Hematopoietic Stem And Progenitor Cells Function Research Articles

Nocturnal Melatonin Renews Bone and Blood Forming Stem Cells Reservoir By Metabolic Reprograming

Bone marrow (BM) residing hematopoietic stem and progenitor cells (HSPC) replenish the blood with mature cells with a finite life span on a daily basis while maintaining the reservoir of undifferentiated stem cells. We recently showed that light/darkness onset induce two different BM HSPC peaks. Morning-induced norepinephrine and TNF secretion metabolically facilitate HSPC differentiation and egress to replenish the circulation with new mature leukocytes. Night augmented BM melatonin renews BM CD150+ hematopoietic stem cell (HSC) reservoir and their long-term repopulation potential (Golan et al, Cell Stem Cell, In Press). How melatonin primes BM HSPC to change their phenotype and function to re-acquire an undifferentiated and primitive state, is poorly understood. The hormone melatonin is an important mediator of bone formation and mineralization, and ultimately regulates the balance of bone remodeling (Cardinali DP et al, J. Pineal Res., 2003). The cross talk between HSPC and their BM stromal microenvironment is tightly regulated and determines HSPC fate. Therefore, we examined whether melatonin plays a role in regulation of murine BM mesenchymal stem and progenitor cells (MSPC, CD45-/Sca-1+/PDGFRα+), known to support HSPC maintenance in their BM niches. Mice treated with melatonin for 5h during the morning had increased levels of BM MSPC endowed with higher colony-forming unit fibroblast (CFU-F) potential in vitro. Interestingly, the metabolic state of these progenitor cells was altered by melatonin demonstrating reduced glucose uptake ability and lower mitochondria content. To test if differences in stromal cells content exist between day and night, we examined BM MSPC and found increased levels at 11PM, the time of melatonin BM peak, with higher Sca-1high surface expression levels, as compared to daylight 11AM. These changes were associated with augmented CFU-F levels by MSPC harvested at 11PM and accompanied by reduced glucose uptake levels and mitochondria content. Our preliminary results suggest that melatonin at night increases BM MSPC levels and reduces their metabolic activity to maintain them in a primitive and undifferentiated state. Moreover, we found that melatonin-elevated HSPC at 11PM also share lower glucose uptake ability with reduced mitochondria content and lower mitochondrial membrane potential (evaluated by TMRE). We hypothesize that melatonin reprograms the metabolic state of both HSPC and their stromal MSPC microenvironment to renew and maintain a primitive state of both populations at night. One of the factors inhibited by melatonin is the bioactive lipid Sphingosine 1-Phosphate (S1P), which in turn inhibits melatonin production. We found that mice with low S1P levels (S1Plow) due to lack of the SPHK1 enzyme have high BM melatonin levels also during the day in contrast to wild type (WT) mice. S1Plow mice had higher levels of primitive stromal progenitor cells including CFU-F and lower levels of differentiating osteoblast precursors compared to WT mice. In addition, these mice had less BM Reactive Oxygen Species (ROS)high committed hematopoietic progenitor cells, but more primitive ROSlow EPCR+ HSC endowed with higher long-term repopulation capacity in both primary and serially transplanted recipients. Next, we examined how light/dark cues affect the homing of transplanted BM HSPC into the BM of irradiated hosts 18h after transplantation. We found that donor HSPC harvested at 11PM have elevated homing ability compared to 11AM harvested cells. Importantly, MSPC also better homed to the BM of irradiated recipients when we transplanted donor BM cells obtained at 11PM compared with 11AM. As a result, accelerated BM repopulation kinetics was documented one week post transplantation in mice transplanted with BM cells harvested at 11 PM.Taken together, our results reveal that in vivo melatonin renews and maintains the BM reservoir and function of primitive MSPC and HSPC by metabolically reprogramming these cells during the night on a daily basis. Since the primed HSPC and MSPC at night showed improved function and BM homing potential, these features might be mimicked by human BM cells in order to harness them for improved clinical transplantation protocols. DisclosuresNo relevant conflicts of interest to declare.

First-in-Human Mitochondrial Augmentation of Hematopoietic Stem Cells in Pearson Syndrome

