High-Density Clonal Analysis Reveals Highly Active Contribution of Multipotent Hematopoietic Stem Cells during Early Phases of Hematopoietic Recovery after Transplantation

Recovery after conditioning and transplantation of hematopoietic stem and progenitor cells (HSPC) is thought to be biphasic, with short-term engrafting progenitors driving the recovery for 6-9 months and multipotent hematopoietic stem cells (HSCs) providing long-term repopulation. Recent clonal tracking data from autologous human gene therapy trials seems to support this model (Biasco et al. 2016, Cell Stem Cell; Six et al. 2020, Blood). These recent reports investigating the contribution of HSCs in patients are based on the longitudinal tracking of thousands of gene-marked cells using retroviral integration site analysis (ISA). While this technology is very reliable to follow gene therapy patients and monitor the potential outgrowth of dominant or malignant clones, low sensitivity and high error rates require significant data exclusion and sophisticated statistical tests to ensure data reliability (Adair et al. 2020, Molecular Therapy MCD). Lack of sensitivity can be overcome by increasing the frequency (high density) of sampling. However, limited material from patients remains a bottleneck for improved data quality and, consequently, correct interpretation of such complex datasets. To overcome the limitations of ISA and determine the onset of HSC contribution we performed high-density sampling for ISA in nonhuman primates (NHPs) transplanted with gene-modified HSCs. In the first month of hematopoietic recovery weekly blood samples were taken to enhance data density and increase the reliablity to detect clones with low abundance. Animals were followed up to 5 years to confirm that identified HSC clones persist long-term. Finally, clonal tracking data from the NHPs was used to inform a simulation of hematopoietic reconstitution, determine the temporal involvement of HSCs, and refine the phases of hematopoietic recovery after myeloablation and HSC transplantation. In contrast to the current biphasic model, contribution of multipotent HSCs clones was detected in the very first blood samples taken 2 to 3 weeks post-transplant during neutrophil recovery. HSC clones found in these early time points persisted long-term throughout the entire follow-up and were detected in bone marrow CD34 + cells 4 years later. Most surprisingly, multipotent HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant. To understand the observed kinetics of HSC contribution and change in clonal diversity in our dataset, we simulated the clonal outgrowth and differentiation of multipotent clones. Simulations predicted that hematopoietic recovery is primarily HSC driven and HSC contribution follows a stochastic pattern. Finally, to confirm that HSCs proliferation and differentiation is a stochastic process, in vitro experiments in colony-forming cell (CFC) assays were carried out. As predicted, the decision of individual HSCs to either grow into a larger pool or differentiate and get lost followed the same kinetics as observed in vivo. Here, we show evidence that long-term persisting multipotent HSCs actively contribute during early hematopoietic reconstitution after myeloablation and HSC transplantation. Enhanced sampling showed that multipotent HSCs produce neutrophils during recovery and become the predominant source of mature blood cells as early as 50 days post-transplant. Most importantly, observed changes in the clonal diversity during early recovery suggest a stochastic engraftment of HSCs rather than a bi-phasic reconstitution initially driven by short-term progenitors. These findings should have important implications for the design of ex vivo and in vivo HSC gene therapy and genome editing approaches. Figure 1 Figure 1. Disclosures Radtke: 47 Inc.: Consultancy; Ensoma Inc.: Consultancy. Kiem: Homology Medicines: Consultancy; VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company.

