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.     

Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media

The unique pluripotential characteristic of human embryonic stem cells heralds their use in fields such as medicine, biotechnology, biopharmaceuticals, and developmental biology. However, the current availability of sufficient quantities of embryonic stem cells for such applications is limited, and generating sufficient numbers for downstream therapeutic applications is a key concern. In the absence of feeder layers or their conditioned media, human embryonic stem cells readily differentiate to form embryoid bodies, indicating that trophic factors secreted by the feeder layers are required for long-term proliferation and maintenance of pluripotency. Adding further complexity to the elucidation of the factors required for the maintenance of pluripotency is the variability of different fibroblast feeder layers (of mouse or human origin) to effectively support human embryonic stem cells. Currently, the deficiency of knowledge concerning the exact identity of factors within the pathways for self-renewal illustrates that a number of factors may be required to support pluripotent, undifferentiated growth of human embryonic stem cells. This study utilized a proteomic analysis (multidimensional chromatography coupled to tandem mass spectrometry) to isolate and identify proteins in the conditioned media of three mitotically inactivated fibroblast lines (human fetal, human neonatal, and mouse embryonic fibroblasts) used to support the undifferentiated growth of human embryonic stem cells. One-hundred seventy-five unique proteins were identified between the three cell lines using a 相似文献   

Establishment of hamster blastocyst-derived embryonic stem (ES) cells

The establishment of four ES cell lines from the Syrian "golden" hamster (Mesocricetus auratus) is described. The cells can be maintained in the undifferentiated state when grown on primary mouse embryonic fibroblast feeder layers. In suspension culture they spontaneously differentiate into embryoid bodies of increasing complexity which contain a variety of tissues including embryonic ectoderm and myocardium. All four lines--one female and three male--are karyotypically normal with 44 chromosomes. Hamster is the second species from which ES cells have been established. As in mouse, the cells should be useful for developmental and transgenic studies.     

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.     

Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells

Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.     

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.     

Comparative study of mouse and human feeder cells for human embryonic stem cells

Various types of feeder cells have been adopted for the culture of human embryonic stem cells (hESCs) to improve their attachment and provide them with stemness-supporting factors. However, feeder cells differ in their capacity to support the growth of undifferentiated hESCs. Here, we compared the expression and secretion of four well-established regulators of hESC pluripotency and/or differentiation among five lines of human foreskin fibroblasts and primary mouse embryonic fibroblasts throughout a standard hESC culture procedure. We found that human and mouse feeder cells secreted comparable levels of TGF beta 1. However, mouse feeder cells secreted larger quantities of activin A than human feeder cells. Conversely, FGF-2, which was produced by human feeder cells, could not be detected in culture media from mouse feeder cells. The quantity of BMP-4 was at about the level of detectability in media from all feeder cell types, although BMP-4 dimers were present in all feeder cells. Production of TGF beta 1, activin A, and FGF-2 varied considerably among the human-derived feeder cell lines. Low- and high-producing human feeder cells as well as mouse feeder cells were evaluated for their ability to support the undifferentiated growth of hESCs. We found that a significantly lower proportion of hESCs maintained on human feeder cell types expressed SSEA3, an undifferentiated cell marker. Moreover, SSEA3 expression and thus the pluripotent hESC compartment could be partially rescued by addition of activin A. Cumulatively, these results suggest that the ability of a feeder layer to promote the undifferentiated growth of hESCs is attributable to its characteristic growth factor production.     

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.     

Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts were used, a mechanical method of passaging led to better cell growth than passaging by trypsin digestion. We also found that exogenous supplementation with leukemia inhibitory factor maintained the embryonic stem cell-like cells in an undifferentiated state, whereas addition of stem cell factor resulted in their differentiation. Our findings provide an experimental basis for the establishment of an effective culture system for bovine embryonic stem cells.     

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.     

A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. Fibroblast feeder layers are used to maintain the undifferentiated growth of hES cells and an examination of the conditioned media (CM) of human neonatal fibroblasts (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin fibroblast line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated growth of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and growth factor functional categories were considered most likely to be involved in the maintenance of hES cell growth, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Wnt signaling and inhibition of bone morphogenetic proteins.     

Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture

Fibroblast feeder cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem cells (hESCs). The feeder cells secrete various growth factors and extracellular matrix (ECM) proteins into extracellular milieu. However, the roles of the feeder cell-secreted factors are largely unclear. Animal feeder cells and use of animal serum also make current feeder cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free feeder cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) feeder cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) feeder cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive feeder cells produce laminin-511 and express laminin-binding integrins α3ß1, α6ß1 and α7ß1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct feeder cells.     

Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. These cells have, therefore, potential for in vitro differentiation studies, gene function, and so on. The aim of this study was to produce a human embryonic stem cell line. An inner cell mass of a human blastocyst was separated and cultured on mouse embryonic fibroblasts in embryonic stem cell medium with related additives. The established line was evaluated by morphology; passaging; freezing and thawing; alkaline phosphatase; Oct-4 expression; anti-surface markers including Tra-1-60 and Tra-1-81; and karyotype and spontaneous differentiation. Differentiated cardiomyocytes and neurons were evaluated by transmission electron microscopy and immunocytochemistry. Here, we report the derivation of a new embryonic stem cell line (Royan H1) from a human blastocyst that remains undifferentiated in morphology during continuous passaging for more than 30 passages, maintains a normal XX karyotype, is viable after freezing and thawing, and expresses alkaline phosphatase, Oct-4, Tra-1-60, and Tra-1-81. These cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers in the presence or absence of recombinant human leukemia inhibitory factor. Royan H1 cells can differentiate in vitro in the absence of feeder cells and can produce embryoid bodies that can further differentiate into beating cardiomyocytes as well as neurons. These results define Royan H1 cells as a new human embryonic stem cell line.     

Derivation and characterization of pluripotent embryonic germ cells in chicken

Embryonic germ (EG) cell lines established from primordial germ cells (PGCs) are undifferentiated and pluripotent stem cells. To date, EG cells with proven germ-line transmission have been completely established only in the mouse with embryonic stem (ES) cells. We isolated PGCs from 5.5-day-old (stage 28) chicken embryonic gonads and established a putative chicken EG cell line with EG culture medium supplemented with stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), interleukin-11 (IL-11), and insulin-like growth factor-I (IGF-I). These cells grew continuously for ten passages (4 months) on a feeder layer of mitotically active chicken embryonic fibroblasts. After several passages, these cells were characterized by screening with the periodic acid-Schiff reaction, anti-SSEA-1 antibody, and a proliferation assay. The chicken EG cells maintained characteristics of gonadal PGCs and undifferentiated stem cells. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types. The chicken EG cells were injected into stage X blastodermal layer and produced chimeric chickens with various differentiated tissues derived from the EG cells. Chicken EG cells will be useful for the production of transgenic chickens and for studies of germ cell differentiation and genomic imprinting.     

Expansion of pluripotent human embryonic stem cells on human feeders

Human embryonic stem cells (HES) hold great potential for regenerative medicine because of their ability to differentiate to any cell type. However, a limitation is that HES cells require a feeder layer to stay undifferentiated. Routinely, mouse embryonic fibroblast is used. However, for therapeutic applications, contamination with mouse cells may be considered unacceptable. In this study, we evaluated three commercially available human foreskin feeder (HF) lines for their ability to support HES cell growth in media supplemented with serum or serum replacer. HES cells on HF in serum replacer-supplemented media were cultured for >30 passages. They remained undifferentiated, maintained a normal karyotype, and continued to be positive for the pluripotent markers Oct-4, SOX-2, SSEA-4, GCTM-2, Tra-1-60, Tra-1-81, and alkaline phosphatase. In vivo, HES cells formed teratomas in SCID mouse models that represent the three embryonic germ layers. In contrast, HES cells cultured on HF in serum-supplemented media differentiated after three passages. Morphologically, the cells became cystic with a loss of intracellular Oct-4. We have successfully adapted and cultured undifferentiated HES cells on three human feeder lines for >30 passages. No difficulties were observed with the exception of serum in the media. This study reveals a safe and accessible source for feeders for HES cell research and potential therapeutic applications.     

wnt3a but not wnt11 supports self-renewal of embryonic stem cells

wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not. Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state.     

Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.     

Derivation, propagation and differentiation of human embryonic stem cells

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug discovery, drug testing and repair of damaged or diseased tissues via transplantation.