细胞系提供研究思路。     

Pluripotency network in porcine embryos and derived cell lines

Huge amounts of work have been dedicated to the establishment of embryonic stem cell lines from farm animal species since the successful isolation of embryonic stem cells from the mouse and from the human. However, no conclusive results have been obtained so far, and validated lines have yet to be established in domestic animals. Many limiting factors have been suggested and need to be studied further to isolate truly pluripotent cell lines from livestock. In this review, we will discuss the difficulties in deriving and maintaining embryonic stem cell lines from farm animal embryos and how can this lack of success be explained. We will summarize results obtained in our laboratory regarding derivation of pluripotent cells in the pigs. Problems related to the identification of standard methods for derivation, maintenance and characterization of cell lines will also be examined. We will focus our attention on the need for appropriate stemness-related marker molecules that can be used to reliably investigate pluripotency in domestic species. Finally, we will review data presently available on functional key pluripotency-maintaining pathways in farm animals.     

Nanog基因的生物学功能研究进展

胚胎干细胞具有无限增殖能力和多向分化潜能决定了它在医学及生物学基础研究中具有巨大的应用潜力。探索维持胚胎干细胞特性的分子机制成为胚胎干细胞的生物学研究中的热点。研究发现与维持胚胎干细胞多能性相关的基因有Oct4、Nanog、Sox2等，其中Nanog是2003年5月末发现的一个基因，它对维持胚胎干细胞多能性起关键性作用，能够独立于L1F/Stats维持ICM和ES细胞的多能性。几年来，Nanog的生物学功能及其与Oct4、Sox2等多能性维持基因之间的相互作用关系已有较为深入的研究。作者在综述Nanog基因的表达特征和功能的基础上，重点探讨Nanog基因表达调控以及Oct4、Sox2等多能性维持基因之间的相互作用关系，并展望其应用前景。     

Livestock induced pluripotent stem cells

Chimeric animals generated from livestock-induced pluripotent stem cells (iPSCs) have opened the door of opportunity to genetically manipulate species for the production of biomedical models, improving traits of agricultural importance and potentially providing a system to test novel iPSC therapies. The potential of pluripotent stem cells in livestock has long been recognized, with many attempts being chronicled to isolate, culture and characterize pluripotent cells from embryos. However, in most cases, livestock stem cells derived from embryonic sources have failed to reach a pluripotent state marked by the inability to form chimeric animals. The in-depth understanding of core pluripotency factors and the realization of how these factors can be harnessed to reprogram adult cells into an induced pluripotent state has changed the paradigm of livestock stem cells. In this review, we will examine the advancements in iPSC technology in mammalian and avian livestock species.     

Recent advances in stem and germ cell research: implications for the derivation of pig pluripotent cells

Pluripotent stem cells have the unique capacity to contribute to all the tissues of an adult animal after transfer into a host embryo. How pluripotency is acquired during early development and how it is maintained in stem cells have attracted the interest of many scientists for over three decades. Much progress in our understanding of how stem cells arise in culture and the signals required for homoeostasis has enabled the derivation of pluripotent cells in multiple species. Here, we discuss recent developments in stem cell biology that will impact the generation of pluripotent cells from different embryonic origins and will contribute to increase our capacity for generating transgenic animals.     

Interspecies cell fusion between mouse embryonic stem cell and porcine pluripotent cell

In the area of stem cell research, fusion of somatic cells into pluripotent cells such as mouse embryonic stem (ES) cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell on the pluripotent state of somatic nucleus. As many other groups, we previously established a porcine pluripotent cell line at a low potential. Therefore, here, we performed experiments to investigate if the fusion with mouse ES cell could improve the pluripotent state of porcine pluripotent cell. Our data showed that resultant mouse–porcine interspecies fused cells are AP positive, and could be passaged up to 20 passages. Different degrees of increases in expression of porcine pluripotent genes proved that pig-origin gene network can be programmed by mouse ES. Further differentiation study also confirmed these fused cells’ potential to form three germ layers. However, unexpectedly, we found that chromosome loss and aberrant (especially in porcine chromosomes) is severe after the cell fusion, implying that interspecies cell fusion may be not suitable to study porcine pluripotency without additional supportive conditions for genome stabilization.     

