Engraftment Of Human Hematopoietic Cells Research Articles

Macrophage depletion overcomes human hematopoietic cell engraftment failure in zebrafish embryo

Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.

The Effects of Busulfan on Xenogeneic Transplantation of Human Peripheral Blood Mononuclear Cells in Recipient Mice

Background. Humanized mouse models with engraftment of human peripheral blood mononuclear cells (PBMCs) or hematopoietic stem cells (HSCs) are effective tools for the study of human immunity. Busulfan has been used as a substitute for irradiation in human hematopoietic stem cell (HSC) transplantation models, but it has not been tested in human peripheral blood mononuclear cell (PBMC) transplantation models.Materials and Methods. This study evaluated PBMC engraftment using cytometry and enzyme-linked immunosorbent assay (ELISA) in female NOD.CB17/Prkdcscid/JKrb/ IL2 receptor γ−/− (NIG) mice treated with busulfan.Results. In this model, the percentage of human CD3+ T cell engraftment in the blood was 28.2%, with dominant infiltration of CD8+ cells in the spleen 3 weeks post PBMC transplantation. Production of human cytokines, including IL-12p70, IL-4, IL-5, IFN-γ, IL-6, IL-8, IL-22, TNF-α, and IL-10, was determined in mice treated with busulfan.Conclusions. Our findings demonstrate that busulfan treatment is a beneficial alternative for simple and efficient PBMC engraftment in a rodent model, possibly helping to evaluate human immunity in preclinical studies.

Humanized mouse model for vaccine evaluation: an overview.

Animal models are essential in medical research for testing drugs and vaccines. These models differ from humans in various respects, so their results are not directly translatable in humans. To address this issue, humanized mice engrafted with functional human cells or tissue can be helpful. We propose using humanized mice that support the engraftment of human hematopoietic stem cells (HSCs) without irradiation to evaluate vaccines that influence patient immunity. For infectious diseases, several types of antigens and adjuvants have been developed and evaluated for vaccination. Peptide vaccines are generally used for their capability to fight cancer and infectious diseases. Evaluation of adjuvants is necessary as they induce inflammation, which is effective for an enhanced immune response but causes adverse effects in some individuals. A trial can be done on humanized mice to check the immunogenicity of a particular adjuvant and peptide combination. Messenger RNA has also emerged as a potential vaccine against viruses. These vaccines need to be tested with human immune cells because they work by producing a particular peptide of the pathogen. Humanized mice with human HSCs that can produce both myeloid and lymphoid cells show a similar immune response that these vaccines will produce in a patient.

Potent and Target Specific Cytotoxicity of PRAME TCR Mimic CAR T Cells in KMT2A-Rearranged AML Patient-Derived Xenograft Model

