MISTRG mice support engraftment and assessment of nonhuman primate hematopoietic stem and progenitor cells
2018; Elsevier BV; Volume: 70; Linguagem: Inglês
10.1016/j.exphem.2018.12.003
ISSN1873-2399
AutoresStefan Radtke, Yan-Yi Chan, Trisha R. Sippel, Hans‐Peter Kiem, Anthony Rongvaux,
Tópico(s)CAR-T cell therapy research
ResumoPreclinical feasibility, safety, and efficacy testing of hematopoietic stem cell (HSC)-mediated gene therapy approaches is commonly performed in large-animal models such as nonhuman primates (NHPs). Here, we wished to determine whether mouse models would allow engraftment of NHP HSPCs, which would enable more facile and less costly evaluation of promising strategies. In this study, we comprehensively tested two mouse strains for the engraftment of NHP CD34+ hematopoietic stem and progenitor cells (HSPCs). No engraftment of NHP HSPCs was observed in NSG mice, whereas the gene-humanized MISTRG model did demonstrate dose-dependent multilineage engraftment of NHP cells in the peripheral blood, bone marrow, spleen, and thymus. Most importantly, and closely mimicking the hematopoietic recovery of autologous stem cell transplantations in the NHP, only HSC-enriched CD34+CD90+CD45RA– cell fractions engrafted and reconstituted the bone marrow stem cell niche in MISTRG mice. In summary, we here report the first "monkeynized" mouse xenograft model that closely recapitulates the autologous hematopoietic reconstitution in the NHP stem and progenitor cell transplantation and gene therapy model. The availability of this model has the potential to pre-evaluate novel HSC-mediated gene therapy approaches, inform studies in the NHP, and improve the overall outcome of large-animal experiments. Preclinical feasibility, safety, and efficacy testing of hematopoietic stem cell (HSC)-mediated gene therapy approaches is commonly performed in large-animal models such as nonhuman primates (NHPs). Here, we wished to determine whether mouse models would allow engraftment of NHP HSPCs, which would enable more facile and less costly evaluation of promising strategies. In this study, we comprehensively tested two mouse strains for the engraftment of NHP CD34+ hematopoietic stem and progenitor cells (HSPCs). No engraftment of NHP HSPCs was observed in NSG mice, whereas the gene-humanized MISTRG model did demonstrate dose-dependent multilineage engraftment of NHP cells in the peripheral blood, bone marrow, spleen, and thymus. Most importantly, and closely mimicking the hematopoietic recovery of autologous stem cell transplantations in the NHP, only HSC-enriched CD34+CD90+CD45RA– cell fractions engrafted and reconstituted the bone marrow stem cell niche in MISTRG mice. In summary, we here report the first "monkeynized" mouse xenograft model that closely recapitulates the autologous hematopoietic reconstitution in the NHP stem and progenitor cell transplantation and gene therapy model. The availability of this model has the potential to pre-evaluate novel HSC-mediated gene therapy approaches, inform studies in the NHP, and improve the overall outcome of large-animal experiments. Autologous hematopoietic stem cell (HSC) gene therapy and editing is currently one of the most promising treatment strategies for a broad variety of hematologic diseases and disorders [1Adair JE Kubek SP Kiem HP Hematopoietic stem cell approaches to cancer.Hematol Oncol Clin North Am. 2017; 31: 897-912Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 2Morgan RA Gray D Lomova A Kohn DB Hematopoietic stem cell gene therapy: progress and lessons learned.Cell Stem Cell. 2017; 21: 574-590Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 3Tran E Longo DL Urba WJ A milestone for CAR T cells.N Engl J Med. 2017; 377: 2593-2596Crossref PubMed Scopus (48) Google Scholar, 4D'Aloia MM Zizzari IG Sacchetti B Pierelli L Alimandi M CAR-T cells: the long and winding road to solid tumors.Cell Death Dis. 2018; 9: 282Crossref PubMed Scopus (217) Google Scholar]. The transfer of therapeutic genes to repair deficiencies or the delivery of nucleases to edit genes has the potential to cure diseases such as Fanconi anemia, severe-combined immunodeficiency (SCID), HIV/AIDS, and hemoglobinopathies [5Becker PS Taylor JA Trobridge GD et al.Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.Gene Ther. 2010; 17: 1244-1252Crossref PubMed Scopus (33) Google Scholar, 6Burtner CR Beard BC Kennedy DR et al.Intravenous injection of a foamy virus vector to correct canine SCID-X1.Blood. 2014; 123: 3578-3584Crossref PubMed Scopus (28) Google Scholar, 7Peterson CW Wang J Norman KK et al.Long-term multilineage engraftment of genome-edited hematopoietic stem cells after autologous transplantation in nonhuman primates.Blood. 2016; 127: 2416-2426Crossref PubMed Scopus (52) Google Scholar, 8Humbert O Peterson CW Norgaard ZK Radtke S Kiem HP A nonhuman primate transplantation model to evaluate hematopoietic stem cell gene editing strategies for beta-hemoglobinopathies.Mol Ther Methods Clin Dev. 2018; 8: 75-86Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. Clinical feasibility, safety, and, in particular, engraftment of genetically modified HSCs is commonly tested in nonhuman primates (NHPs) [9Trobridge GD Kiem HP Large-animal models of hematopoietic stem cell gene therapy.Gene Ther. 2010; 17: 939-948Crossref PubMed Scopus (43) Google Scholar, 10Larochelle A Dunbar CE Genetic manipulation of hematopoietic stem cells.Semin Hematol. 2004; 41: 257-271Crossref PubMed Scopus (25) Google Scholar]. Benefits of this large-animal model include close similarity in size, weight, and physiology to humans; the ability to perform autologous stem cell transplantations with full multilineage support; and the opportunity to take frequent peripheral blood (PB) and bone marrow (BM) samples for a comprehensive long-term follow-up. Most importantly, cross-reactivity of markers, reagents, and drugs between humans and NHPs permits an easy translation of successfully tested approaches into clinical treatment strategies. However, studies in NHPs are expensive and require special facilities and trained staff, making high throughput testing of novel strategies difficult. In many cases, only individual and heavily preselected approaches are tested in autologous NHP transplantations. Unfortunately, preselecting treatment strategies based on ex vivo experiments or human xenograft assays can bear the risk of misinterpretation, lack of translatability, or, in the worst case, lead to failure of long-lasting and expensive large-animal experiments. The availability of a mouse model that supports xenotransplantation of NHP cells, mimics the multilineage engraftment of hematopoietic stem and progenitor cells (HSPCs) in the autologous setting [11Radtke S Adair JE Giese MA et al.A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.Sci Transl Med. 2017; 9: eaan1145Crossref PubMed Scopus (59) Google Scholar], and enables functional assessment of gene-modified NHP cells would allow comprehensive and cost-efficient high-throughput screening of potential treatment strategies upfront. The ability to screen multiple experimental parameters in a mouse xenograft model would provide the ability to objectively select conditions for further testing in large-animal studies and ultimately improve the outcome of NHP transplantation studies. We have previously reported successful but low-level engraftment of gene-modified baboon HSPCs in the NOD/SCID mouse model [12Horn PA Thomasson BM Wood BL Andrews RG Morris JC Kiem HP Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates.Blood. 2003; 102: 4329-4335Crossref PubMed Scopus (67) Google Scholar]. Although we detected multilineage donor chimerism and gene-marked cells for up to 12 weeks in these mice, the observed pattern of engraftment, clonal composition, and frequency of gene marking barely resembled the autologous transplantation situation in the baboon. With the aim of establishing a "monkeynized" mouse model that provides higher levels of engraftment, better multilineage support, and more closely recapitulates autologous hematopoietic recovery, we here performed comprehensive engraftment studies of pigtail macaque (PM) and rhesus macaque (RM) CD34+ in two mouse strains, NSG [13Ishikawa F Yasukawa M Lyons B et al.Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice.Blood. 2005; 106: 1565-1573Crossref PubMed Scopus (690) Google Scholar] and MISTRG [14Rongvaux A Willinger T Martinek J et al.Development and function of human innate immune cells in a humanized mouse model.Nat Biotechnol. 2014; 32: 364-372Crossref PubMed Scopus (459) Google Scholar]. Similar to xenograft studies of human HSPCs, we assessed the multilineage engraftment potential of NHP cells in various tissues, determined the homing and engraftment of NHP HSPCs into the BM stem cell niche, and titrated the number of SCID-repopulating cells (SRCs). To validate the read-out of this new "monkeynized" mouse model, we further determined the engraftment potential of phenotypically and functionally defined NHP HSPC subsets in analogy to our recently reported study in the NHP [11Radtke S Adair JE Giese MA et al.A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.Sci Transl Med. 2017; 9: eaan1145Crossref PubMed Scopus (59) Google Scholar]. NHP CD34+ cells were harvested, enriched, and cultured as described previously [11Radtke S Adair JE Giese MA et al.A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.Sci Transl Med. 2017; 9: eaan1145Crossref PubMed Scopus (59) Google Scholar, 15Trobridge GD Beard BC Gooch C et al.Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors.Blood. 2008; 111: 5537-5543Crossref PubMed Scopus (62) Google Scholar, 16Adair JE Waters T Haworth KG et al.Semi-automated closed system manufacturing of lentivirus gene-modified haematopoietic stem cells for gene therapy.Nat Commun. 2016; 7: 13173Crossref PubMed Scopus (26) Google Scholar]. Briefly, before enrichment of NHP CD34+ cells, red cells were lysed in ammonium chloride lysis buffer, WBCs incubated for 20 minutes with the 12.8 immunoglobulin-M anti-CD34 antibody, washed and incubated for another 20 minutes with magnetic-activated cell-sorting anti-immunoglobulin-M microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The cell suspension was run through magnetic columns enriching for CD34+ cell fractions with a purity of 60–80% confirmed by flow cytometry. Antibodies used for flow cytometric analysis and fluorescence-activated cell sorting (FACS) of NHP and murine cells are listed in Supplementary Table E2 (online only, available at www.exphem.org). Dead cells and debris were excluded via forward scatter/side scatter gating. Flow cytometric analysis was performed on an LSR IIu (BD Biosciences, Franklin Lakes, NJ), Fortessa X50 (BD Biosciences), and FACSAria IIu (BD Biosciences). Cells for in vitro assays were sorted using a FACSAria IIu cell sorter (BD Biosciences). For colony-forming cell (CFC) assays, 1000–1200 sort-purified NHP or CD34 subpopulations were seeded into 3.5 mL of ColonyGEL 1402 (ReachBio, Seattle, WA). Hematopoietic colonies were scored after 12–14 days. Arising colonies were identified as colony-forming unit (CFU)-granulocyte (CFU-G), CFU-macrophage (CFU-M), granulocyte-macrophage (CFU-GM), or burst-forming unit-erythrocyte (BFU-E).Colonies consisting of erythroid and myeloid cells were scored as CFU-MIX. Neonatal MISTRG mice (CSF1h/h IL-3/CSF2h/hSIRPAtg THPOh/h Rag2–/– Il2rg–/–) and NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) within 48–72 hours of birth or adult (8–12 week) NSG mice received a radiation dose of 150 cGy (neonatal MISTRG/NSG) or 275 cGy (adult NSG) followed 4 hours later by a 30 µL intrahepatic (neonatal MISTRG/NSG) or 200 µL intravenous injection (adult NSG) of sort-purified NHP CD34+ cells or CD34 subpopulation. Beginning at 6–8 weeks after injection, blood samples were collected biweekly and analyzed by flow cytometry for expression of NHP lineage markers (see Supplementary Table E2, online only, available at www.exphem.org). After 16–24 weeks, animals were sacrificed and tissues were harvested and dissociated through 70 µm filters for analysis. All animal studies were carried out at Fred Hutchinson Cancer Research Center in compliance with the approved institutional animal care and use committee protocol #1483. Data analysis of limiting dilution experiments was performed as described previously [17Hu Y Smyth GK ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays.J Immunol Methods. 