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Identification and isolation of small CD44-negative mesenchymal stem/progenitor cells from human bone marrow using
Sean R R Hall1, Yajuan Jiang, Elizabeth Leary
1NeoStem Inc., Cambridge, MA, USA.
Abstract:
The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence.
Insights
Researchers identified rare, small mesenchymal stem/progenitor cells (MSPCs) in human bone marrow lacking CD44. These cells give rise to mesenchymal stem/stromal cells (MSCs) with distinct properties compared to standard plastic-adherent MSCs.
Area of Science:
- Stem Cell Biology
- Hematology
- Immunology
Background:
- Mesenchymal stem/stromal cells (MSCs) are crucial for research and therapy, but isolation methods yield heterogeneous populations.
- Current isolation relies on plastic adherence, limiting purity and understanding of true MSC precursors.
- Identifying specific cell surface markers is key to isolating pure MSC populations.
Purpose of the Study:
- To identify and characterize a distinct population of mesenchymal stem/progenitor cells (MSPCs) within human bone marrow.
- To determine the antigenic profile of MSC precursors for prospective isolation.
- To compare the properties of prospectively isolated MSPCs with conventionally cultured MSCs.
Main Methods:
- Utilized polychromatic flow cytometry and counterflow centrifugal elutriation for cell isolation and analysis.
- Characterized cell populations based on expression of key surface markers including CD44, CD45, CD73, CD90, and CD105.
- Assessed in vitro functional properties such as adherence, proliferation, colony formation, and multi-lineage differentiation.
Main Results:
- Identified rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells lacking CD44 as human bone marrow MSPCs.
- These MSPCs adhere to plastic, proliferate, differentiate into osteoblasts, chondrocytes, and adipocytes, and acquire CD44 expression in culture.
- Acquired CD44 expression on MSPCs could be partially downregulated by G-CSF treatment, unlike conventional MSCs.
Conclusions:
- Human bone marrow contains rare, small MSPCs defined by the absence of CD44 and presence of CD73, CD90, and CD105.
- These MSPCs are the precursors to MSCs with distinct phenotypic and functional characteristics compared to plastic-adherent MSCs.
- Prospective identification of MSPCs offers a pathway to highly pure cell populations for research and clinical applications.
