Abstract
Signals via fibroblast growth factor receptors (FGFRs) are involved in mesoderm induction events and may be also critical for early hematopoietic specification and proliferation of the hemangioblast. In vitro differentiated embryonic stem cells represent excellent system for the study of early hematopoietic commitment, particularly for understanding signals regulating the onset of hematopoietic differentiation. We have used human embryonic stem cells (hESCs) to study the expression of FGFR1, 2, 3, and 4 in undifferentiated cells and their differentiated progeny. Culturing hESCs i/ in high densities (protocol 1), ii/ without feeder layer of mouse embryonal fibroblasts and basic fibroblast growth factor (protocol 2), and iii/ in three-dimensional aggregates called embryoid bodies (protocol 3), was used to induce the differentiation. To achieve more directed and homogenous differentiation feeder-free hESCs were first subjected to the aggregation step (formation of embryoid bodies) that resembles the gastrulation process. This was followed by differentiation in monolayer in the presence of basic fibroblast growth factor (protocol 4). Such two-step differentiation protocol (5 + 10 days) was shown to activate ectodermal and mesodermal genes and form ectodermal and mesodermal cells (Schuldiner et al., PNAS97:11307, 2000). The gene expression levels for all FGFRs were determined by quantitative real-time RT-PCR. Real-time RT-PCR results were normalized by comparison to the expression of ABL gene. We revealed that undifferentiated hESCs that were cultured with feeder cells and in low density express all four FGFRs in the following pattern: FGFR1 is highly expressed and dominant; FGFR3 is also strongly expressed; FGFR4 shows lower expression; and FGFR2 is only weakly expressed. This expression pattern was changed when hESCs grew and started to differentiate in high densities (protocol 1) or have initiated differentiation either by feeder cells and basic fibroblast growth factor withdrawal or by aggregation step (protocol 2 and 3). Two-fold upregulation of FGFR1 and FGFR4, and downregulation of FGFR3 characterize such changed expression pattern. Notably, the expression levels for all four FGFRs were increased when hESCc were subjected to the two-step differentiation protocol (protocol 4). Compared to the undifferentiated hESCs, FGFR1 and 4 exhibited 7-fold increase, and FGFR2 and 3 were found to be upregulated more than twice. In summary our results show that the expression of FGFRs tightly follows changing culture conditions that may direct hESCs to differentiate. Furthermore, strong upregulation of FGFR1 and 4 in prospective hESC-derived mesodermal cells suggests their involvement in the earliest stages of hematopoiesis. This research was supported in part by the Grant Agency of the Czech Republic (301/03/1122), Ministry of Health (MZ 00065269705), Ministry of Education, Youth, and Sports (MSM 432100001, LN 00A065), and Academy of Sciences of the Czech Republic (AV 0Z5039906).
Fibroblast Growth Factor-4 Enhances Proliferation of Mouse Embryonic Stem Cells via Activation of c-Jun Signaling
