A multiplex PCR technique to characterize human bone marrow derived mesenchymal stem cells |
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Authors: | Suneel Rallapalli Dillip Kumar Bishi Rama Shanker Verma Kotturathu Mammen Cherian Soma Guhathakurta |
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Institution: | (1) 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;(2) Stem cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology, Madras, Chennai, 600036, India;(3) Frontier Life line, Module No: 109, Ticel Biopark, Taramani, Chennai, 600113, India; |
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Abstract: | 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 β-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. |
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