CELLULAR AND MOLECULAR EVALUATION OF IN VITRO QUALITY PARAMETERS OF HUMAN UMBILICAL CORD BLOOD-DERIVED MESENCHYMAL STEM CELLS

Abstract

Introduction: Establishing a reproducible adult stem cell culture system, such as mesenchymal stem cells (MSCs), is critical for elucidating the function of molecular markers associated with these cells' undifferentiated state. In this study, we describe some important parameters to be considered for a successful isolation, culture, and characterization of MSCs from human umbilical cord blood (hUCB). Methods: Five hUCB samples were collected from healthy female subjects who were free from infectious diseases and genetic disorders. Mononuclear cells (MNCs) were counted, and viability was determined using MTT assay. MNCs were cultured in DMEM supplemented with 10% fetal bovine serum and enriched through culture and characterized using morphometric, and molecular analysis. Results: The minimum number of cells was 12.5 million and highest number of cells was 20.6 million from all ve samples. In initial culture of MSCs from hUCB, various morphologic phenotypes were seen, although the cells eventually developed a homogeneous broblast-like shape at day 14 showing >80% conuency. Spindle-shaped clonogenic MSCs expressed a high level of CD90, CD105, and CD73, while negative expression of CD34. Our study provided evidence of expansion of enriched MSCs in culture from day 1 to day 21 as supported by data of CD90, CD105 and CD73 expression levels in a time-dependent manner. Conclusion: Our results suggest that the expanded hUCB harbor an enriched source of MSCs that express pluripotent stem cell markers and lack hematopoietic markers after culture and forms the basis for using hUCB as eminent source of MSCs, which can be used for different therapeutic applications.