Bone Marrow HSPCs Research Articles

Transient HR enhancement by RAD51-stimulatory compound confers protection on intestinal rather than hematopoietic tissue against irradiation in mice

DNA double-strand breaks (DSBs) are cytotoxic lesions that compromise genomic integrity and trigger cell death. Homologous recombination (HR) is a major pathway for repairing DSBs in cycling cells. However, it remains unclear whether transient modulation of HR could confer protection to adult stem cells against lethal irradiation exposure. In this study, we investigated the radio-protective effect of the RAD51-stimulatory compound RS-1 on adult stem cells and progenitor cells with varying cycling rates in intestinal and hematopoietic tissues. Treatment with RS-1 even at high doses did not induce noticeable cell death or proliferation of intestinal crypt cells in vivo. Pretreatment with RS-1 before irradiation significantly decreased mitotic death, promoted DNA repair and enhanced the survival of intestinal stem cells and progenitor cells and increased the number of regenerative crypt colonies thereby mitigating IR-induced gastrointestinal syndrome. Moreover, RS1 pretreatment could increase the survival and regeneration of irradiated intestinal organoid in vitro, which can be rescued by RAD51 inhibitor. However, pretreatment with RS-1 in vivo did not elevate nucleated cell count or HSPCs in bone marrow after 6Gy irradiation. Additionally, there was no impact on mouse survival due to drug treatment observed. Thus, our data suggest that targeting HR as a strategy to prevent tissue damage from acute irradiation exposure may depend on cell cycling rates and intrinsic DNA repair mechanisms.

NK-2 Homeobox Transcription Factor NKX2-3 Confers Differentiation Block in NPM1-Mutated in Acute Myeloid Leukaemia

Dysregulation of HOX homeobox family transcription factor (TF) gene expression is important in the pathogenesis of AML in particular in NPM1-mutated and MLL rearranged molecular subtypes. However, little is known about the role of NK2-homeobox (NKX2) family TF genes. NKX2 genes have critical roles in normal development of the central nervous system, thyroid, lung and pancreas. We observed high expression of NKX2-3 in patient AML samples and hypothesised the transcription factor might have a functional role in the disease. By qPCR, we found that NKX2-3 is highly expressed in normal HSCs but is rapidly down regulated as cells differentiate. In primary AML samples, it is highly expressed in 21-37% cases, including within the immunophenotypic LSC compartment, in particular in cases with NPM1 and/or FLT3-ITD mutations. Forced expression of NKX2-3 in normal murine KIT + bone marrow (BM) HSPCs transiently enhanced their clonogenic activity and impaired differentiation: NKX2-3-expressing HSPCs expressed higher levels of E2F or MYC target genes, and leukemia stem cell (LSC) maintenance genes. Congenic transplant experiments revealed that forced expression of NKX2-3 in normal BM HSPCs was sufficient to enhance multilineage engraftment across four months of follow up without inducing leukemia. In keeping with this, transplantation of Nkx2-3 -/- knockout BM cells led to significantly inferior engraftment compared with BM cells from litter mate control mice. Thus, while increased expression of Nkx2-3 enhances engraftment of normal stem cells in transplantation assays, loss of Nkx2-3 impairs it. Analysis of NKX2-3 high primary human AML cases demonstrated that HOXA9 was the most highly upregulated transcription factor gene compared with NKX2-3 low cases. We expressed both factors singly and together in murine KIT+ BM HSPCs: Hoxa9/NKX2-3 cells exhibited significantly higher clonogenic activity and reduced morphologic and immunophenotypic differentiation compared with Hoxa9/MTV cells. Congenic BM transplant experiments demonstrated that Hoxa9/NKX2-3 recipients developed leukemias significantly earlier than Hoxa9/MTV recipients (median 82 vs 155 days). BM morphology and immunophenotyping confirmed that Hoxa9/NKX2-3AMLs exhibited a greater degree of differentiation block than Hoxa9/MTV AMLs. Leukemia cells from Hoxa9/NKX2-3 recipients coordinately expressed higher levels of E2F or MYC target genes, and LSC maintenance genes. NKX2-3 KD to ~30% of control values reduced proliferation and induced morphologic and immunophenotypic differentiation in primary patient NPM1-mutated AML cells. NKX2-3 KD led to down regulation of E2F and MYC target genes and normal human CD34 + stem cell genes; and up regulation of myeloid differentiation genes. Using a CRISPR ribonucleofection approach to relocate NPM1c to the nucleus we confirmed that NKX2-3 expression in NPM1-mutant primary AML cells is sustained by mutant NPM1c. ChIPseq in NPM1-mutated primary patient and KO52 AML cells identified NKX2-3 binding peaks. As expected, DNA sequences under peak apices were strongly enriched for NKX2-3 binding motifs; there was also significant enrichment for RUNX and ETS family motifs, but not HOX/MEIS/PBX motifs. To identify genes potentially regulated by NKX2-3, genomic coordinates of strong binding peaks were mapped to the single nearest gene. Remarkably, among genes up regulated following NKX2-3 knockdown in primary human AML cells there was a highly significant enrichment of genes with putative regulatory elements bound by NKX2-3. Likewise, among genes down regulated in NKX2-3-expressing KIT+ murine HSPCs, there was a similar significant enrichment of genes with putative regulatory elements bound by NKX2-3. These data suggest that NKX2-3 functions as a transcription repressor at bound regulatory elements in primary AML cells. In summary, NKX2-3 is highly expressed in NPM1 & FLT3 mutated molecular subtypes of AML and serves to enhance self-renewal and the level of differentiation block through binding to and repressing regulatory elements of myeloid differentiation genes.