▪Background:Pearson Syndrome (PS) is an ultra-rare disease caused by de-novo mitochondrial DNA (mtDNA) deletions. Patients present at infancy with sideroblastic anemia and later develop a multisystem metabolic disorder, leading to death in early or late childhood. No disease-modifying treatments are available for PS. Ex-vivo enrichment of functional mitochondria into various cells has been previously demonstrated, as has inter-cellular mitochondrial transfer. In preclinical models of mitochondrial and lysosomal disorders, hematopoietic stem and progenitor cells (HSCs) have been shown capable of carrying and transferring normal organelles into diseased tissues, thereby altering disease phenotype. Here, we show enrichment of PS-derived HSCs with wild-type mitochondria, a process termed mitochondrial augmentation. We further report on three patients with PS treated with autologous HSCs following ex-vivo mitochondrial augmentation.Methods:Diagnosis of PS was confirmed by MLPA and deletion-specific dPCR. Colony formation assays were performed on PS patient-derived HSCs, prior to and after mitochondrial augmentation. HSC mobilization was performed with GCSF alone (n=1) or with plerixafor (n=2) prior to leukapheresis. Autologous CD34+ cells were positively-selected using a CliniMACS system, followed by ex-vivo mitochondrial augmentation of the cells with maternal cryopreserved mitochondria carrying normal mtDNA as confirmed by MLPA. Enriched cells were intravenously infused without conditioning. Level of heteroplasmy (relative normal to deleted mtDNA) was determined by deletion-specific dPCR of DNA from peripheral blood. Patients were followed for a period of up to 1 year including clinical and metabolic evaluations. Adverse events were reported as per CTCAE v4.03. Cellular mitochondrial function was studied on peripheral blood mononuclear cells (PBMCs) by ATP content, O2 consumption and flow cytometry for TMRE (tetramethylrhodamine ethyl ester) and MTG (mitotracker green).Results:Three patients were treated with production and safety data available, and in two patients efficacy data is available. PS-patient derived HSCs have a diminished capacity to form colonies in vitro (median, 360 colonies per 5x104 cells vs. 1090 in healthy donors). HSC colony forming capacity increased by an average of 30% after mitochondrial augmentation. Target cell dose (4x106 CD34+ cells/kg) was not reached despite two leukapheresis procedures in patients 1 and 2, who received 1.1 and 1.8 million CD34+ cells/kg recipient, respectively. Patient 3 received 2.8 million cells/kg following a single apheresis. Mitochondrial enrichment in the products was 156%, 162% and 114% for patients 1, 2 and 3. To date, the only treatment-related adverse events noted were leukapheresis related, including anemia, hypocalcemia and alkalosis.In two patients with more than 3 months follow-up, we observed in vivo mitochondrial enrichment starting 3-4 months after cellular therapy, and throughout the follow-up period (Figure). Metabolic function of PBMCs showed improvement at 5 months post-treatment in lymphocyte ATP content, O2 consumption and TMRE:MTG ratio, indicating improved mitochondrial respiratory capacity. Improvement in mitochondrial heteroplasmy and function was in line with clinical findings. Following cell therapy, no events of metabolic crisis occurred, along with normalization of a pre-treatment negative base excess in patient 1 and ongoing improvement in baseline lactate levels in patient 2. Aerobic ability and fine motor functions were superior compared to baseline in both patients. Importantly, quality of life, as measured by the International Pediatric Mitochondrial Disease Score (IPMDS), was greatly improved after treatment.Conclusion:We report a first in human study with a novel form of cellular therapy, mitochondrial augmentation, in which we enrich HSCs with organelles encoding non-mutated version of the mtDNA sequence. We show the ability of mitochondrial augmentation to improve in vitro PS-derived HSC function, and improvement in metabolic determinants, aerobic capacity and quality of life of two patients treated. Together, these preliminary clinical data suggest that mitochondrial augmentation therapy is safe, and may alter the clinical course for patients with mitochondrial deletions/mutations including PS. [Display omitted] DisclosuresJacoby:Novartis Israel: Consultancy. Blumkin:Minovia Therapeutics: Employment. Sher:Minovia Therapeutics: Employment. Yivgi Ohana:Minovia Therapeutics: Employment. Toren:Novartis Israel: Consultancy.

Interleukin-10 Signaling in Hematopoietic Stem and Progenitor Cells Maintains Stem Cell Function and Regulates Inflammation-Induced Myeloid Cell Output

Inflammation shapes the immune response to foreign antigens by influencing effector immune function and is dysregulated in many forms of inflammatory bowel disease and other auto-immune conditions. It is increasingly recognized that inflammation also impacts hematopoietic stem and progenitor cell (HSPC) function. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that regulates inappropriate immune responses by suppressing inflammatory programs in mature immune cells. However, the direct role of IL-10 in modulating HSPC function and in suppressing hematopoietic cell output in the context of inflammation has not previously been demonstrated.IL10Rß-deficient (Il10rb-/-) mice have an increased inflammatory cytokine profile and develop overt colitis symptoms between 4-6 months of age. We observed an expansion in the relative frequency of immunophenotypically-defined HSPCs (LSK+: Lineage-c-Kit+Sca-1+) and frequency and absolute numbers of granulocyte-monocyte progenitors (GMPs: Lineage-c-Kit+CD34+FcgRII/III+) in pre-colitic Il10rb-/- mice relative to wildtype mice (WT) (see table). Increased GMPs correlated with increased myeloid and reduced lymphoid frequency and number in the bone marrow (BM). Consistent with the expansion in the Il10rb-/-BM HSPC compartment, transplantation of a 1:1 ratio of Il10rb-/-: WT whole BM also resulted in a 2-fold higher short-term reconstitution of Il10rb-/- CD45+ cells in peripheral blood relative to WT cells. Moreover, in vitro we observed suppressed expansion of WT but not Il10rb-/- lineage- cells after IL-10 treatment, suggesting that IL10 suppresses HSPC proliferation. To determine if IL-10 signaling affects the function of hematopoietic stem cells (HSCs), we compared an equal number of purified HSCs (LSK+CD150+CD48-) from Il10rb-/- and WT mice in repopulation assays. Il10rb-/- HSCs demonstrated a 6-fold (P<0.01) reduced lineage reconstitution in peripheral blood relative to WT HSCs in competitive transplantation, thus demonstrating that, while Il10rb-/- mice have an increased pool of committed progenitor cells, HSC function is reduced relative to WT mice. To confirm direct IL-10 signaling in HSPC populations, purified LSK+ and myeloid progenitors (LK+Sca-1-) were exposed to IL-10. Significant STAT3 phosphorylation was observed in HSPCs and myeloid progenitors, with the highest proportion of phosphorylated cells in the GMP fraction. These data suggest that IL-10 directly acts on multiple stem and progenitor populations and that myeloid progenitors robustly respond to IL-10. To determine if the alterations in Il10rb-/- BM are cell intrinsic or a product of underlying inflammation in the absence of IL-10 signaling in vivo, we generated mixed whole BM WT: Il10rb-/- chimeric mice and induced inflammation through E. coli lipopolysaccharide (LPS) injection. LPS exposure led to a 1.5-fold larger depletion of Il10rb-/- GMPs compared to WT GMPs in mixed chimeric mice 24 hours post-injection relative to vehicle treatment. These data suggest a protective role for IL-10 on progenitors during an acute inflammatory insult. Upon transplantation of BM from primary LPS-treated mixed chimeric mice, a short-term increase in peripheral blood myeloid cells was observed when compared to transplanted BM from vehicle-treated mice. Notably, the enhanced peripheral blood myeloid output from LPS-treated donor BM was skewed 2-fold in favor of Il10rb-/- cells and BM myeloid colony-forming-cells were skewed 4-fold in favor of Il10rb-/- cells relative to WT cells. These data demonstrate that IL-10 attenuates inflammation-induced myeloid output upon BM transplantation.Taken together, this study reveals a previously unrecognized role for IL-10 in suppressing progenitor cell expansion and promoting HSC function at steady state while mitigating myeloid lineage skewing after an inflammatory insult. [Display omitted] DisclosuresWilliams:Bluebird Bio: Research Funding.