Hospitalization and Healthcare Resource Use of Omidubicel Vs Cord Blood Transplantation for Hematological Malignancies in a Global Randomized Phase III Clinical Trial

Introduction Umbilical cord blood (UCB) remains an important source of hematopoietic stem cells for patients, where no matched donor is available and for racial minorities. However, cord blood transplants have been associated with delayed hematopoietic recovery, prolonged hospitalization, and higher costs of transplant compared with other donor sources. Omidubicel is an advanced cell therapy that preserves stem cell function to optimize cell homing, engraftment, differentiation, and self-renewal and is manufactured from an appropriately HLA-matched single UCB unit for each patient. A recent phase III clinical trial (NCT02730299) reported that patients who received omidubicel experienced faster time to neutrophil engraftment, faster platelet recovery, reduced rates of infections and shorter hospitalization time than patients who received standard single (33%) or double (67%) UCB transplantation [Horwitz et al, Blood, 2021]. We report on an analysis of resource utilization including hospital length of stay, hospital care setting, visits by provider type, rates of transfusions and readmissions from the Phase III trial. Methods The Phase III clinical trial included patients aged 12-65 with hematological malignancies and eligible for an allogeneic transplant. The primary endpoint was time to neutrophil engraftment, with secondary endpoints of time to platelet engraftment, first grade 2/3 bacterial or fungal infections and days alive and out of hospital in the first 100 days. 125 patients were randomized in the study. We analyzed resource utilization data for patients treated with omidubicel (n=52) or UCB (n=56) ("as-treated population") focusing on resource utilization within the first 100 days of transplantation. Summary statistics were compared between treatment arms, means and medians used to draw comparison and significance testing was performed. Results In the Phase III clinical trial, omidubicel met its primary endpoint and secondary endpoints with statistical significance. The demographics and patient characteristics were overall well-balanced with slightly more males (52% vs 63%) and a lower median age (40 vs 36) in the UCB arm. The study population was diverse with 17.6% Black, 14.8% Asian, 14% Hispanic or Latino and 12% other/unknown. Most patients had AML (45%) or ALL (34.3%), with MDS, CML and lymphomas making up the rest of the study population. The disease risk index was high/very high in 35% of the patients and 24% of the patients had a Karnofsky/Lansky performance score of <90. The rapid hematopoietic recovery was supported by a reduced rate of infections. The rates of acute and chronic GVHD in the two arms were comparable. Within the first 100 days after transplant, omidubicel patients experienced shorter average total length of hospital stay than UCB recipients (mean 41.2 vs 50.8 days; p = 0.027) and more days alive and out of the hospital (mean 55.8 vs 43.7 days; p=0.023). 12% of patients on omidubicel arm died vs 16% on UCB arm before day 100. During the primary hospitalization (transplant to discharge), fewer omidubicel patients required intensive care unit (ICU) stay (9.6% vs 23.2%) and spent fewer days in the ICU (mean 0.4 vs 4.7 days; p =0.028) and the transplant unit (mean 25.3 vs 32.9 days; p =0.022) compared to UCB recipients. Omidubicel patients required fewer outpatient consultant visits and fewer outpatient non-consultant visits (X-rays, scans, biopsies etc.) and required fewer platelet or other transfusions (RBC, albumin, plasma, and factor product) within 100 days from transplant (Table 1). Conclusions This analysis shows that more rapid hematopoietic recovery in patients transplanted with omidubicel was associated with significantly shorter hospital length of stay and reduced healthcare resource use compared to UCB in the clinical trial. Although economic data were not collected as part of the clinical trial, the costs of providing transplantation care during the first 100 days are likely lower with omidubicel compared to UCB in the real-world setting, as hospital stay, outpatient visits, and blood product transfusions are among the major drivers of costs during this time period. Figure 1 Figure 1. Disclosures Majhail: Incyte Corporation: Consultancy; Anthem, Inc: Consultancy. Manghani: Gamida Cell, Inc.: Current Employment; Tricida, Inc.: Ended employment in the past 24 months. Sivaraman: Incyte Corporation: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Gamida Cell, Inc.: Current Employment. Galamidi-Cohen: Gamida Cell, Ltd: Current Employment. Maziarz: Bristol-Myers, Squibb/Celgene,, Intellia, Kite: Honoraria; CRISPR Therapeutics: Consultancy; Artiva Therapeutics: Consultancy; Intellia: Honoraria; Omeros: Research Funding; Athersys: Other: Data and Safety Monitoring Board, Patents & Royalties; Incyte Corporation: Consultancy, Honoraria; Novartis: Consultancy, Other: Data and Safety Monitoring board, Research Funding; Allovir: Consultancy, Research Funding; Vor Pharma: Other: Data and Safety Monitoring Board.

