Clonal analysis and hierarchy of human bone marrow mesenchymal stem and progenitor cells
Received 19 July 2009; received in revised form 2 November 2009; accepted 3 November 2009. published online 09 November 2009.
Objective
This study was performed to assess adult human bone marrow mesenchymal stem/progenitor cells at a single-cell level and to determine a hierarchy based on proliferative potential.
Materials and Methods
Adult bone marrow mesenchymal cells expressing the enhanced green fluorescent protein (EGFP) were sorted as single cells into 24-well plates, each well confirmed with single EGFP-positive cells by fluorescence microscopy, and counted every 3 days. Colonies derived from single cells were expanded then sorted and evaluated using established differentiation protocols for adipogenic, chondrogenic, and osteogenic lineages. Cells were further analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) (peroxisome proliferator-activated receptor[PPAR]−γ2, LEP, LPL, LUM, COMP, BIG, RUNX2, IBSP, BGLAP) and immunocytochemistry (PPAR−γ1/2, collagen II, bone sialoprotein II) specific for trilineage differentiation.
Results
Bone marrow mesenchymal cells were found to contain high proliferative potential (HPP) mesenchymal colony-forming cells (MCFC) (7%), low proliferative potential (LPP) MCFC (29%), mesenchymal cell clusters (MCC, 26%), and mature mesenchymal cells (MMC, 38%). All LPP-MCFC, MCC, and MMC colonies reached senescence at the end of the evaluation period. However, HPP-MCFC continued to grow, showed differentiation toward all three lineages, and demonstrated the capacity to give rise to secondary HPP-MCFC upon replating at a clonal level.
Conclusion
These findings suggest that there is a low frequency of bone marrow−derived HPP-MCFC that can both self-renew at a single-cell level and differentiate toward multiple lineages of mesenchymal origin.
aCenter of Excellence in Translational Human Stem Cell Research, University of California, Davis, Calif., USA
bCalifornia National Primate Research Center, University of California, Davis, Calif., USA
cDepartment of Pediatrics, Indiana University School of Medicine, Indianapolis, Ind., USA
dDepartments of Pediatrics and Cell Biology and Human Anatomy, University of California, Davis, Calif., USA
Offprint requests to: Alice F. Tarantal, Ph.D., California National Primate Research Center, University of California, Davis, Pedrick and Hutchison Roads, Davis, CA 95616-8542
ABSTRACT