Ex Vivo Gene-Edited Cell Therapy for Sickle Cell Disease: Disruption of the BCL11A Erythroid Enhancer with Zinc Finger Nucleases Increases Fetal Hemoglobin in Plerixafor Mobilized Human CD34+ Cells

Increases in fetal hemoglobin (HbF) are associated with improved clinical outcomes in the inherited hemoglobinopathies. We are developing a novel gene-edited cell therapy to treat patients with sickle cell disease (SCD) and beta-thalassemia (BT) using autologous hematopoietic stem and progenitor cells (HSPCs) genetically modified with zinc finger nucleases (ZFNs) to restore high levels of HbF expression. The ZFNs have been designed to specifically target the GATA motif within an intronic erythroid-specific enhancer (ESE) of BCL11A, the gene encoding a transcriptional regulator of the fetal-to-adult hemoglobin switch. Previously, we reported successful ZFN-mediated, ex vivoBCL11A gene editing in dual mobilized HSPCs, i.e. peripheral stem cells mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and plerixafor1. The editing procedure was optimized for high on-target/low off-target modification levels and increases in HbF in erythroid progeny. This drug product, ST-400, passed extensive safety testing and is currently in a phase 1/2a clinical trial for transfusion-dependent beta-thalassemia (ClinicalTrials.gov number NCT03432364).An analogous drug product, BIVV003, is being developed as a therapeutic for SCD. In patients with SCD, the use of G-CSF for stem cell mobilization is not recommended due to the risk of clinical complications. Therefore, peripheral HSPCs are obtained from SCD patients via single agent, plerixafor mobilization and apheresis2-5. Understanding the effect of this change in mobilization strategy on ZFN editing efficiency and specificity is a key element in preparing for SCD gene-edited cell therapy. In the present studies, we demonstrate comparability of ZFN editing outcomes in single and dual mobilized HSPCs obtained from healthy donors. In plerixafor mobilized HSPCs from five healthy donors at research scale, ZFN-mediated gene editing induced an efficient modification at the BCL11A ESE target site (>75% of alleles modified, as measured by MiSeq deep sequencing) with high post-editing viability (77%). Similar gene editing efficiencies (>70%) were obtained in HSPCs at clinical manufacturing scale (n=2). Further, in vitro HbF protein levels and HbF+ cell frequencies within erythroid progeny of edited cells were increased by >4 and 3-fold respectively - compared to non-edited cells in the same culture conditions, using reverse phase high-performance liquid chromatography and flow cytometry (n=4 healthy donors at research scale). Single cell clone analysis revealed that ZFN-mediated gene editing targeted both alleles of BCL11A at high frequency (91-94% of edited cells within erythroid progeny) with high levels of replicable GATA-disrupting indel patterns. On average, each edited allele contributed an additional 17.6% increase in HbF production in vitro, with a statistically-significant increase in HbF level for biallelic edited vs. unedited controls (3.4 fold).Critical to BIVV003 use in clinical trials, ZFN-mediated gene editing did not impair single agent mobilized HSPC function in vitro based on measurements of colony forming unit (CFU) production and frequencies of long-term HSC (LT-HSC) and common myeloid progenitor (CMP) cells by flow cytometry. In agreement with this data, injection of BIVV003 into immune-deficient NBSGW mice resulted in robust long-term engraftment (21 weeks) without any impact on the number of HSPCs and their differentiated progeny. Overall, these data demonstrate potential efficacy of ZFN-edited HSPCs (BIVV003) as a novel cell therapy for SCD patients.Holmes et al., 2017 (ASH abstract)Fitzhugh et al., 2009Lagresle-Peyrou et al., 2018Hsieh and Tisdale, 2018Yannaki et al., 2012 DisclosuresMoran:Bioverativ, a Sanofi Company: Employment. Ling:Bioverativ, a Sanofi Company: Employment. Lessard:Bioverativ, a Sanofi Company: Employment. Vieira:Bioverativ a Sanofi Company: Employment. Hong:Bioverativ, a Sanofi Company: Employment. Holmes:Sangamo Therapeutics: Employment. Reik:Sangamo Therapeutics: Employment. Dang:Sangamo Therapeutics: Employment. Gray:Sangamo Therapeutics: Employment. Levasseur:Bioverativ, a Sanofi Company: Employment. Rimmele:Bioverativ, a Sanofi Company: Employment.

Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors.