Chimeric antigen receptor (CAR) T cell therapy was a resounding success in CD19+ ALL despite the fact that it is essential for B-cell maturation because it is not expressed during normal myeloid hematopoiesis. Normal and leukemic B-cells are targeted by CD19+ CAR T-cells without causing myeloid hematopoietic toxicity. Advancing such therapy to acute myeloid leukemia (AML) has been challenging in part due to a paucity of “dispensable” targets or targets whose expression is limited to the leukemic cells. Using a large AML and normal hematopoiesis transcriptome database, we pursued discovery of “AML-Restricted Targets”; genes that are silent in normal hematopoiesis but expressed in AML. This broad discovery effort yielded a library of AML-restricted targets that included PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein expressed via MHC on the cell surface, to be a highly expressed in AML including the aggressive KMT2A-rearranged (KMT2A-r) AML. We used a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface, to develop a PRAME directed TCR mimic CAR T (PRAME mTCRCAR T) for pre-clinical studies. Studies in cell lines and CDX models have shown significant efficacy of this CAR T (Kirkey, Bld Adv. 2022). To conduct final IND-enabling studies, we generated a KMT2A-r HLA-A2+/PRAME+ patient-derived xenograft (PDX) model to treat with PRAME mTCRCAR T-cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against this unique PDX AML model. VL and VH sequences from the PRAME specific TCR mimic antibody (Pr20) were used to construct the single-chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME +/HLA-A2 + pediatric patient with KMTA2-r AML. PDX cells were transduced with lentiviral ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. Mice were transplanted with 1x10 6 PDX leukemia cells then 1 week later, PDX leukemia-bearing mice were treated with unmodified T cells or PRAME mTCRCAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse. Leukemia burden was measured by IVIS imaging and regular peripheral blood analysis. Mice who received unmodified T cells had rapid disease progression by day 50 and all died by day 105. In contrast, PRAME mTCRCAR T-treated mice rapidly cleared disease and all remained alive and leukemia-free >180 days post-treatment (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T-cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75- and 429.6-fold increase in radiance by IVIS at 6 and 11 weeks post-treatment, respectively. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. We further evaluated hematopoietic toxicity of PRAME mTCRCAR T cells in humanized mice. Sub-lethally irradiated NSG-SGM3 mice were reconstituted with HLA-A2+ CD34 selected human cord blood stem cells with simultaneous and allowed to engraft. Human hematopoietic cell engraftment was evaluated following treatment with unmodified T cells vs. PRAME mTCRCAR T cells. Evaluation of human CD45+ cells in the peripheral blood via flow cytometry showed no difference in the levels of hematopoietic engraftment in mice treated with PRAME mTCRCAR T cell (8%) vs. unmodified T cells (6%) vs. an untreated control cohort (5%), demonstrating lack of toxicity of PRAME mTCRCAR T cells against HLA-A2 hematopoiesis. We demonstrate that PRAME mTCRCAR T cells can irradicate AML in a target specific manner in aggressive KMT2A-r AML without toxicity. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival, without hematopoietic toxicity. These results provide strong rationale for advancing this therapeutic approach to clinical development.

Prostaglandin E2 as transduction enhancer affects competitive engraftment of human hematopoietic stem and progenitor cells

Exvivo gene therapy (GT) is a promising treatment for inherited genetic diseases. An ideal transduction protocol should determine high gene marking in long-term self-renewing hematopoietic stem cells (HSCs), preserving their repopulation potential during invitro manipulation. In the context of the improvement of a clinically applicable transduction protocol, we tested prostaglandin E2 (PGE2) as a transduction enhancer (TE). The addition of PGE2 shortly before transduction of human CD34+ cells determined a significant transduction increase in the invitro cell progeny paralleled by a significant reduction of their clonogenic potential. This effect increased with the duration of PGE2 exposure and correlated with an increase of CXCR4 expression. Blockage of CXCR4 with AMD3100 (plerixafor, Mozobil) did not affect transduction efficiency but partially rescued CD34+ clonogenic impairment invitro. Once transplanted invivo in a competitive repopulation assay, human CD34+ cells transduced with PGE2 contributed significantly less than cells transduced with a standard protocol to the repopulation of recipient mice, indicating a relative repopulation disadvantage of the PGE2-treated CD34+ cells and a counter-selection for the PGE2-treated cell progeny invivo. In conclusion, our data indicate the need for risk/benefit evaluations in the use of PGE2 as a TE for clinical protocols of GT.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such “hu-NK mice” rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

Human growth hormone supplement promotes human lymphohematopoietic cell reconstitution in immunodeficient mice.

Aim: To investigate the potential of human growth hormone (hGH) to improve human hematopoietic reconstitution in humanized mice. Materials & methods: Immunodeficient mice were conditioned by total body irradiation and transplanted with human CD34+ fetal liver cells. Peripheral blood, spleen and bone marrow were harvested, and levels of human lymphohematopoietic cells were determined by flow cytometry. Results: Supplementation with hGH elevated human lymphohematopoietic chimerism by more than twofold. Treatment with hGH resulted in significantly increased reconstitution of human B cells and myeloid cells in lymphoid organs, enhanced human erythropoiesis in the bone morrow, and improved engraftment of human hematopoietic stem cells. Conclusion: hGH supplementation promotes human lymphohematopoietic reconstitution in humanized mice.

Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis.