2009; 347: 70-78Crossref PubMed Scopus (1221) Google Scholar]. Statistical analysis of data was performed using GraphPad Prism version 5 software. Significance analyses were performed with unpaired, two-sided Student t test (*p < 0.05; **p < 0.01; ***p < 0.001). The NSG mouse model is the most frequently used xenograft assay for human HSPCs [18Ishikawa F Modeling normal and malignant human hematopoiesis in vivo through newborn NSG xenotransplantation.Int J Hematol. 2013; 98: 634-640Crossref PubMed Scopus (14) Google Scholar]. To determine whether NSG mice support multilineage engraftment of PM HSPCs, bulk CD34+ HSPCs from granulocyte-colony stimulating factor (G-CSF)-primed BM aspirates as well as sort-purified CD34 subsets enriched for HSCs (CD90+CD45RA–), multipotent and erythro-myeloid progenitors (MPPs/EMPs: CD90–CD45RA–), or lympho-myeloid progenitors (LMPs: CD90–CD45RA+) were individually transplanted into sublethally irradiated adult and neonatal NSG mice [11Radtke S Adair JE Giese MA et al.A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.Sci Transl Med. 2017; 9: eaan1145Crossref PubMed Scopus (59) Google Scholar]. The frequency of NHP CD45+ cells in the PB was tracked longitudinally and the engraftment of NHP cells in the BM, spleen, and thymus was determined after 16–24 weeks at necropsy. NSG mice did not support engraftment of CD34+ cells or sort-purified NHP CD34 subsets following transplantation in adult or neonatal recipients (Table 1A). Only a single mouse receiving the HSC-enriched cell fraction demonstrated low levels (<0.3%) of CD45+ NHP cells in the PB that was skewed toward the B-lymphoid lineage. However, no engraftment of CD45+ cells or NHP HSPCs was observed in the BM or other tissues in this specific mouse.Table 1Engraftment of NHP HSPCs in NSG miceAdult NSGNeonatal NSGAPM - Primed BM - 1 × 105 cells/mousePBBMPBBMCD90–CD45RA+0/60/60/90/9CD90+/–CD45RA–0/80/8——CD90–CD45RA–0/40/40/70/7CD90+CD45RA–1/60/60/100/10BPM - Primed BM - CD34+ cell dose escalationAdult NSGNeonatal NSG 0.1% by flow cytometry. Open table in a new tab Freshly isolated and sort-purified NHP HSPCs were injected intravenously into NSG mice via the tail vein (adult mice) or intrahepatically (neonatal mice) after sublethal irradiation. PB draws were performed every other week and the terminal end point for tissue harvest was performed 16–20 weeks after transplantation. Mice were considered to be engrafted when showing a distinct population of NHP CD45+ cells >0.1% by flow cytometry. To determine whether the transplantation of higher numbers of NHP CD34+ cells would result in successful engraftment in NSG mice, we injected up to 1 × 106 NHP CD34+ cells in adult mice and 7.5 × 105 CD34+ cells in neonatal mice (Table 1B). Even the highest cell doses did not lead to detectable engraftment in any tissues. Similarly, engraftment of CD34+ cells from an alternative stem cell source (steady-state BM) or from the RM was not supported by the NSG mouse model (Table 1C). Engraftment, stem cell homing, and multilineage differentiation of PM and RM HSPCs are not supported by the NSG mouse model. In addition to immunodeficiency of the host, successful xenotransplantation of HSPCs requires interspecies cross-reactivity of the "don't eat me" signal mediated by the SIRPα/CD47 axis [19Barclay AN Van den Berg TK The interaction between signal regulatory protein alpha (SIRPalpha) and CD47: structure, function, and therapeutic target.Annu Rev Immunol. 2014; 32: 25-50Crossref PubMed Scopus (407) Google Scholar]. NSG mice efficiently support human HSPC engraftment because of a genetic polymorphism stemming from the NOD genetic background that alters the glycosylation of the SIRPα protein and renders it cross-reactive with human CD47 [20Takenaka K Prasolava TK Wang JC et al.Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells.Nat Immunol. 