A finely tuned balance of self‐renewal, differentiation, proliferation, and survival governs the pool size and regenerative capacity of blood‐forming hematopoietic stem and progenitor cells (HSPCs). Here, we report that protein kinase C delta (PKCδ) is a critical regulator of adult HSPC number and function that couples the proliferative and metabolic activities of HSPCs. PKCδ‐deficient mice showed a pronounced increase in HSPC numbers, increased competence in reconstituting lethally irradiated recipients, enhanced long‐term competitive advantage in serial transplantation studies, and an augmented HSPC recovery during stress. PKCδ‐deficient HSPCs also showed accelerated proliferation and reduced apoptosis, but did not exhaust in serial transplant assays or induce leukemia. Using inducible knockout and transplantation models, we further found that PKCδ acts in a hematopoietic cell‐intrinsic manner to restrict HSPC number and bone marrow regenerative function. Mechanistically, PKCδ regulates HSPC energy metabolism and coordinately governs multiple regulators within signaling pathways implicated in HSPC homeostasis. Together, these data identify PKCδ as a critical regulator of HSPC signaling and metabolism that acts to limit HSPC expansion in response to physiological and regenerative demands.

KDM6B overexpression activates innate immune signaling and impairs hematopoiesis in mice

AbstractKDM6B is an epigenetic regulator that mediates transcriptional activation during differentiation, including in bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). Overexpression of KDM6B has been reported in BM HSPCs of patients with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Whether the overexpression of KDM6B contributes to the pathogenesis of these diseases remains to be elucidated. To study this, we generated a Vav-KDM6B mouse model, which overexpresses KDM6B in the hematopoietic compartment. KDM6B overexpression alone led to mild hematopoietic phenotype, and chronic innate immune stimulation of Vav-KDM6B mice with the Toll-like receptor (TLR) ligand lipopolysaccharide (LPS) resulted in significant hematopoietic defects. These defects recapitulated features of MDS and CMML, including leukopenia, dysplasia, and compromised repopulating function of BM HSPCs. Transcriptome studies indicated that KDM6B overexpression alone could lead to activation of disease-relevant genes such as S100a9 in BM HSPCs, and when combined with innate immune stimulation, KDM6B overexpression resulted in more profound overexpression of innate immune and disease-relevant genes, indicating that KDM6B was involved in the activation of innate immune signaling in BM HSPCs. Finally, pharmacologic inhibition of KDM6B with the small molecule inhibitor GSK-J4 ameliorated the ineffective hematopoiesis observed in Vav-KDM6B mice. This effect was also observed when GSK-J4 was applied to the primary BM HSPCs of patients with MDS by improving their repopulating function. These results indicate that overexpression of KDM6B mediates activation of innate immune signals and has a role in MDS and CMML pathogenesis, and that KDM6B targeting has therapeutic potential in these myeloid disorders.

Cross-talk between T Cells and Hematopoietic Stem Cells during Adoptive Cellular Therapy for Malignant Glioma.

Purpose: Adoptive T-cell immunotherapy (ACT) has emerged as a viable therapeutic for peripheral and central nervous system (CNS) tumors. In peripheral cancers, optimal efficacy of ACT is reliant on dendritic cells (DCs) in the tumor microenvironment. However, the CNS is largely devoid of resident migratory DCs to function as antigen-presenting cells during immunotherapy. Herein, we demonstrate that cellular interactions between adoptively transferred tumor-reactive T cells and bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) lead to the generation of potent intratumoral DCs within the CNS compartment.Experimental Design: We evaluated HSPC differentiation during ACT in vivo in glioma-bearing hosts and HSPC proliferation and differentiation in vitro using a T-cell coculture system. We utilized FACS, ELISAs, and gene expression profiling to study the phenotype and function of HSPC-derived cells ex vivo and in vivo To demonstrate the impact of HSPC differentiation and function on antitumor efficacy, we performed survival experiments.Results: Transfer of HSPCs with concomitant ACT led to the production of activated CD86+CD11c+MHCII+ cells consistent with DC phenotype and function within the brain tumor microenvironment. These intratumoral DCs largely supplanted abundant host myeloid-derived suppressor cells. We determined that during ACT, HSPC-derived cells in gliomas rely on T-cell-released IFNγ to differentiate into DCs, activate T cells, and reject intracranial tumors.Conclusions: Our data support the use of HSPCs as a novel cellular therapy. Although DC vaccines induce robust immune responses in the periphery, our data demonstrate that HSPC transfer uniquely generates intratumoral DCs that potentiate T-cell responses and promote glioma rejection in situClin Cancer Res; 24(16); 3955-66. ©2018 AACR.

Oncogenic N-Ras and Tet2 haploinsufficiency collaborate to dysregulate hematopoietic stem and progenitor cells

AbstractConcurrent genetic lesions exist in a majority of patients with hematologic malignancies. Among these, somatic mutations that activate RAS oncogenes and inactivate the epigenetic modifier ten-eleven translocation 2 (TET2) frequently co-occur in human chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias, suggesting a cooperativity in malignant transformation. To test this, we applied a conditional murine model that endogenously expressed oncogenic NrasG12D and monoallelic loss of Tet2 and explored the collaborative role specifically within hematopoietic stem and progenitor cells (HSPCs) at disease initiation. We demonstrate that the 2 mutations collaborated to accelerate a transplantable CMML-like disease in vivo, with an overall shortened survival and increased disease penetrance compared with single mutants. At preleukemic stage, N-RasG12D and Tet2 haploinsufficiency together induced balanced hematopoietic stem cell (HSC) proliferation and enhanced competitiveness. NrasG12D/+/Tet2+/− HSCs displayed increased self-renewal in primary and secondary transplantations, with significantly higher reconstitution than single mutants. Strikingly, the 2 mutations together conferred long-term reconstitution and self-renewal potential to multipotent progenitors, a pool of cells that usually have limited self-renewal compared with HSCs. Moreover, HSPCs from NrasG12D/+/Tet2+/− mice displayed increased cytokine sensitivity in response to thrombopoietin. Therefore, our studies establish a novel tractable CMML model and provide insights into how dysregulated signaling pathways and epigenetic modifiers collaborate to modulate HSPC function and promote leukemogenesis.