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials.

Nuclear translocation of TFE3 under hypoxia enhances the engraftment of human hematopoietic stem cells

Nuclear translocation of TFE3 under hypoxia enhances the engraftment of human hematopoietic stem cells

Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells

SummaryHematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.

S. Aureus Cas9 Leads to Higher Sickle Mutation Correction Compared to S. Pyogenes Cas9 in Patient Hematopoietic Stem and Progenitor Cells

Introduction: Sickle cell disease (SCD) is a red blood cell disorder caused by a single nucleotide mutation in the β-globin gene (HBB). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but is available to only a minority of patients and can be associated with high morbidity and mortality. CRISPR/Cas9 mediated genome editing may provide a permanent cure for SCD patients by correcting the sickle mutation in HBB in hematopoietic stem and progenitor cells (HSPCs). Previously, we achieved ~39% sickle mutation correction in SCD HSPCs by delivering S. pyogenes (Spy) Cas9/R-66S gRNA as ribonucleoprotein (RNP) and single-stranded oligodeoxynucleotides (ssODN) corrective donor template. S. aureus (Sau) Cas9 has potentially advantageous properties to improve therapeutic gene editing efficiency and safety, including smaller size allowing for efficient in vivo delivery and longer Protospacer Adjacent Motif (PAM) sequence for higher specificity. However, although in general, the cutting efficiency of SauCas9 is lower than SpyCas9, the differences in gene correction and other gene-editing outcomes between SpyCas9 and SauCas9 have not been well studied.Methods: With our R-66S gRNA sequence targeting the sickle mutation, the PAM sequence of SauCas9 (NGGRRT) is mutually permissive with that of SpyCas9 (NGG), allowing the same sequence to be targeted by both Cas9 nucleases. We delivered R-66S gRNA with SpyCas9 and SauCas9 respectively as RNP, along with corrective ssODN donor template into SCD HSPCs. We analyzed sickle mutation correction rate and small insertions and deletions (INDELs) profile by Next Generation Sequencing (NGS).Results/discussions: We found that although the INDEL rate of SpyCas9 is higher than SauCas9 at the same molar concentration of RNP, SauCas9 gave 43% sickle mutation correction, slightly higher than SpyCas9 (39%), demonstrating efficient homology-directed repair (HDR) mediated gene correction by SauCas9. To further investigate the potential for clinical translation, we will perform in-depth efficiency and safety characterization comparing SauCas9 and SpyCas9 mediated sickle mutation correction therapy in SCD HSPCs.Conclusion: In this work, we showed that, compared with the highly-optimized and widely-used SpyCas9, SauCas9 leads to a higher sickle mutation correction in SCD HSPCs, demonstrating the therapeutic potential of SauCas9 for treating SCD. We will further investigate the efficiency and safety of gene-edited therapy mediated by these two Cas9 orthologs, including in-depth characterization of off-target effects, chromosomal rearrangement and aberrations, and large genomic modifications. We will differentiate gene-corrected SCD HSPCs to study erythropoiesis and red cell phenotype, including normal hemoglobin production and reduced sickling under hypoxic conditions. Lastly, we will evaluate the engraftment efficiency of gene-edited cells in Nonirradiated NOD,B6.SCID Il2rγ -/- Kit (W41/W41) (NBSGW) mice that support the engraftment of human hematopoietic stem cells. DisclosuresSheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.

IL‐10 induces IgG4 production in NOD‐scid Il2rγnull mice humanized by engraftment of peripheral blood mononuclear cells

IL‐10 induces IgG4 production in NOD‐<i>scid</i> Il2rγ^null mice humanized by engraftment of peripheral blood mononuclear cells

Spleen route accelerates engraftment of human hematopoietic stem cells

Intravenous injections of human hematopoietic stem cells (hHSCs) is routinely used in clinic and for modeling hematopoiesis in mice. However, unspecific dilution in vascular system and non-hematopoietic organs challenges engraftment efficiency. Although spleen is capable of extra medullar hematopoiesis, its ability to support human HSC transplantation has never been evaluated. We demonstrate that intra-splenic injection results in high and sustained engraftment of hHSCs into immune-deficient mice, with higher chimerisms than with intravenous or intra-femoral injections. Our results support that spleen microenvironment provides a niche for HSCs amplification and offers a new route for efficient HSC transplantation.

