Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.     

Feeder-free growth of undifferentiated human embryonic stem cells

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.     

Feeder layer- and serum-free culture of human embryonic stem cells

In addition to their contribution to the research on early human development, human embryonic stem (hES) cells may also be used for cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast feeder layers, which allow their continuous growth in an undifferentiated state. However, the use of hES cells in human therapy requires an animal-free culture system, in which exposure to mouse retroviruses is avoided. In this study we present a novel feeder layer-free culture system for hES cells, based on medium supplemented with 15% serum replacement, a combination of growth factors including transforming growth factor beta1 (TGFbeta1), leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix. Human ES cells grown in these conditions maintain all ES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. The culture system presented here has two major advantages: 1) application of a well-defined culture system for hES cells and 2) reduced exposure of hES cells to animal pathogens. The feeder layer-free culture system reported here aims at facilitating research practices and providing a safer alternative for future clinical applications of hES cells.     

人胚胎干细胞优化培养的进展

人胚胎干细胞(humanembryonicstemcell,hEScell)是来源于着床前人囊胚内细胞团(innercellmass,ICM)的、具有自我更新能力和分化全能性的细胞。由于hES细胞能在一定条件下分化成三个胚层来源的各种细胞,所以它具有重要的基础研究价值和巨大的临床应用前景,可应用于人早期胚胎发育过程的研究、药物毒物筛选、细胞移植治疗、基因治疗等领域。目前,世界上已经建立了多株hES细胞系,最早建立的hES细胞系是生长在小鼠胚胎成纤维(mouseembryonicfibroblast,MEF)细胞上的,培养体系中含血清等动物源性成分,这些成分可能引起动物源性病原体或支原体的污染,从而限制了hES细胞的临床应用。近年来,科学家们在优化hES细胞的体外培养体系方面做出了很大的努力并取得了长足进展,已经开始采用无血清、无饲养层细胞、无外源性蛋白、成分明确的培养体系进行hES细胞建系及培养,从而在一定程度上解决了上述问题。本文主要从饲养层细胞、无饲养层培养体系、培养基质、细胞因子等方面综述了hES细胞建系和维持其未分化状态的优化培养所取得的最新进展和存在的问题。     

无饲养层培养人胚胎干细胞方法的建立

人胚胎干细胞(human embryonic stem cell,hES细胞)是当前医学研究的热点之一．然而hES细胞培养条件苛刻,通常需要采用鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEFs)饲养层来维持其未分化状态,成为目前hES细胞研究的瓶颈之一、本实验成功地将hES细胞接种在细胞外基质包被的六孔板上培养,传代20次后细胞仍然保持良好的未分化状态,各种hES细胞生物学特性(如表面标志物SSEA-3、SSEA-4、TRA-1-60和TRA-1-8l,OCT-4,碱性磷酸酶及体内外分化潜能等)均无改变;其冻存、复苏效果与生长在饲养层上的hES细胞无明显差异．因此,该无饲养层培养体系可以用于培养hES细胞,并为hES细胞转基因研究及大规模培养打下良好的基础．     

Human feeder layers for human embryonic stem cells

Human embryonic stem (hES) cells hold great promise for future use in various research areas, such as human developmental biology and cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast (MEF) feeder layers, which permit continuous growth in an undifferentiated stage. To use these unique cells in human therapy, an animal-free culture system must be used, which will prevent exposure to mouse retroviruses. Animal-free culture systems for hES cells enjoy three major advantages in the basic culture conditions: 1). the ability to grow these cells under serum-free conditions, 2). maintenance of the cells in an undifferentiated state on Matrigel matrix with 100% MEF-conditioned medium, and 3). the use of either human embryonic fibroblasts or adult fallopian tube epithelial cells as feeder layers. In the present study, we describe an additional animal-free culture system for hES cells, based on a feeder layer derived from foreskin and a serum-free medium. In this culture condition, hES cells maintain all embryonic stem cell features (i.e., pluripotency, immortality, unlimited undifferentiated proliferation capability, and maintenance of normal karyotypes) after prolonged culture of 70 passages (>250 doublings). The major advantage of foreskin feeders is their ability to be continuously cultured for more than 42 passages, thus enabling proper analysis for foreign agents, genetic modification such as antibiotic resistance, and reduction of the enormous workload involved in the continuous preparation of new feeder lines.     

Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium

Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen contamination that would exacerbate an immune response to foreign proteins, and the potential risk of transmission of retroviral and other zoonotic pathogens to humans. As a step towards this goal, we derived a new hES cell line (MISCES-01) on human adult skin fibroblasts as feeder cells using serum replacement (SR) medium. The MISCES-01 cells have a normal diploid karyotype (46XX), express markers of pluripotency (OCT4, GCTM-2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and alkaline phosphatase) and following in vitro and in vivo differentiation, give rise to derivatives of the three primary germ layers. This cell line can be obtained for research purposes from the Australian Stem Cell Centre (http://www.stemcellcentre.edu.au).     

Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells

Previous reports have demonstrated the growth of undifferentiated human embryonic stem (HES) cells on mouse embryonic fibroblast (MEF) feeders and on laminin- or Matrigel-coated plastic surfaces supplemented with MEF-conditioned medium. These xenosupport systems run the risk of cross-transfer of animal pathogens from the animal feeder, matrix, or conditioned medium to the HES cells, thus compromising later clinical application. Here we show that human fetal and adult fibroblast feeders support prolonged undifferentiated HES cell growth of existing cell lines and are superior to cell-free matrices (collagen I, human extracellular matrix, Matrigel, and laminin) supplemented with human or MEF feeder-conditioned medium. Additionally, we report the derivation and establishment of a new HES cell line in completely animal-free conditions. Like HES cells cultured on MEF feeders, the HES cells grown on human feeders had normal karyotypes, tested positive for alkaline phosphatase activity, expressed Oct-4 and cell surface markers including SSEA-3, SSEA-4, Tra 1-60, and GCTM-2, formed teratomas in severely combined immunodeficient (SCID) mice, and retained all key morphological characteristics. Human feeder#150;supported HES cells should provide a safer alternative to existing HES cell lines in therapeutic applications.     

A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells

Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs.     

Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells

In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2), and transforming growth factor β (TGF-β), thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.     

A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: Comparative expression profiles of mRNAs,microRNAs and proteins among different feeders and conditioned media

Background  

Human embryonic stem (hES) cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF) feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages.     

Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces

Previous studies have shown that cultivation of undifferentiated human embryonic stem (hES) cells requires human fibroblasts (hF) or mouse embryonic fibroblast (mEF) feeders or a coating matrix such as laminin, fibronectin or Matrigel in combination with mEF or hF conditioned medium. We here demonstrate a successful feeder-free and matrix-free culture system in which undifferentiated hES cells can be cultured directly on plastic surfaces without any supportive coating, in a hF conditioned medium. The hES cells cultured directly on plastic surfaces grow as colonies with morphology very similar to cells cultured on Matrigel(TM). Two hES cell lines SA167 and AS034.1 were adapted to matrix-free growth (MFG) and have so far been cultured up to 43 passages and cryopreserved successfully. The lines maintained a normal karyotype and expressed the expected marker profile of undifferentiated hES cells for Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and SSEA-1. The hES cells formed teratomas in SCID mice and differentiated in vitro into derivates of all three germ layers. Thus, the MFG-adapted hES cells appear to retain pluripotency and to remain undifferentiated. The present culture system has a clear potential to be scaleable up to a manufacturing level and become the preferred culture system for various applications such as cell therapy and toxicity testing.     

Indole derivatives sustain embryonic stem cell self-renewal in long-term culture

Embryonic stem cells (ESCs), which have characteristics such as self-renewal, indefinite proliferation, and pluripotency, are thought to hold great promise for regenerative medicine. ESCs are generally cultured on mouse embryonic fibroblast (MEF) or MEF conditioned medium (MEF-CM). However, for therapeutic applications, it is preferable for ESCs to be cultured under chemically defined conditions. Here, we report synthetic compounds that allow expansion of undifferentiated mouse ESCs in the absence of MEF, Leukemia Inhibitory Factor (LIF), and Fetal Bovine Serum (FBS). ESCs cultured for more than 30 d in a serum-free medium supplemented with indole derivertives retained their characteristic morphology and expressed markers such as SSEA-1, OCT3/4, Rex-1, Sox2, and Nanog. They consistently differentiated into many types of cells, including neurons, muscle cells, and hepatocytes. These results indicate that our compounds provide a more efficient and safer large-scale culture system for pluripotent ESCs, and hence might contribute to the use of ESCs in therapeutic applications.     

Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines

The role of individual supplements necessary for the self-renewal of human embryonic stem (hES) cells is poorly characterized, and furthermore we have found that previously reported feeder cell- and serum-free culture systems used for individual hES cell lines are unable to maintain HUES7 cells for more than one passage. We have therefore derived a feeder/serum-free culture system that can support the long-term (at least 10 passages) self-renewal of several euploid hES cell lines including MAN1, HUES7, and HUES1 with minimal spontaneous differentiation and without the need for manual propagation. This system contains fibroblast growth factor 2, activin A, neurotrophin 4, and the N2, B27 supplements together with a human fibronectin substrate. We demonstrate that these components exert distinct functions: both FGF2 and activin A were necessary to prevent differentiation of hES cells while NT4 promoted cell survival, FGF2 could not be substituted by IGFII, and the fibronectin substrate supported a rapid rate of hES culture expansion. Inhibition studies showed that β1 integrin-dependent attachment of hES cells to fibronectin was at least partially via the α5 subunit but independent of integrin αV.     

Fabrication of synthetic polymer coatings and their use in feeder-free culture of human embryonic stem cells

The culture of human embryonic stem (hES) cells in defined and xenogeneic-free conditions will contribute substantially to future biotechnological and medical applications. To achieve this goal, we developed the first fully defined synthetic polymer coating poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) that sustains long-term growth of hES cells in different culture media. Here we describe a detailed protocol for the reproducible fabrication of PMEDSAH coating on tissue culture polystyrene dishes, and for the feeder-free culture of hES cells on PMEDSAH coating in defined culture medium. This culture system represents a key step toward the fully defined and xenogeneic-free culture of hES cells.     

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

We previously showed that Wnt3a could stimulate human embryonic stem （hES） cell proliferation and affect cell fate determination. In the absence of feeder cell--derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wntl and Wnt5a. While Wntl displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wntl enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wntl gene in immortalized human adult fibroblast （HAFi） cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt tmnsgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells （W3R） that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.     

Human embryonic stem cells: caught between a ROCK inhibitor and a hard place

Since their derivation, human embryonic stem (hES) cells have been used for a variety of applications including developmental biology, pathology, chemical biology, genomics, and proteomics. However, their most important potential application is the generation of cells and tissues, which can be used for cell‐based therapies. One of the main drawbacks of hES cell culture is that they are particularly sensitive to dissociation, which is required for passaging, expansion, cryopreservation, and other applications. Recently, it has been discovered that an inhibitor of Rho kinase (ROCKi; Y‐27632) increases the survival rate of dissociated, single hES cells. This breakthrough has allowed new methods in hES cell culture to be developed, with the promise of increasing hES cell numbers into the realm of clinical relevance. In our studies demonstrating that ROCKi dramatically increases hES cell cryopreservation efficiency, we have observed that ROCKi treatment does not decrease hES cell's susceptibility to apoptosis. Rather, we hypothesize that ROCKi treatment desensitizes single hES cells to their environment reducing the odds that individual cells will undergo anoikis.     

LMP1 of Epstein-Barr virus induces proliferation of primary mouse embryonic fibroblasts and cooperatively transforms the cells with a p16-insensitive CDK4 oncogene

The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-associated malignancies including nasopharyngeal carcinoma and Hodgkin's lymphoma. Previous work demonstrates that the LMP1 gene of EBV is sufficient to transform certain established rodent fibroblast cell lines and to induce the tumorigenicity of some human epithelial cell lines. In addition, LMP1 plays pleiotropic roles in cell growth arrest, differentiation, and apoptosis, depending on the background of the target cells. To examine the roles of LMP1 in cell proliferation and growth regulation in primary culture cells, we constructed a recombinant retrovirus containing an LMP1 gene. With this retrovirus, LMP1 was shown to stimulate the proliferation of primary mouse embryonic fibroblasts (MEF cells). It has a mitogenic activity for MEF cells, as demonstrated by an immediate induction of cell doubling time. In addition, it significantly extends the passage number of MEF cells to more than 30 after retroviral infection, compared with less than 5 for uninfected MEF cells. Furthermore, LMP1 cooperates with a p16-insensitive CDK4(R24C) oncogene in transforming MEF cells. Our results provide the first evidence of the abilities of the LMP1 gene, acting alone, to effectively induce the proliferation of primary MEF cells and of its cooperativity with another cellular oncogene in transforming primary cells.