Engraftment Of Hematopoietic Stem Cells Research Articles

Recovery of Highly Functional Human Hematopoietic Stem and Progenitor Cells from 27-Year Old Cryopreserved Umbilical Cord Blood

Umbilical cord blood (CB) is a promising source of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) for hematopoietic cell transplantation (HCT). CB is cryopreserved following collection, allowing for the amassment of nearly 800,000 CB units in public banks and an estimated 4 million in private banks. Cryopreservation of CB is essential for successful CB transplantation, and it is critical to understand how long CB can be stored with efficient recovery of engraftable HSCs. Prior work demonstrated efficient recovery of engraftable HSCs from 10-, 15-, and 21-year old cryopreserved CB. Here, we demonstrate efficient recovery of highly engraftable HSCs from 27-year old cryopreserved CB units. 3 CB units cryopreserved for >27 years were thawed with clinical protocols, and total nucleated cells were recovered with 76-87% viability. 3-6 fresh CB units were used as a comparison for cryopreserved units. Immunophenotyping assays to determine numbers of HSCs/HPCs showed that cryopreserved CB units had on average 4.25x103 CD34+ cells per 1x106 nucleated low density CB cells (LDCB), similar to fresh CB with 4.55x103CD34+ per 1x106 LDCB. Within the CD34+ population, cryopreserved CB when compared to fresh CB had on average equal or greater numbers of stringently immunophenotypically defined HSCs (9.1x103 vs 6.2x103), multipotent progenitors (MPPs) (2.1x105 vs 8.6x104), and multilymphoid progenitors (MLPs) (1.9x104 vs 1.0x104) per 1x106 CD34+ cells. Conversely, there were fewer numbers of common myeloid progenitors (CMPs) (2.9x104 vs 1.1x105), megakaryocyte-erythroid progenitors (MEPs) (4.5x104 vs 1.0x105), and granulocyte macrophage progenitors (GMPs) (4.8x104 vs 1.4x105). Colony forming unit (CFU) assays demonstrated that cryopreserved CB units exhibited lower functional HPC numbers per 1x106 CD34+ enriched cells including CFU-GM (1.9x104 vs 8.3x104), BFU-E (1.2x104 vs 3.4x104), and CFU-GEMM (1.1x104 vs 5.8x104). In ex vivo analysis of HSC/HPC proliferative capacity, cryopreserved CB units showed similar or greater capacity for expansion in growth stimulating media compared to fresh CB, including HSCs (7.7-fold vs 6.2-fold increase), MPPs (3.5-fold vs 4-fold increase), MLPs (44-fold vs 1.7-fold increase), CFU-GM (29-fold vs 13.3-fold increase), and CFU-GEMM (28.6-fold vs 4-fold increase). For in vivo functional HSC analysis, 10,000 CD34+ enriched cells were transplanted to sublethally irradiated NSG mice. Cryopreserved CB units exhibited early and late human engraftment in peripheral blood (PB) at months 1, 2, 4, and 5. Bone marrow (BM) human chimerism at month 5 ranged from low (0.5%) to robust levels of engraftment (52.7%). Limiting dilution analysis revealed that 2 cryopreserved CB units had similar SCID repopulating cell (SRC) frequency, ranging from 314 to 1392 SRCs per 1x106 CD34+ cells while the third had a much lower 65 to 393 SRCs per 1x106 CD34+ cells. The third CB unit also exhibited lower average human chimerism in the BM (0.55%) than the other 2 units (15% and 16.2%). However, the SRC frequencies in all 3 CB units are similar to those in fresh CB units published by our group and others (range 51 to 929 SRCs per 1x106 CD34+ cells). Secondary transplants showed that all 3 CB units have secondary repopulation capacity demonstrated by human chimerism in PB. These data suggest functional HSCs with short- and long-term engrafting and sustained self-renewal capacity can be cryopreserved for at least 27 years. We show that different cryopreserved CB units contain variable functional HSC frequency. Similar results in ex vivo characterization suggest cryopreservation is not the cause of these functional differences, but rather there were functional differences in the CB units prior to cryopreservation. This underscores the need to have reliable measures of CB functionality prior to cryopreservation for more efficient selection of donor units and to test potential effects of cryopreservation on individual units. Transcriptomic studies are ongoing to determine molecular programs that may contribute to differences in SRC frequency. These data will provide insight into mechanistic explanations for differences in HSC function in different CB units. Authors’ note: This project was conceived by the authors prior to the death of Hal E. Broxmeyer. Dr. Broxmeyer initiated the study as a continuation of his life's work demonstrating the utility of CB for HCT. This work is dedicated to Hal.

Mlkl Mediates Age-Related Attrition of Hematopoietic Stem Cells and Ineffective Hematopoiesis in Myelodysplastic Syndrome