Leukemia cells impair normal hematopoiesis and induce functionally loss of hematopoietic stem cells through immune cells and inflammation

Bone marrow (BM) failure is often seen in leukemia patients, indicating an abnormal hematopoietic process. However, hematopoiesis in leukemic milieus is largely unknown. In the present study, we utilized one of the most frequent leukemogenic translocations MLL-AF9 to induce leukemia and investigated the hematopoiesis and the activity of hematopoietic stem and progenitor cells (HSPCs) in a leukemic milieu. We found that the phenotypes of the non-leukemic population in leukemic BM were drastically different than normal BM, including blockage of differentiation and a drastically reduced Lin-/Sca+/c-kit+ (LSK) population that contains all HSPCs in leukemic BM. Further, transplantation assays demonstrated that stem cell function of HSPCs from leukemic BM was significantly compromised. Intriguingly, BM from a patient-derived xenograft leukemia model and from immunocompromised mice transplanted with murine MLL-AF9 cells, showed comparable percentage of hematopoietic stem cells (HSCs) to normal controls, indicating that an immunocompetent microenvironment is critical for leukemia-induced loss of HSPCs. Mechanistically, we found that the non-leukemic cells from leukemic BM possessed a more inflammatory profile than either leukemic cells or normal BM counterparts. Co-culturing or co-transplantation with non-leukemic cells from leukemic BM impaired the stem cell function of normal HSPCs in vitro and in vivo respectively, suggesting that the highly inflammatory non-leukemic population in leukemic BM not only is functionally abnormal but displayed a ‘leukemia-like’ characteristic. Finally, we tested the effect of the anti-inflammation drug diclofenac on leukemia mice. However, no phenotypic changes of HSPCs were observed upon diclofenac treatment due to only mild repression of inflammatory genes by diclofenac, further indicating that inflammation is a powerful negative regulator of HSPCs. Together, our results suggest that leukemia impairs normal hematopoiesis and inflammation as well as immune cells play a critical role in leukemia-induced BM failure.

Chronic viral infections induce major disruption of bone marrow stromal cell networks and persistent loss of hematopoietic stem cell function

Hematopoiesis is a highly demand-adapted and tightly regulated process that is sustained by a rare population of self-renewing, multipotent hematopoietic stem and progenitor cells (HSPCs) residing in specialized microenvironments within bone marrow (BM) cavities. The basic tissue infrastructure of the BM is provided by stromal cellular networks of mesenchymal, neural and vascular origin, which are critically involved in the fine regulation of different stages of hematopoiesis. Viral infections act as major stressors to the hematopoietic system, inducing massive and adaptive responses in cellular output. Albeit the effects of viral challenge and ensuing inflammatory responses on hematopoietic cells have been studied, whether viral infections alter BM stromal scaffolds and thus shape hematopoietic responses remains poorly defined. By combining conventional in vitro and in vivo assays with 3D confocal imaging, we herein investigated the structural and functional alterations imposed on the BM after a chronic infection with Lymphocytic Choriomeningitis Virus (LCMV). Our data shows that chronic LCMV infections trigger a substantial loss of BM endothelial and mesenchymal CXCL12-abundant reticular cells. Upon viral challenge, conspicuous vasodilation of BM sinusoidal vessels is induced which was followed by intense vascular remodeling and substantial disruption of extracellular matrix networks throughout the BM cavity. Major damage to BM stromal integrity is accompanied by a profound and sustained reduction in the number of both HSPCs as well as hematopoietic stem cells by phenotype. Competitive repopulation assays revealed that the remaining HSCs are additionally impaired in their repopulation capacity for prolonged times after LCMV infection. Finally, our results indicate that the observed alterations in the composition and functionality of cells in the BM are mediated by virus-specific CD8 T cells and partly dependent on the production of specific cytokines. Our observations thus indicate that chronic infections result in persistent damage to HSPC function, which might be partially explained by an impaired regulatory function of the stromal compartment of the BM.

Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells.

Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver-derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine.

Bone marrow niche-mimetics modulate HSPC function via integrin signaling

The bone marrow (BM) microenvironment provides critical physical cues for hematopoietic stem and progenitor cell (HSPC) maintenance and fate decision mediated by cell-matrix interactions. However, the mechanisms underlying matrix communication and signal transduction are less well understood. Contrary, stem cell culture is mainly facilitated in suspension cultures. Here, we used bone marrow-mimetic decellularized extracellular matrix (ECM) scaffolds derived from mesenchymal stromal cells (MSCs) to study HSPC-ECM interaction. Seeding freshly isolated HSPCs adherent (AT) and non-adherent (SN) cells were found. We detected enhanced expansion and active migration of AT-cells mediated by ECM incorporated stromal derived factor one. Probing cell mechanics, AT-cells displayed naïve cell deformation compared to SN-cells indicating physical recognition of ECM material properties by focal adhesion. Integrin αIIb (CD41), αV (CD51) and β3 (CD61) were found to be induced. Signaling focal contacts via ITGβ3 were identified to facilitate cell adhesion, migration and mediate ECM-physical cues to modulate HSPC function.

Trilaciclib (G1T28): A cyclin dependent kinase 4/6 inhibitor, in combination with etoposide and carboplatin (EP) for extensive stage small cell lung cancer (ES-SCLC)—Phase 1b results.

