Isolation, characterization and differentiation potential of rat bone marrow stromal cells.
BACKGROUND
Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with varying degrees of plasticity in humans. However, there are a few reports on rat-derived cells, which could be good models for the research purpose. We describe here a simple method of establishing the rat bone marrow stromal cells by the principle of adhesion and document their phenotype along with their differentiation potential to other lineages.


METHODS
Rat bone marrow stromal cells were isolated by three methods: direct plastic adherence, ficoll hypaque separation and a combination of both. The stromal cells obtained by these methods were characterized by fluorescent activating cell sorting (FACS) for established hematopoietic and non-hematopoietic markers. The cells obtained by combination method (combination of ficoll density gradient centrifugation and plastic adherence) were cultured and serially passaged. Transcriptional confirmation was done by reverse transcription polymerase chain reaction (RT-PCR) for vimentin and collagen type 1 alpha 1. Attempts were made to differentiate the marrow stromal cells into adipocytes, osteocytes and neuronal like cells.


RESULTS
Bone marrow samples from 10 rats yielded 4-5 million bone marrow mononuclear cells /ml per femur. Of the three methods tested, a combination method yielded good growth of spindle cells. The cells obtained by combined method showed high percentage of positivity for vimentin, fibronectin and CD90 and negative for hematopoietic markers. Further, RT-PCR confirmed vimentin and collagen type - 1 alpha 1 expression. Oil red O staining and Alizarin red staining confirmed adipocytic and osteogenic differentiation. On immunocytochemical analysis, the cells expressed nestin, beta-tubulin III, neurofilament and synaptophysin.


CONCLUSIONS
Adequate quantities of rat marrow stromal cell cultures can be established by a simple method based on adhesion properties. Their phenotypic characteristics and plasticity support the evidence that they are mesenchymal stem cells with a distinct tendency for neural lineage.
Polisetti
Naresh
N
Stem Cell Biology Laboratory, L.V. Prasad Eye Institute, Banjara Hills, Hyderabad, India.
Chaitanya
V G
VG
Babu
Phanithi Prakash
PP
Vemuganti
Geeta K
GK
D017667:Adipocytes / Q000502:physiology; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured / Q000502:physiology; D002499:Centrifugation, Density Gradient / Q000379:methods; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000502:physiology; D010011:Osteocytes / Q000502:physiology; D010641:Phenotype; D051381:Rats; D017208:Rats, Wistar; D017154:Stromal Cells / Q000145:classification / Q000502:physiology*; D018800:Thy-1 Antigens / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
C000716129:Col1a1 protein, rat; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D005353:Fibronectins; D009419:Nerve Tissue Proteins; D018800:Thy-1 Antigens; D014746:Vimentin