Isolation of a Highly Purified HSC-enriched CD34+CD90+CD45RA- Cell Subset for Allogeneic Transplantation in the Nonhuman Primate Large-animal Model

Abstract

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a common treatment for patients suffering from different hematological disorders. Allo-HCT in combination with hematopoietic stem cell (HSC) gene therapy is considered a promising treatment option for millions of patients with HIV+ and acute myeloid leukemia. Most currently available HSC gene therapy approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly progenitor cells and only very few HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side effects in nontarget cells. We have previously shown that purified CD34⁺CD90⁺CD45RA^- cells are enriched for multipotent HSCs with long-term multilineage engraftment potential, which can reconstitute the entire hematopoietic system in an autologous nonhuman primate transplant model. Here, we tested the feasibility of transplantation with purified CD34⁺CD90⁺CD45RA^- cells in the allogeneic setting in a nonhuman primate model.

Methods: To evaluate the feasibility of this approach, CD34⁺CD90⁺CD45RA^- cells from 2 fully major histocompatibility complex-matched, full sibling rhesus macaques were sort-purified, quality controlled, and transplanted. Engraftment and donor chimerism were evaluated in the peripheral blood and bone marrow of both animals.

Results: Despite limited survival due to infectious complications, we show that the large-scale sort-purification and transplantation of CD34⁺CD90⁺CD45RA^- cells is technically feasible and leads to rapid engraftment of cells in bone marrow in the allogeneic setting and absence of cotransferred T cells.

Conclusions: We show that purification of an HSC-enriched CD34⁺ subset can serve as a potential stem cell source for allo-HCTs. Most importantly, the combination of allo-HCT and HSC gene therapy has the potential to treat a wide array of hematologic and nonhematologic disorders.

FIGURE 1.

Transplant scheme and pretransplant stem and progenitor cells quality control. A, Experimental setup for the allogenic transplantation of sort-purified CD34⁺CD90⁺CD45RA⁻ cells in rhesus macaques. B, Flow-cytometric quality control of CD34⁺ cells pre-MACS and post-MACS as well as CD34⁺CD90⁺CD45RA⁻ cells presort and postsort. C, Colony-forming cell (CFC) potential of CD34⁺ and CD34⁺CD90⁺CD45RA⁻ cells across the different processing steps. BFU-E, burst-forming unit erythrocyte; BM, bone marrow; CFU, colony-forming unit; G, granulocyte; HSPC, hematopoietic stem and progenitor cell; M, monocyte/macrophage; MACS, magnetic-assisted cell sorting; MIX, erythrocytes, granulocytes, and monocytes/macrophages; TBI, total body irradiation.

FIGURE 2.

Posttransplant follow-up and chimerism analysis in peripheral blood (PB). A, Neutrophil and platelet counts posttransplant until early euthanasia (vertical red dashed line). B, Frequency of donor chimerism in PB white blood cells (WBCs) determined by microsatellite DNA analysis.

FIGURE 3.

Bone marrow (BM) engraftment and chimerism at necropsy. A, Flow-cytometric assessment of total CD34⁺ cells as well as CD34⁺CD90⁺CD45RA⁻ cells in the BM at the d of necropsy. B, Colony-forming cell (CFC) potential of different numbers of BM white blood cells (WBCs) cells at the d of necropsy. C, Donor chimerism in total BM WBCs and BM CFCs at the d of necropsy (d 8 and 9 for A17229 and A17230, respectively) determined by microsatellite DNA analysis. BFU-E, burst-forming unit erythrocyte; CFU, colony-forming unit; G, granulocyte; HSPC, hematopoietic stem and progenitor cell; M, monocyte/macrophage; MIX, erythrocytes, granulocytes, and monocytes/macrophages.