Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

Selective growth and expansion of human corneal epithelial basal stem cells in a three-dimensional-organ culture

We report on a three dimensional (3D)-organotypic culture in vitro for selective growth and expansion of human corneal epithelial stem cells. Limbal corneal explants were cultured on porous collagen sponges submerged in Epilife medium containing 10% fetal bovine serum. The fragments were analyzed by immunohistochemistry for the expression and distribution of a spectrum of corneal epithelium markers: p63, CK-19, CK-3, Ki-67, pan-cytokeratins and vimentin. Early in culture the epithelium began to exfoliate losing its differentiated high-zone layers into the medium, maintaining only basal and few parabasal cells (mostly both p63 and CK-19 positive), which had remained attached to the specimen. After 14 days a new epithelium was formed displaying an increasing prominence of basal and suprabasal cells that, sliding onto the whole explant, showed the tendency to underlay stromal tissue and infiltrate into the underlaying sponge. After 21 days, sponge and fragments were incubated with trypsin-EDTA and dispersed epithelial cells were pipetted on a feeder monolayer of mitomycin-c-treated murine NIH.3T3 fibroblasts. Colonies of undifferentiated epithelial cells (p63, CK-19 and Ki-67 positive, CK-3 negative) were obtained: their cells, if seeded onto a collagen matrix containing embedded primary human corneal fibroblasts as feeder, provided the basic building blocks for reconstructing in vitro a 3D-multilayered corneal epithelium.     

Uniform expression of alcohol dehydrogenase 3 in epithelia regenerated with cultured normal, immortalised and malignant human oral keratinocytes

The human oral epithelium is a target for damage from the inhalation of formaldehyde. However, most experimental studies on this chemical have relied on laboratory animals that are obligatory nose breathers, including rats and mice. Therefore, in vitro model systems that mimic the structure of the human oral epithelium and which retain normal tissue-specific metabolic competence are desirable. Based on the established role of alcohol dehydrogenase 3 (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, as the primary enzyme catalysing the detoxification of formaldehyde, the aim of this study was to investigate the expression of ADH3 in organotypic epithelia regenerated with normal (NOK), immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes. Organotypic epithelia, usually consisting of 5-10 cell layers, were produced at the air-liquid interface of collagen gels containing human oral fibroblasts, after culture for 10 days in a standardised serum-free medium. Immunochemical staining demonstrated uniform expression of ADH3 in these organotypic epithelia, as well as in the epithelial cells of oral tissue. The specificity of the ADH3 antiserum was ascertained from the complete neutralisation of the immunochemical reaction with purified ADH3 protein. Assessment of the staining intensities indicated that the expression levels were similar among the regenerated epithelia. Furthermore, the regenerated epithelia showed similar ADH3 expression to the epithelium in oral tissue. Therefore, a tissue-like expression pattern for ADH3 can be generated from the culture of various oral keratinocyte lines in an organotypic state. Similar expression levels among the various cell lines indicate the preservation of ADH3 during malignant transformation, and therefore that NOK, SVpgC2a and SqCC/Y1 represent functional models for in vitro studies of formaldehyde metabolism in human oral mucosa.     

The effect of 3T3 fibroblasts on the expression of anchorage independence and cornification of oral keratinocytes

This study examined the effect of 3T3 fibroblasts on the expression of anchorage independence and the degree of cornification in early cultures of three carcinoma-derived epithelial cell lines (R59, R63a, R63b) and in one cell line derived from non-malignant dysplastic epithelium where there was no evidence of invasion (R66a). The epithelial cell lines originated from the palatal (R63a, R66a) and the lingual (R59, R63b) mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. In the absence of 3T3 fibroblasts, progressive culture resulted in an increase in the colony forming efficiency (CFE) of R63a, R63b and R59 and a decrease in the percentage of cornified cells in all cell lines. 3T3 fibroblasts caused a decrease in the CFE and the degree of cornification in the 3T3-dependent cell line (R63a), particularly at the lower passages, but these parameters remained essentially unchanged by 3T3 fibroblasts in the 3T3-independent cell lines (R59, R63b). 3T3 fibroblasts did not influence the cornification of R66a and this cell line remained anchorage dependent throughout the study. The results suggest that in malignant cell lines characterised by being independent of 3T3 fibroblasts (R63b, R59) the CFE was inversely correlated to the degree of cornification. However, in the malignant cell line showing a greater dependence on support (R63a) the relationship between CFE and cornification was unclear because these parameters may have been modulated by the presence of 3T3 fibroblasts. The cell line from dysplastic non-invasive tissue (R66a) differed from its malignant counterparts in the fact that CFE and cornification were unaffected by 3T3 fibroblasts despite previous studies showing a dependence on mesenchymal support.     

Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines

Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to 8-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), STO combined with BRL at a 9:1 and 1:1 ratio, STO with BRL-conditioned medium (STO + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:1 and 1:1 ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the STO and the STO with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (1:1), PEF:BRL (1:1), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by STO and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epithelial-like) cell lines over that of STO feeders.     

Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders.     

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.     

Effects of feeder layers made of human,mouse, hamster,and rat cells on the cloning efficiency of transformed human cells

Summary The effect of feeder layers on cloning efficiency of transformed human cells was investigated. Embryonic human skin or lung fibroblasts; adult human skin fibroblasts; early passage cells from embryos of mouse, rat, and hamster; established mouse cell lines; 3T3 and 10T1/2 were used as feeder layers after they were lethally exposed to Co-60 gamma-rays at 3,000 rad. As test cells to study the effect of feeder layers on cloning efficiency, WI-38 CT-1 cells transformed in vitro by Co-60 gamma-rays and HGC cells cultured from a human gastric cancer were used. The effect of feeder layers on the cloning efficiency of the test cells was dependent on cell density of feeder layer cells, sources of the feeder layer cells, and kinds of test cells. An optimal density of feeder cels produced cloning efficiencies 3 to 15 times higher than in cultures without a feeder layer. Generally, high density of cells in feeder layers decreased the cloning efficiency of the test cells, presumably owing to contact inhibition of growth and depletion of essential nutrients by the feeder layer cells. Regarding the effect of the feeder layers made of human fibroblasts, there were no significant differences in population doubling levels; tissue origins of fibroblasts; or fibroblasts derived from normal individuals, patients with cancer, or with a genetically high familial incidence of cancer, hereditary adenomatosis of the colon and rectum. This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan.     

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.     

Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.     

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.     

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.     

Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates.     

人多潜能胚胎生殖细胞的分离和培养（简报）

To establish human pluripotent embryonic germ (EG) cell lines, human primordial germ cells (PGCs) of embryos aborted in 5-9 week were cultured on inactive mouse STO fibroblast feeder. The medium contained human leukemia inhibitory factor (hLIF), human basic fibroblast growth factor (hbFGF) and forskolin. The EG cells could be passaged continuously until 12 generations. Most cells were positive in alkaline phosphatase staining and expressed cell surface antigen SSEA-3 and pluripotent marker Oct-4. These EG cell populations that retained normal karyotype could form embryoid body in culture and differentiate further into neuron-like cells, mucous epithelial cells, epithelial cells and other types of the cells spontaneously. These results indicated the cell clones derived from human PGCs resemble pluripotent EG cells from mouse PGCs in appearance or nature.     

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.     

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.     

Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system

The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 μl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs.     

Junctional Adhesion Molecules (JAMs) are differentially expressed in fibroblasts and co-localize with ZO-1 to adherens-like junctions

Junctional Adhesion Molecules (JAMs) are components and regulators of the well-characterized epithelial and endothelial tight junction. Since the molecular components of native fibroblast adherens-like junctions remain poorly described we determined JAM expression profiles in fibroblasts. We found JAM-C on human dermal, lung, and corneal primary fibroblast cultures. Within murine lines, JAM-A was found in L-cells, JAM-C in 3T3 L1 cells, and both JAM-A and JAM-C were co-expressed in NIH 3T3 fibroblasts. In primary dermal fibroblasts, JAM-C concentrated at zipper-like junctions that formed between apposing cells. Dual immunostaining showed JAM-C co-localization with the ZO-1 intracellular scaffolding molecule at cell contacts that ranged from 7 μm to over 25 μm in length. JAM-C also labeled similar zipper-like junctions detected with N-Cadherin and Cadherin-11 antibodies. We conclude that endogenous JAM-C is an integral component of the dermal fibroblast adherens-like junction, and our data extend the expression and potential function of JAMs into mesenchymal tissues.     

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.