title: Isolation of the stromal-vascular fraction of mouse bone marrow markedly enhances the yield of clonogenic stromal progenitors.
abstract: The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of mouse bone marrow stromal cells (BMSCs), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of stromal cell stem/progenitors, we hypothesized that a potential reason for the low yield of mouse BMSCs is the flushing of the marrow used to remove single-cell suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of BMSCs physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs, which yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1631.8 + 199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32 + 1.9) by at least 2 orders of magnitude (P < .001; N = 8) with an accompanying 113.95-fold enrichment of CFU-F frequency when plated at low oxygen (5%). Purified BMSC populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single-cell suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays.
authors.affiliation: Brown Foundation Institute of Molecular Medicine for Prevention of Human Diseases, Houston, TX, USA.
authors.forename: Colby
authors.initials: C
authors.lastname: Suire
authors.forename: Nathalie
authors.initials: N
authors.lastname: Brouard
authors.forename: Karen
authors.initials: K
authors.lastname: Hirschi
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Simmons
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D004730:Endothelium, Vascular / Q000166:cytology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D013234:Stem Cells / Q000166:cytology*; D017154:Stromal Cells / Q000166:cytology*