Clonal analysis and hierarchy of human bone marrow mesenchymal stem and progenitor cells

Lee, C. Chang I.; Christensen, Jared E.; Yoder, Mervin C.; Tarantal, Alice F.

doi:10.1016/j.exphem.2009.11.001

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Volume 38, Issue 1 , Pages 46-54, January 2010

Clonal analysis and hierarchy of human bone marrow mesenchymal stem and progenitor cells

C. Chang I. Lee
- Center of Excellence in Translational Human Stem Cell Research, University of California, Davis, Calif., USA
- California National Primate Research Center, University of California, Davis, Calif., USA
Jared E. Christensen
- Center of Excellence in Translational Human Stem Cell Research, University of California, Davis, Calif., USA
- California National Primate Research Center, University of California, Davis, Calif., USA
Mervin C. Yoder
- Center of Excellence in Translational Human Stem Cell Research, University of California, Davis, Calif., USA
- Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Ind., USA
Alice F. Tarantal
- Center of Excellence in Translational Human Stem Cell Research, University of California, Davis, Calif., USA
- California National Primate Research Center, University of California, Davis, Calif., USA
- Departments 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

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.