382 ISOLATION AND CHARACTERIZATION OF SIZE-SIEVED MESENCHYMAL STEM CELLS FROM PERIVASCULAR AND INTERVASCULAR WHARTONS JELLY OF HORSE UMBILICAL CORD

Abstract

Horse umbilical cord has recently been suggested as a potential source for mesenchymal stem cells (MSC). Despite their clinical importance for treating injuries to musculoskeletal tissues, there is still not a well-defined protocol for the isolation and expansion of MSC in culture. Literature shows few experiments conducted on equine MSC; in these experiments cells are isolated primarily by their tight adherence to culture plastic dishes. These cells are initially heterogeneous so we aimed to separate homogeneous subpopulations of MSC using multi-dishes with transwell inserts of 8 μm pores. After digestion of perivascular and intervascular Wharton’s jelly with collagenase (0.75 mg mL^-1) for 16 h at 37°C, 12 primary cell cultures from 3 animals were obtained. Cells were plated at a density of 10⁶ cells/cm² on these culture dishes. The lower (8μm) and upper populations of perivascular and intervascular cells, cultured in fetal bovine serum-supplemented DMEM-HG, with EGF, were studied for their morphology, renewal capacity, mesenchymal markers expression, and differentiating potential. Cells with less than an 8 μm diameter that adhered to the lower plate surface were a morphological homogeneous population if compared with the upper larger sized population of either perivascular or intervascular jelly. Every subpopulation steadily proliferated over the passages studied, without spontaneous differentiation, reaching confluence even after 10 passages. The large cells of the perivascular portion propagated slowly and passed 16.58 cell population doublings (PD) after 31 days, whereas in the same time range, the small cells reached 19.49 cell PD. After the seventh passage, the proliferating traits of the 2 cell populations became similar. As a control, the unsieved perivascular portion passed 8.54 cell PD. On the contrary, in the intervascular portion, the large cells propagated more rapidly with respect to the small ones (20.53 v. 13.66 cell PD) and the unsieved control (9.42 cell PD). The fibroblast colony forming unit (CFU-F) assay supported these differences with greater CFU-F for the small perivascular cells and the large intervascular cells (the rates were, respectively, 1:133 and 1:106). As shown by RT-PCR, every subpopulation was positive for MSC markers (CD105, CD 44, CD 29) and CD34 negative. Osteogenic differentiation of each subpopulation was confirmed by von Kossa stain and osteocalcin mRNA expression after 10 days of induction. At this time point, only intervascular cells expressed osteopontin, suggesting an earlier expression of this marker in these cells. We suggest that in the perivascular portion, the large cells are mature MSC and the smaller ones are recycling stem cells and that in the intervascular fraction, MSC do not divide rapidly until after they are separated from non-MSC of whole primary culture. The size-sieving procedure is a simple and effective method to isolate more proliferative MSC.