Osteogenic and hematopoietic functional studies of isolated fractions of OBs. (A) Ca deposition and enzymatic ALP activity of parental populations and groups 1 through 11 shown in Figure 6. Cells were cultured in osteogenic media (αminimal essential medium supplemented with 10% fetal calf serum, 50 μg/mL ascorbic acid, 2 times per week). Starting on day 7, cultures were supplemented with 5mM β-glycerophosphate to induce mineralization and were assayed on day 14. ALP activity and Ca deposition were assessed as described in “ALP activity” and “Quantitative analysis of Ca deposition.” With the exception of the unsorted 2-day C and 2-day LB data (n = 3 in duplicate), results are from 2 experiments each performed in duplicate. (B) Parental populations and cell fractions collected in sufficient numbers were used in cocultures with LSK cells. Cultured cells were harvested on day 7, counted, and assayed for HPC content performed in triplicate. Data shown were collected from 2 sorting experiments. (C-D) Results from Ca deposition (C) and enzymatic ALP activity (D) from 2-day C, 2-day LB, 6- to 8-week C, and 6- to 8-week LB (n = 6-8 for all groups) are reported as box plots depicting the range of data points and the mean (line). Error bars represent SD associated with the mean. Data points more than 2 SD from the mean are identified on the plot and were still used in statistical determinations. Statistical analysis methods used to analyze data shown in panels C and D are described in “Statistical analysis.”