Hematopoietic Stem Cell Regeneration through Paracrine Regulation of the Wnt5a-Prox1 Signaling Axis

The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration after injury. Here we show that deletion of the genes encoding the DNA repair-deficient syndrome Fanconi anemia (FA), Fanca and Fancc, in mice dampens HSC regeneration through both direct effects on HSCs and indirect effects on BM niche cells. Specifically, Fanca- or Fancc-deficiency compromises hematologic recovery and dampens HSC regeneration following irradiation. FA HSCs show persistent upregulation of the Wnt target Prox1, a homeobox transcription factor, in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improves long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in wild-type (WT) HSC mimics the defective repopulation phenotype of FA HSCs. By analyzing paracrine factors in Wnt signaling, we found that WT mice, but not FA mice, show significant induction by TBI of BM stromal Wnt5a protein, which is produced in LepR +CXCL12 + BM stromal cells. Wnt5a treatment of irradiated FA mice enhances hematopoietic recovery and HSC regeneration. Conversely, Wnt5a neutralization in co-cultured LepR + BM stromal cells inhibits HSC regeneration and hematopoietic recovery following TBI. Mechanistically, Wnt5a secreted by LepR +CXCL12 + BM stromal cells inhibits b-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSPCs. The detrimental effect of deregulated Wnt5a-Prox1 signaling on HSC regeneration and hematopoietic recovery is also observed in aged mice. Irradiation induces upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improves HSC regeneration and hematopoietic recovery after irradiation. Finally, treatment of aged mice with Wnt5a enhances hematopoietic repopulation. Collectively, these findings identify the novel paracrine Wnt5a-Prox1 signaling axis in regulating HSC regeneration under conditions of injury and aging. Disclosures No relevant conflicts of interest to declare.

YAP/TAZ Promote Hematopoietic Regeneration Via Accelerating the Recovery of Bone Marrow Niche

