Cytoskeletal Expression and Remodeling in Pluripotent Stem Cells

Many emerging cell-based therapies are based on pluripotent stem cells, though complete understanding of the properties of these cells is lacking. In these cells, much is still unknown about the cytoskeletal network, which governs the mechanoresponse. The objective of this study was to determine the cytoskeletal state in undifferentiated pluripotent stem cells and remodeling with differentiation. Mouse embryonic stem cells (ESCs) and reprogrammed induced pluripotent stem cells (iPSCs), as well as the original un-reprogrammed embryonic fibroblasts (MEFs), were evaluated for expression of cytoskeletal markers. We found that pluripotent stem cells overall have a less developed cytoskeleton compared to fibroblasts. Gene and protein expression of smooth muscle cell actin, vimentin, lamin A, and nestin were markedly lower for ESCs than MEFs. Whereas, iPSC samples were heterogeneous with most cells expressing patterns of cytoskeletal proteins similar to ESCs with a small subpopulation similar to MEFs. This indicates that dedifferentiation during reprogramming is associated with cytoskeletal remodeling to a less developed state. In differentiation studies, it was found that shear stress-mediated differentiation resulted in an increase in expression of cytoskeletal intermediate filaments in ESCs, but not in iPSC samples. In the embryoid body model of spontaneous differentiation of pluripotent stem cells, however, both ESCs and iPSCs had similar gene expression for cytoskeletal proteins during early differentiation. With further differentiation, however, gene levels were significantly higher for iPSCs compared to ESCs. These results indicate that reprogrammed iPSCs more readily reacquire cytoskeletal proteins compared to the ESCs that need to form the network de novo. The strategic selection of the parental phenotype is thus critical not only in the context of reprogramming but also the ultimate functionality of the iPSC-differentiated cell population. Overall, this increased characterization of the cytoskeleton in pluripotent stem cells will allow for the better understanding and design of stem cell-based therapies.     

Circadian Clock Genes Are Essential for Normal Adult Neurogenesis,Differentiation, and Fate Determination

Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during differentiation, but they generated normal percentages of neuronal cells. Neuronal fate commitment therefore appears to be controlled through a non-clock function of BMAL1. This study provides insight into how cell autonomous circadian clocks and clock genes regulate adult neural stem cells with implications for treating neurodegenerative disorders and impaired brain functions by manipulating neurogenesis.     

Ell3 enhances differentiation of mouse embryonic stem cells by regulating epithelial-mesenchymal transition and apoptosis

Ell3 is a testis-specific RNA polymerase II elongation factor whose cellular function is not clear. The present study shows that Ell3 is activated during the differentiation of mouse embryonic stem cells (mESCs). Furthermore, Ell3 plays a critical role in stimulating lineage differentiation of mESCs by promoting epithelial-mesenchymal transition (EMT) and suppressing apoptosis. Mouse ESCs engineered to stably express Ell3 were rapidly differentiated compared with control cells either under spontaneous differentiation or neural lineage-specific differentiation conditions. Gene expression profile and quantitative RT-PCR analysis showed that the expression of EMT markers, such as Zeb1 and Zeb2, two major genes that regulate EMT, was upregulated in Ell3-overexpressing mESCs. Remarkably, knockdown of Zeb1 attenuated the enhanced differentiation capacity of Ell3-overexpressing mESCs, which indicates that Ell3 plays a role in the induction of mESC differentiation by inducing EMT. In contrast to Ell3-overexpressing mESCs, Ell3-knock down mESCs could not differentiate under differentiation conditions and, instead, underwent caspase-dependent apoptosis. In addition, apoptosis of differentiating Ell3-knock out mESCs was associated with enhanced expression of p53. The present results suggest that Ell3 promotes the differentiation of mESCs by activating the expression of EMT-related genes and by suppressing p53 expression.     

Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.     