Engraftment of Human Hematopoietic Cells in Biomaterials Implanted in Immunodeficient Mouse Models.

Over the last 20years, significant progress has been made in the development of immunodeficient mouse models that now represents the gold standard tool in stem cell biology research. The latest major improvement has been the use of biomaterials in these xenogeneic mouse models to generate human "bone marrow like" tissues, which not only provides a more relevant xenograft model but can also potentially enable us to delineate the interactions that are specific between human bone marrow cells. There are a number of biomaterials and strategies to create humanized niches in immunodeficient mouse models, and the methods can also differ significantly among various research institutes. Here, we describe a protocol to create a humanized 3D collagen-based scaffold human niche in immunodeficient mouse model(s). This humanized in vivo model provides a powerful technique for understanding the human BM microenvironment and the role it plays in the regulation of normal as well as malignant hematopoiesis.

Development of a Bioluminescent Human Osteosarcoma Model in Humanized NSG Mice: A Pilot Study.

Osteosarcoma is the most common type of bone cancer, but current therapeutic interventions remain largely insufficient. The development of new treatment strategies is needed, and moreover, optimal rodent models are necessary for testing the efficacy of new treatment modalities of osteosarcoma. Humanized mice carry human hematopoietic and immune systems, and are considered an ideal tool to study human diseases including cancer immunology. Herein, we performed a preliminary study toward developing an in vivo bioluminescent osteosarcoma model using humanized immunodeficient (NSG) mice. To establish the xenograft and orthotopic mouse model, NSG mice engrafted with human CD34+ hematopoietic stem cells were injected with luciferase-expressing KHOS/NP cells at two different time points. Bioluminescence images were obtained to monitor in vivo tumor growth and metastasis. Influence of the degree of human cell engraftment on tumor growth and metastatic behavior was analyzed and compared between the two groups. KHOS/NP-luc cells injected in humanized NSG mice formed macroscopic tumors. The percentage of human CD45+ cells in these models was similar, but the percentage of human CD45+CD3+ and their subset was higher in the late-injection group compared to that of the early-injection group. The rate of KHOS/NP tumor growth was higher in the early-injection group than in the late-injection group. In the present study, human hematopoietic cell engraftment was not influenced by KHOS/NP cell injection, but KHOS/NP osteosarcoma showed more aggressive behavior in the early-injection group than that in the late-injection group, forming larger tumor volumes and earlier metastases. The results indicated that tumor growth and progression in humanized NSG mice may have been influenced by higher levels of human cell engraftment, especially T cells. Although there exist some limitations to our study, our preliminary results can provide the basis for the development of a humanized osteosarcoma mouse model.

830\xa0nm photobiomodulation therapy promotes engraftment of human umbilical cord blood-derived hematopoietic stem cells

Human umbilical cord blood (hUCB)-derived hematopoietic stem cells (HSCs) are an important source for HSCs in allogeneic HSC transplantation, but a limited number and a low efficacy of engraftment greatly restrict their clinical use. Here, we report the ability of photobiomodulation therapy (PBMT) to significantly enhance the engraftment efficacy of hUCB HSCs and progenitor cells (HSPCs). hUCB CD34+ cells were illuminated at a fluence of 2 J/cm2 with a near-infrared light (830 nm) transmitted by an array of light-emitting diodes (LED) prior to infusion of NOD/SCID-IL2Rγ−/− mice. The pre-treatment resulted in a threefold higher of the mean percentage of human CD45+ cells in the periphery of the mice compared to sham-treated CD34+ cells. The enhanced engraftment may result from a PBMT-mediated increase of intracellular reactive oxygen species (ROS) levels and Src protein phosphorylation in CD34+ cells. The two events were causally related as suggested by the finding that elevation of ROS by hydrogen peroxide increased Src phosphorylation, while ROS reduction by N-acetyl cysteine partially reversed the phosphorylation. The investigation demonstrates that PBMT can promote engraftment of hUCB HPSCs, at least in part, via ROS-mediated Src signaling pathway. PBMT can be potentially a safe, convenient, and cost-effective modality to improve hematological reconstitution in patients.