Adult hematopoietic stem cells (HSCs) reside and are protected in a unique bone marrow (BM) microenvironment, termed the HSC niche, which consists mainly of vascular endothelial cells (EC) and EC-associated mesenchymal stromal cells (MSC). Myeloablative stresses, such as ionizing radiation (IR) and chemotherapy, induce not only depletion of hematopoietic cells but also disruption of HSC niche components, as exemplified by dilation and leakiness of BM vasculature and depletion and dysfunction of BM MSCs. These structural and functional changes in the HSC niche restrain efficient hematopoietic recovery, which often compromises the efficacy of HSC transplantation (HSCT) and chemotherapy. YAP/TAZ are the two transcriptional coactivators normally repressed by LATS kinases downstream of the Hippo pathway. Although cumulative evidence has established a critical role of YAP/TAZ activation in tissue regeneration of various solid organs, their role in BM regeneration remains poorly understood. Our quantitive RT-PCR revealed that YAP/TAZ are abundantly expressed in steady-state mouse ECs (CD45 -Ter119 -CD31 +Sca1 +CD105 hi) and MSCs (CD45 -Ter119 -CD31 -PDGFRα +CD51 +LepR +) but scarcely in hematopoietic cells including HSCs (Lin -cKit +Sca1 +Flk2 -CD150 +CD48 -CD34 lo), which was confirmed by reanalysis of the published single cell RNA-seq datasets (GSE128423). Immunofluorescent imaging of BM sections revealed that YAP/TAZ are distributed mainly in the cytoplasm of ECs but evenly in the cytoplasm and nuclei of MSCs, indicating their differential basal activity in these two HSC niche components. Kinetic transcriptome analysis revealed that YAP/TAZ activity is transiently activated in ECs at 24 hours and returns to a basal repressive state by day 3 after sublethal IR. This transient activation of endothelial YAP/TAZ was critical for vascular integrity, as conditional deletion of YAP/TAZ in ECs (Cdh5-Cre ERT2Yap1 f/fTaz f/f) caused 100% lethality of mice within 10 days following sublethal IR. In sharp contrast, the kinetic expression analysis of a YAP/TAZ target gene CTGF indicated their transient inhibition in MSCs after sublethal IR, and the conditional YAP/TAZ deletion in BM MSCs (Ebf3-Cre ERT2Yap1 f/fTaz f/f) led to their reduced colony forming ability when assessed by colony forming unit fibroblast (CFU-F) assay. Recently, we discovered a novel and potent LATS inhibitor GA-003 that selectively induces mouse and human YAP/TAZ activation in vitro (IC 50 against LATS1 = 1.06 ± 0.08 nM). To analyze the effect of pharmacological YAP/TAZ activation on BM regeneration in vivo, we treated mice with intraperitoneal injection of GA-003 (50 mg/kg per day, for 8 days) following sublethal IR. Remarkably, we observed an accelerated recovery of hematopoiesis, with the absolute numbers of BM cellularity, GMP (Lin -cKit +Sca1 -FcγR +CD34 +) and HSC EPCR (Lin -cKit +Sca1 +CD150 +EPCR +) on day 14 increased by 3.50-fold (p=0.0002), 6.49-fold (p=0.0022) and 11.41-fold (p=0.022), respectively in the GA-003-treated group compared to vehicle-treated group. In addition, GA-003 also promoted hematopoietic recovery after 5-FU injection (150 mg/kg) and HSCT. Nonetheless, consistent with the scarce expression of YAP/TAZ in hematopoietic stem and progenitor cells (HSPC), in vitro GA-003 treatment did not enhance HSPC growth, suggesting niche-mediated effects by GA-003. Indeed, in vitro tube formation assay indicated accelerated angiogenesis by GA-003-treated human umbilical vein ECs, and CFU-F assays revealed significant enhancement of colony formation by mouse BM-derived MSCs upon GA-003 treatment. To reveal the effect of GA-003 on the HSC niche components in vivo, we performed whole BM immunofluorescent imaging at various time points following sublethal IR and GA-003 treatment. We observed alleviated vascular dilation and leakiness and earlier restoration of vascular damage in GA-003-treated group compared to vehicle-treated group, which was associated with increased VE-Cadherin expression in ECs. These results suggest that reinforcing YAP/TAZ activity upon myelosuppression promotes HSC niche integrity and recovery and accelerates hematopoietic regeneration. Taken together, our results establish YAP/TAZ as novel regulators of HSC niche and highlight YAP/TAZ as promising therapeutic targets to boost hematopoietic recovery after myeloablative interventions such as chemotherapy and HSCT. Disclosures Aihara: Nissan Chemical Corporation: Current Employment. Iwawaki: Nissan Chemical Corporation: Current Employment. Nishino: Nissan Chemical Corporation: Current Employment. Iwama: Nissan Chemical Corporation: Research Funding.

Direct Intravital Imaging of the Bone Marrow and Splenic Hematopoietic Niches in Individual Mice to Define the Early Engraftment Kinetics Following HSC-Transplant

