Volume 6, Issue 1, Pages 48-58 (January 2010) Intermediate-Term Hematopoietic Stem Cells with Extended but Time-Limited Reconstitution Potential  Patricia Benveniste, Catherine Frelin, Salima Janmohamed, Mary Barbara, Robert Herrington, Deborah Hyam, Norman N. Iscove  Cell Stem Cell  Volume 6, Issue 1, Pages 48-58 (January 2010) DOI: 10.1016/j.stem.2009.11.014 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Erythroid Reconstitution Patterns from Unseparated Marrow Cells at Limiting Dilution Lightly irradiated B6-KitW–41J/W–41J-Gpi1a recipients, 24 in total, received 1000–5000 B6-Gpi1b marrow cells. The upper panel shows 11 recipients with high-level erythoid reconstitutions sustained through 32 weeks posttransplant. The mean proportion donor was 0.71 attained by 16 weeks. The lower panel shows the pattern exhibited by the remaining 13 mice, in which red cells reconstituted to lower levels and then declined to undetectability by 32 weeks. The mean proportion donor at 4 weeks was 0.34. Arrows indicate points where the proportion of donor cells fell below the 3% threshold of detection in the Gpi1 assay. Cell Stem Cell 2010 6, 48-58DOI: (10.1016/j.stem.2009.11.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Isolation and Characterization of RKSL Cells Top: An initial sort was performed to isolate RholoCD3−B220− cells. Flow cytometry showed this fraction to exclude most of the CD34+ and Flt3+ cells present before separation. Bottom: The RholoCD3−B220− fraction was sorted again to isolate c-kit+Sca-1+ cells. Flow cytometry showed that the resulting RKSL fraction contained only a tiny subcomponent of CD34+ (6%) or Flt3+ cells (<3%), whereas most of the residual CD34+ cells from the initial RholoCD3−B220− fraction segregated with Sca-1lo cells. The analysis also suggested that most RKSL cells were CD34lo rather than CD34−. RKSL cells were heterogeneous in expression of CD49b; typical windows chosen for isolating CD49blo and CD49bhi cells are indicated. Cell Stem Cell 2010 6, 48-58DOI: (10.1016/j.stem.2009.11.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Clonal Reconstitutions from Single RKSL Cells Purified B6-Ly5.1 marrow cells were injected into lightly irradiated B6-Ly5.2-KitW–41J/W–41J-Gpi1a recipients, and circulating Ly5.1 leukocytes were analyzed by flow cytometry at the times indicated. All data were collected from mice within cohorts in which fewer than 50% of mice had detectable grafts, ensuring that most grafts arose from a single reconstituting entity. (A) A single reconstitution is shown in which erythroid (Gpi1) reconstitution was sustained at high level through 32 weeks. The corresponding high level of sustained reconstitution of myeloid and lymphoid lineages was representative of numerous other recipients of single cells with sustained erythroid grafts sampled only at 32 weeks. (B) Mean results ± SEMs of reconstitutions tracked in eight recipients of single-cell transplants in which erythroid reconstitution was not sustained and in which donor CD11b+ cells were detectable at 4 or 8 weeks. Grey bars in each panel indicate thresholds of detectability based on counts and their clustering pattern. Total CD11b+ cells are shown but results were similar for highly side-scattering CD11b+ cells preferentially representative of granulocytes (Dai et al., 2002). (C) Proportions of 23 mice with unsustained erythroid grafts detectable at 4 or 8 weeks that also had detectable grafts in the indicated lineages at the time points indicated. “Proportion Donor” indicates in all figures the proportion of total cells of the designated lineage that were donor derived. For each lineage, gates were set to exclude cells expressing markers of the other lineages. For example, “CD11b+” implies “CD11b+CD19−CD3−.” Cell Stem Cell 2010 6, 48-58DOI: (10.1016/j.stem.2009.11.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Expression of α2 Integrin/CD49b Resolves RKSL Cells with Distinct Biological Properties (A) Kinetics of onset of growth of purified reconstituting cells. Single purified cells were cultured with Kit ligand, Flt3 ligand, IL-11, and IL-7 in round-bottom microtiter wells and observed at 2 hr intervals after the first 24 hr. The time at which cells underwent their first division was recorded. Arrows indicate the times at which 50% of cells had divided. The results are representative of three independent experiments scoring multiple time points. (B) Spleen colonies in irradiated recipient mice 12 days after injection of purified marrow cells. Per 80 injected cells, CD49blo cells yielded about 1 ± 0.3 barely visible colony, whereas CD49bhi cells generated 5 ± 0.64 readily counted colonies in two independent experiments (total 18 mice for each cell fraction). SEMs are indicated. Unpurified marrow, 7 × 104 cells, yielded 15 colonies that were significantly larger than those from purified cells (lower left). Spleens of irradiated, uninjected mice (lower right) were essentially devoid of colonies. (C) Kinetics of reconstitution by RKSL-CD49blo and CD49bhi populations. 100 purified Ly5.1-Gpi1a cells were injected with 1 × 106Ly5.2-Gpi1b competitor marrow cells into irradiated Ly5.2-Gpi1b recipients and blood cells analyzed at the indicated times. Means and SEMs of eight independent experiments are shown. Grey bars indicate thresholds of detectability. (D) Expression of CD150 and CD49b on RKSL cells. Top: Control RKSL cells unreacted with CD150 antibody. Bottom: RKSL cells stained with CD150 antibody. Quadrant lines approximately bisect the population. Proportion of the total population in each quadrant is shown. (E) Gene expression profiles in populations enriched for LTRCs or ITRCs. Globally amplified cDNA was prepared from purified RKSL-CD49blo and -CD49bhi cells in six independent experiments. cDNA samples were probed by target-specific PCR for the transcripts indicated. At the left side of the figure, five transcripts were expressed at similar levels in LTRCs and ITRCs. At the right, five transcripts were expressed preferentially in LTRCs. Cell Stem Cell 2010 6, 48-58DOI: (10.1016/j.stem.2009.11.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Present and Modified Views of the Composition of Highly Purified Hematopoietic Stem Cell Populations As illustrated in (A) (purification scheme) and (C) (lineage hierarchy), the c-kit+Sca-1+Lineage− (KSL) fraction has been shown before to be divisible into long-term and short-term reconstituting cells (LTRCs and STRCs, respectively) on the basis of expression of CD34 and Flt3 and of exclusion of Rho123. The present study shows that the LTRC fraction is further divisible (B and D) into LTRC and an intermediate-term (ITRC) class of reconstituting cell on the basis of expression of CD49b. Cell Stem Cell 2010 6, 48-58DOI: (10.1016/j.stem.2009.11.014) Copyright © 2010 Elsevier Inc. Terms and Conditions