1. CD34+ cells from mobilized peripheral blood retain fetal bone marrow repopulating capacity within the Thy-1+ subset following cell division ex vivo 
Judy C Young, Karen Lin, Gun Hansteen, Marilyn Travis, Lesley J Murray, Li Jaing, Roland Scollay, Beth L Hill 
Experimental Hematology 
Volume 27, Issue 6, Pages (June 1999)
DOI: /S X(99)

2. Figure 1
Retention of Thy-1+ cells after 112-hour culture. CD34+ Thy-1+ mobilized peripheral blood cells were cultured for 112 hours in thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), and TPO, FL, and interleukin 6 (IL-6). Cultured cells were harvested, counted, and analyzed for CD34 and Thy-1 antigen expression by flow cytometry. (Top) Percentages of total viable cells that remained Thy-1+. (Bottom) Absolute change in numbers of cells expressing Thy-1, calculated using the formula: Fold increase in total cell number × percent Thy-1+ cells post culture. The average values obtained from seven tissue samples are shown. The error bars represent the standard deviation from the mean
Experimental Hematology  , DOI: ( /S X(99) )

3. Figure 2
Kinetics of cell division of mobilized peripheral blood (MPB) CD34+Thy-1+ cells in 112-hour culture. CD34+Thy-1+ MPB cells were labeled with CFSE dye, cultured in TPO, FL, and KL (TFK) or TPO, FL, and IL-6 (TF6) and then analyzed for Thy-1 expression and CFSE fluorescence by FACS. The histograms shown represent CFSE fluorescence, and each peak represents a distinct number of divisions. The 0-hour control (fluorescence of undivided cells) was obtained by fixing an aliquot of fresh cells after CFSE labeling and holding at 4°C for 112 hours. (Top) Total viable cell events are shown. (Bottom) Thy-1+ cell events only are shown. The percentages of viable cells in each division peak are shown
Experimental Hematology  , DOI: ( /S X(99) )

4. Figure 3
Strategy for isolating Thy-1+ and Thy-1− populations after culture with TPO, FL, and KL. CD34+Thy-1+ MPB cells cultured for 112 hours in TPO, FL, and KL were stained with anti-CD34 and anti-Thy-1 antibodies. (Bottom) Flow cytometric gates for isolating the Thy-1 positive and negative populations. (Top) Thy-1+ gate was set to include events brighter than the isotype control staining. A gap was left between the Thy-1+ and Thy-1+ gates to ensure minimal contamination of one population with the other
Experimental Hematology  , DOI: ( /S X(99) )

5. Figure 4
Strategy for isolating Thy-1+ cell populations having undergone various numbers of divisions during culture. CD34+Thy-1+ mobilized peripheral blood cells were labeled with CFSE dye, cultured in TPO, FL, and KL or TPO, FL, and IL-6 and then stained for Thy-1 expression. Cellular events were analyzed by flow cytometry with Thy-1 fluorescence on the y-axis and CFSE fluorescence on the x-axis. A portion of the tissue sample fixed on day 0 after CFSE labeling and held at 4°C was used as a marker for undivided cells. Thy-1+ cells having undergone 1, 2, 3, and 4 divisions were each sorted. The percentages of Thy-1+ cells out of total viable cells in the cultures that remained undivided or had divided during culture in each cytokine combination in a representative experiment are shown
Experimental Hematology  , DOI: ( /S X(99) )

6. Figure 5
Cobblestone area-forming cell (CAFC) activity of Thy-1+ cells cultured in TPO, FL, and KL is retained after three divisions ex vivo. The sorted cell populations represented in Figure 4 were tested for CAFC activity. The CAFC frequencies are shown (y-axis) for Thy-1+ cell populations having undergone sequential numbers of divisions during 112-hour culture (x-axis). The average CAFC frequency of the freshly isolated, uncultured CD34+Thy-1+ mobilized peripheral blood cells is also shown. Each datapoint represents the average frequency of 72 to 312 wells from 3 to 13 CAFC assays (each assay was performed on a different tissue). The error bars represent the standard deviation from the mean of each datapoint. Because cells divided less rapidly in the TPO, FL, and IL-6 growth condition, insufficient datapoints were obtained for the fourth division to be statistically sound
Experimental Hematology  , DOI: ( /S X(99) )

7. Figure 6
Engraftment of SCID-hu bone with cultured Thy-1+ cells. CD34+ cells were sorted as in Figure 4, segregating individual divisions. Each population was tested at two doses (5,000 and 15,000 cells) for engraftment in the SCID-hu bone assay as described in the legend to Table 2. Each datapoint represents an individual bone graft. Grafts containing less than 1% human donor cells out of total cells in the graft were considered to have no engraftment. Because, typically, at least 10,000 cells (both mouse and human) could be obtained from the grafts, 1% represented at least 100 cellular events. Engraftment rates (number of positive grafts/number of grafts injected) for uncultured CD34+ cells and cultured Thy-1+ cells having undergone various numbers of divisions are shown. Average numbers of donor cells recovered from grafts injected with uncultured CD34+ cells, and TPO, FL, and KL, or TPO, FL, and IL-6 cultured cells are shown
Experimental Hematology  , DOI: ( /S X(99) )

8. Figure 7
Substantial numbers of Thy-1+ cells continue to cycle after division in culture. Postculture cells were analyzed as in Figure 4, except that, before analysis, cells also were stained with Hoechst dye to segregate cells containing a 2N vs >2N DNA content. In the representative plot shown, events have been gated such that only viable Thy-1+ cells are shown. The percentages of Thy-1+ cells that are in G0/G1 or S/G2/M for each division are shown
Experimental Hematology  , DOI: ( /S X(99) )