Hematopoietic stem cells (HSCs) give rise to and maintain the entire hematopoietic system for the life of an organism. This remarkable feat has established HSC transplant as an indispensable tool for treating a variety of hematological disorders. Yet the initial steps of homing, engraftment, and clonal expansion, which lead to eventual long-term hematopoietic recovery after HSC transplant, remain incompletely characterized. Given the determinative effect that early HSC activity has on transplant outcomes, a more complete understanding of initial engraftment dynamics is imperative for bettering HSC therapies. Preliminary studies aimed at functional characterization of classic HSC and hematopoietic stem and progenitor cell (HSPC) populations-namely the CD150 +CD48 -Sca-1 +c-Kit +Lin - (SLAM SKL) and Sca-1 +c-Kit +Lin - (SKL) populations, respectively-revealed that these populations exhibit disparate early engraftment dynamics. Using previously developed intravital imaging techniques, we were able to partially characterize early HSC and HSPC engraftment dynamics in mice competitively transplanted with GFP + SLAM SKL and DsRed + SKL cells. The SKL population was found to primarily engraft in the bone marrow, completely recapitulating engraftment behavior of transplanted whole bone marrow. In contrast, the more purified SLAM SKL population engrafted poorly in the marrow space and instead preferentially engrafted in the spleen, where it produced the majority of donor-derived blood at early stages (7 days) after competitive transplant into lethally irradiated mice. However, by 14 days post-transplant, SLAM SKL-derived cells migrated from the spleen to repopulate the majority of bone marrow space. These results reflect the dynamic nature of hematopoietic recovery in a myeloablative model and highlight the need for in vivo imaging techniques to fully understand hematopoietic reconstitution by the SLAM SKL population. In order to further dissect the interactive processes of bone marrow hematopoiesis and splenic extramedullary hematopoiesis, we have developed a novel, multi-organ intravital imaging technique that allows for simultaneous analysis of defined hematopoietic compartments in a single animal. Our multimodal imaging approach combines direct visualization of fluorescently labeled hematopoietic cells in the spleen via our recently developed spleen window, with concomitant observation of hematopoietic cells in tibia marrow environment. Our spleen window is a specially engineered biocompatible ring with an affixed coverslip to allow for direct, non-invasive microscopic visualization of labeled hematopoietic cells in the spleen. The spleen window can be installed with the tibia window in an individual mouse. Multimodal mice can be visualized repeatedly over a minimum of 7 days post-HSC transplant to follow individual cell behaviors within the living recipient. Preliminary results from competitive repopulation assays utilizing our multimodal imaging approach suggest that the SLAM SKL population is an active one that confers rapid hematopoietic recovery in lethally irradiated recipients primarily from extramedullary hematopoiesis in the spleen (CFU-S). The results of ongoing work characterizing the active use of the splenic and marrow niches will be presented. Disclosures Cogle: Celgene: Membership on an entity's Board of Directors or advisory committees; Aptevo therapeutics: Research Funding.

Hematopoietic recovery after transplantation is primarily derived from the stochastic contribution of hematopoietic stem cells

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

Microbiota-derived lactate promotes hematopoiesis and erythropoiesis by inducing stem cell factor production from leptin receptor+ niche cells

Although functional interplay between intestinal microbiota and distant sites beyond the gut has been identified, the influence of microbiota-derived metabolites on hematopoietic stem cells (HSCs) remains unclear. This study investigated the role of microbiota-derived lactate in hematopoiesis using mice deficient in G-protein-coupled receptor (Gpr) 81 (Gpr81−/−), an established lactate receptor. We detected significant depletion of total HSCs in the bone marrow (BM) of Gpr81−/− mice compared with heterogenic (Gpr81+/−) mice in a steady state. Notably, the expression levels of stem cell factor (SCF), which is required for the proliferation of HSCs, decreased significantly in leptin receptor-expressing (LepR+) mesenchymal stromal cells (MSCs) around the sinusoidal vessels of the BM from Gpr81−/− mice compared with Gpr81+/− mice. Hematopoietic recovery and activation of BM niche cells after irradiation or busulfan treatment also required Gpr81 signals. Oral administration of lactic acid-producing bacteria (LAB) activated SCF secretion from LepR+ BM MSCs and subsequently accelerated hematopoiesis and erythropoiesis. Most importantly, LAB feeding accelerated the self-renewal of HSCs in germ-free mice. These results suggest that microbiota-derived lactate stimulates SCF secretion by LepR+ BM MSCs and subsequently activates hematopoiesis and erythropoiesis in a Gpr81-dependent manner.