Aging causes anemia, perturbed immunity, and increased propensity to age-related hematological diseases such as myelodysplastic syndrome (MDS). Age-dependent functional decline in hematopoietic stem cells (HSCs), which is accompanied by morphological and functional changes in their organelles as well as sterile inflammation in the bone marrow (BM), is one of the main reasons why the hematopoietic system ages. However, molecular mechanisms by which HSCs alter their function during aging remain unclear. Necroptosis is a form of programmed necrosis that underlies sterile inflammation and can take place upon exposure to inflammatory stimuli in apoptosis-resistant cells including HSCs. MLKL, the effector molecule of necroptosis that can form channel-like structure on lipid bilayers upon activation, not only induces cell death but also injures membrane-bound organelles such as mitochondria, though its significance in HSC aging remains elusive. Our quantitative RT-PCR revealed abundant expression of MLKL in murine BM immature hematopoietic cells including HSCs (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150+), which was validated by reanalysis of the published proteome datasets (Zaro et al., eLife 2020). Using FRET-based necroptosis reporter mice and Western blotting analysis, we found that while MLKL was inactivated at the steady state, it was readily activated in HSCs upon poly I:C injection. To study the effect of MLKL activation on HSCs, we assessed cell death of HSCs isolated from poly I:C-treated MLKL-deficient (Mlkl-/-) mice. Unexpectedly, Annexin V/DAPI staining revealed no changes in the frequencies of dying and dead cells between wild-type (WT) and Mlkl-/- HSCs. Nonetheless, PVA-based HSC culture revealed that the growth suppression observed in poly I:C-treated WT HSCs largely depended on MLKL (p=0.0003). Furthermore, HSC transplantation assay revealed that reduced engraftment and skewed myeloid differentiation were observed in poly I:C-treated WT HSCs but largely alleviated by MLKL deficiency (p=0.011). These results indicate that inflammation-induced MLKL activation does not cause HSC death but impairs their growth and lymphopoietic potential. To assess the relevance of MLKL activation in HSC aging, we treated WT and Mlkl-/- mice with serial rounds of 5-FU treatment, which were shown to induce age-related alterations in HSCs (Beerman et al., Cell Stem Cell 2013). Of note, we observed marked attenuation of age-related features such as myeloid-skewed hematopoiesis and expansion of phenotypic HSCs in 5-FU-treated Mlkl-/- mice (p=0.0051). HSC transplantation experiments further revealed that MLKL deficiency alleviated 5-FU-induced decrease in engraftment and lymphopoietic potential of HSCs (p=0.013). Indeed, analysis of 18-month-old WT and Mlkl-/- mice demonstrated that MLKL deficiency largely rescued myeloid-skewed hematopoiesis and loss of BM CLPs (Lin-/cKitlo/Sca1lo/Flk2+/IL-7Rα+) (p=0.0047), and serial HSC transplantation revealed better self-renewal and lymphopoietic potential of aged Mlkl-/- HSCs (p=0.0055). Importantly, multiplex ELISA assay using BM supernatants revealed no changes in concentrations of inflammatory cytokines, indicating that the observed effects in Mlkl-/- HSCs were likely caused by cell intrinsic changes. In line with this, electron microscopy analysis revealed that age-related changes in mitochondria, such as elongated and swollen structure with disorganized cristae, were significantly attenuated in aged Mlkl-/- HSCs (p<0.0001). JC-1 dye staining further revealed that age-related accumulation of damaged mitochondria was significantly attenuated in Mlkl-/- HSCs (p=0.049). Finally, to evaluate the effect of MLKL on development of age-related hematological disease, we established MDS mouse models by transplantation of RUNX1S291fs-transduced WT and Mlkl-/- HSCs. We found that WT MDS mice mainly died of severe anemia due to ineffective hematopoiesis, whereas Mlkl-/- MDS mice showed milder anemia and better overall survival (p=0.044), indicating that MLKL promotes ineffective hematopoiesis in MDS. Taken together, our results identify MLKL as a key mediator of age-related functional decline in HSCs and ineffective hematopoiesis in MDS. They also suggest that pharmacological inhibition of MLKL activity is a promising strategy to better control aging of the hematopoietic system and age-related hematological disease.

NLRP6 in the Bone Marrow Microenvironment Regulates HSPC Quiescence Via MGP/RUNX2

Inflammatory response in the bone marrow microenvironment affects hematopoietic stem cell (HSC) engraftment and hematopoietic reconstruction. NLR Family Pyrin Domain Containing 6 (NLRP6) involves in multiple signaling pathways to regulate inflammatory response and maintain cell homeostasis. However, the mechanism of NLRP6 in bone marrow microenvironment regulating hematopoiesis remains unclear. To analysis of the effect of NLRP6 deficiency on bone marrow microenvironment and HSC activity, bone marrow chimeras with NLRP6 deficiency in the microenvironment was performed to analyze HSPC quiescence and differentiation in the microenvironment with Nlrp6 deficiency. Single-cell sequencing technology was utilized to analyze the cellular and molecular abnormalities of NLRP6-deficient stromal cells in the bone marrow and to furtherly explore the regulatory mechanism of the NLRP6-deficient microenvironment to resident HSPCs. The results showed that NLRP6 deficiency in bone marrow microenvironment increased proportion of LSK-HSPCs in G0 phase but not effect on the homing of HSPCs. LSK-HSPCs sorted from NLRP6-/- recipients at week 8 after 1st transplantation for 2nd transplantation had higher frequency of CD45.1+ cells in the PBMCs or in total myeloid cells compared with control. NLRP6 deficiency in bone marrow microenvironment reduced CMP and GMP proportion. CMPs sorted from NLRP6-/- recipients at week 8 after 1st transplantation had lower regeneration potential through 2nd transplantation and cell proliferation assay in vitro. Transcriptome analysis of LSK-HSPC, CMP, GMP and MEP from two groups at 8 weeks after transplantation showed that NLRP6 deficiency in the microenvironment modulated cell cycle of HSPCs and is associated with NOD-like receptor signaling pathway. Single-cell sequencing of bone marrow CD45-Ter119- cells showed that NLRP6 deficiency increased CAR cells of bone marrow stroma. The molecule expression of CAR cells (including Cxcl12 and Kitl) contributing to hematopoiesis was upregulated which was associated with MAPK and NOD-like receptor signaling pathway. NLRP6 deletion in the microenvironment upregulated MGP expression of CAR cell which were furtherly supported by western blot and immunofluorescence staining. Recombinant mouse MGP protein (rmMGP) decreased Colony-Forming Units (CFUs) of WT LSK-HSPCs. Single-cell sequencing of LSK-HSPCs at week 8 after 1st transplantation showed that HSCs differentiated into Mpps which separately kept dormant or entered cell cycling. NLRP6 deficiency in the microenvironment markedly increased the quiescent cellular proportion of LSK-HSPCs compared with control, especially Flt3+ CD48- MPPs which were dormant MPPs differentiated early from HSCs. LSK-HSPCs resident in the microenvironment with NLRP6 deletion kept lower transcription of genes about cell cycling and NOD like receptor signaling pathway. Transcription factor Runx2 and its target inhibitors of cell cycle were upregulated and RUNX2 phosphorylation increased by western blot compared with control. In a word, this study suggests that NLRP6 in the bone marrow microenvironment promotes HSPC quiescence induced by RUNX2 through upregulating MGP from CAR cells.

Intracellular Iron Overload Induces Metabolic Switching in Active Hematopoietic Stem Cells in Beta-Thalassemia