Noncoding GATA2 Genetic Variation As a Determinant of Hematopoietic Stem/Progenitor Cell Activity and Mobilization Efficiency

Germlinecoding and enhancer variants within the hematopoietic regulator GATA2 create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). We generated a mouse model of GATA2 deficiency syndrome by engineering mice with a single-nucleotide variant in an intronic enhancer (+9.5) from GATA2 deficiency patients combined with deletion of the enhancer from the second allele (compound heterozygous; CH). This system models patients harboring a germline pathogenic allele accompanied by epigenetic silencing of the normal allele. Unlike homozygous Gata2 coding or regulatory deletions that are embryonic lethal, CH represents a unique system to analyze postnatal GATA2 function. We demonstrated that adult germline Gata2 variation alters steady-state HSPCs, blocks HSPC expansion and differentiation upon chemotherapy, attenuates long-term repopulating activity following bone marrow transplantation, leads to bone marrow failure following chronic inflammation by the viral mimetic polyI:C, alters HSPC response to the bacterial cell wall component LPS and attenuates HSPC mobilization in response to granulocyte colony stimulating factor (G-CSF) (Soukup et al., 2019, Soukup et al., 2021). As short-term G-CSF treatment induces HSPC metabolic gene signatures (Fast et al., 2021) and expansion and cell cycle entry (Schuettpelz et al., 2014), and CH have decreased MPPs (LSKCD150 -CD48 -) and common myeloid progenitors (CMPs) (Lin −Sca1 −Kit +FcγR −CD34 +) (Soukup et al., 2021), we asked if mobilization defects reflected decreased functional HSPCs in bone marrow. CH had 2.2-fold fewer colony forming units (CFU) from bone marrow (p = 0.007) than wild type (WT), reflecting a paucity of functional HSPCs. 16 hours following a single G-CSF dose, WT bone marrow immunophenotypic HSCs (LSKCD150 +CD48 -) and MPPs expanded 1.8-fold (p = 0.001 and 0.01, respectively); CH HSCs and MPPs were unchanged. CH led to decreased HSPCs and failed to induce rapid HSPC expansion post-G-CSF. We tested whether Gata2 variation impairs mobilization in response to different stimuli, or if select pathways are impacted via disrupting the CXCR4/CXCL12 chemokine axis with the CXCR4 antagonist plerixafor, stromal/neutrophil CXCR2 activation with rhIL-8, or VLA-4/VCAM1 binding with VLA-4 inhibitor BIO5192. IL-8 or BIO5192 resulted in 3.9- and 2.2-fold increased WT CFU, respectively (p < 0.0001 and 0.0014) without altering CH CFU. By contrast, mobilization with plerixafor resulted in a 4.3-fold increase in WT CFU (p < 0.0001) and 4.5-fold increase in CH CFU (p = 0.004). Targeting these pathways collectively with G-CSF was insufficient to restore normal mobilization in CH. Competitive transplantations with peripheral blood revealed G-CSF + plerixafor increased WT donor-derived hematopoiesis 24- and 20-fold at 8 and 12 weeks, respectively, versus vehicle control. Treatment of CH did not increase donor-derived contributions. Since GATA2 deficiency syndrome and somatic GATA2 variation involve heterozygous alleles, we tested whether monoallelic +9.5 Ets motif variation disrupts mobilization. G-CSF increased CFU 28.5-fold (p < 0.0001) relative to vehicle-treated WT. Mobilization of Gata2 Ets +/- HSPCs were reduced significantly (p = 0.003). CH variation severely disrupted mobilization, with 7.3-fold fewer CFU than Gata2 Ets +/- (p = 0.02). Synergistic G-CSF/plerixafor treatment increased CFU 89-fold in WT versus untreated (p < 0.0001), and Gata2 Ets +/- mobilization was reduced (p = 0.006) and not improved relative to Gata2 Ets -/- or CH, resembling G-CSF/BIO5192. Thus, Gata2 heterozygosity impaired G-CSF-induced HSPC mobilization with all regimens. Although GATA2 noncoding variation is not commonly evaluated, analysis of human data will be performed to assess relationships between variation and poor mobilization outcomes. Gata2 variation depletes functional HSPCs in the bone marrow, reducing the available HSPCs to mobilize and rendering most mobilization regimens inefficient. Ongoing studies are elucidating molecular mechanisms underlying the mobilization blockade. Our mobilization-defective system offers unique utility for elucidating fundamental HSPC mechanisms e.g., in the context of additional environmental and/or genetic insults. Future knowledge linking functionality of mobilization regimens in these contexts may unveil insights into mechanisms operational in poor mobilizers.

Early Molecular Steps in Human Bone Marrow Stem Cell Differentiation Trajectories

