Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant, and infectious diseases. We have shown in a nonhuman primate gene therapy and transplantation model that the CD34 + CD90 + cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC enrichment strategies include the purification of CD133 + cells or CD38 low/– subsets of CD34 + cells from human blood products. We directly compared these strategies to the isolation of CD90 + cells using a good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90 + cell selection results in about 30-fold fewer target cells in comparison to CD133 + or CD38 low/– CD34 + hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential in vivo . Single-cell RNA sequencing confirmed nearly complete depletion of lineage-committed progenitor cells in CD90 + fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90 + cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and gene-engineered cell products.

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Hematopoietic stem cells (HSCs) are assumed to be rare, infrequently dividing, long-lived, and not involved in immediate recovery after transplantation. Here we performed unprecedented high-density clonal tracking in nonhuman primates and found long-term persisting HSC clones to actively contribute during early neutrophil recovery and be the main source of blood production as early as 50 days post-transplant. Most surprisingly, we observed a rapid decline in the number of unique HSC clones, while persisting HSCs expanded undergoing symmetric divisions to create identical siblings and formed clonal pools ex vivo as well as in vivo. In contrast to the currently assumed model of hematopoietic reconstitution, we provide evidence for contribution of HSCs in short-term recovery as well as symmetric expansion of individual clones into pools. These findings provide novel insights into HSC biology informing the design of HSC transplantation and gene therapy studies.

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Reconstitution after hematopoietic stem cell (HSC) transplantation is assumed to occur in two distinct phases: initial recovery mediated by short-term progenitors and long-term repopulation by multipotent HSCs which do not contribute to hematopoietic reconstitution during the first 6-9 months. We have previously reported the transplantation and exclusive engraftment of the HSC-enriched CD34+CD45RA-CD90+ phenotype in a nonhuman primate model. Here, we closely followed the clonal diversity and kinetics in these animals. Enhanced sampling and high density clonal tracking within the first 3 month revealed that multipotent HSCs actively contributed to the early phases of neutrophil recovery and became the dominant source for blood cells as early as 50 days after transplant. Longitudinal changes in clonal diversity supported a stochastic engraftment of HSCs with the majority of HSCs clones vanishing early during neutrophil recovery and a smaller fraction of HSC clones expanding into bigger pools to support long-term hematopoiesis. In contrast to the bi-phasic model, we propose that hematopoietic recovery after myeloablation and transplantation is primarily derived from HSCs in a stochastic manner rather than in two phases by independent cell populations.