2007; 8: 1313-1323Crossref PubMed Scopus (357) Google Scholar]. We reasoned that the NOD-derived SIRPα receptor may not be cross-reactive with NHP CD47, but that the SIRPα/CD47 pair may be cross-reactive between humans and NHPs. Indeed, these proteins are highly conserved among primates (Table 2 and Supplementary Table E1, online only, available at www.exphem.org). Therefore, we evaluated NHP hematopoiesis following xenotransplantation in MISTRG recipient mice. In MISTRG mice, the gene encoding human SIRPA was knocked-in in the endogenous Sirpa locus, resulting in physiological expression of the human SIRPα protein [21Herndler-Brandstetter D Shan L Yao Y et al.Humanized mouse model supports development, function, and tissue residency of human natural killer cells.Proc Natl Acad Sci U S A. 2017; 114: E9626-E9634Crossref PubMed Scopus (89) Google Scholar, 22Deng K Pertea M Rongvaux A et al.Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations.Nature. 2015; 517: 381-385Crossref PubMed Scopus (377) Google Scholar]. MISTRG mice also harbor knock-in humanization of several cytokine-encoding genes (Csf1, encoding CSF-1 aka macrophage colony-stimulating factor [M-CSF]; Il-3, encoding interleukin-3; Csf2, encoding granulocyte-macrophage colony-stimulating factor [GM-CSF]; and Thpo, encoding thrombopoietin) [14Rongvaux A Willinger T Martinek J et al.Development and function of human innate immune cells in a humanized mouse model.Nat Biotechnol. 2014; 32: 364-372Crossref PubMed Scopus (459) Google Scholar]. The absence of these mouse cytokines affects mouse hematopoiesis, resulting in a form of genetic preconditioning that opens the BM niche for transplanted cells. Furthermore, the human cytokines may support the maintenance and differentiation of NHP HSPCs, since most of these cytokines are highly conserved between humans and NHPs (Table 2 and Supplementary Table E1, online only, available at www.exphem.org).Table 2Cross-species conservation of amino acid sequences% HomologyMouse → HumanMouse → PMHuman → PMSIRPα66.5%67.8%*Rhesus mulatta (Macaca nemestrina sequence not available).91.9%*Rhesus mulatta (Macaca nemestrina sequence not available).CD4770.3%70.3%99.8%IL-332.1%32.9%82.1%M-CSF69.9%69.4%99.4%GM-CSF54.9%53.5%99.3%TPO74.3%73.9%95.1%Categories: red, 90%. Rhesus mulatta (Macaca nemestrina sequence not available). Open table in a new tab Categories: red, 90%. We initially sort-purified PM CD34+ cells and injected them intrahepatically into neonatal preconditioned MISTRG mice. We longitudinally tracked engraftment of NHP CD45+ cells in the PB starting in week 8 and analyzed tissues 16–24 weeks after transplantation. In contrast to NSG mice (Table 1), NHP HSPCs successfully engrafted MISTRG mice (Figure 1). Development of phenotypical NHP granulocytes (CD11b+CD14–), monocytes (CD11b+CD14+), B cells (CD20+), natural killer (NK) cells (CD16+), and T cells (CD4+, CD8+) was detected in the PB, BM, spleen, and thymus of MISTRG mice (Figures 1A and 1B). In addition, NHP CD34+ cells, as well as phenotypical CD34 subsets enriched for HSCs, MPPs/EMPs, and LMPs, were detected in the BM of multilineage engrafted MISTRG mice (Figure 1C). Intrahepatic injection of 1–20 × 105 NHP CD34+ cells into MISTRG mice demonstrated dose-dependent multilineage engraftment in the PB, BM, and spleen (Figures 2A and 2D, Table 3A). Engraftment of NHP T cells in the thymus was dose independent and showed either full or no chimerism (Figure 2E). Mice receiving >20 × 105 NHP CD34+ cells became anemic or demonstrated significant weight loss and were euthanized 8–10 weeks after transplantation. In contrast to human HSPC transplantation in MISTRG [14Rongvaux A Willinger T Martinek J et al.Development and function of human innate immune cells in a humanized mouse model.Nat Biotechnol. 