Research ArticleStem Cells

A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates

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Science Translational Medicine  01 Nov 2017:
Vol. 9, Issue 414, eaan1145
DOI: 10.1126/scitranslmed.aan1145
  • Fig. 1. Persistence of early-engrafting stem cell–like clones after transplant in nonhuman primates.

    (A) In vivo hematopoietic cell clone tracking data for five retrospectively analyzed pig-tailed macaques followed for more than 2 years after myeloablative transplant of lentivirus gene–modified autologous CD34+ cells. Bars represent the contribution of clones identified in peripheral blood <3 months after transplant (first bar in each graph) over time. The number above each bar is the total number of clones identified in the time period; n, number of individual blood samples analyzed during that time period. NA, no samples available within this time period. (B) Longitudinal contributions of abundant HSC clones (maximum clone contribution > mean maximum contribution for all HSC clones identified in the animal). Each colored band represents one HSC clone. Bandwidth corresponds to the clone frequency (y axis). To be called an HSC clone, the clone signature (lentivirus insertion locus) had to be present in one short-lived and one long-lived mature blood cell lineage and present at more than a single time period of analysis, one of which had to be later than 1 year after transplant.

  • Fig. 2. Identification of phenotypically defined ssBM-derived CD34+ subpopulations in the pig-tailed macaque.

    (A) Cell surface expression of CD45 versus CD34, CD45RA versus CD117, and CD90 versus CD123 on pig-tailed macaque ssBM–derived white blood cells. CD45RA/CD117 expression is gated on CD34highCD45int (I) populations; CD90/CD123 analysis is shown for CD34highCD45intCD45RACD117+ (IV) populations. FSC, forward scatter; SSC, side scatter. (B) Phenotypically distinct subpopulations were labeled I to IX (int, intermediate staining). Corresponding data can be found in figs. S2 and S3. (C) Summary of the in vitro functional properties observed in each subpopulation. (n.d., not determined). Corresponding data can be found in figs. S3 to S9. CAFC, cobblestone area–forming cells; CFC, colony-forming cells.

  • Fig. 3. Multilineage engraftment is exclusively driven by CD34+CD45RACD90+ cell fractions.

    (A) Outline for autologous nonhuman primate stem cell transplantation experiments including time points for peripheral blood and bone marrow analysis. PB, peripheral blood; BM, bone marrow; G-CSF, granulocyte colony-stimulating factor; SCF, stem cell factor; MOI, multiplicity of infection; TBI, total body irradiation; QC, quality control. (B) Bone marrow–derived nonhuman primate CD34+ HSPCs were separated into fractions i, ii, and iii by fluorescence-activated cell sorting (FACS) based on the expression of CD45RA and CD90. These fractions contained all CD34highCD45int subpopulations as follows: fraction i (population VII), fraction ii (populations IV, VIII, and IX), and fraction iii (populations V and VI). The dashed box highlights presumed multipotent cell phenotypes expected to contribute to engraftment in vivo. (C) A total of four animals were used for this study. FACS-purified HSPC fractions were transduced in different combinations with lentivirus encoding GFP (green), mCherry (red), or mCerulean (blue), as indicated. (D) Long-term follow-up of gene marking in nonhuman primate white blood cells. CMV, cytomegalovirus. (E) Frequency of gene-marked granulocytes (CD11b+CD14+), monocytes (CD11b+CD14+), B cells (CD20+), T cells (CD3+), and NK cells (CD16+) in the peripheral blood of nonhuman primates.

  • Fig. 4. Clone tracking by insertion site analysis confirms early, multilineage hematopoietic engraftment of CD34+CD45RACD90+ cells.

    Venn diagrams illustrate the number of shared clone signatures between fluorophore and cell surface marker–sorted peripheral blood cell lineages in two animals at 4 months (Z13264) and 6.5 months (Z14004) after transplant. Fluorophore+ fraction i cells were mCherry+ (Z13264) or GFP+ (Z14004). Subsets include B cells (CD20+), T cells (CD3+), granulocytes (CD11b+CD14), and monocytes (CD11b+CD14+).

  • Fig. 5. Correlation between CD34+CD45RACD90+ cell dose, engraftment success, and onset of neutrophil/platelet recovery in nonhuman primates.

    (A) Statistical comparison of transplanted CD34+ and CD34+CD45RACD90+ cells/kg body weight in animals with engraftment failure or long-term engraftment (unpaired, two-sided t test). (B) Logarithmic correlation of transplanted CD34+CD45RACD90+ cells/kg body weight with the day of neutrophil and platelet recovery, as calculated using Spearman’s rank correlation coefficient. The linear regression and the 95% confidence interval for each correlation are indicated with solid and dotted lines, respectively.

  • Fig. 6. Phenotypic and transcriptomic similarities observed between human and nonhuman primate HSC-enriched cell fractions.

