Related Experiment Video
Updated: May 19, 2026
Purification, Expansion, and Flow Cytometry-Based Phenotyping of Mouse Derived Bone Marrow Mesenchymal Stem Cells
Published on: July 11, 2025
Differentiation potential and GFP labeling of sheep bone marrow-derived mesenchymal stem cells
Marta Czernik1, Antonella Fidanza, Martina Sardi
1Department of Comparative Biomedical Science, University of Teramo, 64100 Teramo, Italy. mczernik@unite.it
Abstract:
Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow-derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34- and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre-clinical sheep model to test the efficiency and safety of cell replacement therapy.
Insights
This study isolated sheep bone marrow mesenchymal stem cells (MSCs) and confirmed their ability to differentiate into multiple cell types. Reporter gene expression in cloned embryos suggests potential for stem cell research and cell therapy models.
Area of Science:
- Stem Cell Biology
- Regenerative Medicine
- Animal Models
Background:
- Mesenchymal stem cells (MSCs) reside in bone marrow and exhibit multipotency.
- MSCs can differentiate into various connective tissues, including bone, cartilage, and fat.
- Sheep models are valuable for preclinical studies in cell therapy.
Purpose of the Study:
- To isolate, expand, and characterize sheep bone marrow-derived MSCs.
- To assess the differentiation potential of these MSCs.
- To demonstrate stable reporter gene expression in MSCs for future research.
Main Methods:
- Bone marrow collection and mononuclear cell isolation via density gradient centrifugation.
- Characterization of MSCs (CD44+, CD34-) and transfection with a green fluorescent protein (GFP) reporter gene.
- Nuclear transfer of GFP+ MSCs into cloned blastocysts and assessment of GFP expression.
Main Results:
- Sheep MSCs were successfully isolated and characterized.
- Transfected MSCs showed stable GFP expression in approximately 46.6% of cloned blastocysts.
- MSCs demonstrated differentiation into adipocytes, chondrocytes, and osteoblasts under specific culture conditions.
Conclusions:
- Sheep bone marrow-derived MSCs possess significant multipotency and plasticity.
- The study establishes a foundation for a preclinical sheep model using genetically modified MSCs for cell therapy research.
- This model can facilitate comparative studies on adult stem cell versus embryonic stem cell differentiation capacity.