8568 Background: Chemotherapy (chemo) has significant clinical utility, however consequent damage to hematopoietic stem and progenitor cells (HSPCs) and the immune system may limit activity. If chemo-mediated anti-tumor activity was maximized, while minimizing myelosuppression and immunosuppression, patient outcomes would be improved. Trilaciclib (T) is an intravenous CDK4/6 inhibitor in development to reduce myelosuppression and preserve immune system function during chemo. HSPCs are dependent on CDK4/6 for proliferation. Preclinical data demonstrated that transient T-induced G1 cell cycle arrest renders HSPCs resistant to chemo cytotoxicity, allowing faster hematopoietic recovery, preservation of long-term function, and enhancement of anti-tumor immunity and activity. Methods: Objectives of this ongoing multicenter Phase 1b/2a study are to assess dose limiting toxicities (DLTs), safety, tolerability, hematological profile, PK, and anti-tumor activity of T administered prior to EP. Phase 1b was open-label, dose-finding, and the ongoing Phase 2a is randomized (1:1), double-blind. Eligible pts had confirmed diagnosis of ES-SCLC, adequate organ function, ECOG PS 0-2, no prior chemo, and no symptomatic brain metastases. Results: 19 pts were enrolled in the Phase 1b: 10 pts received T 200 mg/m2 + EP and 9 pts received T 240 mg/m2 + EP. T + EP was well tolerated. 2 pts at T 200 mg/m2 and 1 pt at T 240 mg/m2 experienced asymptomatic DLTs in cycle 1. 2 pts (1 at each dose) had an ANC &lt; 1500 on cycle 2 day 1, delaying the start of cycle 2, and 1 pt at the T 200 mg/m2dose had grade 4 thrombocytopenia. There were no cases of febrile neutropenia or bleeding. PK analysis showed no drug interactions between T and EP. 17/19 pts were evaluable: 1 pt had CR, 14 had PR (confirmed ORR = 88%); 1 pt had SD (clinical benefit rate = 94%). Conclusions: In the Phase 1b part of the study, T + EP was well tolerated. Early activity results are promising with a confirmed objective response rate of 88%. This novel approach allowing the administration of chemotherapy while preserving HSPC and immune system function could potentially improve treatment outcomes for SCLC pts. Clinical trial information: NCT02499770.

MRNA-mediated glycoengineering ameliorates deficient homing of human stem cell-derived hematopoietic progenitors.

Generation of functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) has been a long-sought-after goal for use in hematopoietic cell production, disease modeling, and eventually transplantation medicine. Homing of HSPCs from bloodstream to bone marrow (BM) is an important aspect of HSPC biology that has remained unaddressed in efforts to derive functional HSPCs from human PSCs. We have therefore examined the BM homing properties of human induced pluripotent stem cell-derived HSPCs (hiPS-HSPCs). We found that they express molecular effectors of BM extravasation, such as the chemokine receptor CXCR4 and the integrin dimer VLA-4, but lack expression of E-selectin ligands that program HSPC trafficking to BM. To overcome this deficiency, we expressed human fucosyltransferase 6 using modified mRNA. Expression of fucosyltransferase 6 resulted in marked increases in levels of cell surface E-selectin ligands. The glycoengineered cells exhibited enhanced tethering and rolling interactions on E-selectin-bearing endothelium under flow conditions in vitro as well as increased BM trafficking and extravasation when transplanted into mice. However, glycoengineered hiPS-HSPCs did not engraft long-term, indicating that additional functional deficiencies exist in these cells. Our results suggest that strategies toward increasing E-selectin ligand expression could be applicable as part of a multifaceted approach to optimize the production of HSPCs from human PSCs.

Long-Term Engraftment and Fetal Globin Induction upon BCL11A Gene Editing in Bone-Marrow-Derived CD34+ Hematopoietic Stem and Progenitor Cells

To develop an effective and sustainable cell therapy for sickle cell disease (SCD), we investigated the feasibility of targeted disruption of the BCL11A gene, either within exon 2 or at the GATAA motif in the intronic erythroid-specific enhancer, using zinc finger nucleases in human bone marrow (BM) CD34+ hematopoietic stem and progenitor cells (HSPCs). Both targeting strategies upregulated fetal globin expression in erythroid cells to levels predicted to inhibit hemoglobin S polymerization. However, complete inactivation of BCL11A resulting from bi-allelic frameshift mutations in BCL11A exon 2 adversely affected erythroid enucleation. In contrast, bi-allelic disruption of the GATAA motif in the erythroid enhancer of BCL11A did not negatively impact enucleation. Furthermore, BCL11A exon 2-edited BM-CD34+ cells demonstrated a significantly reduced engraftment potential in immunodeficient mice. Such an adverse effect on HSPC function was not observed upon BCL11A erythroid-enhancer GATAA motif editing, because enhancer-edited CD34+ cells achieved robust long-term engraftment and gave rise to erythroid cells with elevated levels of fetal globin expression when chimeric BM was cultured ex vivo. Altogether, our results support further clinical development of the BCL11A erythroid-specific enhancer editing in BM-CD34+ HSPCs as an autologous stem cell therapy in SCD patients.

The Role of Runx1 in Embryonic Blood Cell Formation.

The de novo generation of hematopoietic stem and progenitor cells (HSPC) occurs solely during embryogenesis from a population of epithelial cells called hemogenic endothelium (HE). During midgestation HE cells in multiple intra- and extraembryonic vascular beds leave the vessel wall as they transition into HSPCs in a process termed the endothelial to hematopoietic transition (EHT). Runx1 expression in HE cells orchestrates the transcriptional switch necessary for the transdifferentiation of endothelial cells into functional HSPCs. Runx1 is widely considered the master regulator of developmental hematopoiesis because it plays an essential function during specification of the hematopoietic lineage during embryogenesis. Here we review the role of Runx1 in embryonic HSPC formation, with a particular focus on its role in hemogenic endothelium.