    (A) Gating of the HSC-enriched CD34+CD45RACD90+ cell fraction in nonhuman primate ssBM and primed bone marrow. (B) Gating of an HSC-enriched cell fraction in human G-CSF–mobilized PBSCs using classical gating (left) and our new gating strategy (right) for a CD34+CD45RACD90+ subfraction. (C) Comparison of transcript expression in human and nonhuman primate HSC-enriched cell fractions to that of bulk CD34+ cells. The log2 fold change in gene expression was calculated for each species and gene individually. The number of genes included in each quadrant for all genes (gray), differentially expressed genes in human or nonhuman primate (black), and differentially expressed genes in both species (red) with a P value <0.05 is given next to the scatterplot, with genes showing the same pattern (lower left and upper right quadrants), highlighted in bold. Key genes of HSCs and hematopoietic differentiation are tagged and labeled.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/414/eaan1145/DC1

    Methods

    Fig. S1. Engraftment kinetics in transplanted nonhuman primates.

    Fig. S2. HSPC composition in nonhuman primate stem cell populations from different sources.

    Fig. S3. Ex vivo culture of nonhuman primate HSPCs.

    Fig. S4. T cell potential of nonhuman primate HSPCs.

    Fig. S5. Lympho-myeloid potential of nonhuman primate HSPCs.

    Fig. S6. Colony-forming cell potential of nonhuman primate HSPCs.

    Fig. S7. Megakaryocyte potential of nonhuman primate HSPCs.

    Fig. S8. Cobblestone area–forming cell potential of nonhuman primate HSPCs.

    Fig. S9. Secondary colony-forming cell potential of nonhuman primate HSPCs.

    Fig. S10. Quality control for competitive repopulation experiments.

    Fig. S11. Neutrophil recovery in transplanted nonhuman primates.

    Fig. S12. Platelet recovery in transplanted nonhuman primates.

    Fig. S13. Bone marrow engraftment of gene-modified HSPCs.

    Fig. S14. Long-term engraftment versus failure.

    Fig. S15. Correlation of recovery with infused HSPCs and colony-forming cells.

    Fig. S16. Gating of HSC-enriched cell fractions in human umbilical cord blood.

    Table S1. Identification of clones displaying HSC biology in vivo.

    Table S2. Cross-species reactive antibodies in nonhuman primates.

    Table S3. MS-5 assay colonies.

    Table S4. Characteristics of nonhuman primate transplants.

    Table S5. Characteristics of nonhuman primate transplants II.

    Table S6. Differentially expressed genes in human and nonhuman primate HSCs.

    References (4054)

  • Supplementary Material for:

    A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates

    Stefan Radtke, Jennifer E. Adair, Morgan A. Giese, Yan-Yi Chan, Zachary K. Norgaard, Mark Enstrom, Kevin G. Haworth, Lauren E. Schefter, Hans-Peter Kiem*

    *Corresponding author. Email: hkiem{at}fredhutch.org

    Published 1 November 2017, Sci. Transl. Med. 9, eaan1145 (2017)
    DOI: 10.1126/scitranslmed.aan1145

    This PDF file includes:

    • Methods
    • Fig. S1. Engraftment kinetics in transplanted nonhuman primates.
    • Fig. S2. HSPC composition in nonhuman primate stem cell populations from different sources.
    • Fig. S3. Ex vivo culture of nonhuman primate HSPCs.
    • Fig. S4. T cell potential of nonhuman primate HSPCs.
    • Fig. S5. Lympho-myeloid potential of nonhuman primate HSPCs.
    • Fig. S6. Colony-forming cell potential of nonhuman primate HSPCs.
    • Fig. S7. Megakaryocyte potential of nonhuman primate HSPCs.
    • Fig. S8. Cobblestone area–forming cell potential of nonhuman primate HSPCs.
    • Fig. S9. Secondary colony-forming cell potential of nonhuman primate HSPCs.
    • Fig. S10. Quality control for competitive repopulation experiments.
    • Fig. S11. Neutrophil recovery in transplanted nonhuman primates.
    • Fig. S12. Platelet recovery in transplanted nonhuman primates.
    • Fig. S13. Bone marrow engraftment of gene-modified HSPCs.
    • Fig. S14. Long-term engraftment versus failure.
    • Fig. S15. Correlation of recovery with infused HSPCs and colony-forming cells.
    • Fig. S16. Gating of HSC-enriched cell fractions in human umbilical cord blood.
    • Table S1. Identification of clones displaying HSC biology in vivo.
    • Table S2. Cross-species reactive antibodies in nonhuman primates.
    • Table S3. MS-5 assay colonies.
    • Table S4. Characteristics of nonhuman primate transplants.
    • Table S5. Characteristics of nonhuman primate transplants II.
    • Table S6. Differentially expressed genes in human and nonhuman primate HSCs.
    • References (4054)

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