Understanding the molecular signatures of human hematopoietic stem/progenitor cells (HSCPs) and gene network changes leading to differentiation induction are of utmost importance to achieve therapeutic hematopoietic stem cell (HSC) expansion. The identification of surface markers has improved the prospective isolation of human bone marrow HSCs over the last decades, but the isolated population still remains very heterogeneous. Single cell sequencing approaches have the potential to unbiased assort stem cells along their differentiation stages and trajectories, and to decipher molecular markers in differentiation hierarchies. However, whole transcriptome single cell sequencing often suffers from inadequate detection of low-expressed genes with increased drop-outs. To address this limitation and to provide sensitive mRNA quantification in human HSPCs, we developed a selected targeted gene panel (600 genes) in combination with 50 protein markers to cover most relevant genes/proteins of early HSPC differentiation and cell cycle status. We employed single cell CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) based multi-omic profiling to >60.000 high-quality FACS-enriched human bone marrow HSPCs (CD34 + and CD34 +CD38 - cells) from 15 healthy donors representing young, mid and old age groups. Here, we demonstrate the most comprehensive molecular map of early human HSC differentiation and provide evidence of early branching points into lineage trajectories across different age groups using Slingshot pseudotime analysis. Our data demonstrate that the HSC branched into two main lineages, with the first branching point giving rise to megakaryocytic and erythroid progenitors (MKP/ERP), followed by lymphoid and myeloid progenitors (LMP/MDP). As expected, the most immature HSCs were defined by high expression of CD34, CD90, CRHBP, HOPX, HLF, MLLT3. However, we observed a number of hitherto undescribed HSC markers. The MKP/ERP lineage was defined by GATA1, TFRC, CD36, MPL and VWF expression. Furthermore, we found CD274, the Programmed Cell Death receptor ligand 1, to be selectively expressed in the MKP cluster. Re-clustering and trajectory analysis of the most immature CD34 +CD38 -/low cells confirmed early lineage commitment, suggesting that an MKP/ERP development does not include myeloid or lymphoid progenitors. A similar pattern was observed for all age groups. Interestingly, we observed increased expression of novel marker genes ( HLA-E, DLK1, ADGRG6, ADGRG1, MMRN1) and proteins (CD137 and CD273) in the most immature HSCs. Besides a gradual upregulation of CD38 along differentiation, differentiated cells showed lower CD45 levels. Furthermore, MKP/ERP committed cells showed a downregulation of SPINK2. TradeSeq analysis defined the continuous single cell expression changes in genes and markers upon early differentiation and lineage commitment, allowing the detection of decision-making gene networks. Our data adds further evidence to the revised model of hematopoiesis that challenges the dogma of a common myeloid progenitor giving rise to erythroid, megakaryocytic and myeloid cells. Furthermore, we see a structured hierarchical organization with a sequential loss of lineage potential and not an early commitment of uni-lineage restricted precursors. We report on the upregulation of ADGRG6, ADGRG1, DLK1, HLA-E, MMRN1 CD137 and CD273 in the most immature HSCs. Molecular and functional characterization of CD273 hi and low HSCs are presented in an adjacent report by our group at this meeting (Schmachtel et al.). In conclusion, we provide a valuable resource for the continuous regulation of genes and surface proteins along early differentiation of very immature human HSCs.

Megakaryocyte membrane-wrapped nanoparticles for targeted cargo delivery to hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are desirable targets for gene therapy but are notoriously difficult to target and transfect. Existing viral vector-based delivery methods are not effective in HSPCs due to their cytotoxicity, limited HSPC uptake and lack of target specificity (tropism). Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are attractive, nontoxic carriers that can encapsulate various cargo and enable its controlled release. To engineer PLGA NP tropism for HSPCs, megakaryocyte (Mk) membranes, which possess HSPC-targeting moieties, were extracted and wrapped around PLGA NPs, producing MkNPs. In vitro, fluorophore-labeled MkNPs are internalized by HSPCs within 24 h and were selectively taken up by HSPCs versus other physiologically related cell types. Using membranes from megakaryoblastic CHRF-288 cells containing the same HSPC-targeting moieties as Mks, CHRF-wrapped NPs (CHNPs) loaded with small interfering RNA facilitated efficient RNA interference upon delivery to HSPCs in vitro. HSPC targeting was conserved in vivo, as poly(ethylene glycol)-PLGA NPs wrapped in CHRF membranes specifically targeted and were taken up by murine bone marrow HSPCs following intravenous administration. These findings suggest that MkNPs and CHNPs are effective and promising vehicles for targeted cargo delivery to HSPCs.

TLR9-Selective Therapeutic Targeting of MDS Clones with a CpG Payload-Delivery Strategy

Inflammatory molecules and their receptors serve as regulatory cues driving the proliferation of hematopoietic stem and progenitors (HSPC) in myelodysplastic syndromes (MDS). Recent studies indicate that inflammaging (inflammation in aging) in the bone marrow (BM) microenvironment contributes to hematopoietic impairment and MDS clone expansion. However, attempts to develop therapeutics targeting these processes have been hampered by potential off target effects due to the lack of identification of selective biomarkers to specifically target the MDS malignant clone. Therefore, a better understanding of the mechanisms of MDS inflammaging processes for selection and target of malignant clones remains a key goal. Several groups, including ours, have found that innate immune receptors are over expressed in MDS BM HSPC, including Toll-like receptors (TLRs). Overexpression of these receptors was associated with secretion of inflammatory cytokines and mediators including S100A9. S100A9, a Damage-Associated Molecular Pattern (DAMPs) molecule, causes pyroptosis of HSPC and induces genomic instability leading to MDS clonal expansion in an autocrine amplification loop. However, most of the TLRs and associated factors are hard to target due to their wide expression on healthy immune cells. We found that TLR9, usually an intracellular receptor, is upregulated and translocated to the surface of MDS progenitors, which thereby presents an opportunity for disease-specific targeting through its natural ligand, CpG oligonucleotides. TLR9 is one of the main pathogen recognition receptors expressed intracellularly in innate immune cells and recognizes bacterial nucleic acid moieties. Its gene expression has been previously found to be upregulated in MDS and now our work demonstrates a functional upregulation and translocation in low-risk disease. Flow cytometric sorting of low risk MDS BM cells with labeled CpG separated cells based on mutational burden suggesting that surface TLR9 is specific to malignant clones. Furthermore, pyroptotic release of nucleic acid DAMPs, such as RNA:DNA hybrids and our recently published biomarker oxidized mitochondrial DNA, is linked to the translocation of TLR9 from intracellular endosomes to the surface of these cells. This overexpression was validated in our unique inflammaging animal model, the S100A9 transgenic (Tg) mice, that phenocopies human MDS. Moreover, treatment of human BM mononuclear cells (MNC) with recombinant human S100A9 induced surface expression of TLR9, through the accumulation of S100A9-induced RNA:DNA hybrids and oxidized mitochondrial DNA. Therefore, this paradoxical surface expression of TLR9 in MDS BM HSPCs provides an opportunity to selectively target malignant cells through the targeted delivery of cytotoxins using our newly developed CpG-linked payload deliver strategy. CpG has been used safely in phase I and Phase II clinical trials where it has been shown to be well tolerated, although not effective as a stand-alone therapy. Therefore, we linked CpG to dendrimer particles (CpG-Den) providing a cationic lipid surface structure capable of disrupting cells only after internalization through TLR9. The CpG-Den linkage ratio can be manipulated to modify cytotoxicity strength and the particle size avoids leukocyte engulfment. Using both primary MDS BM MNC and BM MNC from S100A9Tg mice we demonstrate that treatment in vitro and in vivo with CpG-Den selectively targets surface TLR9+ HSPC, increasing healthy hematopoiesis in colony forming assays, and BM expression of HSPC, as well ameliorating anemia in vivo in the S100A9Tg mice. Use of this therapy also led to a decrease in suppressive myeloid cells likely aiding in the reduction of the inflammaging cycle. Localized targeting was confirmed in vivo by labeling CpG-Den with IR800 revealing specific accumulation in the mice BM. Our data supports TLR9-targeting potential in a proof-of-concept strategy to selectively target primary MDS malignant cells to restore and enhance hematopoiesis while avoiding toxicities.

