Autogenic feeder free system from differentiated mesenchymal progenitor cells, maintains pluripotency of the MEL-1 human embryonic stem cells

Human embryonic stem cells (hESc) are known for its pluripotency and self renewal capability, thus possess great potential in regenerative medicine. However, the lack of suitable xenofree extracellular matrix substrate inhibits further applications or the use of hESc in cell-based therapy. In this study, we described a new differentiation method, which generates a homogeneous population of mesenchymal progenitor cells (hESc–MPC) from hESc via epithelial–mesenchymal transition. The extracellular matrix (ECM) proteins from hESc–MPC had in turn supported the undifferentiated expansion of hESc. Immunocytochemistry and flow cytometry characterization of hESc–MPC revealed the presence of early mesenchymal markers. Tandem mass spectometry analysis of ECM produced by hESc–MPC revealed the presence of a mixture of extracellular proteins which includes tenascin C, fibronectin, and vitronectin. The pluripotency of hESc (MEL-1) cultured on the ECM was maintained as shown by the expression of pluripotent genes (FoxD3, Oct-4, Tdgf1, Sox-2, Nanog, hTERT, Rex1), protein markers (SSEA-3, SSEA-4, TRA-1-81, TRA-1-60, Oct-4) and the ability to differentiate into cells representative of ectoderm, endoderm and mesoderm. In summary, we have established a xeno-free autogenic feeder free system to support undifferentiated expansion of hESc, which could be of clinical relevance.

Graphical AbstractFigure optionsDownload high-quality image (367 K)Download as PowerPoint slideHighlights
► Our study described a protocol for direct differentiation of hESc into hESc–MPC.
► Expression of lineage-specific genes along differentiation pipeline was revealed.
► Autogenic ECM from hESc–MPC supports xeno and feeder free expansion of hESc.
► The hESc grown on autogenic ECM was functionally pluripotent.
► ECM proteins compositions revealed by LC/MS/MS tandem mass spectrometry.