Characterization of circulating osteoblast lineage cells in humans

Abstract

We recently identified circulating osteoblastic cells using antibodies to osteocalcin (OCN) or alkaline phosphatase (AP). We now provide a more detailed characterization of these cells. Specifically, we demonstrate that 46% of OCN positive (OCN(pos)) cells express AP, and 37% also express the hematopoietic/endothelial marker CD34. Using two different anti-OCN antibodies and forward/side light scatter characteristics by flow cytometry, we find that OCN(pos) cells consist of two distinct populations: one population exhibits low forward/side scatter, consistent with a small cell phenotype with low granularity, and a second population has higher forward/side scatter (larger and more granular cell). The smaller, low granularity population also co-expresses CD34, whereas the larger, more granular cells are CD34 negative. Using samples from 26 male subjects aged 28 to 68 years, we demonstrate that the concentration of circulating OCN(pos) cells increases as a function of age (R=0.59, P=0.002). By contrast, CD34(pos) cells tend to decrease with age (R=-0.31, P=0.18); as a consequence, the ratio of OCN(pos):CD34(pos) cells also increase significantly with age (R=0.54, P=0.022). These findings suggest significant overlap between circulating cells expressing OCN and those expressing the hematopoietic/endothelial marker CD34. Further studies are needed to define the precise role of circulating OCN(pos) cells not only in bone remodeling but rather also potentially in the response to vascular injury.

Figure 1

Detection of OCN by immunohistochemistry in normal human bone using the SC V-19 anti-OCN (A, × 40) or the HT AON-5031 anti-OCN (B, × 40) antibodies. Note the brown staining of the matrix and cells with both antibodies. The inserts in A and B show a high power image (× 100) of a positively stained osteocyte in the human bone section. In the same sections, neither antibody stained cells or matrix in the adjacent muscle or connective tissue (C, SC V-19; D, HT AON-5031). Serial sections were also stained with secondary antibody only (E, F). Note the absence of any brown staining in panels C–F; the blue staining is from the Schmidt’s hematoxylin, used to visualize the cells.

Figure 2

Analysis of cell phenotypes using the SC V-19 anti-OCN and anti-CD34 antibodies. OCN^pos cells were first isolated using MACS, then stained with the anti-CD34 antibody, and subsequently analyzed by FACS. OCN^pos/CD34^neg and OCN^pos/CD34^pos cells were then back-gated to analyze their forward/side scatter characteristics, with higher forward scatter indicating larger size and higher side scatter indicating more granularity. Shown are density plots, with darker color representing more cells.

Figure 3

Phase contrast microscopy of cells (× 63) sorted first using MACS and the SC V-19 anti-OCN antibody and then separated by FACS based gating for positive cells and on low forward/side scatter (A) or higher forward/side scatter (B). Consistent with the forward/side scatter characteristics, cells in panel A are smaller and exhibit less granularity than the cells in panel B.

Figure 4

(A–E), confocal microscopy (× 63) using the SC V-19 anti-OCN labeled with a PE-conjugated secondary antibody (red, panel A), the anti-CD34 FITC-conjugated antibody (green, panel B), nuclear stain (DAPI, panel C), the merged image (panel D), and the merged image at higher power (panel E). (F–J), confocal microscopy (x 63) using the HT AON-5031 anti-OCN labeled with a PE-conjugated secondary antibody (red, panel A), the anti-CD34 FITC-conjugated antibody (green, panel B), nuclear stain (DAPI, panel C), the merged image (panel D), and the merged image at higher power (panel E).

Figure 5

Confocal microscopy (× 63) using the anti-AP antibody labeled with a PE-conjugated secondary antibody (red, panel A), the SC V-19 anti-OCN labeled with a FITC-conjugated secondary antibody (green, panel B), nuclear stain (DAPI, panel C), the merged image (panel D).

Figure 6

Representative dot plots showing co-staining of (A) OCN with AP; (B) OCN with CD34; and (C) AP with CD34. Percentages refer to the percent of gated cells in each quadrant.

Figure 7

Percent OCN^pos cells as a function of age. R = 0.59, P = 0.002.

Figure 8

Model of the bone remodeling compartment (BRC) as described by Hauge and colleagues [24]. In this model, osteoblastic cells enter the BRC principally via the afferent capillary and could include non-adherent (or adherent) bone marrow (BM) cells, circulating cells, or osteoblastic cells originating from precursor cells in the vasculature. MSC, marrow stromal cell.