Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture

Abstract

Summary: We isolate and characterize osteoblasts from humans without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders.

Introduction: There is currently no data regarding the expression of specific genes or pathways in human osteoblasts that have not been subjected to extensive in vitro culture. Thus, we developed methods to rapidly isolate progressively enriched osteoblast populations from humans and characterized these cells.

Methods: Needle bone biopsies of the posterior iliac crest were subjected to sequential collagenase digests. The cells from the second digest were stained with an alkaline phosphatase (AP) antibody, and the AP+ cells were isolated using magnetic cell sorting.

Results: Relative to AP- cells, the AP+ cells contained virtually all of the mineralizing cells and were enriched for key osteoblast marker genes. The AP+ cells were further purified by depletion of cells expressing CD45, CD34, or CD31 (AP+/CD45/34/31- cells), which represented a highly enriched human osteoblast population devoid of hematopoietic/endothelial cells. These cells expressed osteoblast marker genes but very low to undetectable levels of SOST. We next used high-throughput RNA sequencing to compare the transcriptome of the AP+/CD45/34/31- cells to human fibroblasts and identified genes and pathways expressed only in human osteoblasts in vivo, but not in fibroblasts, including 448 genes unique to human osteoblasts.

Conclusions: We provide a detailed characterization of highly enriched human osteoblast populations without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders.

Figure 1

a Example of needle biopsies obtained from the iliac crest and b µCT image of one of the human bone biopsies

Figure 2

Alizarin Red staining of sequential fractions of cells from human bone biopsies and bone marrow

Figure 3

Relative expression of osteoblastic genes in a first versus second digest cells; b AP− versus AP+ cells; and c AP+/CD45/34/31+ versus AP+/CD45/34/31− cells. *P <0.05; **P <0.01; ***P <0.001; ^†P =0.08. ALPL alkaline phosphatase, BGLAP osteocalcin, IBSP bone sialoprotein, SPP1 osteopontin

Figure 4

a Relative expression of hematopoietic/endothelial marker genes in AP+/CD45/34/31+ versus AP+/CD45/34/31− cells. *P <0.05; **P <0.01. b Relative expression of osteocyte genes in the stripped bone fragments, AP+ cells, and AP+/CD45/34/31− cells. *P <0.05; **P <0.01; ^†P = 0.06 versus the stripped bone samples