Beta-thalassemia (BThal) is a complex monogenic disorder, causing severe anemia, ineffective erythropoiesis and multi-organ secondary defects. Iron overload (IO), associated with ineffective erythropoiesis and therapeutic blood transfusions, is one of the main complications of the disease, leading to morbidities in various organs. Despite the improvement in chelation therapies, IO toxicity is still a relevant issue. Correction of BThal is achieved by transplantation of hematopoietic stem cells (HSC) from healthy donors or autologous HSC from patients upon gene therapy. The quality and the engraftment of HSC depend on the bone marrow (BM) microenvironment, thus niche-HSC crosstalk plays a crucial role for transplantation outcome. We provided the first demonstration of impaired function of HSC caused by an altered BM niche in BThal (Aprile et al., Blood 2020). HSC from Hbbth3/+ (th3) BThal mice showed more active cycling profile and response to stress. We reported that IO reduces the hematopoietic supportive capacity of BM mesenchymal stromal cells from patients and recent evidence highlighted that both IO and iron deficiency are sources of cellular stress detrimental to HSC maintenance. Thus, we hypothesized that accumulation of iron might directly affect HSC function in BThal, as a model of chronic IO. Transcriptome analysis of sorted HSC from th3 mice showed a positive enrichment of genes involved in iron homeostasis, such as TfR1, Steap3, Fth1 and Ftl1, suggesting iron uptake and storage. Consistently, BThal HSC displayed higher levels of intracellular free reactive iron, as compared to wild-type (wt) controls (1.7±0.2 calcein MFI fold to wt, p<0.0001). We found that iron specifically accumulates in the mitochondria causing mitochondrial defects, as reduced size and lower membrane potential (0.4±0.05 TMRE/MTG fold to wt, p<0.001). These data, along with the downregulation of mitochondrial biogenesis and mitophagy genes, indicate an accumulation of damaged mitochondria in BThal HSC. In vivo administration of iron dextran to wt mice generated intracellular IO in HSC and was sufficient to decrease the mitochondrial membrane potential at levels comparable to those of th3 HSC, thus suggesting a direct effect of iron on mitochondrial activity. Since mitochondria are the powerhouse of the cell, we wondered if the mitochondrial dysfunction of BThal HSC could affect their production of metabolic energy. Metabolism indeed control HSC function: active HSC are more cycling and, because of their higher energy request, change the metabolism from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, we found that ATP levels in th3 HSC are lower compared to controls (wt 9.8±2.6 vs. th3 3.7±0.3 ATP nM, p<0.01). In line with mitochondrial dysfunction, in vitro inhibition of OXPHOS did not affect ATP content of BThal HSC since they switch to glycolysis for their energy demand. Consistently, th3 HSC showed positive enrichment of glycolytic genes and higher glucose uptake (wt 1639±163 vs. th3 2401±85 MFI 2NBDG, p<0.05). To unravel the basis of mitochondrial damage caused by IO, we investigated the role of reactive oxygen species (ROS). Iron is one of the sources of ROS and consistently, th3 HSC showed increased activation of antioxidant genes and higher levels of mitochondrial ROS (wt 2507±756 vs. th3 5205±459 MFI MitoSOX, p<0.01). In vivo reduction of mitochondrial ROS by MitoQ rescued mitochondrial activity (th3 0.3±0.07 vs. th3+MitoQ 0.9±0.2 TMRE/MTG fold to wt, p<0.05), thus demonstrating that mitochondrial dysfunction is reversible in BThal. Ongoing experiments will assess the metabolite profile, the restoration of HSC metabolism and in vivo function upon MitoQ and will also test HSC from BThal patients for mitochondrial defects. Overall, our study revealed that the multisystemic complications of IO in BThal also affect HSC. Iron has a direct impact on HSC metabolism by inducing oxidative stress and mitochondrial dysfunction. Despite the active cycling state, BThal HSC switch their metabolism to glycolysis because of the mitochondrial defect. Targeting mitochondrial iron or ROS might provide new tools to preserve HSC function in the setting of autologous transplantation upon gene therapy/editing for BThal. Moreover, this research will add novel insight about the regulation of HSC biology linking iron to mitochondrial metabolism.

Endothelial mTOR Preserves Hematopoietic Stem Cell Fitness By Suppressing Thrombospondin1

Hematopoietic stem cells (HSC) reside in close proximity to blood vessels within the bone marrow (BM) termed 'perivascular niches' which provide instructive cues for maintaining HSC self-renewal potential. The perivascular niche is comprised of endothelial cells (ECs) lining the BM blood vessels (BMECs), and an array of perivascular stromal cells that collectively serve to regulate balanced HSC self-renewal and differentiation. While young HSCs are endowed with robust self-renewal potential, aging is associated with a significant decline in HSC fitness including a loss of their self-renewal ability and a biased differentiation towards the myeloid lineages at the expense of lymphopoiesis. These aging related HSC defects result in anemias, impaired response to immunizations, and an increased risk for developing clonal disorders including acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). However, mechanisms that drive HSC defects during aging remain poorly understood, and represents a critical barrier towards developing strategies for preservation of hematopoietic fitness during aging. While HSC aging was generally assumed as a predominantly cell-intrinsic process, recent studies have identified that aging of the BM niche plays an instructive role in promoting age-related HSC defects. Supporting this, we recently discovered that aging is associated with a significant decline in mTOR activity within BMECs that impairs their capacity to serve as a HSC-supportive niche. Our findings showed that deletion of mTOR specifically within ECs of young (4 month old) mice (Endothelial Cell specific mTOR Knock Out; mTORECKO) is sufficient to recapitulate HSC aging phenotypes including loss of HSC self-renewal, and a myeloid-biased differentiation. Here, we sought to identify the mechanisms by which endothelial mTOR preserves HSC fitness. To identify candidate factors that promote HSC aging in mTORECKO mice, we performed a plasma proteomic analysis that revealed a putative role for Thrombospondin1 (Thbs1) wherein mTORECKO mice displayed elevated levels of Thbs1. Analysis of cultured BMECs revealed that inhibition of mTOR signaling within BMECs was sufficient to increase Thbs1 expression. Thbs1 is a well-defined antiangiogenic factor that has recently been identified to play a critical role in promoting a multitude of age-related cardiovascular and inflammatory disorders. However, whether Thbs1 regulates HSC activity is not known. To this end, we performed ex vivo HSC expansion transplantation assays which revealed that Thbs1 causes a significant decline in long term HSC engraftment. RNA Seq analysis of HSC expansions revealed that Thbs1 activates chronic inflammatory responses within HSCs that have been suggested to promote aging related HSC defects. To determine whether Thbs1 promotes HSC aging in mTORECKO mice, we crossed them with Thbs1 global knockout mice (Thbs1KO) to generate mTORECKOThbs1KO mice. Analysis of mTORECKOThbs1KO mice revealed that deletion of Thbs1 was sufficient to restore hematopoietic defects in mTORECKO mice including restoration of peripheral blood myeloid bias, BM HSC frequency and hematopoietic progenitor activity. Furthermore, serial HSC transplantation analysis confirmed that deletion of Thbs1 was sufficient to restore HSC self-renewal and lymphoid output in mTORECKO mice, identifying a putative role for Thbs1 as a pro-geronic factor for HSCs. To determine whether Thbs1 promotes HSC dysfunction during physiological aging, we aged Thbs1KO mice for 18 months and analyzed their hematopoietic parameters. Our analysis revealed that deletion of Thbs1 was able to prevent the age-associated increase in peripheral blood myeloid bias and BM HSC frequency. Notably, HSCs derived from aged (18 month old) Thbs1KO mice demonstrated a suppression of inflammatory responses by RNA Seq analysis, and a restoration of self-renewal ability and balanced lineage output in serial HSC transplantation assays, that are indistinguishable from HSCs derived from young (3 month old) mice. Our findings identify Thbs1 as a novel pro-geronic factor for HSC aging that can be targeted to preserve hematopoietic healthspan.

