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Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice
Published on: January 6, 2018
CD45-CD14 +CD34 + murine bone marrow low-adherent mesenchymal primitive cells preserve multilineage differentiation
Krzysztof Szade1, Ewa Zuba-Surma, Andrzej J Rutkowski
1Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
Abstract:
Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45(-)CD14(+)CD34(+) phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45(-)CD14(+) CD34(+) bone marrow-derived cells that could be maintained in long-term culture without losing this potential.
Insights
Researchers isolated a novel multipotent stem cell population from bone marrow. These CD45(-)CD14(+)CD34(+) cells can be expanded long-term and differentiate into endothelial and neuronal lineages.
Area of Science:
- Stem cell biology
- Hematology
- Regenerative medicine
Background:
- Mesenchymal stem cells (MSCs) derived from bone marrow are a heterogeneous population.
- Expansion of primitive MSC subsets remains challenging.
- Understanding MSC heterogeneity is crucial for therapeutic applications.
Purpose of the Study:
- To optimize a protocol for propagating a specific low-adherent subfraction of bone marrow-derived cells.
- To characterize the phenotype and multipotency of the expanded cell population.
- To assess the differentiation potential and functional properties of these cells.
Main Methods:
- Optimized culture protocol for low-adherent bone marrow cells.
- Flow cytometry analysis for cell surface markers (CD45, CD14, CD34, MSC markers).
- In vitro differentiation assays towards endothelial and neuronal lineages.
- Assessment of angiogenic marker expression and functional endothelium properties.
- In vitro sphere formation assay.
Main Results:
- Successfully established long-term expansion of a CD45(-)CD14(+)CD34(+) cell population expressing common MSC markers.
- Expanded cells demonstrated multipotency, differentiating into endothelial cells with angiogenic properties and neuronal cells.
- These cells formed characteristic cellular spheres in vitro, indicative of primitive stem cell features.
- The isolated subpopulation maintained its potential during long-term culture.
Conclusions:
- A novel multipotent subpopulation of bone marrow-derived cells (CD45(-)CD14(+)CD34(+)) was isolated and characterized.
- This subpopulation can be expanded long-term while retaining multipotency and primitive stem cell characteristics.
- These findings offer potential for new therapeutic strategies in regenerative medicine and cell-based therapies.
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