Figure 4
Figure 4. Cytokine mobilized osteoblasts promote HSC proliferation and maintain HSC reconstituting potential. (A) Experimental strategy. A total of 100 000 lineage-negative (Lin−) BM cells, which include HSC and hematopoietic progenitor cells (HPCs) from untreated mice, were exposed for 12 hours in vitro to 2000 OPN+ osteoblasts isolated from untreated mice (white) and analyzed for KTLS frequency and/or proliferation by loss of CFSE by FACS. (B) Frequency of c-kit+Thy1.1loLin−Sca1+ (KTLS) HSC among Lin-depleted BM cells (HPC) maintained alone () or with untreated () or cytokine-modified (D2; ■) osteoblasts for 12 hours. KTLS HSC frequency among input HPCs is also shown (□). Data are presented as the average frequency of KTLS HSC (± SD) as determined by FACS in short-term (12 hours) HPC:osteoblast cocultures (*P < .05). Data are compiled from 6 independent experiments performed. (C) Increased proliferation of CFSE-labeled KTLS HSC among Lin-depleted BM cells (HPC) cultured with cytokine-modified (HPC:D2, panel 3) compared with untreated (HPC:untreated, panel 2) osteoblasts after 36 hours. Histograms represent CFSE labeling of only those cells falling within the KTLS gate and are representative of 7 independent experiments. (D) Average frequency (± SD) of divided KTLS cells per peak (1, 2, 3, as indicated in panel C) in coculture experiments (*P < .05). (E) Increased cell survival in HPC cultures containing untreated or D2 osteoblast, compared with HPC cultured alone. Cultures were performed as described previously, and stained with KTLS surface markers in addition to markers of cell death and viability (annexin V (early apoptosis marker) and PI (late apoptosis marker)). Data are representative of 2 independent experiments. (F) Increased total numbers of KTLS HSCs in HPC:D2 cultures. HSC numbers were determined for each culture by multiplying the frequency of KTLS cells (determined by FACS) by the total number of cells present. Data are shown as the mean absolute cell number for each condition over 7 independent experiments plus or minus the SD (*P < .05).

Cytokine mobilized osteoblasts promote HSC proliferation and maintain HSC reconstituting potential. (A) Experimental strategy. A total of 100 000 lineage-negative (Lin−) BM cells, which include HSC and hematopoietic progenitor cells (HPCs) from untreated mice, were exposed for 12 hours in vitro to 2000 OPN+ osteoblasts isolated from untreated mice (white) and analyzed for KTLS frequency and/or proliferation by loss of CFSE by FACS. (B) Frequency of c-kit+Thy1.1loLin−Sca1+ (KTLS) HSC among Lin-depleted BM cells (HPC) maintained alone () or with untreated () or cytokine-modified (D2; ■) osteoblasts for 12 hours. KTLS HSC frequency among input HPCs is also shown (□). Data are presented as the average frequency of KTLS HSC (± SD) as determined by FACS in short-term (12 hours) HPC:osteoblast cocultures (*P < .05). Data are compiled from 6 independent experiments performed. (C) Increased proliferation of CFSE-labeled KTLS HSC among Lin-depleted BM cells (HPC) cultured with cytokine-modified (HPC:D2, panel 3) compared with untreated (HPC:untreated, panel 2) osteoblasts after 36 hours. Histograms represent CFSE labeling of only those cells falling within the KTLS gate and are representative of 7 independent experiments. (D) Average frequency (± SD) of divided KTLS cells per peak (1, 2, 3, as indicated in panel C) in coculture experiments (*P < .05). (E) Increased cell survival in HPC cultures containing untreated or D2 osteoblast, compared with HPC cultured alone. Cultures were performed as described previously, and stained with KTLS surface markers in addition to markers of cell death and viability (annexin V (early apoptosis marker) and PI (late apoptosis marker)). Data are representative of 2 independent experiments. (F) Increased total numbers of KTLS HSCs in HPC:D2 cultures. HSC numbers were determined for each culture by multiplying the frequency of KTLS cells (determined by FACS) by the total number of cells present. Data are shown as the mean absolute cell number for each condition over 7 independent experiments plus or minus the SD (*P < .05).

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