PDGFB-expressing mesenchymal stem cells improve human hematopoietic stem cell engraftment in immunodeficient mice

The bone marrow (BM) niche regulates multiple hematopoietic stem cell (HSC) processes. Clinical treatment for hematological malignancies by HSC transplantation often requires preconditioning via total body irradiation, which severely and irreversibly impairs the BM niche and HSC regeneration. Novel strategies are needed to enhance HSC regeneration in irradiated BM. We compared the effects of EGF, FGF2, and PDGFB on HSC regeneration using human mesenchymal stem cells (MSCs) that were transduced with these factors via lentiviral vectors. Among the above niche factors tested, MSCs transduced with PDGFB (PDGFB-MSCs) most significantly improved human HSC engraftment in immunodeficient mice. PDGFB-MSC-treated BM enhanced transplanted human HSC self-renewal in secondary transplantations more efficiently than GFP-transduced MSCs (GFP-MSCs). Gene set enrichment analysis showed increased antiapoptotic signaling in PDGFB-MSCs compared with GFP-MSCs. PDGFB-MSCs exhibited enhanced survival and expansion after transplantation, resulting in an enlarged humanized niche cell pool that provide a better humanized microenvironment to facilitate superior engraftment and proliferation of human hematopoietic cells. Our studies demonstrate the efficacy of PDGFB-MSCs in supporting human HSC engraftment.

In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice

The murine host has remained a readily available and ethically acceptable model for the study of human diseases and therapeutic testing. Immunodeficient mouse models support engraftment of human hematopoietic stem cells (HSC) but with limitation in efficiency and mature lineage representation. Combined knock-in of several non-crossreactive human cytokines (M-CSF, IL3/GM-CSF, and Thrombopoietin) into the corresponding murine loci in the SRG strain (in short termed "MISTRG") has enhanced engraftment and maintenance of human HSCs with multi-lineage differentiation (Rongvaux et al. Annu Rev Immunol 2013, Deng et al. Nature 2015, Saito et al. Blood 2016, Theocharides et al. Haematologica 2016). Despite robust HSC engraftment and myelo- and erythropoiesis in bone marrow (BM), all humanized immunodeficient mouse models lack of mature human red blood cells (RBC), platelets, and myeloid cells in peripheral blood (PB) (Rahmig et al. Stem Cell Reports 2016, Yurino et al. Stem Cell Reports 2016, Song et al. Nat Commun 2019). Yet, full maturation and representation of all myeloid lineages in PB is essential to study diseases of the HSC, such as MDS, and of the RBC, such as sickle cell anemia or malaria. With universal absence of a murine adaptive immune system the culprit is likely the murine host's innate immune system. Previous studies have shown that treatment of engrafted mice with liposomal clodronate that abrogates murine (and human) macrophages, with or without cobra venom factor that eliminates complement, can increase mature human circulating RBC, but only transiently and with significant toxicity. We first sought to determine the site of huRBC sequestration and destruction. Intravital imaging after injection of CFSE labelled huRBC identified the murine liver as the major site of RBC destruction. While muRBC rapidly circulate through the liver circulation, huRBC have greatly increased transit times and are sequestered in liver vessels. We hypothesized that humanization of the murine host's liver could potentially alleviate huRBC sequestration and significantly increase circulating huRBC. In previous studies deletion of fumarylacetoacetate hydrolase (Fah) in the Rag-/-Il2rg-/- background has allowed humanization of the liver and served to study diseases such as malaria (Vaughan et al. J Clin Invest 2012). The liver is the site of synthesis of numerous proteins, some of which directly impact hematopoiesis and blood cells, such as complement. We deleted the Fah gene via CRISPR/Cas9 in MISTRG mice and crossed MISTRG-Fah-/- mice to homozygosity (MISTRGFah). MISTRGFah are viable, fertile, and healthy when maintained on drinking water supplemented with 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC), that blocks tyrosine metabolism upstream of Fah and prevents buildup of hepatotoxic metabolites. At 8 weeks of age we implanted MISTRGFah mice with commercially available, adult human hepatocytes (HuHep) via direct injection into the splenic vein, followed by gradual withdrawal of NTBC water. Regulated buildup of intracellular fumarylacetoacetate results in death of murine Fah-/- hepatocytes and regeneration with HuHep with up to 90% repopulation by HuHep (Azuma et al. Nature biotechnology 2007). When plasma human albumin levels reached 2mg/dL, indicative of significant (~80%) HuHep repopulation, we sublethally (80cGy) irradiated HuHepMISTRGFah mice and engrafted each mouse with 105 fetal liver (FL) derived CD34+ cells. 10 weeks post transplantation, mice were analyzed for engraftment and specifically erythroid maturation in PB. HuHepMISTRGFah mice had significantly higher levels of BM and interestingly spleen erythropoiesis and circulating huRBC in PB (Fig.1 a). CD235a+ huRBC in HuHepMISTRGFah mice are enucleated (Hoechst neg) and mature as evident by loss of CD49d (ITGA4) and gain of Band3 staining (Hu et al. Blood 2013) (Fig.1 b). Interestingly, human erythroid cells in MISTRG but not HuHepMISTRGFah mice are coated with murine complement C3 (muC3) (Fig.1 c) suggesting that liver humanization results in loss of muC3 expression. In conclusion, we have generated the first humanized mouse model with fully mature, circulating huRBC when engrafted with human CD34+ stem and progenitor cells. Ongoing studies are testing the applicability of this model to MDS and sickle cell disease. Disclosures Flavell: SMOC: Equity Ownership; Zai labs: Consultancy; GSK: Consultancy; Artizan Biosciences: Equity Ownership; Troy: Equity Ownership; Rheos Biomedicines: Equity Ownership.