3107 – MULTI-ORGAN FUNCTIONS OF YOLK SAC DURING HUMAN EARLY DEVELOPMENT

Background Model organisms have provided valuable insights into the functions of yolk sac (YS), a conserved extraembryonic structure which provides haematopoietic and metabolic support, but species-specific differences demand a systematic examination of human YS. Methods We profiled n=6 human YS and matched liver samples (CS14-CS23) by droplet-based scRNAseq and leveraged additional data sets on YS at gastrulation (CS7) and CS10-11, aorta gonad mesonephros (AGM) at CS14-17, fetal liver and bone marrow (BM). We incorporated data from a time course of induced pluripotent stem cells (iPSCs) undergoing macrophage differentiation. Results The proportional representation of YS cell states varied over gestational age. Progenitors and megakaryocytes were over-represented early, while dendritic cells and microglia-like cells emerged later. B-lineage cells (including B1 cells) were exclusive to matched liver, while NFIL3, ACY3-expressing innate lymphoid progenitors and PTGS2, MSL1-expressing pre-macrophages were exclusive to YS. CS7-11 YS progenitors expressed SPINK2 and HLF in common with later YS (CS14-23), AGM, liver and BM samples. Liver and BM HSPCs expressed HOXA7, PROM1 and HLA-DRA. Monocytes emerged in YS only after CS14. In force-directed graph embedding, there were two routes to macrophages; a monocyte-independent route prior to CS14 and a monocyte-dependent trajectory after CS14. These pathways were recapitulated during macrophage differentiation from iPSCs. YS endoderm and matched liver shared functions in lipid and glucose metabolism and coagulation. YS endoderm cells also expressed EPO and THPO, critical for erythropoiesis and megakaryopoiesis, and F3-activated coagulation pathway components. Conclusions These findings support the concept of the human YS providing multiorgan functions, which are subsequently subsumed by the liver, kidney and BM. Model organisms have provided valuable insights into the functions of yolk sac (YS), a conserved extraembryonic structure which provides haematopoietic and metabolic support, but species-specific differences demand a systematic examination of human YS. We profiled n=6 human YS and matched liver samples (CS14-CS23) by droplet-based scRNAseq and leveraged additional data sets on YS at gastrulation (CS7) and CS10-11, aorta gonad mesonephros (AGM) at CS14-17, fetal liver and bone marrow (BM). We incorporated data from a time course of induced pluripotent stem cells (iPSCs) undergoing macrophage differentiation. The proportional representation of YS cell states varied over gestational age. Progenitors and megakaryocytes were over-represented early, while dendritic cells and microglia-like cells emerged later. B-lineage cells (including B1 cells) were exclusive to matched liver, while NFIL3, ACY3-expressing innate lymphoid progenitors and PTGS2, MSL1-expressing pre-macrophages were exclusive to YS. CS7-11 YS progenitors expressed SPINK2 and HLF in common with later YS (CS14-23), AGM, liver and BM samples. Liver and BM HSPCs expressed HOXA7, PROM1 and HLA-DRA. Monocytes emerged in YS only after CS14. In force-directed graph embedding, there were two routes to macrophages; a monocyte-independent route prior to CS14 and a monocyte-dependent trajectory after CS14. These pathways were recapitulated during macrophage differentiation from iPSCs. YS endoderm and matched liver shared functions in lipid and glucose metabolism and coagulation. YS endoderm cells also expressed EPO and THPO, critical for erythropoiesis and megakaryopoiesis, and F3-activated coagulation pathway components. These findings support the concept of the human YS providing multiorgan functions, which are subsequently subsumed by the liver, kidney and BM.

3114 – FROM MECHANISMS OF LEUKAEMOGENIC DYSREGULATION TO NEW CANDIDATE THERAPEUTICS

Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities. Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities.

