CD90 (Thy-1)-Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells

Abstract

Background:

Stem cell-based bone tissue engineering with adipose-derived stromal cells (ASCs) has shown great promise for revolutionizing treatment of large bone deficits. However, there is still a lack of consensus on cell surface markers identifying osteoprogenitors. Fluorescence-activated cell sorting has identified a subpopulation of CD105^low cells with enhanced osteogenic differentiation. The purpose of the present study was to compare the ability of CD90 (Thy-1) to identify osteoprogenitors relative to CD¹⁰⁵.

Methods:

Unsorted cells, CD90⁺, CD90⁻, CD105^high, and CD105^low cells were treated with an osteogenic differentiation medium. For evaluation of in vitro osteogenesis, alkaline phosphatase (ALP) staining and alizarin red staining were performed at 7 days and 14 days, respectively. RNA was harvested after 7 and 14 days of differentiation, and osteogenic gene expression was examined by quantitative real-time polymerase chain reaction. For evaluation of in vivo osteogenesis, critical-sized (4-mm) calvarial defects in nude mice were treated with the hydroxyapatite-poly(lactic-co-glycolic acid) scaffold seeded with the above-mentioned subpopulations. Healing was followed using micro-CT scans for 8 weeks. Calvaria were harvested at 8 weeks postoperatively, and sections were stained with Movat's Pentachrome.

Results:

Transcriptional analysis revealed that the CD90⁺ subpopulation was enriched for a more osteogenic subtype relative to the CD105^low subpopulation. Staining at day 7 for ALP was greatest in the CD90⁺ cells, followed by the CD105^low cells. Staining at day 14 for alizarin red demonstrated the greatest amount of mineralized extracellular matrix in the CD90⁺ cells, again followed by the CD105^low cells. Quantification of in vivo healing at 2, 4, 6, and 8weeks postoperatively demonstrated increased bone formation in defects treated with CD90⁺ ASCs relative to all other groups. On Movat's Pentachrome-stained sections, defects treated with CD90⁺ cells showed the most robust bony regeneration. Defects treated with CD90⁻ cells, CD105^high cells, and CD105^low cells demonstrated some bone formation, but to a lesser degree when compared with the CD90⁺ group.

Conclusions:

While CD105^low cells have previously been shown to possess an enhanced osteogenic potential, we found that CD90⁺ cells are more capable of forming bone both in vitro and in vivo. These data therefore suggest that CD90 may be a more effective marker than CD105 to isolate a highly osteogenic subpopulation for bone tissue engineering.