Thrombopoietin from hepatocytes promotes hematopoietic stem cell regeneration after myeloablation

The bone marrow niche plays a critical role in hematopoietic recovery and hematopoietic stem cell (HSC) regeneration after myeloablative stress. However, it is not clear whether systemic factors beyond the local niche are required for these essential processes in vivo. Thrombopoietin (THPO) is a critical cytokine promoting hematopoietic rebound after myeloablation and its transcripts are expressed by multiple cellular sources. The upregulation of bone marrow-derived THPO has been proposed to be crucial for hematopoietic recovery and HSC regeneration after stress. Nonetheless, the cellular source of THPO in myeloablative stress has never been investigated genetically. We assessed the functional sources of THPO following two common myeloablative perturbations: 5-fluorouracil (5-FU) administration and irradiation. Using a Thpo translational reporter, we found that the liver but not the bone marrow is the major source of THPO protein after myeloablation. Mice with conditional Thpo deletion from osteoblasts and/or bone marrow stromal cells showed normal recovery of HSCs and hematopoiesis after myeloablation. In contrast, mice with conditional Thpo deletion from hepatocytes showed significant defects in HSC regeneration and hematopoietic rebound after myeloablation. Thus, systemic THPO from the liver is necessary for HSC regeneration and hematopoietic recovery in myeloablative stress conditions.

Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome

Background Acute radiation syndrome (ARS) is caused by acute exposure to ionizing radiation that damages multiple organ systems but especially the bone marrow (BM). We have previously shown that human macrophages educated with exosomes from human BM-derived mesenchymal stromal cells (MSCs) primed with lipopolysaccharide (LPS) prolonged survival in a xenogeneic lethal ARS model. The purpose of this study was to determine if exosomes from LPS-primed MSCs could directly educate human monocytes (LPS-EEMos) for the treatment of ARS. Methods Human monocytes were educated by exosomes from LPS-primed MSCs and compared to monocytes educated by unprimed MSCs (EEMos) and uneducated monocytes to assess survival and clinical improvement in a xenogeneic mouse model of ARS. Changes in surface molecule expression of exosomes and monocytes after education were determined by flow cytometry, while gene expression was determined by qPCR. Irradiated human CD34+ hematopoietic stem cells (HSCs) were co-cultured with LPS-EEMos, EEMos, or uneducated monocytes to assess effects on HSC survival and proliferation. Results LPS priming of MSCs led to the production of exosomes with increased expression of CD9, CD29, CD44, CD146, and MCSP. LPS-EEMos showed increases in gene expression of IL-6, IL-10, IL-15, IDO, and FGF-2 as compared to EEMos generated from unprimed MSCs. Generation of LPS-EEMos induced a lower percentage of CD14+ monocyte subsets that were CD16+, CD73+, CD86+, or CD206+ but a higher percentage of PD-L1+ cells. LPS-EEMos infused 4 h after lethal irradiation significantly prolonged survival, reducing clinical scores and weight loss as compared to controls. Complete blood counts from LPS-EEMo-treated mice showed enhanced hematopoietic recovery post-nadir. IL-6 receptor blockade completely abrogated the radioprotective survival benefit of LPS-EEMos in vivo in female NSG mice, but only loss of hematopoietic recovery was noted in male NSG mice. PD-1 blockade had no effect on survival. Furthermore, LPS-EEMos also showed benefits in vivo when administered 24 h, but not 48 h, after lethal irradiation. Co-culture of unprimed EEMos or LPS-EEMos with irradiated human CD34+ HSCs led to increased CD34+ proliferation and survival, suggesting hematopoietic recovery may be seen clinically. Conclusion LPS-EEMos are a potential counter-measure for hematopoietic ARS, with a reduced biomanufacturing time that facilitates hematopoiesis.