A Novel Highly Conserved Hematopoietic Stem/Progenitor Cell Population for Stem Cell Transplantation and Gene Therapy

Hematopoietic stem cell (HSC)-mediated gene therapy or editing holds tremendous promise for many genetic diseases, HIV and cancer. Currently, most if not all approaches focus on the gene modification of CD34+ cells. However, CD34+enriched cell products predominantly contain lineage-committed progenitors and only very few “true” long-term engrafting HSCs which are ultimately the cells needed for sustained correction of diseases. Here we have evaluated a novel HSC-enriched phenotype for HSC transplantation and gene therapy.Most recently reported mouse xenograft transplants of purified and gene-modified human CD34+CD38- HSCs led to delayed myeloid engraftment suggesting the loss of crucial short-term engrafting progenitor cells. With the goal to identify a more refined HSC-enriched target cell population for transplantation and gene therapy, we recently performed comprehensive in vivo studies of CD34 subpopulations in our nonhuman primate (NHP) stem cell transplantation model. Comparing lentivirus (LV) gene-modified candidate cell fractions in competitive reconstitution experiments, we found an HSC-enriched phenotype (CD34+CD45RA-CD90+) demonstrating both rapid short-term recovery as well as robust long-term engraftment after myeloablative conditioning and autologous transplantation. Surprisingly and in contrast to published engraftment studies of equivalent human CD34 subpopulations in the mouse xenograft model, cell fractions enriched for multipotent progenitor cells (MPPs, CD34+CD45RA-CD90-) or lympho-myeloid progenitors (LMPs, CD34+CD45RA+CD90-) did not measurably contribute to short-term hematopoietic reconstitution in the NHP model.To determine whether the observed lack of MPP/LMP-contribution in the NHP may have been caused by ex vivo culture and/or LV gene modification conditions, engraftment studies of freshly isolated and sort-purified NHP CD34 subpopulations were performed in a novel “monkeynized” mouse model. Similar to our studies in the autologous setting, multi-lineage engraftment was exclusively driven by CD34+CD45RA-CD90+ cell fractions even freshly-isolated and transplanted without co-administration of MPPs/LMPs. Maturation of all blood lineages was found in the peripheral blood (PB), bone marrow (BM), spleen and thymus of successfully engrafted mice. Furthermore, NHP CD34+CD45RA-CD90+ cells were exclusively capable of homing into and repopulating the BM stem cell niche giving rise to phenotypical MPPs, LMPs as well as downstream progenitors.We next performed comprehensive mouse xenograft studies of freshly isolated and phenotypically identical human CD34+ subpopulations (Figure 1A) to evaluate this HSC-enrichment strategy with human stem cell sources. Similar to our studies with NHP HSPCs, multi-lineage engraftment in PB, BM, spleen and thymus was primarily restricted to CD34+CD45RA-CD90+ cell fractions (Figure 1A-E). Furthermore, engraftment and repopulation of the BM stem cell compartment was only observed for transplanted CD34+CD45RA-CD90+ HSPCs (Figure 1F). Surprisingly, we did not see engraftment of transplanted MPP- and LMP-enriched human cell fractions.In summary, these data demonstrate and confirm that both human and NHP CD34+CD45RA-CD90+ HSPCs are primarily responsible for multilineage hematopoietic reconstitution. In contrast to other HSC-enrichment strategies, the identified phenotype contains both short-term as well as long-term engraftment potential demonstrating no measurable delay in the onset of hematopoietic recovery upon transplantation in the NHP model. Most importantly, this phenotype is maintained throughout ex vivo culture and/or gene-modification, reduces the number of cells for gene-modification 20 to 30-fold, and likely increases targeting/editing efficiency as well as safety, making it a highly attractive target cell population for HSC-mediated gene and cell therapy approaches.Figure Legend - Multi-lineage engraftment potential of human CD34+ subpopulations in NSG mice: (A) Human CD34 subpopulations from GCSF-mobilized apheresis products were sort-purified and injected intravenously into sub-lethally irradiated adult NSG mice (left). Frequency of human cells in the PB over time in transplanted NSG mice (right). (B-E) Multi-lineage engraftment of human cells in the (B) PB, (C) spleen, (D) thymus and (E) BM. (F) Engraftment of human HSPCs in the murine BM. [Display omitted] DisclosuresAdair:CalImmune Inc.: Consultancy; Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Transcriptome reprogramming and myeloid skewing in haematopoietic stem and progenitor cells in systemic lupus erythematosus

ObjectivesHaematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to both myeloid and lymphoid cell lineages. We reasoned that the aberrancies of immune cells in systemic lupus erythematosus...

Inhibiting Matrix Metalloproteinases Hinders Acute Myeloid Leukaemia and Prevents Healthy Stem Cell Loss