人类及小鼠胚胎干细胞的不同多能性状态

胚胎干细胞(embryonic stem cells,ESCs)是来源于早期胚胎的全能性细胞,在合适条件下具有分化为任何一类成体细胞的潜力。在小鼠中,根据细胞来源的胚胎发育时间,ESCs可以被分为原始态多能性(na(?)ve pluripotency)和始发态多能性(primed pluripotency)两种状态。这两种状态的细胞在发育上相互联系,具有不同的形态、信号依赖、发育性质、基因表达及表观遗传学性质,并且在特定的条件下可以相互转化。人类胚胎干细胞(human embryonic stem cells,hESCs)的发育潜能曾一度被认为低于小鼠胚胎干细胞(mouse embryonic stem cells,mESCs),直到人类原始态胚胎干细胞的发现证明了hESCs可以表现出与mESCs相似的性质。这对于人类胚胎发育的研究及ESCs在临床治疗上的实际应用都具有重要的意义。     

Clock controls timing of mouse pancreatic differentiation through regulation of Wnt- and Notch-based and cell division components

The oscillations of circadian genes control the daily circadian clock, regulating a diverse array of physiologies with the 24-hour light/dark cue across a wide variety of organisms. Here we first show that before embryonic circadian rhythms occur, the oscillation (nucleocytoplasmic shuttling) of core circadian gene Clock is tissue-specific and correlated with the state of differentiation during both early development and later pancreas organogenesis. Disruption of Clock as well as Timeless in the embryonic pancreas does not block pancreatic differentiation but alters the balance and maturity of endocrine and exocrine cells. Molecular analysis indicates that inhibition of Clock or Timeless expression disturbs not only cell cycle regulators, but also Wnt- and Notch-signaling components, whose oscillations establish the timing mechanism in somitogenesis. Thus, our results provide new insights about circadian genes' function in control of the timing of differentiation during embryonic development.     

Blocking autocrine VEGF signaling by sunitinib,an anti-cancer drug,promotes embryonic stem cell self-renewal and somatic cell reprogramming

Maintaining the self-renewal of embryonic stem cells (ESCs) could be achieved by activating the extrinsic signaling, i.e., the use of leukemia inhibitory factor (LIF), or blocking the intrinsic differentiation pathways, i.e., the use of GSK3 and MEK inhibitors (2i). Here we found that even in medium supplemented with LIF, mESCs still tend to differentiate toward meso-endoderm lineages after long-term culture and the culture spontaneously secretes vascular endothelial growth factors (VEGFs). Blocking VEGF signaling with sunitinib, an anti-cancer drug and a receptor tyrosine kinase (RTK) inhibitor mainly targeting VEGF receptors (VEGFRs), is capable of maintaining the mESCs in the undifferentiated state without the need for feeder cells or LIF. Sunitinib facilitates the derivation of mESCs from blastocysts, and the mESCs maintained in sunitinib-containing medium remain pluripotent and are able to contribute to chimeric mice. Sunitinib also promotes iPSC generation from MEFs with only Oct4. Knocking down VEGFR2 or blocking it with neutralizing antibody mimicks the effect of sunitinib, indicating that blocking VEGF/VEGFR signaling is indeed beneficial to the self-renewal of mESCs. We also found that hypoxia-inducible factor alpha (HIF1α) and endoplasmic reticulum (ER) stress are involved in the production of VEGF in mESCs. Blocking both pathways inhibits the expression of VEGF and prevents spontaneous differentiation of mESCs. Interestingly, LIF may also exert its effect by downregulating HIF1α and ER stress pathways and subsequent VEGF expression. These results indicate the existence of an intrinsic differentiation pathway in mESCs by activating the autocrine VEGF signaling. Blocking VEGF signaling with sunitinib or other small molecules help to maintain the mESCs in the ground state of pluripotency.     

Induced pluripotent stem cells’ self-renewal and pluripotency is maintained by a bovine granulosa cell line-conditioned medium

Induced pluripotent stem cells (iPSCs) are a promising type of stem cells, comparable to embryonic stem cells (ESCs) in terms of self-renew and pluripotency, generated by reprogramming somatic cells. These cells are an attractive approach to supply patient-specific pluripotent cells, for producing in vitro models of disease, drug discovery, toxicology and potentially treating degenerative disease circumventing immune rejection. In spite of the great advance since iPSCs’ establishment, their obtention and propagation is an increasing area of great interest.In a recent work, we have shown that the conditioned medium from a bovine granulosa cell line (BGC-CM) is able to preserve the basic properties of mESCs. Therefore, based on our previous results and the reported resemblance between iPSCs and ESCs, we hypothesized that BGC-CM could provide a favorable context to culturing iPSCs. In this work, we have reprogrammed mouse embryonic fibroblasts obtaining iPSC lines, and showed that they can be propagated in BGC-CM while maintaining self-renewal and pluripotency, evidenced by expression of specific gene markers and capability of in vitro and in vivo differentiation to cell types from the three germ layers. We believe that these findings may provide a novel context to propagate iPSCs to study the molecular mechanisms involved in self-renewal and pluripotency.     

