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. 2017 Nov 1;9(414):eaan1145.
doi: 10.1126/scitranslmed.aan1145.

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

Stefan Radtke  1   2 , Jennifer E Adair  1   3 , Morgan A Giese  1 , Yan-Yi Chan  1 , Zachary K Norgaard  1 , Mark Enstrom  1 , Kevin G Haworth  1 , Lauren E Schefter  1 , Hans-Peter Kiem  4   3   5
Affiliations

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

Stefan Radtke et al. Sci Transl Med. .

Abstract

Hematopoietic reconstitution after bone marrow transplantation is thought to be driven by committed multipotent progenitor cells followed by long-term engrafting hematopoietic stem cells (HSCs). We observed a population of early-engrafting cells displaying HSC-like behavior, which persisted long-term in vivo in an autologous myeloablative transplant model in nonhuman primates. To identify this population, we characterized the phenotype and function of defined nonhuman primate hematopoietic stem and progenitor cell (HSPC) subsets and compared these to human HSPCs. We demonstrated that the CD34+CD45RA-CD90+ cell phenotype is highly enriched for HSCs. This population fully supported rapid short-term recovery and robust multilineage hematopoiesis in the nonhuman primate transplant model and quantitatively predicted transplant success and time to neutrophil and platelet recovery. Application of this cell population has potential in the setting of HSC transplantation and gene therapy/editing approaches.

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Figures

Fig. 1.
Fig. 1.. Persistence of early-engrafting stem-cell like clones after transplant.
(A) In vivo hematopoietic cell clone tracking data for five retrospectively analyzed pigtailed 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, means 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. Band width 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 post-transplant.
Fig. 2.
Fig. 2.. Identification of phenotypically defined steady state bone marrow-derived CD34+ subpopulations in the pigtailed macaque.
(A) Cell surface expression of CD45 vs. CD34, CD45RA vs. CD117, and CD90 vs. CD123 on pigtailed macaque steady state bone marrow-derived white blood cells. CD45RA/CD117 expression is gated on CD34highCD45int (I) populations; CD90/CD123 analysis is shown for CD34highCD45intCD45RA-CD117+ (IV) populations. (B) Phenotypically distinct subpopulations were labeled I-IX (int = intermediate staining). Corresponding data can be found in fig. 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 fig. S3-S9.
Fig. 3.
Fig. 3.. Multilineage engraftment is exclusively driven by CD34+CD45RA-CD90+ cell fractions.
(A) Outline for autologous nonhuman primate stem cell transplantation experiments including time points for peripheral blood and bone marrow analysis. (B) Bone marrow-derived nonhuman primate CD34+ HSPCs were separated into fractions i, ii and iii by 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) and mCerulean (blue) as indicated. (D) Long-term follow-up of gene-marking in nonhuman primate white blood cells. (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.
Fig. 4.. Clone tracking by insertion site analysis confirms early, multilineage hematopoietic engraftment of CD34+CD45RA-CD90+ 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-) or monocytes (CD11b+CD14+).
Fig. 5.
Fig. 5.. Correlation between CD34+CD45RA-CD90+ cell dose, engraftment success and onset of neutrophil/platelet recovery in nonhuman primates.
(A) Statistical comparison of transplanted CD34+ and CD34+CD45RA-CD90+ cells/kg body weight in animals with engraftment failure or long-term engraftment (unpaired, two-sided t-test). (B)Logarithmic correlation of transplanted CD34+CD45RA-CD90+ 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 the solid and the dotted lines, respectively.
Fig. 6.
Fig. 6.. Phenotypic and transcriptomic similarities observed between human and nonhuman primate HSC-enriched cell fractions.
(A) Gating of the HSC-enriched CD34+CD45RA-CD90+ cell fraction in nonhuman primate steady-state bone marrow and primed bone marrow. (B) Gating of an HSC-enriched cell fraction in human G-CSF-mobilized peripheral blood stem cells using classical gating (left) and our new gating strategy (right) for a CD34+CD45RA-CD90+ 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 (grey), 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 scatter plot, with genes showing the same pattern (lower left and upper right quadrant), highlighted in bold. Key genes of HSCs and hematopoietic differentiation are tagged and labeled.
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