Clonal Dynamics of HDR-Edited HSPCs Targeting the CD33 Locus in Rhesus Macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. Loss or damage of LT-HSCs with resultant oligoclonal hematopoiesis can increase the risk of malignant transformation or long-term hematopoietic and immune dysfunction. HDR has adverse impacts on LT-HSC function as revealed by ex vivo studies and murine xenograft models, likely due to p53 activation, intracellular innate immune activation, and/or other pathways. However, the impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not previously been studied. We tracked the clonal dynamics of CRISPR/HDR-edited barcoded HSPCs following autologous transplantation in rhesus macaques (RMs). HDR-favored gRNAs targeting the CD33 gene locus and an ssODN containing a 6 bp barcode ID and 14 bp random barcode sequences and flanked by CD33 homology arms were designed. Autologous CD34+ cells were electroporated with the Cas9 RNP and HDR cassette in the presence of i53 mRNA to inhibit p53-mediated cell death and reinfused into animal 1 following myeloablation. In animal 1, CD33 KO neutrophils were prevalent early following engraftment and then rapidly decreased over the next two months. Similar to CD33 expression level, we revealed that HDR knock-ins as well as total indel frequency drastically decreased following transplantation. This was in marked contrast to macaques transplanted with CD34+ HSPCs edited at the CD33 locus only for NHEJ, without inclusion of an HDR cassette (Kim et al, Cell, 2018, and Figure 1 below). We next performed a competitive transplantation comparing clonal dynamics of edited cells to barcoded lentiviral (LV)-transduced cells in two macaques. For each animal, CD34+ cells were fractionated into 2 aliquots, and half were transduced with a barcoded GFP-expressing LV vector and half electroporated with the Cas9 RNP and HDR cassette with higher concentrations of i53 mRNA to enhance protection of CRISPR edited LT-HSCs. The two cell populations were combined and reinfused into myeloablated animals. CD33 KO cells in both animals decreased rapidly, however the higher concentration of i53 mRNA facilitated a less steep reduction in CD33 total indel frequency in vivo over time. However, HDR in these two animals also markedly decreased, similar to the pattern in animal 1. In contrast, LV-transduced GFP+ cell engraftment stabilized at 20% after 2 months. We retrieved embedded barcodes from CRISPR/HDR cells and revealed that a diversity of HDR clones contributed to early hematopoietic reconstitution, with restriction to a much smaller set of long-term clones at steady state, proportional in the 3 animals to the level of HDR overall. The diversity of HDR-edited contributing clones was far less than LV-transduced contributing clones long-term post-transplantation. In conclusion, CRISPR/HDR edited cells were found to decrease rapidly after autologous transplantation in rhesus macaques despite substantial gene editing assessed in the infusion product prior to infusion, in contrast to much more stable LV-transduced cells. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which was somewhat improved by use of high dose i53 mRNA during editing. It will be important to find improved approaches to retain LT-HSC functional activity during the HDR editing process, or explore targeted gene correction approaches such as base or PRIME editing that may better preserve HSC activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lnp-Targeting Hematopoietic Stem Cells and Lentiviral Gene Transfer to Generate and Rescue a Novel Mouse Model of Lethal Alpha-Thalassemia

α-Thalassemia (α-thal) is caused by insufficient production of the α-globin protein. Clinical presentation of α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to Bart's Hydrops Fetalis Syndrome/BHFS (complete knockout). In patients with severe α-thal, allogeneic bone marrow transplantation may be required for survival. Alternatively, patients could be treated through autologous BMT following ex vivo transduction with a human α-globin expressing lentiviral vector. However, a proper model system and vector must be established for the development of such a treatment. We crossed animals that had deletion of both Hba-a1 and Hba-a2 in heterozygosity (Hba-a1+/ko/Hba-a2+/ko) to generate knockout (KO) embryonic mice lacking all the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko). Although these embryos die perinatally, their fetal liver cells (FLC) at 13.5 dpc are still viable and represent a source of engraftable hematopoietic stem cells (HSC). KO-FLC were transplanted into congenic myeloablated WT mice to generate mice carrying Hba-a1ko/ko/Hba-a2ko/ko HSC. These animals developed a severe phenotype ~7 weeks post engraftment. As these mice lack α-globin expression, we observed β-tetramer formation (β4, HbH, Fig1A), resulting in aberrant red blood cell (RBC) morphology, β-globin precipitates, high RBC oxygen binding affinity, elevated erythropoietin, hematocrit and hemoglobin levels, splenomegaly and vaso-occlusive episodes, due to the large number of abnormal RBCs. Furthermore, we observed iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver. However, FLC engraftment is cumbersome and requires extensive breeding and many animals to generate enough FLC for few recipients. To facilitate the generation of α-thal mice, we created conditional knockout (cKO) mice that have been edited to remove Hba-a2, while inserting loxP sites flanking Hba-a1 (Hba-a1fl/Hba-a2ko). Homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) demonstrate a mild phenotype. To achieve complete deletion of the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko) in HSC, we used lipid nanoparticles (LNPs), which are one of the most promising delivery systems to package and make mRNA into a useful therapeutic. We developed a novel LNP preparation with an antibody to target CD117 and deliver nucleoside-modified mRNA to HSC, (see abstract Breda et al). We treated HSC from homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) ex vivo with CD117-LNP embedded with Cre mRNAs (Cre-CD117-LNP). Cells showed the expected deletion of the α-globin genes and were injected into myeloablated recipient mice. These chimeras recapitulated the phenotype observed following transplantation of constitutive α-globin-KO FLC, including elevated hematocrit, splenomegaly, and culminating in lethality ~7 weeks post-transplant. We screened multiple potential erythroid specific lentiviral vectors in both mouse and human cell lines and identified a promising candidate, ALS20αI, which at VCN=1 produced human α-globin protein equivalent to that of a single endogenous α-globin gene. Myeloablated recipient mice transplanted with Cre-CD117-LNP-treated cKO BM die roughly 7 weeks post-transplant with the expected pathological phenotype (Fig1A). In contrast, mice receiving BM treated with Cre-CD117-LNP and ALS20αI (VCN>1) all survived long term (>4-month) with normalization of erythropoiesis and iron metabolism. VCNs were proportional to chimeric hemoglobin (human α/mouse β) levels and concurrently decreased or absent HbH (Fig1B-C-D). Additionally, we tested ALS20αI in erythroid progenitors derived from CD34 cells isolated from patients with deletional and non-deletional HbH disease. Preliminary data demonstrates improvement in α-globin/β-globin mRNA ratio and reduction in formation of HbH by HPLC. In summary, we have developed new mouse models for α-thal with a lethal and reproducible phenotype. Remarkably, use of LNP technology accelerated the generation of α-thal mice and their characterization. Furthermore, our new vector ALS20αI rescued these animals with stable expression of the human α-globin gene. These data strongly support the use of ALS20αI for the cure of severe forms of α-thal, as 2 copies may be sufficient to rescue patients affected by BHFS, while 1 copy may be sufficient to cure patients affected by HbH disease. HP, SR corresponding Authors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal Analysis Reveals Functional and Transcriptional Heterogeneity of Self-Renewing Fetal Liver Hematopoietic Stem Cells