Haematopoietic stem cells (HSCs), despite being very rare (<0.015% of bone marrow haematopoietic cells), maintain the turnover of all blood cells through a balance of quiescence, self-renewal and differentiation. Disruption of HSCs function and of the bone marrow (BM) microenvironment are key aspects of Acute Myeloid Leukaemia (AML). AML develops in adults and symptoms arise due to the loss of healthy haematopoietic cells. It is unknown exactly what factors contribute to this although it is clear that there is a progressive loss of BM HSCs in this disease. One hypothesis is that HSCs are pushed out of the BM niche by competing leukaemic blasts. To explore this, we used intravital 2-photon confocal microscopy in a live mouse model of leukaemia which allows us to visualise the dynamics of healthy haematopoietc cells at various stages of the disease. We monitored the development of extramedullary haematopoiesis (EMH) during AML growth and tested the function of HSCs found in these alternative sites to determine whether EMH acts as an alternative mechanism of blood cell maintenance in response to AML. Furthermore, we investigated the influence of an extracellular matrix metalloproteinase inhibitor, Prinomastat, on the loss of HSCs in this model. Prinomastat has been studied extensively in solid organ cancers as it has been shown capable of inhibiting cancer metastasis. In this study, we examined whether it might have a function in preventing the loss of HSCs from the bone during leukaemia infiltration. C57BL/6 mice were injected with 100k of YFP-AML blasts and peripheral blood (PB) checked every four days for AML progression. Cellular dynamics were assessed by intravital microscopy (IVM) of the mouse calvarium and spleen at early (10%), medium (25%) and late (>25%) PB infiltration. To calculate the number of circulating HSCs and progenitors (HSPCs) blood was taken by cardiac puncture and analysed by flow cytometry for HSCPs absolute number. The same was done for BM, spleen and liver HSPCs. HSC functionality was determined by transplanting sorted Lin- c-Kit+ Sca-1+ CD48- CD150+ (LKS Slam) cells from CD45.1 BM, spleen and liver of AML-burdened mice into lethally irradiated C57BL/6 mice. BM reconstitution was then analysed every four weeks. To analyse the role of extracellular matrix remodelling, C57BL/6 mice were transplanted with 100k AML cells tagged with yellow-fluorescent-protein (YFP) and then administered intravenous prinomastat daily. These mice were imaged and had bone marrow analysed using flow cytometry together with a control group at early, medium and late AML based on PB infiltration. AML progression leads to a dramatic and progressive loss HSCPs in the BM. Intravital imaging showed an enhanced egress of healthy cells from the BM into the circulation. Conversely, we found a clear association between the extent of infiltration of the marrow and the number of HSCs found in the spleen and liver. Our transplantation experiments show that the extramedullary HSCs are functional and able to reconstitute the BM of lethally irradiated mice irrespective of the organ from which they were sorted. Treatment with prinomastat significantly reduced the number of HSCs and progenitors leaving the bone marrow (P=0.0001). In the treated mice, the number of BM HSCs was consistently higher at every infiltration time point when compared to untreated mice. In addition, prinomastat caused a reduction in the extent of extramedullary haematopoesis in both the spleen and liver. This study provides a unique insight into the effect of AML on the dynamics of HSCs as disease progresses. Contrary to expectations, HSCs are not lost, but rather a majority appear to migrate from the bone marrow to sites of extramedullary haematopoiesis. These cells remain functional and are capable of regenerating haematopoiesis when transplanted into a recipient mouse. Further to this, we have demonstrated that by inhibiting the function of metalloproteinases using Prinomastat it is possible to prevent this loss of HSCs thus retain these cells within the bone marrow. These findings highlight the importance of the extracellular matrix in acute myeloid leukaemia and suggest that metalloproteinase inhibitors could potentially have a significant role in resisting the perturbations caused by AML on haematopoiesis. Disclosures No relevant conflicts of interest to declare.

A single-cell hematopoietic landscape resolves 8 lineage trajectories and defects in Kit mutant mice

AbstractHematopoietic stem and progenitor cells (HSPCs) maintain the adult blood system, and their dysregulation causes a multitude of diseases. However, the differentiation journeys toward specific hematopoietic lineages remain ill defined, and system-wide disease interpretation remains challenging. Here, we have profiled 44 802 mouse bone marrow HSPCs using single-cell RNA sequencing to provide a comprehensive transcriptional landscape with entry points to 8 different blood lineages (lymphoid, megakaryocyte, erythroid, neutrophil, monocyte, eosinophil, mast cell, and basophil progenitors). We identified a common basophil/mast cell bone marrow progenitor and characterized its molecular profile at the single-cell level. Transcriptional profiling of 13 815 HSPCs from the c-Kit mutant (W41/W41) mouse model revealed the absence of a distinct mast cell lineage entry point, together with global shifts in cell type abundance. Proliferative defects were accompanied by reduced Myc expression. Potential compensatory processes included upregulation of the integrated stress response pathway and downregulation of proapoptotic gene expression in erythroid progenitors, thus providing a template of how large-scale single-cell transcriptomic studies can bridge between molecular phenotypes and quantitative population changes.

Spatiotemporal Regulation of Hematopoietic Stem Cell Niches By Dual Cholinergic Signaling

Hematopoietic stem cells (HSCs) are known to be heterogeneous, but it is unclear whether, or how, different bone marrow (BM) microenvironments can imprint distinct HSC states. In the BM, quiescent HSCs have been found enriched in endosteal niches, whereas activated HSCs traffic in and out the BM through sinusoids localized further from bone. However, how these separate niches are integrally regulated to maintain BM homeostasis remains largely unknown. We have shown previously that sympathetic adrenergic nerve terminals innervating nestin+ mesenchymal stem cells (MSCs) regulate HSC egress from BM to circulation (Méndez-Ferrer S et al, Nature 2008 and 2010). This regulatory network is required to control HSC proliferation and migration, since its damage caused by mutated HSCs can lead to the manifestation of diseases such as myeloproliferative neoplasms and its protection can block the progression of these disorders (Arranz L et al, Nature 2014). Here we aimed to study the possible cooperation of both branches of the autonomic nervous system, the parasympathetic and the sympathetic nervous systems, which have antagonistic actions in other systems, in HSC regulation in endosteal and sinusoidal niches. We describe for the first time different cholinergic neural signals derived from both autonomic branches that regulate spatially and temporally distinct BM HSC niches. We used mice deficient in cholinergic nerve fibers (secreting acethylcholine, the main postsynaptic neurotransmitter of the parasympathetic nervous system), due to the lack of the neurturin receptor Gfrα2, a member of the glial-cell-derived family of neurotrophic factors (Rossi J et al. Neuron 1999). We found that Gfrα2-/- mice exhibited a circadian-specific defect in HSC traffic. Parasympathetic deficiency caused exacerbated sympathetic tone, manifested by ~2-fold increased BM sympathetic adrenergic fibers and nocturnal urine norepinephrine. Circulating HSCs, measured by long-term competitive repopulating assays, were 3-fold higher in Gfrα2-/- mice only during the resting period, and this was rescued by deletion of the β3-adrenergic-receptor. Therefore, systemic parasympathetic cholinergic signals antagonize BM sympathetic adrenergic activity during the resting phase, contributing to β3-adrenergic-receptor-orchestrated circadian HSC traffic through sinusoidal niches. On the other hand, sympathetic cholinergic nerve fibers, described here for the first time in the BM and running along Haversian canals of bone, regulate HSC maintenance and quiescence in endosteal niches. In neonatal mice, we found that some endosteal sympathetic nerve fibers, sensitive to chemical sympathectomy by 6-hydroxydopamine, switch from catecholaminergic to cholinergic fate and help direct developmental HSC migration to BM. This migration, dependent in perinatal life on Cxcl12 produced by BM nestin+ MSCs (Isern et al, eLife 2014), was impaired in Gfrα2-/- mice, which exhibited one week after birth a reversible ~40% reduction in BM HSCs, associated with the lack of BM sympathetic cholinergic fibers. In adult mice, these sympathetic cholinergic fibers activate nicotinic receptors and induce Cxcl12 expression in bone-associated nestin+ MSCs, an effect reproduced in vitro with the MS-5 stromal cell line. Cxcl12 expression was 2.5-fold higher in Nes-GFP+ cells associated with the bone, compared with those that were not, and was 4-fold lower in Gfrα2-/- Nes-GFP+ cells localized only in the endosteal BM. Concomitantly, Gfrα2-/- mice exhibited reduced allocation of quiescent HSPCs in the endosteal BM, compared with control mice, in which the HSPC fraction in the G0 cell cycle phase was 2-fold higher in the endosteal than in non-endosteal BM. Moreover, HSCs from Gfrα2-/- mice showed increased proliferation, even 6 months after transplantation into secondary wild-type recipients. As a result, Gfrα2-/- mice exhibit accelerated hematopoietic recovery after myeloablation, a phenotype mimicked by mice deficient in the α7-nicotinic receptor. Increased HSC proliferation was associated with loss of self-renewal, since bone-associated HSCs from Gfrα2-/- mice exhibited 50% reduced reconstituting capacity and myeloid potential 24 weeks after competitive transplantation into wild-type recipients. Thus, both branches of the autonomic nervous system regulate HSC maintenance and function in temporally and spatially separate niches. DisclosuresNo relevant conflicts of interest to declare.