2014; 32: 364-372Crossref PubMed Scopus (459) Google Scholar], the frequency of phenotypical NHP monocytes (CD11+CD14+ cells) in the PB and spleen was low but remained stable over time. Among lymphoid cells, B cells were gradually replaced by emerging T cells starting at weeks 12–14 (Supplementary Figures E1A–E1D, online only, available at www.exphem.org).Table 3Engraftment of NHP HSPCs in MISTRG miceNeonatal MISTRGAPM CD34+PBBM25 × 1047/76/750 × 1049/99/9100 × 1046/66/6200 × 1043/33/3BPM CD34 SubpopulationsCD90–CD45RA+0/90/9CD90–CD45RA–0/170/17CD90+CD45RA–11/1511/15CLimiting DilutionCD34+5 × 1040/40/47.5 × 1040/30/310 × 1044/53/525 × 1047/76/7CD90+5 × 1040/40/47.5 × 1041/21/210 × 1047/77/725 × 104——Freshly isolated and sort-purified NHP HSPCs were injected intrahepatically into neonatal MISTRG mice after sublethal irradiation. PB draws were performed every other week and the terminal end point for tissue harvest was performed 16–24 weeks after transplantation. Mice were considered to be engrafted when showing a distinct population of NHP CD45+ cells >0.1% by flow cytometry. Open table in a new tab Freshly isolated and sort-purified NHP HSPCs were injected intrahepatically into neonatal MISTRG mice after sublethal irradiation. PB draws were performed every other week and the terminal end point for tissue harvest was performed 16–24 weeks after transplantation. Mice were considered to be engrafted when showing a distinct population of NHP CD45+ cells >0.1% by flow cytometry. Healthy mice did undergo planned euthanasia 16–24 weeks after transplantation to analyze multilineage engraftment of NHP cells in different tissues. NHP granulocytes were predominantly found in the BM, monocytes in the BM and spleen, B cells in the spleen, NK cells in the BM and thymus, and T cells in the thymus of engrafted mice (Figures 1B, 2B–2E). Most importantly, NHP CD34+ cells were able to home and repopulate the BM stem cell niche harboring a phenotypically distinct HSC-enriched subset as well as downstream progenitors (Figures 1C and 2F). In addition, BM-resident NHP CD34+ cells were capable of giving rise to myeloid, erythroid, and erythro-myeloid colonies in CFC assays ex vivo (Figure 2G). In summary, the MISTRG mouse model supports the engraftment of NHP CD34+ HSPCs and their multilineage differentiation to immune cell lineages in the PB and tissues. Furthermore, NHP CD34+ cells can home into the BM stem cell niche of MISTRG mice and successfully reconstitute phenotypically distinct CD34+ cell subsets. The availability of this novel "monkeynized" MISTRG mouse model enabled us to study the engraftment potential of NHP CD34+ subpopulations. Our previous studies performing competitive repopulation experiments of cultured and gene-modified CD34+ subpopulations in the NHP model showed that CD90+CD45RA– HSC-enriched cell fractions exclusively contain hematopoietic reconstitution potential [11Radtke S Adair JE Giese MA et al.A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.Sci Transl Med. 2017; 9: eaan1145Crossref PubMed Scopus (59) Google Scholar]. To determine the engraftment potential of MPP/EMP- and LMP-enriched cell fractions and whether co-administration of committed progenitor cells is required for HSC engraftment, freshly isolated, sort-purified, and unmanipulated HSC-enriched (I), MPP/EMP-enriched (II), and LMP-enriched (III) cell fractions were transplanted into individual neonatal MISTRG mice (Figure 3A). In agreement with our observations in the autologous NHP transplantation setting [11Radtke S Adair JE Giese MA et al.A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.Sci Transl Med. 2017; 9: eaan1145Crossref PubMed Scopus (59) Google Scholar], robust multilineage engraftment of NHP cells in the PB of MISTRG mice was exclusively restricted to the sort-purified HSC-enriched cell fraction (I: CD90+CD45RA–), whereas no NHP cells were detected in mice injected with the MPP/EMP-enriched (II) or LMP-enriched (III) cell fraction (Figure 3B). Upon necropsy, NHP multilineage engraftment in the BM, spleen, and thymus was only observed for mice transplanted with CD90+CD45RA– cells (Figures 3C–3F). HSC-enriched CD90+CD45RA– cell fractions successfully gave rise to multilineage hematopoietic cells in the PB, BM, spleen, and thymus, similar to previous experiments using bulk CD34+ cells. Likewise, homing of CD34+ cells and reconstitution