Study of transforming growth factor alpha for the maintenance of human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) have great potential for regenerative medicine as they have self-regenerative and pluripotent properties. Feeder cells or their conditioned medium are required for the maintenance of hESC in the undifferentiated state. Feeder cells have been postulated to produce growth factors and extracellular molecules for maintaining hESC in culture. The present study has aimed at identifying these molecules. The gene expression of supportive feeder cells, namely human foreskin fibroblast (hFF-1) and non-supportive human lung fibroblast (WI-38) was analyzed by microarray and 445 genes were found to be differentially expressed. Gene ontology analysis showed that 20.9% and 15.5% of the products of these genes belonged to the extracellular region and regulation of transcription activity, respectively. After validation of selected differentially expressed genes in both human and mouse feeder cells, transforming growth factor α (TGFα) was chosen for functional study. The results demonstrated that knockdown or protein neutralization of TGFα in hFF-1 led to increased expression of early differentiation markers and lower attachment rates of hESC. More importantly, TGFα maintained pluripotent gene expression levels, attachment rates and pluripotency by the in vitro differentiation of H9 under non-supportive conditions. TGFα treatment activated the p44/42 MAPK pathway but not the PI3K/Akt pathway. In addition, TGFα treatment increased the expression of pluripotent markers, NANOG and SSEA-3 but had no effects on the proliferation of hESCs. This study of the functional role of TGFα provides insights for the development of clinical grade hESCs for therapeutic applications.

Fig. 1

a–c Relative mRNA levels of NANOG, OCT4 and KRT18 of H9 cultured on hFF-1 or WI-38 feeder layers (n=4). d, e Representative images of BG01V cultured on hFF-1 or WI-38 for 6 days. Bars 5 μm. f–k Immunofluorescent staining of pluripotent markers (NANOG, OCT4, TRA-1-60, TRA-1-81, SSEA4) and early differentiation marker (KRT18) in BG01V cultured on hFF-1 and WI-38 (F fluorescence, M merged images of fluorescence and propidium iodide). Bars 5 μm. l–o Relative mRNA levels of the early differentiation markers (KRT8, n=7; KRT18, n=9) and pluripotent markers (NANOG, n=5; OCT4, n=9) in BG01V cultured on hFF-1 and WI-38. *P<0.05, **P<0.01

Fig. 2

a Principal component analysis diagram of the microarray data to examine the differences in the overall gene expression profiles of hFF-1 (n=3, red dots) and WI-38 (n=3, blue dots). b Tree diagram of unsupervised cluster analysis of the microarray data on three independent samples of hFF-1 (a–c) and WI-38 (a–c). Red regions Highly expressed genes. Green/brown regions Genes expressed at a lower level

Fig. 3

a–i Quantitative polymerase chain reaction analysis for the validation of selected gene expression between hFF-1 (n=10) and WI-38 (n=6). j Relative mRNA levels of transforming growth factor alpha (Tgfa) in mouse feeder cells, namely STO and NIH/3T3 (n=5). *P<0.05

Fig. 4

a, b Relative attachment rates of H9 after seeding on hFF-1 transfected with scramble small interfering RNA (si-RNA) or TGFA si-RNA or on hFF-1 pre-treated with goat IgG or antibody against TGFα (n=4). c–e Relative mRNA levels of NANOG, OCT4 and KRT18 in H9 cultured on hFF-1 transfected with scramble si-RNA or TGFA si-RNA (n=4). f–h Relative mRNA levels of NANOG, OCT4 and KRT18 in H9 cultured on hFF-1 pre-treated with goat IgG or antibody against TGFα (n=4). *P<0.05)

Fig. 5

a Relative attachment rates of H9 after cultured in hFF-1 conditioned medium (hCM), WI-38 CM (wCM), or WI-38 CM supplemented with TGFα (wCMt), n=5. b–d Relative mRNA levels of NANOG, OCT4 and KRT18 of H9 cultured in hCM, wCM, or wCMt (n=4). e, f Relative mRNA levels of three germ layer markers (mesoderm: REN and T; ectoderm: NEFH; endoderm: AMY2A), pluripotent markers (NANOG, OCT4) and early differentiation marker (KRT18) in embryoid body formed from differentiation of H9 cultured in hCM (hCM-EB), wCM (wCM-EB), or wCMt (wCMt-EB)

Fig. 6

a, b Effects of TGFα on the relative expression levels of phosphorylated p44/42 in H9 after treated with (a) TGFA for 10, 30, or 60 mins (n=5; ctrl control) and (b) basic fibroblast growth factor (bFGF), TGFα and/or U0126 (n=5). c Relative level of phosphotyrosine in H9 after TGFα treatment (n=7). d, e Relative level of phosphorylated Akt at Thr308 and Ser473 in H9 after TGFA treatment (n=6). Statistical analysis was performed by one way analysis of variance. *P<0.05

Fig. 7

a, b Effect of TGFα on the expression of the pluripotent marker, NANOG, by Western blotting analysis (n=5) and the percentage of cells expressing the pluripotent marker, SSEA-3, by flow cytometry analysis (n=7), respectively. c, d Effect of TGFα on the relative expression levels of proliferation markers, namely proliferating cell nuclear antigen (PCNA) protein by Western blotting analysis (n=4) and BrdU incorporation by flow cytometric analysis (n=5). *P<0.05)