Paracrine Induction of Stem Cell Renewal by LIF-Deficient Cells: A New ES Cell Regulatory Pathway

Abstract

The propagation of pluripotential mouse embryonic stem (ES) cells is sustained by leukemia inhibitory factor (LIF) or related cytokines that act through a common receptor complex comprising the LIF receptor subunit (LIF-R) and the signal transducer gp130. However, the findings that embryos lacking LIF-R or gp130 can develop beyond gastrulation argue for the existence of an alternative pathway(s) governing the maintenance of pluripotency in vivo. In order to define those factors that contribute to self-renewal in ES cell cultures, we have generated ES cells in which both copies of the lif gene are deleted. These cells showed a significantly reduced capacity for regeneration of stem cell colonies when induced to differentiate, confirming that LIF is the major endogenous regulatory cytokine in ES cell cultures. However, self-renewal was not abolished and undifferentiated ES cell colonies were still obtained in the complete absence of LIF. A differentiated, LIF-deficient, parietal endoderm-like cell line was derived and shown to support ES cell propagation via production of a soluble, macromolecular, trypsin-sensitive activity. This activity, which we name ES cell renewal factor (ESRF), is distinct from members of the IL-6/LIF family because (i) it is effective on ES cells lacking LIF-R; (ii) it is not blocked by anti-gp130 neutralizing antibodies; and (iii) it acts without activation of STAT3. ES cells propagated clonally using ESRF alone can contribute fully to chimaeras and engender germline transmission. These findings establish that ES cell pluripotency can be sustained via a LIF-R/gp130-independent, STAT-3 independent, signaling pathway. Operation of this pathway in vivo could play an important role in the regulation of pluripotency in the epiblast and account for the viability of lifr -/- and gp130 -/- embryos.

Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells

“…Therefore, in order to assess the actual level of gene expression and protein activities in self-renewing -undifferentiated -ES cells, we made use of the IOUD2 ES cell line which expresses the LacZ -neomycin -phosphotransferase fusion gene (beta -geo) driven off the oct-4 promoter (Dani et al, 1998). The promoter of the oct-4 gene is active in undifferentiated ES cells and it is switched off at the onset of differentiation in vitro (Mountford et al, 1994).…”

“…IOUD2, a feeder-independent ES cell line, was used. IOUD2 cells express the LacZ-neomycin-phosphotransferase fusion gene (beta-geo) driven off the oct-4 promoter (Dani et al, 1998). They were cultured in the presence of G418 (250 mg/ml) in order to kill spontaneously differentiating cells.…”

Changes in embryonic stem cell colony morphology and early differentiation markers driven by colloidal crystal topographical cues

“…Additionally, the increased paracrine growth-factor signalling between ESCs in more three-dimensional, tightly packed colonies on the SCC substrates may also stimulate self-renewal. Dani et al showed that even in the absence of LIF, paracrine factors in ESC colonies can stimulate self-renewal (Dani et al, 1998). Of course, this is also true in EBs, but since EBs are in the region of 300-1000 μm in thickness (compared to approximately 80 μm for colonies on the SCC substrates), other factors such as increased cell migration, nutrient depletion, and distance-dependent paracrine signalling antagonistic to self-renewal may obscure this effect.…”

Self-renewal of teratocarcinoma and embryonic stem cells

“…Nevertheless, the lack of a phenotype in the unperturbed embryo suggest that other mechanisms are normally responsible for the maintenance of pluripotent identity. The ability of lif À/À ES cells to self-renew in the presence of neutralizing gp130 antibody provided evidence of an additional pathway for ES cell maintenance (Dani et al, 1998). This ES cell selfrenewal factor (ESRF) was detectable in the conditioned medium produced by a parietal endoderm-like cell line.…”