Study of extracellular matrix for the culture of human embryonic stem cells

Abstract

Background: The development of clinical grade human embryonic stem cells (hESC) suitable for cell therapy requires defined components for hESC culture. We hypothesize that the feeder cells used for hESC culture produce extracellular matrix molecules supporting the growth of hESC. Materials and Methods: The profiles of mRNA and extracellular matrix production of two human feeder cell lines, human foreskin fibroblast (hFF-1) and human lung fibroblast (WI-38) were correlated with their ability to maintain the pluripotency of a hESC line, BG01V. The mRNA expression profile and matrix production were determined by Affymetrix microarray chip and liquid chromatography/mass spectrometry/mass spectrometry analysis, respectively. Results: Using a morphological grading system, hFF-1 but not WI-38 supported the pluripotency of BG01V. The observation was supported by over-expression of early differentiation markers KRT-8 and -18 mRNA in BG01V cells cultured on WI-38, though the mRNA expression of pluripotent markers Nanog, Oct4 and TRA-1-81 in BG01V cultured with hFF-1 and WI-38 was similar. Microarray analysis showed that the expression of 530 transcripts was 2-fold higher (P<0.05) in hFF-1 than in WI-38. The microarray data were validated by real-time PCR on 12 differentially expressed genes related to secretory and extracellular matrix molecules. Proteomic profiling of the extracellular matrix molecules also demonstrated differences between the two feeder cell lines. Both microarray and proteomic analysis identified sulfatase 1 (SUFL1) and chemokine (C-X-C Motif) ligand 12 (CXCL12) were higher in hFF-1. Conclusion: The difference in the ability of feeder cells in maintaining the pleuripotency of hESC may be related to the differential secretion of matrix molecules by the feeder cells.