Hyperactive Ptpn11 Mutations Alters Leukemic Stem Cell Frequency through Mcl-1 Overexpression

Shp2 is a non-receptor protein-tyrosine phosphatase encoded by PTPN11 and implicated in the Ras, JAK-STAT and PI3K pathways. Activating mutations in Shp2 are found in patients with developmental disorders such as Noonan and LEOPARD syndrome, as well as, hematologic malignancies. Although rare in most other solid tumors, Shp2 mutations are common in juvenile myelomonocytic leukemia (JMML) accounting for ~35% of cases. To understand its role as a cooperating mutation in AML we sequenced PTPN11 in human samples. Here we report that Shp2 mutations are present in human AML at a rate of 6.6% (6/91) in the ECOG E1900 dataset. To investigate the biological function of Shp2 mutations we asked how this functions in a cooperative model of leukemogenesis with the MLL-AF9 fusion protein. Despite showing increased genetic stability compared to other leukemias, MLL leukemias commonly contain type I mutations that can functionally cooperate resulting in more aggressive leukemias. These mutations often occur in genes encoding components of the Ras pathway including mutually exclusive mutations of NRAS, KRAS, PTPN11 and NF1 and account for ~37% of MLL rearranged leukemias. However, the mechanisms of cooperation with MLL fusion proteins are poorly understood. We found that the Shp2E76K activating mutation commonly found in humans significantly accelerates MLL-AF9 mediated leukemogenesis. The E76K mutation results in structural changes that confer increased phosphatase activity to the Shp2 protein and increased Ras signaling. We attribute the MLL-AF9/Shp2E76K cooperation to a more rapid leukemic initiation as evidenced in colony formation assays using mouse bone marrow HSPCs. Cells transduced with MLL-AF9/Shp2E76K expanded faster than MLL-AF9 cells at early stages following transduction, indicating more efficient transformation of myeloid progenitors than MLL-AF9 alone. Cytokine independent growth is achieved in MLL-AF9 cells following expression of Shp2E76K through the constitutive activation of the IL3 signaling pathway and ERK phosphorylation. Importantly, addition of Shp2E76K significantly accelerated MLL-AF9 mediated acute myeloid leukemia in mice, indicating activated Shp2 cooperates with MLL-AF9 in vivo. In addition, leukemic stem cell frequency was increased by greater than 4 fold due to Shp2E76K expression. As Shp2 is reported to regulate an anti-apoptotic gene program, we investigated these in the context of MLL-AF9 leukemic cells with and without Shp2E76K. While Bcl2, BclXL and Mcl-1, were upregulated in Shp2E76K cells, Mcl-1 showed the highest upregulation in response to Shp2E76K. Further, expression of Mcl-1 with MLL-AF9 in colony assays phenocopies expression of Shp2E76K suggesting that, mechanistically, Shp2 mutations may cooperate through activation of an anti-apoptotic gene program, primarily through Mcl-1. Finally, we asked how leukemic cells bearing Shp2E76K would respond to small molecule inhibition of Mcl-1. MLL-AF9 leukemic cells expressing Shp2E76K were desensitized to small molecule mediated Mcl-1 inhibition consistent with increased Mcl-1 protein. These data were confirmed in human cells where U937 cells, which contain an activating Shp2 mutation, exhibited resistance to Mcl-1 inhibition compared to ML2 or K562 cell which both bear wild type Shp2. Together, these data suggest patients with hyperactive Shp2 signaling may respond poorly to drugs targeting Mcl-1 due to an overabundance of the protein. DisclosuresNo relevant conflicts of interest to declare.

Challenging Dogmas - Or How Much Evidence Is Necessary To Claim That There Is a Direct Developmental and Functional Link Between The Primordial Germ Cell (PGC) Lineage and Hematopoiesis?

