Enhanced Expansion of Human Pluripotent Stem Cells and Somatic Cell Reprogramming Using Defined and Xeno-Free Culture Conditions

Abstract

Human embryonic stem cells and induced pluripotent stem cells (hPSC) have an unprecedented opportunity to revolutionize the fields of developmental biology as well as tissue engineering and regenerative medicine. However, their applications have been significantly limited by the lack of chemically defined and xeno-free culture conditions. The demand for the high-quality and scaled-up production of cells for use in both research and clinical studies underscores the need to develop tools that will simplify the in vitro culture process while reducing the variables. Here, we describe a systematic study to identify the optimal conditions for the initial cell attachment of hPSC to tissue culture dishes grafted with polymers of N-(3-Sulfopropyl)-N-Methacryloxyethyl-N, N-Dimethylammoniun Betaine (PMEDSAH) in combination with chemically defined and xeno-free culture media. After testing multiple supplements and chemicals, we identified that pre-conditioning of PMEDSAH grafted plates with 10% human serum (HS) supported the initial cell attachment, which allowed for the long-term culture and maintenance of hPSC compared to cells cultured on Matrigel-coated plates. Using this culture condition, a 2.1-fold increase in the expansion of hPSC was observed without chromosomal abnormalities. Furthermore, this culture condition supported a higher reprogramming efficiency (0.37% vs. 0.22%; p < 0.0068) of somatic cells into induced pluripotent stem cells compared to the non-defined culture conditions. This defined and xeno-free hPSC culture condition may be used in obtaining the large populations of hPSC and patient-derived iPSC required for many applications in regenerative and translational medicine.