Leukemia Inhibitory Factor as an Anti-Apoptotic Mitogen for Pluripotent Mouse Embryonic Stem Cells in a Serum-Free Medium Without Feeder Cells

Abstract

We have developed a serum-free medium, designated ESF7, in which leukemia inhibitory factor (LIF) clearly stimulated murine embryonic stem (ES) cell proliferation accompanied by increased expression of nanog and Rex-1 and decreased FGF-5 expression. These effects were dependent on the concentration of LIF. The ES cells maintained in ESF7 medium for more than 2 yr retained an undifferentiated phenotype, as manifested by the expression of the transcription factor Oct-3/4, the stem cell marker SSEA-1, and alkaline phosphatase. Withdrawal of LIF from ESF7 medium resulted in ES cell apoptosis. Addition of serum to ESF7 medium promoted ES cell differentiation. Addition of BMP4 promoted ES cell differentiation into simple epithelial-like cells. In contrast, FGF-2 promoted ES cell differentiation into neuronal and glial-like cells. Under serum-free culture conditions, LIF was sufficient to stimulate cell proliferation, it inhibited cell differentiation, and it maintained self-renewal of ES cells. Because this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent ES cells in vitro, it will allow the elucidation of ES cell responses to growth factors under defined conditions.

Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium

“…We first tested the ability of ESF7 medium [supporting information (SI) Table S1], which we had developed for use with MESCs (25), to support the growth of two HESC lines, HUES-1 (Fig. 1A) (26) and Shef1 (Fig.…”

“…Although this medium shares a number of features with those described by others, including culture on defined ECM attachment factors, it differs in a number of important respects. In particular, we have used a base medium, ESF (25) that we previously developed for use with MESCs, in contrast to DMEM:F12 commonly used in other formulations (25). This medium also excludes Hepes, but includes heparin, a cofactor for FGF-2, which is required for HESC maintenance.…”

Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

“…Furthermore, the establishment of serum-and feeder-free culture conditions is significant to overcome the ethical issues related to animal experiments. Furue et al (2005) previously developed a serumfree, chemically defined medium called ''ESF7'' (now available as ESF-C), which maintains mouse ES cells on collagen-coated dishes. In this manuscript, we show that mouse iPS cells can be cultured in feeder-free conditions as well as on feeder cells using the ESF-C medium containing different kinds of low-molecular-weight cell differentiation inhibitors.…”

Evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse embryonic stem cells

“…In most cases, the growth factors were applied to aggregates of ES cells after removal of leukemia inhibitory factor (LIF), a cytokine that inhibits differentiation. In the absence of LIF, mES cells create intracellular contacts and initiate signaling and spontaneous differentiation (Furue et al, 2005). Keller et al (2004) describe the induction of epithelial-or epidermal-specific gene expression and differentiation using a combination of GFs plus extracellular matrix (ECM) components in human ES cells.…”