Netting Novel Regulators of Hematopoiesis and Hematologic Malignancies in Zebrafish.

Zebrafish are one of the preeminent model systems for the study of blood development (hematopoiesis), hematopoietic stem and progenitor cell (HSPC) biology, and hematopathology. The zebrafish hematopoietic system shares strong similarities in functional populations, genetic regulators, and niche interactions with its mammalian counterparts. These evolutionarily conserved characteristics, together with emerging technologies in live imaging, compound screening, and genetic manipulation, have been employed to successfully identify and interrogate novel regulatory mechanisms and molecular pathways that guide hematopoiesis. Significantly, perturbations in many of the key developmental signals controlling hematopoiesis are associated with hematological disorders and disease, including anemia, bone marrow failure syndromes, and leukemia. Thus, understanding the regulatory pathways controlling HSPC production and function has important clinical implications. In this review, we describe how the blood system forms and is maintained in zebrafish, with particular focus on new insights into vertebrate hematological regulation gained using this model. The interplay of factors controlling development and disease in the hematopoietic system combined with the unique attributes of the zebrafish make this a powerful platform to discover novel targets for the treatment of hematological disease.

EphA5 and EphA7 forward signaling enhances human hematopoietic stem and progenitor cell maintenance, migration, and adhesion via Rac1 activation.

The proliferation, differentiation, adhesion, and migration of hematopoietic stem and progenitor cells (HSPCs) are dependent upon bone marrow stromal cells (BMSCs). In this study, we found that human primitive HSPCs (CD34+CD38-), but not lineage-committed hematopoietic cell populations, express the tyrosine kinase receptors EphA5 and EphA7. Moreover, we found that the ephrinA5 ligand, the high-affinity binding partner of EphA5 and EphA7, is highly expressed by primary human BMSCs. Previous studies have reported that interactions between EphA and ephrinA play important roles in hematopoietic cell trafficking; however, their role in BMSC support of hematopoiesis had not been described previously. Herein, we show that stimulating EphA5 and/or EphA7 forward signaling in HSPCs using soluble ephrinA5-Fc molecules promoted human HSPC-derived colony formation significantly and was associated with increased expression of granulocyte macrophage colony-stimulating factor receptor on HSPCs. Studies using functional blocking peptides to EphA5/7 found that disruption of EphA5/ephrinA5 and/or EphA7/ephrinA5 interactions inhibited HSPC function in BMSC-dependent long-term culture-initiating cell assays. Furthermore, the adhesion and migration of HSPCs was increased significantly in the presence of ephrinA5-Fc molecules compared with human immunoglobulin G-treated controls. Conversely, blocking EphA5 activation led to a reduction of HSPC adhesion, whereas inhibiting EphA5 and/or EphA7 activation hindered HSPC migration. Analysis of HSPC cultured in the presence of ephrinA5-Fc showed that EphA forward signaling stimulated Rac1 gene and protein expression and the Rac1 target molecule WAVE1. Moreover, a significant reduction of ephrinA5-mediated HSPC adhesion and migration was observed in the presence of Rac1 inhibitor. These findings suggest that interactions between EphA and ephrinA5 are important in maintaining the HSPC niche mediated in part by activation of Rac1 signaling.

Serine/Arginine-Rich Splicing Factor 1 (SRSF1) Is Required for Adult and Embryonic Hematopoiesis and Has Non-Overlapping Roles with SRSF2 in Hematopoiesis