Pluripotent stem cells--model of embryonic development,tool for gene targeting,and basis of cell therapy

Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity. They are derived from pre-implantation embryos and can be propagated as a homogeneous, uncommitted cell population for an almost unlimited period of time without losing their pluripotency and their stable karyotype. Murine ES cells are able to reintegrate fully into embryogenesis when returned into an early embryo, even after extensive genetic manipulation. In the resulting chimeric offspring produced by blastocyst injection or morula aggregation, ES cell descendants are represented among all cell types, including functional gametes. Therefore, mouse ES cells represent an important tool for genetic engineering, in particular via homologous recombination, to introduce gene knock-outs and other precise genomic modifications into the mouse germ line. Because of these properties ES cell technology is of high interest for other model organisms and for livestock species like cattle and pigs. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have yet been established for vertebrate species other than the mouse (Evans and Kaufman, 1981; Martin, 1981) and chicken (Pain et al., 1996). The in vitro differentiation capacity of ES cells provides unique opportunities for experimental analysis of gene regulation and function during cell commitment and differentiation in early embryogenesis. Recently, pluripotent stem cells were established from human embryos (Thomson et al., 1998) and early fetuses (Shamblott et al., 1998), opening new scenarios both for research in human developmental biology and for medical applications, i.e. cell replacement strategies. At about the same time, research activities focused on characteristics and differentiation potential of somatic stem cells, unravelling an unexpected plasticity of these cell types. Somatic stem cells are found in differentiated tissues and can renew themselves in addition to generating the specialized cell types of the tissue from which they originate. Additional to discoveries of somatic stem cells in tissues that were previously not thought to contain these kinds of cells, they also appear to be capable of developing into cell types of other tissues, but have a reduced differentiation potential as compared to embryo-derived stem cells. Therefore, somatic stem cells are referred to as multipotent rather than pluripotent. This review summarizes characteristics of pluripotent stem cells in the mouse and in selected livestock species, explains their use for genetic engineering and basic research on embryonic development, and evaluates their potential for cell therapy as compared to somatic stem cells.     

Of stem cells and germ cells

Stem cells have an intrinsic capacity to self-renew and can differentiate to at least one specialized cell type. Different types of stem cells exist that can be cultured in vitro. The identity of the stem cells is marked by their origin and differentiation potential. Germ cells have similarities with pluripotent stem cells but are of a special order: They do not self-renew and are already differentiated, but they have the capacity to form a complete new organism after fertilization. This review focuses on pluripotent stem cells and discusses possibilities of generating pluripotent stem cells from germ cell precursors and possibilities of generating germ cells from stem cells. As it accompanies a plenary lecture at the 15th annual ESDAR Conference 2011, the overview is focused on stem cells from farm animal species and on results from my own research group.     

Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Generation of Human β-thalassemia Induced Pluripotent Stem Cells from Amniotic Fluid Cells Using a Single Excisable Lentiviral Stem Cell Cassette

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may have a wide range of applications in cell and gene therapy. However, the safety issues and the low efficiency associated with generating human iPSCs have limited their usage in clinical settings. The cell type used to create iPSCs can significantly influence the reprogramming efficiency and kinetics. Here, we show that amniotic fluid cells from the prenatal diagnosis of a β-thalassemia patient can be efficiently reprogrammed using a doxycycline (DOX)-inducible humanized version of the single lentiviral "stem cell cassette" vector flanked by loxP sites, which can be excised with Cre recombinase. We also demonstrated that the patient-derived iPSCs can be characterized based on the expression of pluripotency markers, and they can be differentiated into various somatic cell types in vitro and in vivo. Moreover, microarray analysis demonstrates a high correlation coefficient between human β-thalassemia iPS cells and human embryonic stem (hES) cells but a low correlation coefficient between human β-thalassemia amniotic fluid cells and human β-thalassemia iPS cells. Our data suggest that amniotic fluid cells may be an ideal human somatic cell resource for rapid and efficient generation of patient-specific iPS cells.     

Enhancing effects of serum-rich and cytokine-supplemented culture conditions on developing blastocysts and deriving porcine parthenogenetic embryonic stem cells

The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.     

