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Protocol to analyze and validate transcriptomic changes in PDGFRβ-KO mesenchymal stem cell osteogenic potential in
Alastair Morris Kilpatrick1, Madalena Marques1, Mario Gomez-Salazar1
1Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK.
STAR Protocols
|January 14, 2023
Summary
This study details a protocol to analyze mesenchymal stem/stromal cells (MSCs) osteogenic potential without PDGFRβ signaling. It covers cell preparation, transcriptomic analysis, and functional validation for MSC differentiation.
Area of Science:
- Stem cell biology
- Molecular biology
- Genomics
Background:
- Mesenchymal stem/stromal cells (MSCs) possess osteogenic potential, differentiating into osteoblasts.
- Platelet-derived growth factor receptor beta (PDGFRβ) signaling critically regulates MSC osteogenic capacity at both transcriptomic and cellular levels.
- Understanding PDGFRβ's role is crucial for controlling MSC differentiation for therapeutic applications.
Purpose of the Study:
- To present a "computer to the bench" protocol for analyzing MSC osteogenic potential.
- To investigate transcriptomic and cellular changes in MSCs lacking PDGFRβ signaling.
- To provide a comprehensive method for functional validation of MSC osteogenic capacity.
Main Methods:
- Isolation and preparation of cells from mouse embryos.
- Single-cell RNA sequencing (scRNA-seq) for transcriptomic analysis.
- MSC derivation, culture, and osteogenic differentiation assays.
Main Results:
- Detailed protocol for MSC preparation and culture.
- Methodology for analyzing transcriptomic alterations in the absence of PDGFRβ.
- Functional validation of osteogenic potential through assays.
Conclusions:
- The protocol enables detailed analysis of MSC osteogenic potential.
- It facilitates the study of PDGFRβ's role in MSC differentiation.
- This research provides a framework for investigating stem cell behavior and differentiation pathways.
