Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients by Anna Jetmore, P. Artur Plett, Xia Tong, Frances M. Wolber, Robert Breese, Rafat Abonour, Christie M. Orschell-Traycoff, and Edward F. Srour Blood Volume 99(5):1585-1593 March 1, 2002 ©2002 by American Society of Hematology

Flow cytometric analysis of murine BM cells of NOD/SCID recipients of CFSE-stained xenografts of human CD34+ cells.Human BM CD34+ cells were stained with CFSE and transplanted into conditioned NOD/SCID recipients. Flow cytometric analysis of murine BM cells of NOD/SCID recipients of CFSE-stained xenografts of human CD34+ cells.Human BM CD34+ cells were stained with CFSE and transplanted into conditioned NOD/SCID recipients. At 40 hours AT, mice were killed, and BM cells were stained with PE-conjugated immunoglobulin G1 (isotype control, dot plot A) or PE-conjugated CD34 (dot plot B) and analyzed flow cytometrically. CFSE fluorescence was detected on the x-axis and that of PE on the y-axis. Dot plot A shows CFSE+ cells with background level of PE fluorescence (identified by the horizontal cursor) clearly distinguishable from murine CFSE− BM cells. In dot plot B, which displays light scatter gated events from a listmode file containing 1.5 × 105 events, a prominent population of CFSE+CD34+ cells can be identified. The percentage of cells contained in pertinent quadrants is given below each dot plot. Sort windows R1 and R2 in dot plot B were used to isolate CFSE+CD34+ and CFSE+CD34− cells, respectively. Please note that the width of the sorting windows R1 and R2 was sufficient to include all detectable CFSE+ cells regardless of their proliferative history in the BM of recipient mice. Anna Jetmore et al. Blood 2002;99:1585-1593 ©2002 by American Society of Hematology

Cell cycle analysis of fresh, ex vivo–cultured and BM- and spleen-homed MPB and BM CD34+ cells.CD34+ cells from fresh MPB (light bars) or BM (dark bars) were enriched by immunomagnetic selection and stained with CFSE as described in “Materials and methods.”... Cell cycle analysis of fresh, ex vivo–cultured and BM- and spleen-homed MPB and BM CD34+ cells.CD34+ cells from fresh MPB (light bars) or BM (dark bars) were enriched by immunomagnetic selection and stained with CFSE as described in “Materials and methods.” A sample was retained for cell cycle analysis at time zero and for initiating short-term ex vivo cultures. Cells in culture were maintained with a 6-cytokine cocktail containing human SCF at 100 ng/mL, Flt-3 ligand at 50 ng/mL, MGDF at 50 ng/mL, IL-3 at 100 ng/mL, IL-6 at 100 ng/mL, and GM-CSF at 20 ng/mL. Remaining cells were transplanted into conditioned NOD/SCID mice, and 40 hours AT bone marrow (AT/BM) and spleen (AT/Spl) cells were recovered from individual mice and human CFSE+ cells were isolated by flow cytometric cell sorting. Sorted cells and cells maintained in culture were stained with PI and analyzed for cell cycle status. Each bar represents the mean ± SD of 2 to 6 measurements for MPB samples (from 3 independent experiments) and 4 to 5 measurements for BM samples (from 5 independent experiments). Differences between fresh and cultured BM and MPB cells,P < .01; differences between ex vivo–cultured BM and MPB and BM- or spleen-homed cells, P < .01; differences between fresh and BM- or spleen-homed BM and MPB cells,P > .01. Anna Jetmore et al. Blood 2002;99:1585-1593 ©2002 by American Society of Hematology

Cell cycle analysis of fresh, ex vivo–cultured and BM- and spleen-homed MPB and BM CD34+ cells recovered from control and cytokine-treated NOD/SCID recipients.CD34+ cells were isolated from fresh MPB or BM and stained with CFSE as described in “Materials an... Cell cycle analysis of fresh, ex vivo–cultured and BM- and spleen-homed MPB and BM CD34+ cells recovered from control and cytokine-treated NOD/SCID recipients.CD34+ cells were isolated from fresh MPB or BM and stained with CFSE as described in “Materials and methods.” A sample was retained for cell cycle analysis at time zero and for initiating short-term ex vivo cultures maintained as described in the legend of Figure 2. Remaining cells were transplanted into control and cytokine-treated, conditioned NOD/SCID mice. Cytokine treatment of recipient mice consisted of 4 daily intraperitoneal injections on days −2, −1, 0 (day of transplantation), and +1 of a cytokine cocktail delivering per mouse 10 μg SCF, 5 μg Flt-3 ligand, 5 μg MGDF, 6 μg IL-3, 2 μg IL-6, 6 μg GM-CSF, and 10 U erythropoietin. At 24 and 48 hours AT, BM and spleen (Spl) cells were recovered from individual mice, and human CFSE+ cells were isolated by flow cytometric cell sorting. Cell cycle status of all groups of cells was determined by PI staining. Each bar represents the mean of 1 to 3 measurements of MPB or BM samples at the 24-hour time point and 2 to 3 measurements at the 48-hour time point from 3 independent experiments. Statistical analysis of measurements made at 48 hours between control and cytokine-treated recipients was not significant (P > .2). Anna Jetmore et al. Blood 2002;99:1585-1593 ©2002 by American Society of Hematology

