Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth
Ayona Gupta1, Risani Mukhopadhyay1, Himanshi Khandelwal1
1Manipal Institute of Regenerative Medicine, Bengaluru, Manipal Academy of Higher Education, Manipal.
Journal of Visualized Experiments : Jove
|November 27, 2023
Summary
This study introduces a method using flow cytometry to isolate pure stem cells from deciduous teeth. This technique enhances cell viability and proliferation for regenerative medicine applications.
Area of Science:
- Regenerative Biology
- Cell Biology
- Immunology
Background:
- Mesenchymal stem cells (MSCs) have broad differentiation potential and immunomodulatory properties crucial for regenerative biology.
- Cellular ambiguities in MSCs, stemming from diverse sources and in vitro conditions, lead to functional heterogeneity, hindering therapeutic applications.
- Standardized methods are needed to obtain purified, homogeneous MSC populations for safe and effective regenerative medicine.
Purpose of the Study:
- To develop and validate a protocol for identifying and purifying stem cells from human exfoliated deciduous teeth (SHEDs) using fluorescence-assisted single-cell sorting.
- To address the functional heterogeneity of MSCs by isolating specific subpopulations for therapeutic use.
- To establish a reliable method for obtaining viable and proliferative MSCs for downstream applications like lineage-specific differentiation.
Main Methods:
- Utilized multiparametric flow cytometry to analyze surface marker expression (CD90-FITC, CD73-PerCP-Cy5.5, CD105-APC, CD44-V450) on SHEDs.
- Employed fluorescence-assisted single-cell sorting, defining inclusion criteria based on specific positive and negative marker expression to isolate desired MSC subpopulations.
- Assessed cell viability and proliferation post-sorting to ensure the quality of the purified stem cell populations.
Main Results:
- Identified MSCs by simultaneous expression of CD90, CD73, CD105, and CD44 markers.
- Observed a decrease in the percentage of cells expressing all four markers from passage 7 onwards.
- Successfully sorted viable and proliferative MSC subpopulations using defined flow cytometry criteria, demonstrating the efficacy of the isolation protocol.
Conclusions:
- Fluorescence-assisted single-cell sorting provides a robust method for purifying homogeneous MSC populations from SHEDs.
- The developed protocol ensures high cell viability and proliferation, making sorted cells suitable for regenerative medicine and further research.
- This approach can be adapted for other single-cell systems to improve isolation and gather detailed cell surface marker information.
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