The fetal liver (FL) is a key organ supporting the expansion of hematopoietic stem cells (HSCs) during development. The ability to expand transplantable HSCs in vitro is a long-standing challenging; thus, decoding the gene pathways that regulate proliferation and self-renewal during FL-HSC development is highly relevant to engineering HSCs for therapeutic applications. Moreover, while heterogeneity in self-renewal and engraftment properties of HSCs in adult bone marrow has been extensively studied, these properties remain largely uncharacterized in FL-HSCs. To explore FL-HSC properties at the single cell level, we engineered a serum-free culture platform designed to recapitulate the murine FL endothelial niche (FL-EC) (Figure1), and sorted single FL-HSC (CD45+GR1-F4/80-SCA1highEPCRhigh) into individual wells containing FL-EC. Following co-culture, we observed emergence of immunophenotypically distinct types of hematopoietic colonies as determined by flow cytometry. Only colonies containing cells predominantly expressing high levels of HSC markers SCA1 and EPCR (Figure1, HSC colonies) provided multilineage engraftment as determined by transplantation assays, whereas colonies containing cells expressing HSC markers and more differentiated progeny (mixed colonies) were unable to provide serial multilineage engraftment. Furthermore, cellular progeny from a single HSC colony transplanted into multiple recipients demonstrated serial engraftment, indicating the FL-EC niche could amplify single FL-HSC with long-term reconstitution capacity. These studies suggest that transplantable FL-HSCs are uniquely distinguished by their propensity for symmetric self-renewal in this assay. However, not all HSC colonies provided engraftment in secondary recipients, suggesting heterogeneity in self-renewal properties of immunophenotypically defined FL-HSCs that required deeper analysis. Thus, to further assess transcriptional heterogeneity in HSC colonies, we next integrated single-cell RNA sequencing (scRNAseq) into the clonal FL-HSC assay (Figure 2). Residual cells from six HSC colonies were divided for simultaneous scRNAseq and transplantation. Consistent with experiments above, we observed heterogeneity in long-term serial engraftment from individual colonies. Remarkably, cells from the colony capable of serial engraftment were distinctly enriched in genes associated with dormant HSCs in adult marrow (Cabezas-Wallscheid et.al., Cell 2017). Conversely, cells from HSC colonies that lacked serial engraftment capacity exhibited increased expression of Myc target genes by Gene Set Enrichment Analysis (GSEA), and ribosome biogenesis and mitochondrial translation by Gene Ontogeny (GO) analysis, which are hallmarks of biosynthetically activated HSCs in adult marrow that are primed for differentiation to progenitor states. To further assess the transcriptional heterogeneity of primary FL-HSC, we next performed scRNAseq with freshly isolated FL-HSC. Unsupervised clustering and co-regulated modules of differentially expressed genes identified a rare subset of cells highly enriched for genes associated with HSC dormancy, while the other cells featured higher expression of genes involved in biosynthetic processes via GO analysis (e.g. cytoplasmic translation, rRNA processing). This data further supports transcriptional heterogeneity within the FL-HSC compartment, which includes a unique biosynthetically dormant subset. Collectively, our studies elucidate previously unrecognized heterogeneity in FL-HSCs, highlighting a state of relative biosynthetic dormancy as a key to symmetric self-renewal of serially engraftable FL-HSCs. These findings complement recent studies demonstrating distinct developmental origins for multipotent progenitors and functional engraftable HSCs in definitive hematopoiesis (Dignum et.al., Cell Reports 2021; Patel et.al., Nature 2022), suggesting functional heterogeneity in immunophenotypically defined FL-HSCs in our study may in part reflect these distinct ontogenies. Building on these studies, we are currently using CRISPR-Cas9 gene knockout to define the roles of candidate genes identified by our scRNAseq analysis that are implicated in regulating dormancy and symmetric self-renewal of FL-HSCs, which would provide essential insight for future strategies for ex vivo amplification of HSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extracellular DEK Activates NRF2 Antioxidant Targets and Drives Expansion of Functional and ROS-Low Human Hematopoietic Stem Cells

Umbilical cord blood (CB) is a promising source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation (HCT), a life-saving treatment, due to lower incidence of graft versus host disease and its availability. CB HCT is limited by low numbers of HSCs, thus there is a strong need to increase numbers of engraftable HSCs, for instance by expanding functionally competent HSCs ex vivo. We have shown that extracellular DEK protein, an epigenetic regulator that is secreted in response to stress and inflammation, is a novel hematopoietic regulatory chemokine. Treatment with extracellular DEK in growth stimulating media significantly enhances the expansion of engraftable HSCs in mouse bone marrow (BM) and human CB (Capitano, et al. JCI 2019; 129(6):2555-2570). To examine mechanisms driving enhanced expansion of functional HSCs after DEK treatment, we performed RNA-sequencing on CB HSCs, multipotent progenitor cells (MPPs), common myeloid progenitors (CMPs), and granulocyte macrophage progenitors (GMPs) after overnight DEK treatment in expansion media. All cell populations upregulated gene programs related to antioxidant responses upon DEK treatment. Indeed, 7/10 genes upregulated by DEK in all HSC/HPC populations (NMRAL2P, PIR, NQO1, SELENOW, FTL, RIT1, and QPCT) promote antioxidant activity. Overnight DEK treatment significantly reduced mean fluorescence of the activated reactive oxygen species (ROS) indicator CM-H2DCFDA in HSCs, MPPs, CMPs, and GMPs. Thus, DEK may induce antioxidant activity. We next found that all 10 genes commonly upregulated in HSCs/HPCs are either direct transcriptional targets of NRF2 (NMRAL2P, LUCAT1,PIR, NQO1, FTL, and QPCT) or are directly affected by changes in NRF2 activation (SELENOW, EBI3, TNFRSF9, and RIT1). Further, several genes highly upregulated by DEK treatment in HSCs (i.e., CCL2, KYNU, and CXCL8) are NRF2 targets. To examine if DEK activates NRF2 transcriptional activity, we utilized a transgene reporter assay where expression of red fluorescent protein (RFP) is driven by activation of promoter antioxidant response elements (ARE), to which active NRF2 binds. DEK treatment of hematopoietic derived cell lines (mouse MLL-AF9 AML and human HL-60 APL) expressing the transgene significantly increased RFP signal, indicating that DEK could activate NRF2. We further examined whether DEK treatment affects mitochondrial metabolism, a source of ROS and tightly regulated process in hematopoiesis. Seahorse XFe analysis, which measures metabolic flux, showed DEK treatment of MLL-AF9 cells significantly reduced mitochondrial oxygen consumption rate, suggesting a reduced reliance on oxidative phosphorylation as an energy source in DEK treated cells. These data suggest that DEK treatment during expansion of primitive hematopoietic cells drives activation of NRF2 transcription, upregulation of antioxidant response genes, reduction of intracellular ROS levels, and may reduce the mitochondrial metabolic rate. These effects in a sense "mimic” the in vivo niche of HSCs, where they reside in low oxygen tensions, have low intracellular ROS, and exhibit low levels of oxidative phosphorylation. To test this hypothesis, we examined if DEK treatment has similar effects to maintaining HSCs in physiologic oxygen tensions (3%) compared to HSCs exposed to extraphysiologic ambient air (21%), which drives oxidative stress (Mantel, et al. Cell 2015; 161(7):1553-65). Isolating and maintaining mouse BM in physiologic oxygen tensions or treating mice in vivo with recombinant DEK preserved HSC numbers compared to exposing the BM cells to ambient air or treating with vehicle. However, combining in vivo DEK treatment with physiologic isolation of HSCs/HPCs had no additive or synergistic effect, suggesting that the overall effects of DEK treatment and maintaining low oxygen tensions may be similar. Thus, extracellular DEK treatment leads to expansion of functionally competent HSCs and reduction of oxidative stress. These data have important implications for the regulation of HSCs/HPCs in stress conditions, where DEK is most likely to be secreted. Further, DEK may present a novel way to mimic physiologic oxygen conditions for enhancement of functional HSC expansion, and the elucidated mechanisms may be exploited for more efficient targeting of molecular programs critical for functional HSC competency, potentially providing new ways to enhance HCT.

