Defining the Role of Wnt/β‐Catenin Signaling in the Survival, Proliferation, and Self‐Renewal of Human Embryonic Stem Cells

TLDR

The study aimed to identify growth factors that support human embryonic stem cell survival, proliferation, and self‑renewal, and to test whether Wnt/β‑catenin signaling can maintain the undifferentiated state. The authors used human and mouse fibroblast panels to screen for supportive factors and performed experiments blocking or adding recombinant Wnt proteins to assess effects on undifferentiated hESCs. Feeder‑derived factors promote hESC survival and block spontaneous differentiation, but the antidifferentiation factor is not Wnt; Wnt3a enhances proliferation yet induces differentiation, and canonical Wnt/β‑catenin activation is minimal in undifferentiated cells but rises during differentiation, indicating that Wnt/β‑catenin alone cannot sustain pluripotency.

Abstract

We used a panel of human and mouse fibroblasts with various abilities for supporting the prolonged growth of human embryonic stem cells (hESCs) to elucidate growth factors required for hESC survival, proliferation, and maintenance of the undifferentiated and pluripotent state (self‐renewal). We found that supportive feeder cells secrete growth factors required for both hESC survival/proliferation and blocking hESC spontaneous differentiation to achieve self‐renewal. The antidifferentiation soluble factor is neither leukemia inhibitory factor nor Wnt, based on blocking experiments using their antagonists. Because Wnt/β‐catenin signaling has been implicated in cell‐fate determination and stem cell expansion, we further examined the effects of blocking or adding recombinant Wnt proteins on undifferentiated hESCs. In the absence of feeder cell–derived factors, hESCs cultured under a feeder‐free condition survived/proliferated poorly and gradually differentiated. Adding recombinant Wnt3a stimulated hESC proliferation but also differentiation. After 4–5 days of Wnt3a treatment, hESCs that survived maintained the undifferentiated phenotype but few could form undifferentiated hESC colonies subsequently. Using a functional reporter assay, we found that the β‐catenin–mediated transcriptional activation in the canonical Wnt pathway was minimal in undifferentiated hESCs, but greatly upregulated during differentiation induced by the Wnt treatment and several other methods. Thus, Wnt/β‐catenin activation does not suffice to maintain the undifferentiated and pluripotent state of hESCs. We propose a new model for the role of Wnt/β‐catenin signaling in undifferentiated hESCs.