Several forms of evidence suggest that heterologous transplantation of porcine adipose-derived stem cells (ADSC) enhances bone healing. Freshly harvested ADSC are a heterogeneous population, which contain several types of cells other than stem cells. The isolation of highly purified ADSC can be of clinical importance. The CD34 protein is a known marker of hematopoietic stem cells. In this study, we compared the in vitro growth characteristics and in vivo healing potential of ADSC cells separated using CD34 as a marker.
ADSC were extracted from back fat of 4 male pigs at 6 months of age. Immediately after extraction aliquots of the ADSC were sorted by flow cytometry into CD34 positive (CD34+) and CD34 negative (CD34-) cells. For the in vitro experiment, the unsorted ADSC (UADSC), plus the CD34+, CD34– , and a 50:50 mixture of CD34+ and CD34- (MIX) were plated in 24 well plates, cultured to ~80% confluence, and differentiated into osteocytes. The number of bone nodules was counted and nodule dimension and density was assessed by Image J software. Two wells of each cell type and from each pig were harvested (before cell plating, on several time points during expansion, at confluence, and at 3, 6, and 18 days [d] of differentiation) for cell counting and RNA extraction. Real-time RTPCR was performed for CD34 and COL1A1 genes. Data were statistically analyzed using a Mixed model (SAS), with time and cell type as fixed effect and pig as the random effect. Significance was set with P<.05. For the in vivo experiment, freshly isolated ADSC and sorted CD34+ and CD34- cells were transplanted, in duplicate, into 10 or 25 mm porcine mandible defects. Mandibles were harvested after 8 weeks for evaluation of healing by DEXA scanning.
Sorting of freshly harvested ADSC yielded 42.3±11.0% CD34+ cells. No differences in viability were observed between any cell type (82.3±18.0%). The number of cells and pg RNA/cell were affected by time and by the interaction between cell type x time. The UADSC reached confluence at 6 d after plating while CD34-, CD34+ and MIX cells took 16 d. The pg RNA/cell decreased during the first 6 d after plating. All cells types had an increase in formation of bone nodules after starting osteogenic differentiation. The size of the nodules was not different among cell types and was not affected by time. The number of nodules/well was greater in UADSC than the other cell types but the density of the nodules was numerically lower for this group compared to other cell types. The ratio of cells/nodule decreased through time and was lower in UADSC compared to other cell types. Expression of CD34 gene was numerically higher in the CD34+ vs. CD34- cells and UADSC prior to plating. Expression of CD34 was negatively affected by time, but not affected by differentiation, with only a numerical higher expression in the CD34+ vs. CD34- cells and UADSC prior to plating. Expression of COL1A1 was negatively affected during expansion but did not increase significantly during the first week of differentiation in all cell types. The in vivo healing was measured as % bone density relative to original bone. Fresh isolated ADSC tended (P=0.09) to have greater bone density (1.14±0.23%, mean±SE) compared with CD34+ (0.33±0.28%) cells. The CD34- had a numerically greater (0.82±0.28%), but not significantly different bone density compared with CD34+ cells.
These results indicate that CD34+ cells do not differ significantly from CD34- in both in vitro osteogenic differentiation and in vivo healing capacity. The freshly isolated ADSC appear to have a greater healing capacity than isolated cells, as indicated by both in vitro and in vivo experiments.