Antibody based conditioning for allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic option for many patients with hematological malignancies and nonmalignant hematopoietic disorders. To achieve stable engraftment of donor hematopoietic stem cells (HSCs), recipient HSC deletion is needed to create space for incoming donor HSCs and donor HSCs must escape immune rejection by the recipient. Conventional allo-HSCT requires high dose of irradiation and/or chemotherapy to produce sufficient host stem cell and immune system ablation to permit donor HSC engraftment. However, these procedures also result in nonspecific tissue injury that can cause short- and long-term adverse effects as well as incite and amplify graft-versus-host-disease (GVHD). The delivery of targeted radiotherapy to hematopoietic tissues with the use of a radioimmunoconjugate (ROIC) as a part of transplant preparative regimen has shown clinical benefits. ROIC clinical data provide evidence for decreased relapse without increased transplant-related mortality by delivering higher targeted radiation to sites of malignancy than when given in a nontargeted fashion. An alternative approach to allo-HSCT has been developed and tested in preclinical mouse models in which nonmyeloablative preconditioning with low dose of the alkylating agent (busulfan) or lower systemic dose of irradiation combined with co-stimulatory pathway blockade (CTLA4-Ig, anti-CD40L monoclonal antibody) and/or immunosuppressive drugs have been used. Under these conditions, mixed chimerism and transplantation tolerance to fully MHC mismatched donor marrow was observed. Recently, several novel proof-of-concept antibody-mediated preconditioning methods have been developed that can selectively target hematopoietic stem and immune cells with minimal overall toxicity. Antibody-drug-conjugate (ADC) combined with reduced intensity conditioning or high dose ADC as single dose monotherapy have shown promise for allo-HSCT in preclinical models. The purpose of the current review is to discuss the literature exploring antibody-based conditioning that includes native antibody, radiolabeled antibody conjugates, and ADC for allo-HSCT.

Autologous stem cell transplantation in tandem with Anti-CD30 CAR T-cell infusion in relapsed/refractory CD30+ lymphoma

BackgroundLong-term outcome is unfavourable for relapsed/refractory (r/r) lymphoma patients who are resistant to salvage chemotherapy, even after subsequent autologous stem-cell transplantation (ASCT). Although anti-CD30 chimeric antigen receptor (CAR30) T-cell therapy induces high response rates in these patients, the duration of response is relatively limited.MethodsThis open-label, single-center and single-arm pilot study investigated the safety and efficacy of ASCT in tandem with CAR30 T-cell infusion in r/r CD30+ lymphoma. The primary endpoint was safety and key secondary endpoint was overall response rate, overall survival, progression-free survival, and duration of response.ResultsFive classical Hodgkin lymphoma (cHL) patients and 1 anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) patient were enrolled. The median age was 24 years. No patient had prior ASCT. Three patients (50.0%) relapsed for ≥ 2 times and 3 patients (50.0%) had primary refractory diseases. All had a Deauville score of 4 or 5, and 5 patients (83.3%) had a stable or progressive disease (SD/PD) at enrollment. All patients received myeloablative chemotherapy and infused CD34-positive hematopoietic stem cells (HSCs) and CAR30 T cells in tandem, with a median dose of 3.9 × 106/kg and 7.6 × 106/kg, respectively. Five paitents presented with cytokine release syndrome (CRS), all of which were grade 1. No neurotoxicity was observed. All patients had successful HSCs engraftment and reached an objective response, including 5 (4 cHL and 1 ALCL, 83.3%) with a complete response (CR) and 1 with a partial response (PR). With a median follow-up of 20.4 (range, 12.1–34.4) months, all remained alive and maintained their responses.ConclusionOur work demonstrates the combined administration of ASCT and CAR30 T-cell therapy is well-tolerate and highly effective in r/r cHL and ALCL, even in PET-positive or chemorefractory patients who are expected to have inferior outcome after ASCT, although further large-scaled validation in prospective clinical trial is warranted.Trial registration The trial was registered with the Chinese Clinical Trial Registry (ChiCTR, number ChiCTR2100053662).

Case Report: Unique patterns of lymphocyte recovery post-hematopoietic stem cell transplant associated with hyperbaric oxygen therapy: A case series

The utilization of hematopoietic stem cell transplantation (HSCT) has been rapidly growing due to multiple factors, including better availability of donors and improved supportive care. Hyperbaric oxygen has been associated with the improvement of hematopoietic stem cell (HSC) homing at the time of transplant through lowering erythropoietin levels in preclinical studies. We studied the role of hyperbaric oxygen (HBO) in the enhancement of engraftment of HSC when utilized prior to umbilical cord HSCT and autologous HSCT in two pilot clinical trials with excellent safety profiles. In these two pilot studies, we observed an uncommon phenomenon post-transplant, particularly a significant peripheral blood lymphocytosis and lymphocyte infiltration of different tissues in 3/34 of HBO-treated patients. This peripheral blood lymphocyte expansion was associated with various clinical manifestations that can be confused with infections, inflammatory conditions, or disease relapse. We hypothesize that this observation is related to different immune reconstitution dynamics related to the use of HBO. While the incidence is ~9%, this may have implications as HBO is being investigated in larger clinical trials. This case series highlights the clinical presentation, course, outcome, and potential implications of this significant rise in lymphocytes when utilizing HBO before HSCT.

