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

Charles C Lee, Jared E. Christensen, Mervin C. Yoder, Alice F Tarantal

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)46-54
Number of pages9
JournalExperimental Hematology
Volume38
Issue number1
DOIs
StatePublished - Jan 2010

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Mesenchymal Stromal Cells
Bone Marrow
Peroxisome Proliferator-Activated Receptors
Bone Marrow Cells
Integrin-Binding Sialoprotein
Fluorescence Microscopy
Reverse Transcription
Collagen
Immunohistochemistry

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Molecular Biology
  • Hematology

Cite this

Clonal analysis and hierarchy of human bone marrow mesenchymal stem and progenitor cells. / Lee, Charles C; Christensen, Jared E.; Yoder, Mervin C.; Tarantal, Alice F.

In: Experimental Hematology, Vol. 38, No. 1, 01.2010, p. 46-54.

Research output: Contribution to journalArticle

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abstract = "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.",
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