Self Renewal of Embryonic Stem Cells in the Absence of Feeder Cells and Exogenous Leukaemia Inhibitory Factor

Abstract

To evaluate the role of leukaemia inhibitory factor (LIF) for maintaining pluripotent embryonic stem (ES) cells in culture, we established several exogenous LIF-independent ES cell lines by continuous passaging in culture. The newly established ES cells, Kli and CBli, sustained their growth and remained undifferentiated in LIF-deficient medium. Analysis of chimaeric animals, produced with the p-galactosidase transgenic Kli ES cells, revealed that LDF-independent ES cells can contribute to all embryonic germ layers. There was no detectable LIF protein in ES cell conditioned medium, and no upregulation of LIF mRNA was found. The addition of neutralising anti-LIF antibodies was not sufficient to abrogate the self renewal of the Kli ES cells. These studies suggest that the signalling pathway involving diffusible LIF can be bypassed for maintaining the pluripotency in culture, and indicate a considerable heterogeneity in growth factor dependence and differentiation of different ES cells.     

Collagen IV differentially regulates planarian stem cell potency and lineage progression

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway.

Across the animal kingdom, stem cell function is regulated by the microenvironment in the surrounding niche (1), where the concentration of molecular signals for self-renewal and differentiation can be precisely regulated (2). The niche affects stem cell biology in many processes, such as aging and tissue regeneration, as well as pathological conditions such as cancer (3). Most studies have been done in tissues with large stem cell populations, such as the intestinal crypt (4) and the hair follicle (5) in mice. Elucidation of the role of the stem cell niche in tissue regeneration requires the study of animals with high regenerative potential, such as freshwater planarians (flatworms) (6). Dugesia japonica and Schmidtea mediterranea are two well-studied species that possess the ability to regenerate any missing body part (6, 7).Adult S. mediterranea maintain a high number of stem cells (neoblasts)—∼10 to 30% of all somatic cells in the adult worm—with varying potency, including pluripotent cells (8–14). Neoblasts are the only proliferating somatic cells: they are molecularly heterogeneous, but all express piwi-1 (15–18). Lineage-committed neoblasts are “progenitors” that transiently express both piwi-1 and tissue-specific genes (15, 19). Examples include early intestinal progenitors (γ neoblast, piwi-1⁺/hnf4⁺) (8, 10, 15, 19–21) and early epidermal progenitors (ζ neoblast, piwi-1⁺/zfp-1⁺) (8, 15). Other progenitor markers include collagen for muscles (22), ChAT for neurons (23), and cavII for protonephridia (24, 25). During tissue regeneration, neoblasts are recruited to the wound site, where they proliferate then differentiate to replace the missing cell types (16, 26). Some neoblasts express the pluripotency marker tgs-1, and are designated as clonogenic neoblasts (cNeoblasts) (10, 11). cNeoblasts are located in the parenchymal space adjacent to the gut (11).Neoblasts are sensitive to γ-irradiation and can be preferentially depleted in the adult planarian (27). After sublethal γ-irradiation, remaining cNeoblasts can repopulate the stem cell pool within their niche (10, 11). The close proximity of neoblasts to the gut suggests gut may be a part of neoblast niche (28, 29). When gut integrity was impaired by silencing gata4/5/6, the egfr-1/nrg-1 ligand-receptor pair, or wwp1, maintenance of non–γ-neoblasts were also disrupted (20, 30, 31), but whether that indicates the gut directly regulates neoblast remains unclear. There is evidence indicating the dorsal-ventral (D/V) transverse muscles surrounding the gut may promote neoblast proliferation and migration, with the involvement of matrix metalloproteinase mt-mmpB (32, 33). The central nervous system has also been implicated in influencing neoblast maintenance through the expression of EGF homolog neuregulin-7 (nrg-7), a ligand for EGFR-3, affecting the balance of neoblast self-renewal (symmetric or asymmetric division) (34).In other model systems, an important component of the stem-cell niche is the extracellular matrix (ECM) (35). Germline stem cells in Drosophila are anchored to niche supporting cells with ECM on one side, while the opposite side is exposed to differentiation signals, allowing asymmetric cell fate outcomes for self-renewal or differentiation following division (36–38). Few studies have addressed the ECM in planarians, largely due to the lack of genetic tools to manipulate the genome, the absence of antibodies to specific planarian ECM homologs, or the tools required to study cell fate changes. However, the genomes of D. japonica (39–41) and S. mediterranea (41–45), and single-cell RNA-sequencing (scRNA-seq) datasets for S. mediterranea are now available (11, 46–50). A recent study of the planarian matrisome demonstrated that muscle cells are the primary source of many ECM proteins (51), which, together with those produced by neoblasts and supporting parenchymal cells, may constitute components of the neoblast niche. For example, megf6 and hemicentin restrict neoblast’s localization within the parenchyma (51, 52). Functional studies also implicate ECM-modifiers, such as matrix metalloproteases (MMPs) in neoblast migration and regeneration. For example, reducing the activity of the ECM-degrading enzymes mt-mmpA (26, 33), mt-mmpB (53), or mmp-1 (33) impaired neoblast migration, proliferation, or overall tissue growth, respectively. Neoblasts are also likely to interact with ECM components of the niche via cell surface receptors, such as β1 integrin, inactivation of which impairs brain regeneration (54, 55).Here, we identified planarian ECM homologs in silico, followed by systematic functional assessment of 165 ECM and ECM-related genes by RNA interference (RNAi), to determine the effect on neoblast repopulation in planarians challenged by a sublethal dose of γ-irradiation (10). Surprisingly, multiple classes of collagens were shown to have the strongest effects. In particular, we show that the type IV collagens (COLIV) of basement membranes (BMs), were required to regulate the repopulation of neoblasts as well as lineage progression to progenitor cells. Furthermore, our data support an interaction between COLIV and the discoidin domain receptor (DDR) in neurons that activates signaling of NRG-7 in the neoblasts to regulate neoblast self-renewal versus differentiation. Together, these data demonstrate multifaceted regulation of planarian stem cells by ECM components.     

Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad-) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad- cells. Similarly, proliferation of the 3+Ecad- cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3-Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad- subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad- cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics.     

Unique and redundant roles of SOX2 and SOX17 in regulating the germ cell tumor fate

Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma-like TCam-2 cells to SOX17 in somatic cells and SOX2 in EC-like 2102EP cells. In seminoma-like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17-binding sites overlap in seminoma-like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma-like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC-like cells SOX2 regulates pluripotency-associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.     

干细胞维持多潜能性的能力与机制

背景：近年研究发现,成体细胞能够重新编程回到胚胎状态,这为利用成体干细胞治疗疾病提供了可能。现在虽然有不少关于干细胞增殖能力及自我更新的研究报道,但对其机制还缺乏深入的了解。 目的：对国内外关于干细胞维持多潜能性的能力与机制研究作一综述。 方法：应用计算机检索PubMed数据库中1993-01/2010-03关于干细胞维持多潜能性能力机制的文章,在标题和摘要中以“stem cells, pluripotency, mechanism”为检索词进行检索。选择文章内容与干细胞多潜能性能力维持机制有关者,同一领域文献则选择择近期发表或发表在权威杂志文章。初检得到122篇文献,根据纳入标准选择33篇文献进行综述。 结果与结论：干细胞主要通过一些主要的转录因子的表达、表观遗传学的调控和一些microRNAs(miRNAs)的作用来维持其多潜能性和自我更新能力。转录因子调节网络中的主要成员Oct4、Nanog及Sox2和一些表观遗传学修饰如组蛋白和DNA的甲基化以及miRNAs共同作用来抑制那些促进干细胞分化的基因表达和激活那些有助于维持干细胞多能性维持的基因表达,进而形成一个相互调控和依存的网络来维持干细胞的多能性和自我更新。     

Human embryonic stem cells as a model for early human development

Although, the study of human development--from fertilized egg to mature embryo--is extremely important, the early differentiation of human tissues remains an enigma. Moreover, the relatively high percentage of unexplained pregnancy loss--a major concern of human embryologists and gynaecologists--emphasizes the need for an appropriate model for studying early human development. The availability of human pluripotent stem cells might allow us to study previously inaccessible basic processes that occur during human embryogenesis, such as gastrulation and organogenesis.     

Novel triblock co‐polymer nanofibre system as an alternative support for embryonic stem cells growth and pluripotency

Conventionally, embryonic stem cells (ESCs) are cultured on gelatin or over a mitotically inactivated monolayer of mouse embryonic fibroblasts (MEFsi). Considering the lack of versatile, non‐animal‐derived and inexpensive materials for that purpose, we aimed to find a biomaterial able to support ESC growth in a pluripotent state that avoids the need for laborious and time‐consuming MEFsi culture in parallel with mouse ESC (mESC) culture. Undifferentiated mESCs were cultured in a new nanofibre material designed for ESC culture, which is based on the self‐assembly of a triblock co‐polymer, poly(ethyleneglycol‐β‐trimethylsilyl methacrylate‐β‐methacrylic acid), conjugated with the peptide glycine–arginine–glycine–aspartate–serine, to evaluate its potential application in ESC research. The morphology, proliferation, viability, pluripotency and differentiation potential of mESCs were assessed. Compared to conventional stem cell culture methodologies, the nanofibres promoted a higher increase in mESCs number, enhanced pluripotency and were able to support differentiation after long‐term culture. This newly developed synthetic system allows the elimination of animal‐derived matrices and provides an economic method of ESC culture, made of a complex network of nanofibres in a scale similar to native extracellular matrices, where the functional properties of the cells can be observed and manipulated. Copyright © 2013 John Wiley & Sons, Ltd.