CD90 (Thy-1)-positive selection enhances osteogenic capacity of human adipose-derived stromal cells - PubMed (original) (raw)

. 2013 Apr;19(7-8):989-97.

doi: 10.1089/ten.TEA.2012.0370. Epub 2013 Jan 28.

Chunjun Liu, Jeong S Hyun, David D Lo, Daniel T Montoro, Masakazu Hasegawa, Shuli Li, Michael Sorkin, Robert Rennert, Michael Keeney, Fan Yang, Natalina Quarto, Michael T Longaker, Derrick C Wan

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CD90 (Thy-1)-positive selection enhances osteogenic capacity of human adipose-derived stromal cells

Michael T Chung et al. Tissue Eng Part A. 2013 Apr.

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 CD(105).

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.

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Figures

FIG. 1.

FIG. 1.

(A) Preliminary cell selection was performed through size and complexity gating to exclude cell debris and to interrogate a focused population. (B) Fluorescence-activated cell sorting analysis of CD90 single-sorted (left) and CD105 single-sorted adipose-derived stromal cells (ASCs) (right) 36 h after ASC harvest. Color images available online at

www.liebertpub.com/tea

FIG. 2.

FIG. 2.

(A) Alkaline phosphatase staining (top) and quantification (bottom) of ASCs at 7 days differentiation. (B) Alizarin red stain (top) and quantification (bottom) of ASCs at 14 days differentiation demonstrating increased extracellular matrix mineralization for CD90+ ASCs compared to CD90− (*p<0.05) and CD105low cells (#p<0.05). Color images available online at

www.liebertpub.com/tea

FIG. 3.

FIG. 3.

Gene expression of (A) early (RUNX2), (B) intermediate (OPN), and (C) late (OCN) osteogenic markers. Across all genes at each time point, CD90+ cells had greater expression relative to CD90− (*p<0.05) and CD105low cells (#p<0.05). Color images available online at

www.liebertpub.com/tea

FIG. 4.

FIG. 4.

(A) Three-dimensional reconstruction of calvarial defects. Mice were scanned at 2, 4, 6, and 8 weeks following surgery. At each time point, the CD90+-treated defects demonstrated improved bone healing. (B) Quantification of osseous healing by micro-CT revealed significantly more healing with CD90+ cells relative to CD90− (*p<0.05) and CD105low cells (#p<0.05) cells at the 2-, 4-, 6-, and 8-week time points. Color images available online at

www.liebertpub.com/tea

FIG. 5.

FIG. 5.

Calvarial defects 4 mm in size were allowed to heal for 8 weeks before histological analysis by Movat's Pentachrome staining. Pictures were taken in the middle of the defect site. In pentachrome stains, bone appears yellow. Color images available online at

www.liebertpub.com/tea

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References

    1. Friedenstein A.J. Chailakhyan R.K. Latsinik N.V. Panasyuk A.F. Keiliss-Borok I.V. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation. 1974;17:331. - PubMed
    1. Friedenstein A.J. Deriglasova U.F. Kulagina N.N. Panasuk A.F. Rudakowa S.F. Luria E.A., et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol. 1974;2:83. - PubMed
    1. Zuk P.A. Zhu M. Mizuno H. Huang J. Futrell J.W. Katz A.J., et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211. - PubMed
    1. Gronthos S. Franklin D.M. Leddy H.A. Robey P.G. Storms R.W. Gimble J.M. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol. 2001;189:54. - PubMed
    1. Halvorsen Y.C. Wilkison W.O. Gimble J.M. Adipose-derived stromal cells—their utility and potential in bone formation. Int J Obes Relat Metab Disord. 2000;24(Suppl 4):S41. - PubMed

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