Role of leukaemia inhibitory factor in the induction of pluripotent stem cells in mice

iPS (induced pluripotent stem) cells can be induced from somatic cells in mice by genetic manipulation. Most previously established mouse iPS cell lines have been derived using feeder layers supplemented with exogenous LIF (leukaemia inhibitory factor). Although a feeder‐free induction system has been developed in recent studies, LIF is still required for reprogramming, but its role in the generation of mouse iPS cells has remained elusive. In this study, we investigated its contribution to the induction of pluripotency. Our results showed that LIF activates AP (alkaline phosphatase) through a c‐Myc‐dependent mechanism. Moreover, it acts as a protective factor during the transition from AP‐positive colonies to Oct3/4‐positive cells. These findings illustrate a mechanism by which LIF may integrate signalling into reprogramming.     

A Robust Approach to Identifying Tissue-Specific Gene Expression Regulatory Variants Using Personalized Human Induced Pluripotent Stem Cells

Normal variation in gene expression due to regulatory polymorphisms is often masked by biological and experimental noise. In addition, some regulatory polymorphisms may become apparent only in specific tissues. We derived human induced pluripotent stem (iPS) cells from adult skin primary fibroblasts and attempted to detect tissue-specific cis-regulatory variants using in vitro cell differentiation. We used padlock probes and high-throughput sequencing for digital RNA allelotyping and measured allele-specific gene expression in primary fibroblasts, lymphoblastoid cells, iPS cells, and their differentiated derivatives. We show that allele-specific expression is both cell type and genotype-dependent, but the majority of detectable allele-specific expression loci remains consistent despite large changes in the cell type or the experimental condition following iPS reprogramming, except on the X-chromosome. We show that our approach to mapping cis-regulatory variants reduces in vitro experimental noise and reveals additional tissue-specific variants using skin-derived human iPS cells.     

c-Myc对诱导性多能干细胞的诱导效率及全能性影响的研究

该研究探讨转录因子c—Myc对多能诱导干细胞（inducedpluripotentstemcells,iPS）诱导效率及形成的iPS克隆全能性的影响。将Yam,dnaka四因子（Oct4、Sox2、Klf4和C—Myc,OSKM）和不舍C。Myc的三因子（OSK）病毒分别感染OG2小鼠成纤维细胞（mouseembryofibroblast,MEF）,诱导成为iPSN胞,通过计数iPS克隆形成数目和流式细胞仪分析iPS克隆的绿色荧光蛋白（GFP）阳性比例。比较OSKM和OSK诱导iPS的效率。分别挑取三株OSK和OSKM诱导的iPS克隆,采用荧光定量PCR法、碱性磷酸酶fAP）染色法和免疫荧光法检测iPS克隆的干性相关蛋白的表达;采用畸胎瘤实验、嵌合体形成实验和四倍体补偿实验检测iPS克隆的全能性。结果显示：在该实验室诱导体系里,OSK病毒感染MEF后形成的iPS克隆数目明显增多,GFP阳性克隆的比例增加。OSK和OSKM诱导iPS克隆的干细胞基本特征,包括形态、AP染色、干细胞特异性基因表达、三胚层形成均没有明显差异。然而,OSK诱导iPS克隆在形成嵌合体小鼠的全能性评估实验中的阳性比例高于OSKM克隆,且只有OSK克隆株能够获得生殖系传递小鼠和四倍体补偿小鼠。该研究表明,在实验诱导体系里去除C—Myc的三因子OSK诱导iPS的效率显著高于OSKM四因子,且三因子诱导iPS克隆具有更好的全能性。     

Morphologic and Gene Expression Criteria for Identifying Human Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells can be generated from somatic cells by the forced expression of four factors, Oct3/4, Sox2, Klf4, and c-Myc. While a great variety of colonies grow during induction, only a few of them develop into iPS cells. Researchers currently use visual observation to identify iPS cells and select colonies resembling embryonic stem (ES) cells, and there are no established objective criteria. Therefore, we exhaustively analyzed the morphology and gene expression of all the colonies generated from human fibroblasts after transfection with four retroviral vectors encoding individual factors (192 and 203 colonies in two experiments) and with a single polycistronic retroviral vector encoding all four factors (199 and 192 colonies in two experiments). Here we demonstrate that the morphologic features of emerged colonies can be categorized based on six parameters, and all generated colonies that could be passaged were classified into seven subtypes in colonies transfected with four retroviral vectors and six subtypes with a single polycistronic retroviral vector, both including iPS cell colonies. The essential qualifications for iPS cells were: cells with a single nucleolus; nucleus to nucleolus (N/Nls) ratio ∼2.19: cell size ∼43.5 µm²: a nucleus to cytoplasm (N/C) ratio ∼0.87: cell density in a colony ∼5900 cells/mm²: and number of cell layer single. Most importantly, gene expression analysis revealed for the first time that endogenous Sox2 and Cdx2 were expressed specifically in iPS cells, whereas Oct3/4 and Nanog, popularly used markers for identifying iPS cells, are expressed in colonies other than iPS cells, suggesting that Sox2 and Cdx2 are reliable markers for identifying iPS cells. Our findings indicate that morphologic parameters and the expression of endogenous Sox2 and Cdx2 can be used to accurately identify iPS cells.     

Efficient Generation of Rat Induced Pluripotent Stem Cells Using a Non-Viral Inducible Vector

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.     

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.     

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors

Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors.     

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13^highCD133⁺ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.     

Activation of the Imprinted Dlk1-Dio3 Region Correlates with Pluripotency Levels of Mouse Stem Cells

Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency levels of stem cells. A mammalian conserved cluster of microRNAs encoded by this region exhibited significant expression differences between full and partial pluripotent stem cells. Several microRNAs from this cluster potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels; therefore, the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or embryonic stem cells from partial pluripotent cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy.     

Generation of Corneal Epithelial Cells from Induced Pluripotent Stem Cells Derived from Human Dermal Fibroblast and Corneal Limbal Epithelium

Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6⁺/K12⁺ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.     

Generation of Induced Pluripotent Stem Cells in Rabbits: POTENTIAL EXPERIMENTAL MODELS FOR HUMAN REGENERATIVE MEDICINE*

Human induced pluripotent stem (iPS) cells have the potential to establish a new field of promising regenerative medicine. Therefore, the safety and the efficiency of iPS-derived cells must be tested rigorously using appropriate animal models before human trials can commence. Here, we report the establishment of rabbit iPS cells as the first human-type iPS cells generated from a small laboratory animal species. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2, and OCT3/4) were introduced successfully into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells; they formed flattened colonies with sharp edges and proliferated indefinitely in the presence of basic FGF. They expressed the endogenous pluripotency markers c-MYC, KLF4, SOX2, OCT3/4, and NANOG, whereas the introduced human genes were completely silenced. Using in vitro differentiating conditions, rabbit iPS cells readily differentiated into ectoderm, mesoderm, and endoderm. They also formed teratomas containing a variety of tissues of all three germ layers in immunodeficient mice. Thus, the rabbit iPS cells fulfilled all of the requirements for the acquisition of the fully reprogrammed state, showing high similarity to their embryonic stem cell counterparts we generated recently. However, their global gene expression analysis revealed a slight but rigid difference between these two types of rabbit pluripotent stem cells. The rabbit model should enable us to compare iPS cells and embryonic stem cells under the same standardized conditions in evaluating their ultimate feasibility for pluripotent cell-based regenerative medicine in humans.     

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.