The discovery that mRNA splicing factors are frequently mutated in clonal blood disorders represented one of the most unexpected findings from cancer genome sequencing projects. While initial functional studies of these mutations have suggested that alterations in constitutive and alternative splicing may directly contribute to malignant hematopoiesis, they also highlighted limitations in our knowledge of how splicing factors regulate differential splicing and usage of mRNA isoforms in normal hematopoietic development. Interestingly, the splicing factor SRSF2, a member of the serine/arginine-rich (SR) protein family that regulates alternative splicing in a tissue-specific manner, is recurrently mutated only in myeloid leukemias, whereas other splicing factors are also often mutated in lymphoid leukemias and epithelial cancers. These data suggest a specific role for SRSF2 in hematopoiesis and/or myelopoiesis and previous studies have shown that Srsf2 is required for T-lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) function. At the same time, SRSF2s functions in regulating splicing have been suggested to be at least partially overlapping with those of SRSF1, the founding member of the SR protein family. We therefore set out to understand the role and function of SRSF1 in normal fetal and adult hematopoiesis and to directly compare SRSF1s role in normal hematopoiesis to that of SRSF2.To understand the role of Srsf1 in adult hematopoiesis, we utilized mice for conditional deletion of 1 or 2 copies of Srsf1 or Srsf2 and performed non-competitive and competitive bone marrow transplantation (BMT) assays. BM from adult Mx1-Cre/Srsf1+/+, Mx1-Cre/Srsf1fl/+, Mx1-Cre/Srsf1fl/fl and Mx1-Cre/Srsf2fl/fl mice (together with the same number of wildtype CD45.1 marrow in competitive BMT) were transplanted into lethally irradiated CD45.1 recipients, followed by polyinosinic:polycytidylic acid (pIpC) administration 4 weeks after reconstitution to induce Srsf1 deletion. In both competitive and non-competitive BMT, peripheral blood (PB) chimerism showed that while heterozygous deletion of Srsf1 did not affect PB chimerism, homozygous deletion of Srsf1 resulted in compromised multi-lineage reconstitution similar to homozygous Srsf2 deletion (Figure 1A). Analysis of hematopoietic organs 20 weeks post BMT revealed no contribution to hematopoiesis by Srsf1 or Srsf2 homozygous knockout cells. More specifically, the contribution of HSPC-enriched Lineage- Sca-1+ c-Kit+ (LSK) and myeloid progenitor-enriched Lineage- Sca-1- c-Kit+ (LK) fractions were significantly reduced in mice transplanted with Srsf1- and Srsf2-deficient BM (Figure 1B). These observations identify an absolute requirement for both splicing factors in HSPC function in adult mice.To determine if Srsf1 or Srsf2 are required for fetal hematopoiesis, we crossed Vav-CreTg/Srsf1fl/+ to Srsf1fl/fl mice and Vav-CreTg/Srsf2fl/+ to Srsf2fl/fl mice followed by HSPC analysis at embryonic day 13~14. At this time point, both Vav-CreTg/Srsf1fl/fl and Vav-CreTg/Srsf2fl/fl embryos were found at the approximate expected Mendelian ratio (p=0.77 for Srsf1 crosses and p=0.22 for Srsf2 crosses; Fishers exact test). Interestingly, the numbers of HSPCs and erythroid progenitors (determined by flow cytometry using Ter119 and CD71 antibodies) were similar in Vav-CreTg/Srsf1fl/fl fetal livers compared to control embryos, while Vav-CreTg/Srsf2fl/fl fetal livers had drastically reduced fetal liver cellularity, HSPC, and erythroid progenitors relative to control embryos. However, both Vav-CreTg/Srsf1fl/fl and Vav-CreTg/Srsf2fl/fl HSPCs had reduced colony-forming capacities in vitro relative to control, suggesting severe functional defects in homozygous mutants that are otherwise not observed in heterozygous mice.The above data identify that both Srsf1 and Srsf2 are essential for normal hematopoietic function in both embryonic and adult life. Although SRSF1 and SRSF2 have been suggested to regulate splicing by binding to similar sequences of pre-mRNA to promote splicing, here we find that both are absolutely required for hematopoiesis in a haplosufficient, non-overlapping manner. We are now performing transcriptomic analyses to delineate the common as well as distinct molecular targets of how SR proteins regulate HSPC function. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Protection from UV-Light Is an Evolutionarily Conserved Feature of the Hematopoietic Niche

Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow cavity in all mammals, but other locations are used in non-mammalian animals, such as the kidney marrow in zebrafish. The niche location is thought to be selected based on unique advantages for HSPC function and protection in each organism. Studying the zebrafish HSPC niches during development and adulthood, we observed that melanocytes occupy the location above the kidney prior to the arrival of HSPCs. To elucidate whether these particular melanocytes play a role for HSPCs, we compared HSPCs in wildtype and nacre-mutant embryos (that lack all melanocytes) by cmyb in situ at different time-points. There were no obvious differences, suggesting that melanocytes are dispensable for steady state hematopoiesis. Since the position of the melanocytes above the HSPCs was very consistent in different strains and at various ages, we asked whether the melanocytes protect the HSPC niche from UV light. Indeed, after irradiation with UV light, levels of UV-induced DNA damage were approximately 50% higher in HSPCs isolated from kidneys of unpigmented embryos compared to their pigmented siblings as assessed by immunocytochemistry with a cyclobutane pyrimidine dimer antibody (TDM-2). In addition to acquiring more DNA damage, the unpigmented embryos also showed a significantly more pronounced decrease of HSPCs as assessed by cmyb in situ: 55% of control embryos had high cmyb staining. Upon irradiation 44% of irradiated pigmented embryos retained high c-myb staining compared to only 28% of irradiated unpigmented embryos (total embryos analyzed: n= 29, 27 and 36 respectively; p=0.01 between the latter two groups). The reduction of HSPCs could not be rescued in unpigmented p53-mutant embryos, indicating a p53-independent mechanism of HSPC loss after UV irradiation. The protective effect of melanocytes could either be due to a physical shielding of the HSPCs or due to a paracrine mechanism, such as secretion of cytokines or uptake of reactive oxygen species. The paracrine mechanism could be dependent on melanin. To address this question, we focused the path of light at a different angle in which the melanocytes would not be able to block the UV light from reaching the kidney marrow. This abrogated the protective effect of pigment containing melanocytes and caused a reduction of HSPCs by cmyb in situ to the levels of unpigmented siblings: pigmented embryos irradiated at an angle now also had high cmyb staining in only 28% of the embryos (n= 25). This suggests that the melanocytes only provide shade for the HSPCs. We then asked if this protective pigmentation pattern is specific to zebrafish and traced the origin of this trait by comparative histology. We found that other teleost fish but also evolutionarily older fish such as sturgeon and polypterus have melanocytes covering the kidney marrow. Even in lamprey, an ancient jawless vertebrate, the HSPC niche (located in the supraspinal organ) is covered by melanocytes, which allows us to date the evolutionary origin of UV protection back to at least 500 million years. We performed histological studies in frogs and found that the HSPCs reside under a melanocyte shield in the tadpole stage, while it is known that in adult frogs the HSPCs move into the bone marrow cavity. Considering that all terrestrial animals such as reptiles, birds and mammals host hematopoiesis in the bone marrow, we suggest that during the transition from aquatic to terrestrial life, the HSPCs were driven out of their melanocyte covered niche due to the high level of irradiation and relocated into a more protective niche, the bone marrow. DisclosuresZon:Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.