Characterization of Human Bone Marrow Stromal Cells During Multi-passaging

Objectives:

Human bone marrow stromal cells (hBMSCs) are adherent fibroblast-like cells found in the bone marrow. They are a heterogeneous population of cells that includes a subset of osteoprogenitors. BMSCs have been widely used for tissue engineering, especially for bone regeneration. However, for clinical application currently, large quantities of hBMSCs are usually required for transplantation which is typically produced by serial passages of the cells ex vivo. We examined the effects of in vitro expansion on hBMSCs’ proliferation, multidifferentiation, and gene expression profiles.

Methods:

hBMSCs were harvested from surgical waste bone specimens from 3 healthy adults with IRB approval. The hBMSCs were cultured in α-MEM with 10% FBS and 1% penicillin-streptomycin. hBMSCs were trypsinized and passaged when they reached 70-80% confluence. Cells from early passage (p2 or 3) were compared with late passage (p7 or 8). MTT assay was used to determine the growth kinetics of hBMSCs. Multidifferentiation ability was assayed by culturing cells in osteogenic or adipogenic induction medium and stained with alizarin red or oil red. For gene expression comparison, total RNA was extracted from hBMSCs using RNeasy Kit (Qiagen) and gene expression was examined with human Mesenchymal Stem Cell PCR Array (SABiosciences).

Result:

The proliferation rate of the hBMSCs dramatically diminished (p<0.01) from p2 to p7. When induced in vitro, the osteogenic differentiation ability greatly decreased in late passages. 15 of 84 genes were differentially expressed by more than 3-fold between p2 and p7. Adipogenesis marker, PPARγ, increased and chondrogenesis marker, TGFβ1, decreased in p7. Specific markers for mesenchymal stem cells, such as MCAM, VCAM1, MMP2 and Nestin, decreased with passaging (p<0.05).

Conclusion:

Expansion of hBMSCs results in gradual loss of stem cell qualities that may support osteoprogenitor function and proliferation. Culture conditions are crucial to clinical application and methods should be developed to maintain cell stemness.