Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells

Abstract

Embryonic stem (ES) cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein-tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent Src-family kinase inhibitor A-419259 retained the morphology of domed, pluripotent colonies and continued to express the self-renewal marker TRA-1-60 despite culture under differentiation conditions. Taken together, these observations support a role for Src-family kinase signaling in the regulation of human ES cell fate, and suggest that the activities of individual Src-family members are required for the initiation of the differentiation program.

Figure 1. Profile of SFK gene expression in hES cells

A) The hES cell lines H1, H7 and H9 were maintained under feeder-free conditions on Matrigel-coated plates using mTeSR1 medium. All three lines grew as large domed colonies with well-circumscribed boundaries. B) H1 hES cells were harvested 5 days after passage for RNA isolation. SFK (Blk, Fgr, Fyn, Hck, Lck, Lyn, c-Src, c-Yes), Src-related kinase (Brk, Frk, Srm) and pluripotency marker (Oct4) expression were determined by RT-PCR with GAPDH as a positive control. Images of agarose gels of the PCR products are shown after 30 and 35 PCR cycles. C) Relative SFK (Fyn, Hck, Lck, Lyn, c-Src, c-Yes) and pluripotency marker (Oct4, Nanog) expression levels across three hES cell lines. Transcript levels for the indicated genes were determined by quantitative real-time RT-PCR for the H1, H7 and H9 hES cell lines. The results are expressed as the average fold-change in mRNA level in H7 and H9 cells relative to H1 cells ± SEM (n = 3, *p < 0.05; pairwise xed reallocation randomization test). SFK expression varied by less than 2-fold across these hES cell lines (gray area), with the exception of Hck expression in H7 cells.

Figure 2. SFK expression during EB formation from H7 hES cells

A) EB formation was initiated from H7 hES cells using Aggrewell plates as described under Materials and Methods. Representative images of H7 hES cells and EBs after 3, 6 and 12 days of culture later are shown; all images recorded at same magnification (100 ×). B) Total RNA was extracted from renewing hES cells and 3, 6, and 12-day EBs. Expression of self-renewal (Oct4, Nanog) and differentiation markers (T, mesoderm; Pax6, Sox1, Msx1, ectoderm; and Sox17, endoderm) as well as SFKs (Fyn, Hck, Lck, Lyn, c-Src, c-Yes) was determined by qPCR relative to control H7 hES cells maintained in mTeSR medium. Results are expressed as the average fold change relative to the undifferentiated hES cell population ± S.E.M. (n = 3; *p < 0.05; Pairwise Fixed Reallocation Randomization Test).

Figure 3. SFK protein expression and activity in ES cells and 6 day EBs

Lysates were prepared from mouse ES cells (D3 line), human ES cells (H1 and H7 lines) and 6-day EBs derived from them. Endogenous SFKs (c-Src, c-Yes, Fyn, Lck, Hck, Lyn) were immunoprecipitated and blotted with a phosphospecific antibody for the active form of each kinase (pY418 antibody, which recognizes the conserved phosphotyrosine residue in the activation loop of the kinase domain) as well as the respective SFK protein. Cell lysates were also blotted for Oct4 as marker of ES cell self-renewal plus actin as a loading control. This experiment was repeated twice with comparable results; a representative example is shown.

Figure 4. hES cells treated with the small molecule SFK inhibitor A-419259 retain undifferentiated colony morphology

The H1 and H7 hES cell lines were grown in mTeSR self-renewal medium or switched to differentiation medium in the absence or presence of the pan-SFK inhibitor, A-419259, at the concentrations shown. Note that both hES cells lines maintained undifferentiated colony morphology when cultured in the presence of A-419259 under differentiation conditions.

Figure 5. A-419259 inhibits endogenous SFK activity in hES cells

Self-renewing hES cells (H1 and H7 lines) were cultured in the absence or presence of A-419259 (1.0 μM) for 16 h, followed by lysis and immunoprecipitation of c-Src and Lck. Each immunoprecipitate was then blotted with a phosphospecific antibody for the active form of each kinase (pY418 antibody, which recognizes the conserved phosphotyrosine residue in the activation loop of the kinase domain) as well as the respective SFK protein. Cell lysates were also blotted for pY418 as well as Oct4 as a marker of ES cell self-renewal. This experiment was repeated twice with comparable results; a representative example is shown.

Figure 6. hES cells retain Tra-1-60 expression following SFK inhibition

The H1 line of hES cells was grown in mTeSR self-renewal medium or switched to differentiation medium in the absence (DMSO carrier) or presence of the pan-SFK inhibitor, A-419259, at the concentrations shown. Cells were fixed and immunostained for the cell-surface pluripotency marker Tra-1-60 three days later (magnification 400×). This experiment was repeated three times with comparable results; representative fields of cells from each condition are shown.

Figure 7. Loss of Lck expression accompanies hES cell differentiation

The H7 line of hES cells was grown in mTeSR self-renewal medium or switched to differentiation medium in the absence (Control) or presence of A-419259 (1 μM). Cells were fixed, permeabilized and immunostained for the SFK Lck and the nuclear pluripotency factor Oct4 four days later. DAPI-stained nuclei are also shown. The merged image combines cells in the Lck and Oct4 panels only. This experiment was repeated three times with comparable results; a representative field of cells is shown for each condition.