A complex role for FGF-2 in self-renewal, survival, and adhesion of human embryonic stem cells

Abstract

The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor-2 (FGF-2), which activates FGF receptors (FGFRs) and stimulates the mitogen-activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin-like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF-2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF-2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation-related genes and downregulation of stem cell genes. Thus, exogenous FGF-2 reinforced the pluripotency maintenance program of intracrine FGF-2 signaling. Consistent with this hypothesis, expression of endogenous FGF-2 decreased during hESC differentiation and FGF-2 knockdown-induced hESC differentiation. In addition, FGF-2 signaling via FGFR2 activated MAPK kinase/extracellular signal-regulated kinase and AKT kinases, protected hESC from stress-induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self-renewal, cell survival, and adhesion by exogenous and endogenous FGF-2 may synergize to maintain the undifferentiated growth of hESCs.

Figure 1

Regulated expression of endogenous FGF-2 correlates with differentiation of hESCs and their binding capacity to exogenous FGF-2. (A): RT-PCR of FGF-2 gene and immunoblotting of 18-, 22-, 22.5-, and 24-kDa FGF-2 isoforms in undifferentiated and differentiating hESCs. (B): Peripheral differentiation after FGF-2 withdrawal (arrows) and downregulated nuclear FGF-2, Oct4, and Nanog immunofluorescence. (C): Biotinylated FGF-2 (green) binding to undifferentiated hESCs (+FGF-2) or peripherally differentiated hESCs (−FGF-2). Cells are counterstained with 4′,6-diamidino-2-phenylindole (blue). Representative results of triplicate experiments. Abbreviations: EB, embryoid body; FGF-2, fibroblast growth factor-2; GADPH, glyceraldehyde 3-phosphate dehydrogenase; hESCs, human ESCs; RT-PCR, reverse transcriptase-polymerase chain reaction.

Figure 2

shRNA-mediated knockdown of FGF-2 in human ESCs (hESCs). (A): Immunoblot of 18-, 22-, 22.5-, and 24-kDa isoforms of FGF-2. (B): hESC morphology after FGF-2 depletion; arrows mark differentiation. Undifferentiated morphology of hESCs was not rescued by addition of 10 ng/ml FGF-2 (+exogenous FGF-2). Representative results of duplicate experiments. Abbreviations: FGF-2, fibroblast growth factor-2; shRNA, small hairpin RNA.

Figure 3

FGF-2 signaling via FGF receptor 2, mitogen-activated protein kinases, and Akt. (A): Phospho-receptor kinases protein array of FGF-2 starved human ESCs (hESCs) or (B) after FGF-2 treatment. (C): Immunoblot analysis of MEK and ERK in time course FGF-2-treated hESCs. MAPK protein array of hESCs treated with (D) FGF-2, (E) SU5402, or vehicle (DMSO). Representative results of more than five experiments are shown. Abbreviations: DMSO, dimethylsulfoxide; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FGF-2, fibroblast growth factor-2; IGF1R, insulin-like growth factor 1 receptor; MEK, mitogen-activated protein kinase kinase.

Figure 4

Human ESC (hESC) apoptosis and increased expression of FGF-2 in response to cellular stress. (A): Immunoblot of PARP and lamin B cleavage (3 hours after treatment), and (B) quantitative TUNEL (12 hours after treatment) of hESCs FGF-2 prestarved (6 hours) and incubated with 10 ng/ml FGF-2 (2 hours) and irradiated (1 Gy) or exposed to H₂O₂ (50 μM, 1 hour). Control cells were irradiated or exposed to H₂O₂ in the absence of exogenous FGF-2. (C): hESCs were treated with 25 μM H₂O₂ without exogenous FGF-2 for 12 and 24 hours and immunoblotted for endogenous FGF-2. Control cells were maintained without H₂O₂. Representative results of triplicate experiments. Abbreviations: FGF-2, fibroblast growth factor-2; IR, irradiated; PARP, poly(ADP-ribose) polymerase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 5

FGF-2 affects the cloning efficiency of human ESC (hESC) single cell suspensions. hESCs were preincubated and seeded in medium with FGF-2 (100 ng/ml) or without FGF-2. (A): Number of alkaline phosphatase-positive colonies; numbers represent the mean of 24 wells of one 24-well plate. (B): Number and viability of nonattached hESCs. Representative results of more than five experiments are shown. Abbreviations: FGF-2, fibroblast growth factor-2.