SC1 sustains the self‐renewal capacity and pluripotency of chicken blastodermal cells by inhibiting the phosphorylation of ERK1 and promoting the phosphorylation of Akt

Small molecules discovered during the recent years can be used to regulate the growth of embryonic stem cells (ES cells). Chicken blastodermal cells (cBCs) play an important role in both basic and transgenic researches as an important ES cell. However, the regulatory mechanism of small molecules involved in the self‐renewal and pluripotency of cBCs remains unknown. This study revealed that the small molecule, SC1, can maintain cBCs in an undifferentiated, pluripotent state in serum‐ and feeder‐free E8 media without leukaemia inhibitory factor. Furthermore, SC1 inhibits downregulation of pluripotency‐related genes caused by retinoic acid and promotes the proliferation of cBCs. Furthermore, the results of this study indicated that SC1 functions by inhibiting ERK1 phosphorylation and promoting Akt phosphorylation, thus promoting the expression of pluripotency‐related genes and maintaining the pluripotency of cBCs. The results also demonstrated that SC1 sustains the self‐renewal capacity and pluripotency of cBCs cells by inhibiting ERK1 phosphorylation and promoting Akt phosphorylation. This kind of regulatory mechanism might be conserved in avian ES cells. Other molecules, similar to SC1, might provide insights into the molecular mechanisms that control the fate of stem cells and ultimately help in‐vivo stem cell biology and therapy.     

miRNA对干细胞重编程的影响

miRNA在胚胎干细胞(ES)细胞的自我更新及多能性中扮演重要角色,国内外研究结果表明,在体细胞形成诱导性多能干细胞(IPS)的过程中,许多miRNA与Sox2、Oct4和Nanog等调控因子组成调控网络。miRNA在细胞周期、重编程及表型建立过程中也起着重要的调控作用。另外,在IPS细胞形成中,miRNA很有可能代替关键转录因子。     

Immunocytochemical analysis of vimentin expression patterns in porcine embryos suggests mesodermal differentiation from day 9 after conception

The expression of the intermediate filament proteins vimentin and keratin in porcine embryos was studied by whole-mount immunocytochemistry between day 7 and day 11 after conception. Expression of vimentin was first detected in the inner cell mass of about 50% of the 9-day-old embryos. In elongated 11-day-old embryos, cells expressing vimentin were observed in the epiblast (after disappearance of Rauber's membrane) and in cells migrating from the epiblast between the trophoblast and the underlying hypoblast layer. A keratin-positive response was observed in trophectoderm cells at all stages. These findings suggest that inner cell mass cells in the pig start differentiating into mesodermal cells not later than day 9 after conception. While the delamination of the mesodermal germ layer is known to correlate with the loss of pluripotency of the inner cell mass cells, the early onset of mesodermal differentiation in the porcine embryo, characterized by vimentin expression and in contrast to the mouse, could in part be responsible for the lack of success in establishing pluripotent embryonic stem cell lines in this species. Our results suggest that further attempts to isolate inner cell mass-derived pluripotent cells should be attempted well before day 9 after conception.     

禽类多能性干细胞研究进展

胚胎干细胞是未分化的具有增殖和自我更新能力的细胞,并且能分化成所有类型的体细胞以及生殖细胞。它们提供了早期胚胎分化的体外模型,也是基因操作的重要靶细胞。禽类多能性干细胞培养最重要的应用领域是以干细胞体外遗传修饰、鉴定为技术平台的家禽转基因技术。通过此技术对禽类基因进行遗传修饰与操作,在胚胎发育基础研究、转基因禽类生产及家禽育种等方面有巨大的应用前景。但是禽类多能性干细胞培养的许多基本问题仍亟待解决,如探索其建系的培养条件、揭示其维持多能性和增殖能力的分子机制等。文章综述了禽类多能性干细胞的分离方法、体外分化能力、嵌合体形成以及基因修饰方面的研究进展及目前的研究局限。     

Generation of induced pluripotent stem cells from Asian patients with chronic neurodegenerative diseases

Induced pluripotent stem (iPS) cells derived from disease patients are an invaluable resource for biomedical research and may provide a source for replacement therapies. In this study, we have generated iPS cells from Asian patients with chronic degenerative diseases of the nervous system, including spinal muscular atrophy (SMA), Parkinson disease (PD) and amyotrophic lateral sclerosis (ALS) by transduction with four factors (KLF4, SOX2, OCT4 and c-MYC). All of the iPS cells showed pluripotency similar to that of human embryonic stem cells (hESCs) and were able to differentiate into various somatic cell types in vitro and in vivo. Furthermore, the iPS cells also can be committed to differentiate into neural cells, the cell type that is affected in chronic degenerative diseases. Therefore, the patient-specific iPS cells we generated offer a cellular model in which to investigate disease mechanisms, discover and test novel drugs and develop new therapies for chronic neurodegenerative diseases.