Selection of RNA aptamers against mouse embryonic stem cells

Embryonic stem cells (ESCs) are capable of unlimited self-renewal and differentiation into multiple cell types. Recent large-scale analyses have identified various cell surface molecules on ESCs. Some of them are considered to be beneficial markers for characterization of cellular phenotypes and/or play an essential role for regulating the differentiation state. Thus, it is desired to efficiently produce affinity reagents specific to these molecules. In this study, to develop such reagents for mouse ESCs (mESCs), we selected RNA aptamers against intact, live mESCs using several selection strategies. The initial selection provided us with several anti-mESC aptamers of distinct sequences, which unexpectedly react with the same molecule on mESCs. Then, to isolate aptamers against different surface markers on mESCs, one of the selected aptamers was used as a competitor in the subsequent selections. In addition, one of the selections further employed negative selection against differentiated mouse cells. Consequently, we successfully isolated three classes of anti-mESC aptamers that do not compete with one another. The isolated aptamers were shown to distinguish mESCs from differentiated mouse cell lines and trace the differentiation process of mESCs. These aptamers could prove useful for developing molecular probes and manipulation tools for mESCs.     

Cell growth arrest and apoptosis induced by Oct4 or Nanog knockdown in mouse embryonic stem cells: a possible role of Trp53

It has been clear that both Oct4 and Nanog play essential roles in maintaining embryonic stem cells (ESCs) undifferentiation. However, the roles of Oct4 and Nanog in ESCs growth and apoptosis have been much less explored. In this study, we systematically examined the effects of Oct4 or Nanog knockdown on mouse ESCs (mESCs) growth and apoptosis as well as potential mechanisms. Our results show that Oct4 or Nanog knockdown induces growth arrest and apoptosis in mESCs, indicating that the two genes also play important roles in mESCs survival and growth. Moreover, upregulation in Trp53 and its downstream genes expression was detected in Oct4 or Nanog knockdown mESCs, suggesting a possible role of Trp53 in Oct4 or Nanog knockdown induced mESCs growth arrest and apoptosis.     

印迹基因修饰使孤雌胚胎干细胞获得四倍体补偿能力

孤雌胚胎干细胞(Parthenogenetic embryonic stem cells,pESCs)的遗传物质全部来源于母源基因组,因缺失父源基因而不具备四倍体补偿的能力。为了使pESCs也具备发育到个体的能力,呈现与受精卵来源ESCs类似的多能性,文中借助CRISPR/Cas9系统对孤雌来源的pESCs中的2个重要母源印迹基因的差异甲基化区域(Differentially methylated region,DMR)进行单等位基因敲除(H19-DMR,IG-DMR),获得双基因敲除的(DKO)pESCs。结果表明,pESCs虽然来源于母源基因组,但是其形态特征、多能干性标记分子的表达水平、体外神经分化能力与受精卵来源的ESCs基本一致。最后,通过基因修饰的DKOpESCs可以通过四倍体补偿获得发育到期的胎儿,表明经过印迹基因修饰的pESCs也具有发育到一个完整个体的多能性。从而为再生医学研究提供了一类具有主要组织相容性复合基因匹配且多能性良好的资源细胞。     

