Abstract
BackgroundThe growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Methodology/Principal FindingsHere, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Conclusion/SignificanceOur results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.
Highlights
Stem cells are invaluable tools for research, drug screening, to study diseases and can potentially serve as a resource for regenerative therapies
We developed a xeno-free medium (RegES) composed of a knockout-Dulbecco’s modified Eagle’s medium (KO-DMEM, Invitrogen, Carlsbad, CA) base supplemented with human serum albumin, amino acids, vitamins, antioxidants, trace minerals, and growth factors (Table S1)
The xeno-free RegES medium was evaluated with human embryonic stem cells (hESC) lines HS237, HS346 and HS401 [25,26] and it was noticed that the colonies were thinner and the growth of the hESCs was slower than in hESC lines derived and cultured in the conventional medium containing KO-SR
Summary
Stem cells are invaluable tools for research, drug screening, to study diseases and can potentially serve as a resource for regenerative therapies. Even more expectations on clinical applicability in diverse fields of cell- and tissue-replacement therapies are focused on pluripotent stem cells. Human iPSCs are a potential source of patientspecific pluripotent stem cells that could be used to treat a number of human degenerative diseases without evoking immune rejection. From these stem cell types, only patient specific ASCs have so far been used in clinical cell therapy while clinical trials using hESCs is at the very beginning (http://www.geron.com). For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable
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