32 (PB022) - Humanized mouse models for preclinical evaluation of novel immune therapies, checkpoint inhibitors and immune cell engagers

Background: The preclinical evaluation of novel immune therapies demands humanized mouse models with functional human immune cells. In previous studies we have established a humanized immune system with functional T- B- and NK cells, as well as monocytes in immunodeficient mice by transfer of hematopoietic stem cells (HSCs) or peripheral blood mononuclear cells (PBMC). By transplantation of cell-line-derived (CDX) or patient-derived (PDX) tumor xenografts on humanized mice, we successfully generated a full human tumor-immune-cell model for different tumor entities. Finally, we validated the functionality of these models using checkpoint inhibitors or immune cell engagers. Methods: HSC-humanized mice were generated by single i.v. transplantation of CD34+ stem cells to immunodeficient NOG mice. Engraftment of immune cells was monitored by FACS analysis of blood samples. CDX and PDX from different entities were transplanted on the humanized mice. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections into tumor-bearing mice or with simultaneous tumor cell transplantation. These models were used to evaluate immune checkpoint inhibitors. Blood and tumor samples were analysed by FACS and immunohistochemistry for immune cell infiltration and activation. Results: The transplanted HSCs engrafted in mice and established a functional human differentiated immune system with proliferating immune cells. Up to 20% of the human immune cells in the blood were functional T cells, characterized by a high PD-1 expression 14 weeks after HSC inoculation. Selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Checkpoint inhibitor treatments induced tumor growth delay in selected models. FACS analysis of xenograft tumors revealed an increased percentage of tumor-infiltrating T cells. In addition, we identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other immune therapeutics. Conclusions: We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSCs on immunodeficient mouse strains generating mice with a functional human hematopoiesis. These models have been employed for preclinical evaluation of novel checkpoint inhibitors and immune cell engagers. Our human tumor-immune-cell models allow preclinical, translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. No conflict of interest.

Human kidney-derived hematopoietic stem cells can support long-term multilineage hematopoiesis

Long-term multilineage hematopoietic donor chimerism occurs sporadically in patients who receive a transplanted solid organ enriched in lymphoid tissues such as the intestine or liver. There is currently no evidence for the presence of kidney-resident hematopoietic stem cells in any mammal species. Graft-versus-host-reactive donor T cells promote engraftment of graft-derived hematopoietic stem cells by making space in the bone marrow. Here, we report full (over 99%) multilineage, donor-derived hematopoietic chimerism in a pediatric kidney transplant recipient with syndromic combined immune deficiency that leads to transplant tolerance. Interestingly, we found that the human kidney-derived hematopoietic stem cells took up long-term residence in the recipient's bone marrow and gradually replaced their host counterparts, leading to blood type conversion and full donor chimerism of both lymphoid and myeloid lineages. Thus, our findings highlight the existence of human kidney-derived hematopoietic stem cells with a self-renewal ability able to support multilineage hematopoiesis.

231.2: Selective Bcl-2 Inhibition Promotes Hematopoietic Chimerism and Allograft Tolerance Without Myelosuppressive Complications

Introduction: Hematopoietic stem cell transplantation (HSCT) has been an only approach that reproducibly achieved renal allograft tolerance in humans. However, severe myelosuppression and toxicities after myeloablative conditioning have hampered wider clinical application. To achieve hematopoietic stem cell (HSC) engraftment, it is essential to make space in the bone marrow (BM) niches by depleting host HSCs. This has been achievable only by nonselective treatments such as irradiation or chemotherapeutic drugs. Therefore, a novel approach more selectively capable of depleting HSCs is needed to widen the clinical application of HSCT. Based on murine studies that showed successful induction of mixed chimerism and allograft tolerance without irradiation by Bcl-2 inhibition, we evaluated an FDA approved Bcl-2 inhibitor (Venetoclax, Vntx) in our mixed chimerism inducing conditioning regimen. Methods: Cynomolgus monkeys received kidney and bone marrow transplantation after conditioned with various nonmyeloablative regimens which included total body irradiation (TBI), thymic irradiation (TI) and anti-thymocyte globulin (ATG), followed by a short post-transplant course of costimulatory blockade (CoB) and Cyclosporine. The study groups were consisted of 3Gy TBI, no Vntx (Group A), 1.5Gy TBI, no Vntx (Group B), TBI 1.5Gy with Vntx(10 mg/kg x 11 on days -4 to 6) (Group C), TBI 1.5Gy with Vntx but Belatacept as CoB (Group D), no TBI with Vntx (Group E), no TI with Vntx (Group F), no CoB with Vntx (Group G). Results: Most recipients treated with 3Gy TBI (Group A) developed mixed chimerism and long-term immunosuppression (I.S.)-free renal allograft survival (Table 1), but suffered from transient but severe pancytopenia. With reduced TBI (1.5Gy), all recipients failed to develop chimerism, and 4/5 developed acute rejection. By adding Vntx, despite the dose of TBI being reduced to half (1.5Gy), all six recipients (Group C) developed significantly superior chimerism (Figure 1) than regimens without Bcl-2 inhibition (Groups A and B), and 5/6 achieved long-term I.S.-free allograft without pancytopenia (Figure 2). With Belatacept in place of anti-CD154 mAb, 2/3 achieved chimerism and long-term I.S. free allograft survival. The current studies also revealed that a minimal non-toxic dose of TBI, TI and CoB remain to be essential for long-term I.S.-free renal allograft survival. Without TBI (Group E) or CoB (Group G), the recipients failed to develop chimerism and rejected allografts despite administration of Vntx. Without TI (Group F), all recipients developed mixed chimerism but failed to achieve tolerance. Conclusion: Bcl-2 inhibition with Vntx significantly promoted mixed chimerism and renal allograft tolerance without myelosuppressive complications. A minimal non-toxic dose of TBI, TI, and CoB were still required for chimerism and allograft tolerance induction even with Bcl-2 inhibition.

How mesenchymal stem cell cotransplantation with hematopoietic stem cells can improve engraftment in animal models.

