A multiplex PCR technique to characterize human bone marrow derived mesenchymal stem cells.
Human mesenchymal stem cells (MSCs), with capacity to differentiate into adipocytes, osteoblasts and chondrocytes, offer potential for the development of novel treatments. A critical question in MSCs biology is whether this cell population possesses a relatively uniform differentiation capability or is comprised of distinct subsets of progenitors committed to differentiate in particular pathways. To quantify the changes during growth of MSCs, we analyzed the mesenchymal phenotype and differentiation ability using a multi-marker PCR with six primer sets specific for CD73, CD90, CD105, CD166, CD45 and beta-actin allowing a gel-based differential detection of the PCR products. To determine degree of variability of MSCs populations in terms of proliferation, cell proliferation assays were performed on expanded MSCs up to the sixth passage. At each passage, the osteogenic and adipogenic differentiation potentials of MSCs were verified by culture in inductive media. RT-PCR and cytochemical analysis revealed that, despite the loss of multipotentiality during expansion, certain markers remain expressed, indicating that these markers are unlikely to be reflective of the MSC's true 'stem cell' nature. Our results suggest that decrease in the expression of MSCs specific markers correlates with down-regulation of proliferation ability and differentiation efficiency of MSCs.
Rallapalli
Suneel
S
International Centre for Cardio Thoracic and Vascular Diseases, A unit of Frontier Lifeline Pvt. Ltd. & Dr. K. M. Cherian Heart Foundation, R-30C, Ambattur Industrial Estate Road, Chennai, 600101, India.
Bishi
Dillip Kumar
DK
Verma
Rama Shanker
RS
Cherian
Kotturathu Mammen
KM
Guhathakurta
Soma
S
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics; D001854:Bone Marrow Cells / Q000502:physiology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D003470:Culture Media / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods*
D000199:Actins; D015703:Antigens, CD; D003470:Culture Media