HMGA2 promotes long-term engraftment and myeloerythroid differentiation of human hematopoietic stem and progenitor cells

AbstractLarsson et al tackle the critical issue of fate choice in hematopoietic stem cells. They demonstrate that HMGA2 is highly expressed on the most immature progenitor cell subsets of fetal, neonatal, and adult human hematopoiesis. They also demonstrate that modulation of this gene's expression significantly affects hematopoietic reconstitution after transplant.

Improved multilineage human hematopoietic reconstitution and function in NSGS mice.

Genetic manipulation of NOD/SCID (NS) mice has yielded numerous sub-strains with specific traits useful for the study of human hematopoietic xenografts, each with unique characteristics. Here, we have compared the engraftment and output of umbilical cord blood (UCB) CD34+ cells in four immune-deficient strains: NS, NS with additional IL2RG knockout (NSG), NS with transgenic expression of human myeloid promoting cytokines SCF, GM-CSF, and IL-3 (NSS), and NS with both IL2RG knockout and transgenic cytokine expression (NSGS). Overall engraftment of human hematopoietic cells was highest in the IL2RG knockout strains (NSG and NSGS), while myeloid cell output was notably enhanced in the two strains with transgenic cytokine expression (NSS and NSGS). In further comparisons of NSG and NSGS mice, several additional differences were noted. NSGS mice were found to have a more rapid reconstitution of T cells, improved B cell differentiation, increased levels of NK cells, reduced platelets, and reduced maintenance of primitive CD34+ cells in the bone marrow. NSGS were superior hosts for secondary engraftment and both strains were equally suitable for experiments of graft versus host disease. Increased levels of human cytokines as well as human IgG and IgM were detected in the serum of humanized NSGS mice. Furthermore, immunization of humanized NSGS mice provided evidence of a functional response to repeated antigen exposure, implying a more complete hematopoietic graft was generated in these mice. These results highlight the important role that myeloid cells and myeloid-supportive cytokines play in the formation of a more functional xenograft immune system in humanized mice.