Assessment of Bone Marrow Mesenchymal Stem Cells Biological Characteristics in Patients with Polycythemia Vera

Objective: To isolate and culture bone marrow mesenchymal stem cell (MSCs) from polycythemia vera (PV) patients and identify their in-vitro biological characteristics.

Methods: Bone marrow was extracted from 9 patients with PV and 6 normal individuals. MSCs were isolated by density gradient centrifugation. The adherent cells were cultured and passaged. The cultured cells harvested from the third passage were assessed. The morphology of the cells was observed by Wright’s staining and the growth curve of the cells was plotted by cell counting. The ultrastructure of MSCs was observed with electron microscopy. The cell cycle and immunophenotype of the expanded MSCs were detected by flow cytometry (FCM). Different agents were used to induce MSCs to differentiate into osteocyte and adipocyte. Von Kossa staining and oil-red staining were used to examine the differentiation. Conventional cytogenetics (CC) was carried out to detect karyotype. Alleles specific polymerase chain reaction (AS-PCR) was used to detect JAK2V617F mutation. RT-PCR were used to analyze the expression of multiple hematopoietic growth factors at mRNA level for MSCs.

Results: MSCs from patients and normal individuals displayed a fibroblast-like morphology adhering to the culture plate and the doubling time of expand MSC was about 43h. The cells were CD73, CD90 positive, and CD34, CD45, CD133, HLA-DR negative. Under suitable conditions, these MSCs could differentiate into osteocytes and adipocytes. MSCs from patients and normal individuals showed normal karyotype. And JAK2V617F mutation was negative in both MSCs. The same expression pattern was seen in patients and normal individuals that they expressed mRNA of SCF, Flt3-ligand, TPO, LIF, IL-6, IL-11 and did not express G-CSF, GM-CSF and IL-3.

Conclusion: MSCs from patients with PV are similar to that from normal individuals.