Cell cycle analysis of ex vivo–expanded MPB and BM CD34+ cells recovered from the BM and spleen of NOD/SCID transplantation recipients 40 hours AT.CD34+ cells were isolated from fresh MPB (light bars) and BM (dark bars) and cultured in vitro as described in... Cell cycle analysis of ex vivo–expanded MPB and BM CD34+ cells recovered from the BM and spleen of NOD/SCID transplantation recipients 40 hours AT.CD34+ cells were isolated from fresh MPB (light bars) and BM (dark bars) and cultured in vitro as described in “Materials and methods” and in the legend of Figure 2. On day 5, cells were harvested, washed, stained with CFSE, and transplanted into conditioned NOD/SCID mice. A sample was retained for cell cycle analysis of day 5 cells (d 5 ex vivo cult), while another was maintained in culture for an additional 40 hours (d 5 cult + 40 h). Human CFSE+cells were isolated by flow cytometric cell sorting 40 hours AT from BM (AT/BM) and spleen (AT/Spl) cells. Cell cycle status of all groups of cells was determined by PI staining. Each bar represents the mean ± SD of 2 measurements for MPB and 3 to 4 measurements for BM samples from 4 independent experiments. Anna Jetmore et al. Blood 2002;99:1585-1593 ©2002 by American Society of Hematology

Cell cycle analysis of mitotically defined groups of ex vivo–expanded MPB and BM CD34+ cells recovered from the BM and spleen of NOD/SCID transplant recipients 40 hours AT.CD34+ cells were isolated from fresh MPB and BM and cultured in vitro as described in... Cell cycle analysis of mitotically defined groups of ex vivo–expanded MPB and BM CD34+ cells recovered from the BM and spleen of NOD/SCID transplant recipients 40 hours AT.CD34+ cells were isolated from fresh MPB and BM and cultured in vitro as described in “Materials and methods.” On day 5, cells were harvested, washed, stained with Hst, and sorted to obtain cells in G0/G1 and S/G2+M. These fractions were stained with CFSE and transplanted into conditioned NOD/SCID mice. A sample was retained to confirm, with PI staining, the cell cycle status of sorted G0/G1 (light bars) and S/G2+M (dark bars) cells prior to transplantation (d 5 sorted G0/G1 & S/G2+M), while another was maintained in culture for an additional 40 hours (d 5 cult + 40h). Human CFSE+ cells were isolated by flow cytometric cell sorting 40 hours AT from BM (AT/BM) and spleen (AT/Spl) cells. Cell cycle status of all groups of cells was determined by PI staining. Each bar represents the mean ± SD of 3 to 7 G0/G1 and 3 to 6 S/G2+M measurements from MPB and BM samples from 6 independent experiments. Anna Jetmore et al. Blood 2002;99:1585-1593 ©2002 by American Society of Hematology

Cell cycle analysis of fresh, ex vivo–cultured and BM- and spleen-homed MPB G0CD34+ and G1CD34+ cells.Immunomagnetically selected MPB CD34+ cells were stained with Hst and PY, and CD34+ cells in G0 or G1 were isolated by cell sorting as described in “Materi... Cell cycle analysis of fresh, ex vivo–cultured and BM- and spleen-homed MPB G0CD34+ and G1CD34+ cells.Immunomagnetically selected MPB CD34+ cells were stained with Hst and PY, and CD34+ cells in G0 or G1 were isolated by cell sorting as described in “Materials and methods.” A sample from each sorted group was retained for cell cycle analysis at time zero (Fresh) and for initiating a short-term ex vivo culture (Ex vivo) that was maintained as described in the legend of Figure 2. Remaining cells from each group were transplanted into conditioned NOD/SCID mice, and 24, 48, and 72 hours AT, marrow (BM) and spleen (Spl) cells were recovered from individual mice, and human CFSE+ cells were isolated by flow cytometric cell sorting. Cell cycle status of all groups of cells was determined by PI staining. Number of measurements from 6 independent experiments for fresh samples is 4, for G0CD34+ cells at 24 hours is 3, and for G1CD34+ is 2; for G0 and G1CD34+ cells at 48 hours is 8 and for both groups at 72 hours is 1. Insufficient cell recovery (because of low cellularity after irradiation) precluded obtaining a measurement for spleen-homed G1CD34+ cells 72 hours AT. Anna Jetmore et al. Blood 2002;99:1585-1593 ©2002 by American Society of Hematology