BACKGROUNDBone marrow transplantation (BMT) can be applied to both hematopoietic and nonhematopoietic diseases; nonetheless, it still comes with a number of challenges and limitations that contribute to treatment failure. Bearing this in mind, a possible way to increase the success rate of BMT would be cotransplantation of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) to improve the bone marrow niche and secrete molecules that enhance the hematopoietic engraftment. AIMTo analyze HSC and MSC characteristics and their interactions through cotransplantation in murine models.METHODSWe searched for original articles indexed in PubMed and Scopus during the last decade that used HSC and MSC cotransplantation and in vivo BMT in animal models while evaluating cell engraftment. We excluded in vitro studies or studies that involved graft versus host disease or other hematological diseases and publications in languages other than English. In PubMed, we initially identified 555 articles and after selection, only 12 were chosen. In Scopus, 2010 were identified, and six were left after the screening and eligibility process.RESULTSOf the 2565 articles found in the databases, only 18 original studies met the eligibility criteria. HSC distribution by source showed similar ratios, with human umbilical cord blood or animal bone marrow being administered mainly with a dose of 1 × 107 cells by intravenous or intrabone routes. However, MSCs had a high prevalence of human donors with a variety of sources (umbilical cord blood, bone marrow, tonsil, adipose tissue or fetal lung), using a lower dose, mainly 106 cells and ranging 104 to 1.5 × 107 cells, utilizing the same routes. MSCs were characterized prior to administration in almost every experiment. The recipient used was mostly immunodeficient mice submitted to low-dose irradiation or chemotherapy. The main technique of engraftment for HSC and MSC cotransplantation evaluation was chimerism, followed by hematopoietic reconstitution and survival analysis. Besides the engraftment, homing and cellularity were also evaluated in some studies. CONCLUSIONThe preclinical findings validate the potential of MSCs to enable HSC engraftment in vivo in both xenogeneic and allogeneic hematopoietic cell transplantation animal models, in the absence of toxicity.

Non-genotoxic Restoration of the Hematolymphoid System in Fanconi Anemia

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for patients with many different blood and immune diseases; however, current treatment regimens contain non-specific chemotherapy and/or irradiation conditioning, which carry both short-term and long-term toxicities. The use of such agents may be particularly harmful for patients with Fanconi anemia (FA), who have genetic mutations resulting in deficiencies in DNA repair, leading to increased sensitivity to genotoxic agents. mAb-based conditioning has been proposed as an alternative conditioning strategy for HSCT that minimizes these toxicities by eliminating collateral tissue damage. Given the high need for improved treatments for FA patients, we aimed to evaluate the efficacy of different αCD117 mAb agents and immunosuppression on hematopoietic stem cell (HSC) depletion and explored their ability to safely establish therapeutic donor hematopoiesis post-HSCT in FA disease models. We evaluated the effects of different concentrations of αCD117 mAbs in vitro and in vivo on HSC growth and depletion. To further assess the efficacy of mAb-based conditioning, Fancd2-/- animals were treated with αCD117 mAb and combination agents with αCD47 mAb and antibody-drug-conjugates (ADCs) for syngeneic HSCT. Immunosuppression αCD4 mAb was added to all in vivo experiments due to a slightly mismatched background between the donor grafts and recipients. Immunosuppressant cocktails were also given to Fancd2-/- animals to evaluate the efficacy of mAb-based conditioning in the haploidentical setting. Statistical analyses were done using the unpaired t-test. We found that antagonistic αCD117 mAbs alone do not deplete host HSCs or enhance HSCT effectively in FA mouse models; however, the potency of αCD117 mAbs can be safely augmented through combination with αCD47 mAbs and with ADCs, both of which lead to profound HSC depletion and establishment of long-term donor engraftment post-syngeneic HSCT. This is the first time these approaches have been tested in parallel in any disease setting, with the greatest donor engraftment observed after CD117-ADC conditioning. Interestingly, our data also suggest that HSC-targeted conditioning is not necessary in HSCT for FA, as high donor HSC engraftment was observed with mAb-based immune suppression alone with immunologically matched and mismatched haploidentical grafts. These results demonstrate the safety and efficacy of several different non-genotoxic mAb-based conditioning strategies in the FA setting. In addition, they show that if sufficient immunosuppression is given to obtain initial donor HSC engraftment, turnover of a majority of the hematolymphoid system can result, likely owing to the survival advantage of wild-type HSCs over FA HSCs. Such non-toxic all-mAb-based conditioning strategies could be transformative for FA patients and those with other hematolymphoid diseases.

Enhanced development of functional human NK cells in NOD-scid-IL2rgnull mice expressing human IL15.

Human innate immunity plays a critical role in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that have opposing roles in the tumor microenvironment, including NK cell subsets that mediate tumor cell cytotoxicity and subsets with regulatory function that contribute to the tumor immune suppressive environment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However, human NK cell development and survival in humanized NOD-scid-IL2rgnull (NSG) mice are severely limited. To enhance NK cell development, we have developed NSG mice that constitutively expresses human Interleukin 15 (IL15), NSG-Tg(Hu-IL15). Following hematopoietic stem cell engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of functional human CD56+ NK cells are detectable in blood and spleen, as compared to NSG mice. Hematopoietic stem cell (HSC)-engrafted NSG-Tg(Hu-IL15) mice also supported the development of human CD3+ T cells, CD20+ B cells, and CD33+ myeloid cells. Moreover, the growth kinetics of a patient-derived xenograft (PDX) melanoma were significantly delayed in HSC-engrafted NSG-Tg(Hu-IL15) mice as compared to HSC-engrafted NSG mice demonstrating that human NK cells have a key role in limiting the tumor growth. Together, these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells, which limit the growth of PDX tumors.

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.

Effects of Soy-Whey Protein Nutritional Supplementation on Hematopoiesis and Immune Reconstitution in an Allogeneic Transplanted Mice.

Profound malnutrition and immunodeficiency are serious negative effects of radiotherapy and bone marrow transplantation for hematologic malignancy patients. This study aimed to evaluate the effects of nutritional supplementation with a soy–whey protein mixture on hematopoietic and immune reconstitution in an allogeneic transplant mouse model. Male BALB/c (H-2Kd) mice, 6–8 weeks-old, were divided randomly into five groups and then provided with different protein nutrition support. After 28 days, blood samples, bone marrow, spleen, and thymus were harvested to measure the effects. The results showed that soy–whey blended protein supplements promoted hematopoietic stem cell engraftment, body weight recovery, and the recovery of white blood cells, lymphocytes, and neutrophils; triggered the expansion of hematopoietic stem cells and progenitor cell pools by increasing the numbers of the c-kit+ progenitor, Lin-Sca1+c-kit+, short-term hematopoietic stem cells, and multipotent progenitors; enhanced thymus re-establishment and splenic subset recovery in both organ index and absolute number; improved overall nutritional status by increasing total serum protein, albumin, and globulin; protected the liver from radiation-induced injury, and increased antioxidant capacity as indicated by lower concentrations of alanine aminotransferase, aspartate aminotransferase, malondialdehyde, and 4-hydroxynonenal. This study indicated that soy–whey blended protein as important nutrients, from both plant and animal sources, had a greater positive effect on patients with hematological malignancies to accelerate hematopoiesis and immune reconstitution after bone marrow transplantation.