Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine

Abstract

BACKGROUND

Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages. In humans, their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs. Studies suggested that mesenchymal stem cells (MSCs), necessary for repair and regeneration via transplantation, require doses ranging from 10 to 400 million cells. Furthermore, the limited expansion of MSCs restricts their therapeutic application.

AIM

To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols.

METHODS

Human umbilical cord (hUC) tissue derived MSCs were obtained and re-cultured. These cultured cells were subjected to the following evaluation procedures: Immunophenotyping, immunocytochemical staining, trilineage differentiation, population doubling time and number, gene expression markers for proliferation, cell cycle progression, senescence-associated β-galactosidase assay, human telomerase reverse transcriptase (hTERT) expression, mycoplasma, cytomegalovirus and endotoxin detection.

RESULTS

Analysis of pluripotent gene markers Oct4, Sox2, and Nanog in recultured hUC-MSC revealed no significant differences. The immunophenotypic markers CD90, CD73, CD105, CD44, vimentin, CD29, Stro-1, and Lin28 were positively expressed by these recultured expanded MSCs, and were found negative for CD34, CD11b, CD19, CD45, and HLA-DR. The recultured hUC-MSC population continued to expand through passage 15. Proliferative gene expression of Pax6, BMP2, and TGFb1 showed no significant variation between recultured hUC-MSC groups. Nevertheless, a significant increase (P < 0.001) in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs. Cellular senescence markers (hTERT expression and β-galactosidase activity) did not show any negative effect on recultured hUC-MSCs. Additionally, quality control assessments consistently confirmed the absence of mycoplasma, cytomegalovirus, and endotoxin contamination.

CONCLUSION

This study proposes the development of a novel protocol for efficiently expanding stem cell population. This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.

Keywords: Human umbilical cord; Mesenchymal stem cells; Expansion; Cell proliferation; In vitro expansion; Senescence

Core Tip: Therapeutic transplantation of mesenchymal stem cells (MSCs) requires one to four million cells/kg of body weight. Ex vivo expansion of stable MSCs at higher passage numbers limits their clinical applications due to senescence, variation in genetic stability and short life span. This study results in the development of a cutting-edge protocol for scaling the stem cell population ex vivo in less time. It rapidly increases the cell number in vitro to fulfill in vivo therapeutic cell doses. This method might decrease immune rejection. Since these MSCs were isolated from the same recultured human umbilical cord, they have persistent MSC stemness and may decrease tissue vs graft rejection due to the less rigorous HLA screening required in allogenic transplantation, which could make it more cost-effective. More studies that are exploratory should be carried out to further elucidate the mechanism via preclinical and clinical applications.