of the BM HSPC compartment including all CD34 subsets was exclusively observed for mice receiving HSC-enriched cell fractions (Figure 3G). Most importantly, engrafted NHP CD34+ cells maintained their differentiation potential, giving rise to erythroid and myeloid colonies ex vivo (Figure 3H). These data demonstrate that NHP CD90+CD45RA– cells exclusively contain multipotent HSCs with multilineage engraftment and BM reconstitution potential. We have shown previously in the autologous NHP transplantation setting that the number of transplanted CD90+CD45RA– cells correlates with the onset of neutrophil and platelet recovery and allows the prediction of successful long-term engraftment [11Radtke S Adair JE Giese MA et al.A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates.Sci Transl Med. 2017; 9: eaan1145Crossref PubMed Scopus (59) Google Scholar]; we have determined that a minimum of 122,000 CD90+CD45RA– cells per kilogram of body weight is required to prevent engraftment failure. To determine whether the xenograft potential of NHP cells recapitulates the autologous NHP transplantation setting and to quantify the frequency of cells with mouse reconstitution potential, we next performed limiting dilution experiments of NHP CD34+ HSPCs and CD90+CD45RA– cell fractions. Three different cell doses (10 × 104, 7.5 × 104, and 5 × 104) were transplanted into MISTRG mice and engraftment of NHP cells in the PB as well as other tissues were analyzed as described previously (Table 3C). Robust multilineage engraftment of NHP cells in the PB and tissues was observed in all mice transplanted with 1 × 105 CD90+CD45RA– cells, whereas chimerism was lower in animals receiving the same number of bulk CD34+ cells (Figures 4A–4C). NHP chimerism in both groups gradually decreased with lower numbers of cells injected and the average frequency of NHP blood cells in all tissues was consistently higher in mice transplanted with CD90+CD45RA– cells (Figures 4A–4C). Similarly, greater engraftment of NHP CD34+ cells, phenotypical CD34 subsets, as well as CD34+ cells with CFC potential was seen in mice receiving CD90+CD45RA– cells compared with bulk CD34+ fractions (Figures 4D and 4E). Limiting dilution calculation [17Hu Y Smyth GK ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays.J Immunol Methods. 2009; 347: 70-78Crossref PubMed Scopus (1221) Google Scholar] for both cell fractions determined that NHP CD34+ cell fractions contained one SRC in 131 × 103 (range 64–267 × 103) transplanted cells (Figures 5A and 5B). CD90+CD45RA– HSPCs were enriched for SRCs compared with CD34+ bulk cells, with an estimate of 1 in 72.2 × 103 cells (range 35–147 × 103) demonstrating MISTRG engraftment potential (Figures 5A and 5B). In summary, CD90+CD45RA– HSPCs are enriched for SRC potential and reliably engraft in MISTRG mice when transplanting a minimum dose of 73 × 103 cells per mouse. In this study, we demonstrate for the first time successful high-level multilineage engraftment of NHP hematopoietic cells in the PB, spleen, thymus, and BM in a xenogeneic transplantation mouse model. This novel "monkeynized" MISTRG mouse model supports dose-dependent engraftment as well as multilineage differentiation of NHP blood cells, enables homing of NHP HSPCs into the BM stem cell niche, and supports complete reconstitution of phenotypically and functionally distinct NHP HSPC subpopulations. Most importantly, this mouse model recapitulates our recent findings obtained by autologous NHP transplantation, thus confirming exclusive enrichment of NHP stem and progenitor cells with multilineage engraftment potential in the CD34+CD90+CD45RA– phenotype. The analysis of primary NHP HSPCs using the mouse xenograft model has rarely been performed in the last decades. One of the first studies reporting successful but low-level engraftment (0.13–2%) of gene-modified and transplanted baboon HSPCs (2 × 106 cells per mouse) was reported in 2003 by Horn et al. using the NOD/SCID mouse model [12Horn PA Thomasson BM Wood BL Andrews RG Morris JC Kiem
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