Development of Chemically Defined Culture Conditions for in vitro Expansion of Human Wharton’s Jelly Stem Cells

Abstract

Mesenchymal stem cells (MSCs) are multi-potent and capable of differentiating into a variety of cell lineages. While MSCs have commonly been isolated from bone marrow for treatment of various diseases, Wharton’s Jelly (WJ), an extra-embryonic umbilical cord tissue rich from hyaluronic acid (HA), represents an alternative source for a safer and less invasive isolation of MSCs. Typically, WJ-MSCs are isolated and cultured in undefined media containing fetal bovine serum (FBS), of which use has been associated with different complications, including reproducibility of studies, transmission of infectious agents, and induction of immunologic reactions. To overcome these complications, and thus to facilitate clinical applications of WJ-MSCs, this project aimed to develop chemically defined and safe culture conditions for human WJ-MSCs. We hypothesize that undifferentiated growth of WJ-MSCs will be supported by an HA-based extracellular matrix and fortified DMEM/F12 supplemented with defined macromolecules, antioxidants, lipids and growth factors found in platelet lysate. This hypothesis was tested by comparing the growth kinetics and morphology of WJ-MSCs cultured in defined and undefined media. WJ-MSCs were isolated via enzymatic digestion from discarded human umbilical cords. Following phenotyping and sorting by evaluating expression of relevant markers (i.e., CD105, CD73, and CD90) using flow cytometer, WJ-MSCs were randomly distributed and cultured in five different defined media plus an undefined control medium. The best alternative in terms of cell morphology and proliferation was the medium 3 consisting of DMEM/F12 supplemented with glutamine, ITS (define), antioxidant mixture, lipid mixture, and growth factor mixture. Medium 3 was further improved by adding increasing concentrations of ethanolamine. These results are of significance for therapeutic applications of MSCs. Further research is needed to optimize compositions of extracellular matrix and growth factors while examining the plasticity of MSCs.