Inhibition of MAGEA2 regulates pluripotency,proliferation, apoptosis,and differentiation in mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) exhibit self-renewal and pluripotency, can differentiate into all three germ layers, and serve as an essential model in stem cell research and for potential clinical application in regenerative medicine. Melanoma-associated antigen A2 (MAGEA2) is not expressed in normal somatic cells but rather in different types of cancer, especially in undifferentiated cells, such as in the testis, differentiating cells, and ESCs. However, the role of MAGEA2 in mESCs remains to be clarified. Accordingly, in this study, we examined the expression and functions of MAGEA2 in mESCs. MAGEA2 messenger RNA (mRNA) expression was decreased during mESCs differentiation. MAGEA2 function was then evaluated in knockdown mESC. MAGEA2 knockdown resulted in decreased pluripotency marker gene expression in mESCs consequent to increased Erk1/2 phosphorylation. Decreased MAGEA2 expression inhibited mESC proliferation via S phase cell cycle arrest with a subsequent decrease in cell cycle-associated genes Cdk1, Cdk2, Cyclin A1, Cyclin D1, and Cdc25a. Apoptotic mESCs markedly increased along with cleaved forms of caspases 3, 6, and 7 and PARP expression, confirming caspase-dependent apoptosis. MAGEA2 knockdown significantly decreased embryoid body size in vitro when cells were differentiated naturally and teratoma size in vivo, concomitant with decreased ectoderm marker gene expression. These findings suggested that MAGEA2 regulates ESC pluripotency, proliferation, cell cycle, apoptosis, and differentiation. The enhanced understanding of the regulatory mechanisms underlying diverse mESC characteristics will facilitate the clinical application of mESCs.     

miR-290家簇在小鼠胚胎干细胞中的表达与调控功能

微RNA（microRNA,miRNA）是一类约22nt的非编码小分子RNA,主要在转录后水平负调控基因表达,其在生物发商、疾病、肿瘤中行使着重要调控作用。胚胎干细胞（embryonic stem cell,ESC）具有发育的全能性,能分化出成体动物的所有组织和器官。研究和利用ESC是当前生物工程领域的热点之一。近年来,越来越多的研究表明,miRNA在ESC的自我更新、分化、命运决定等方面行使着重要的调控作用。其中,miR-290家簇是在鼠科动物ESC中特异且高表达的miRNA。本文综述了miR-290家簇在ESC中的表达、功能及其分子调控网络方面的研究进展。     

Equine embryos and embryonic stem cells: Defining reliable markers of pluripotency

Cartilage and tendon injuries are a significant source of animal wastage and financial loss within the horse-racing industry. Moreover, both cartilage and tendon have limited intrinsic capacity for self-repair, and the functionally inferior tissue produced within a lesion may reduce performance and increase the risk of reinjury. Stem cells offer tremendous potential for accelerating and improving tissue healing, and adult mesenchymal stem cells (MSCs) are already used to treat cartilage and tendon injuries in horses. However, MSCs are scarce in the bone marrow isolates used, have limited potential for proliferation and differentiation in vitro, and do not appear to noticeably improve long-term functional repair. Embryonic stem cells (ESCs) or induced pluripotent stem (iPS) cells could overcome many of the limitations and be used to generate tissues of value for equine regenerative medicine. To date, six lines of putative ESCs have been described in the horse. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit pluripotency in vivo. Moreover, it is becoming clear that the markers used to characterize the putative ESCs were inadequate, primarily because studies in domestic species have revealed that they are not specific to ESCs or the pluripotent inner cell mass, but also because the function of most in the maintenance of pluripotency is not known. Future derivation and validation of equine embryonic or other pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing horse embryo.     

The effect of extracellular matrix on the differentiation of mouse embryonic stem cells

Embryonic stem cells (ESCs) are promising research materials to investigate cell fate determination since they have the capability to differentiate. Stem cell differentiation has been extensively studied with various microenvironment mimicking structures to modify cellular dynamics associated with the cell-extracellular matrix (ECM) interactions and cell-cell communications. In the current study, our aim was to determine the effect of microenvironmental proteins with different concentrations on the capacity and differentiation capability of mouse ESCs (mESCs), combining the biochemical assays, imaging techniques, Fourier transform infrared (FTIR) spectroscopy, and unsupervised multivariate analysis. Based on our data, coating the surface of mESCs with Matrigel, used as an acellular matrix substrate, resulted in morphological and biochemical changes. mESCs exhibited alterations in their phenotype after growing on the Matrigel-coated surfaces, including their differentiation capacity, cell cycle phase pattern, membrane fluidity, and metabolic activities. In conclusion, mESCs can be stimulated physiologically, chemically, or mechanically to convert them a new phenotype. Thus, identification of ESCs’ behavior in the acellular microenvironment could be vital to elucidate the mechanism of diseases. It might also be promising to control the cell fate in the field of tissue engineering.