Percentage of CD34+ cells contained in fresh and ex vivo–expanded BM and MPB grafts and that detected in BM- and spleen-homed cells 40 hours AT.BM and MPB cells partially immunomagnetically enriched for CD34+ cells (to prepare grafts containing approximatel... Percentage of CD34+ cells contained in fresh and ex vivo–expanded BM and MPB grafts and that detected in BM- and spleen-homed cells 40 hours AT.BM and MPB cells partially immunomagnetically enriched for CD34+ cells (to prepare grafts containing approximately 50% CD34+ cells) were either stained with CFSE and transplanted fresh into conditioned NOD/SCID recipients or expanded in vitro as described in the legend of Figure 2. Expanded cells were then stained with CFSE on day 5 and transplanted into conditioned NOD/SCID recipients. Percentage of CD34+ cells was determined in fresh (Fresh BM and Fresh MPB) and ex vivo–expanded (Exp BM and Exp MPB) grafts before transplantation (In Graft) and as a percentage of human CFSE+ cells detected flow cytometrically in the marrow (In BM) and spleen (In Spl) of transplant recipients 40 hours later. Each bar represents the mean ± SD (where applicable) of 3 to 4 measurements for fresh BM and MPB and expanded BM cells and one measurement for expanded MPB cells. Data were collected from 8 separate experiments. Anna Jetmore et al. Blood 2002;99:1585-1593 ©2002 by American Society of Hematology

Percentage of recovery of total, CD34+, and CD34− cells from the BM and spleen of NOD/SCID recipients 40 hours AT.BM cells partially immunomagnetically enriched for CD34+cells (to prepare grafts containing approximately 50% CD34+ cells) were stained with CF... Percentage of recovery of total, CD34+, and CD34− cells from the BM and spleen of NOD/SCID recipients 40 hours AT.BM cells partially immunomagnetically enriched for CD34+cells (to prepare grafts containing approximately 50% CD34+ cells) were stained with CFSE and transplanted into conditioned NOD/SCID recipients. Percentage of CD34+ cells was determined in the graft and among CFSE+ cells recovered from the BM (light bars) and spleens (dark bars) of recipient mice 40 hours AT. These percentages, the number of cells in the graft, and the number of cells recovered from each tissue were used to calculate the percentage of recovery of total, CD34+, and CD34− cells. Each bar represents the mean ± SD of 5 to 7 independent measurements in 6 experiments. No statistically significant differences were detected in any comparison between all 3 measurements from each tissue. Anna Jetmore et al. Blood 2002;99:1585-1593 ©2002 by American Society of Hematology

Percentage of BM-homed G0/G1 or S/G2+M cells undergoing apoptosis 40 hours AT compared with the level of programmed cell death in ex vivo cultures maintained with and without cytokine supplementation.CD34+ cells were isolated from one fresh MPB and 2 BM sam... Percentage of BM-homed G0/G1 or S/G2+M cells undergoing apoptosis 40 hours AT compared with the level of programmed cell death in ex vivo cultures maintained with and without cytokine supplementation.CD34+ cells were isolated from one fresh MPB and 2 BM samples and cultured in vitro as described in “Materials and methods.” Between days 5 and 7, cells were harvested, washed, stained with Hst, and sorted to obtain cells in G0/G1(light bars) and S/G2+M (dark bars). Both G0/G1 (87.5 ± 5.3 in G0/G1, n = 3) and S/G2+M (28.5 ± 4.5 in G0/G1, n = 3) fractions were stained with CFSE and transplanted into conditioned NOD/SCID mice. Samples of G0/G1 and S/G2+M cells were maintained in culture with or without cytokine supplementation as described in the legend of Figure 2 for an additional 40 hours. BM cells were recovered from individual mice and stained with Annexin V as described in “Materials and methods.” CFSE+, PI− cells expressing Annexin V were considered apoptotic, and their percentage was calculated from total CFSE+ cells. Each bar represents the mean ± SD of 4 measurements for transplanted cells and 3 for cells maintained in vitro in 3 separate experiments. Anna Jetmore et al. Blood 2002;99:1585-1593 ©2002 by American Society of Hematology