BackgroundThe recent hot debate on the existence in bone marrow (BM) of developmentally early stem cells with broader specification challenged the hierarchy within the stem cell compartment in murine BM. Evidence has accumulated that hematopoietic stem cells (HSCs) can become specified from a population of migrating primordial germ cells (PGCs) during embryogenesis. In support of this intriguing possibility, HSCs and PGCs are highly migratory cells, and specification of the first primitive HSCs in yolk sac blood islands as well as the origin of definitive HSCs in the aorta–gonado–mesonephros (AGM) region are chronologically and anatomically correlated with the developmental migration of PGCs in extra- and intra-embryonic tissues. Furthermore, several papers have described the sharing of chromosomal aberrations between germline tumors and leukemias or lymphomas, which suggests their clonal origin. Moreover, our recent work demonstrated the presence of quiescent, small, Oct-4+Nanog+Sca-1+Lin–CD45– stem cells in adult murine BM that express several markers shared with migratory PGCs (Leukemia 2010;24:1450) and can be specified into the hematopoietic lineage (Exp.Hematology 2011;39:225). These cells were named very small embryonic-like stem cells (VSELs). HypothesisThe aim of our study was to test the hypothesis that VSELs are related to PGCs, which would support a potential developmental link between hematopoiesis and the germ line. Experimental strategiesWe employed transmission electron microscopy (TEM), immunohistochemical staining, RQ-PCR analysis of mRNA and miRNA expression, gene array studies, and promoter methylation analysis to evaluate the expression of genes characteristic of PGC specification. We evaluated the expression of sex hormone receptors in VSELs and HSCs, and by direct in vitro and in vivo studies, we studied the effect of androgens and pituitary gonadotropins on proliferation and expansion of VSELs and HSCs. Salient ResultsThe TEM studies revealed VSELs to be small cells with a high nuclear/cytoplasmic ratio, euchromatin, and few mitochondria. VSELs isolated under steady-state conditions from BM highly express, at the mRNA and protein levels, genes involved in specification of the epiblast (e.g., Stella, Fragilis, Blimp1) in addition to genes involved in PGC specification, such as Dppa2, Dppa4, and Mvh, which characterize late-migratory PGCs. The expression of some of these genes has been confirmed at the protein level and at the promoter level to confirm chromatin structure characteristic of actively transcribed genes. To explain highly quiescent state of VSELs, we observed that VSELs, like migrating PGCs, modify imprinting of some early-development parentally imprinted gene loci, including Igf2-H19 and KCNQ1/p57Kip2, which results in their resistance to Igf-1/Igf-2 signaling and upregulation of the cyclin-dependent kinase inhibitor p57KIP2. In parallel, VSELs express several miRNAs that attenuate Igf-1/Igf-2 signaling in these cells (mir681, mir470, mir669b) as well as upregulate expression of p57KIP2 (mir25.1, mir19b, mir92). More importantly, we observed that VSELs and HSCs express mRNA for several pituitary and gonadal hormone receptors as well as highly express Sall4, an early-development marker shared by germ and hematopoietic cells. Finally, in direct in vitro and in vivo experiments, we confirmed that the quiescent population of BM-residing VSELs responds to stimulation by androgens (danazol) and pituitary gonadotropins (PSMG, FSH, and LH). In particular, we found that 10-day administration of all the sex hormones evaluated in this study directly stimulated expansion (∼2–3x) of VSELs and HSPCs in BM and enhanced BrdU incorporation (Figure 1). ConclusionsOur data support the challenging, alternative concept that HSCs can be specified during development from epiblast/migrating PGCs and that VSELs that express several unique PGCs markers, are the most primitive population of stem cells in BM. Moreover, changes in the epigenetic signature of imprinted genes as well as the miRNA network involved in resistance of these cells to Igf-1/Igf-2 signaling keeps these cells quiescent in adult tissues and prevents teratoma formation. Finally, our in vitro and in vivo data clearly show that both VSELs and HSCs proliferate in response to sex hormones. Thus, we conclude that the PGC origin of HSCs warrants further study. [Display omitted] Disclosures:Ratajczak:Neostem Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

MyD88 Signaling in CD4 T Cells Promotes IFN-γ Production and Hematopoietic Progenitor Cell Expansion in Response to Intracellular Bacterial Infection

Hematopoietic stem and progenitor cell (HSPC) phenotype and function can change in response to infectious challenge. These changes can be mediated by cytokines, IFNs, and pathogen-associated molecules, via TLR, and are thought to promote tailored immune responses for particular pathogens. In this study, we investigated the signals that activate HSPCs during ehrlichiosis, a disease characterized by profound hematopoietic dysfunction in both humans and mice. In a mouse model of ehrlichiosis, we observed that infection-induced proliferation of bone marrow HSPCs was dependent on IFN-γ signaling and was partially dependent on MyD88. However, MyD88 was not required in HSPCs for their expansion during infection, because similar frequencies of MyD88-deficient and wild-type HSPCs proliferated in mixed bone marrow chimeric mice. MyD88-deficient mice exhibited low serum and bone marrow concentration of IFN-γ compared with wild-type mice. We next identified CD4 T cells as the primary cells producing IFN-γ in the bone marrow and demonstrated a nonredundant role for CD4-derived IFN-γ in increased HSPCs. Using mixed bone marrow chimeric mice, we identified a requirement for MyD88 in CD4 T cells for increased T-bet expression, optimal IFN-γ production, and CD4 T cell proliferation. Our data demonstrate an essential role for CD4 T cells in mediating HSPC activation in response to bacterial infection and illustrate a novel role for MyD88 signaling in CD4 T cells in this process. These findings further support the idea that IFN-γ production is essential for HSPC activation and hematopoietic responses to infection.

A Novel Perspective On Hematopoietic Stem/Progenitor Cell Homing - an Expanding Family of Bone Marrow Homing Factors That Can Support SDF-1-Mediated Homing or Even Replace SDF-1

Abstract 1247 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4, also known as α 4β1integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady-state conditions is undisputed, its role in stem cell homing needs further clarification. For many years, it has been assumed that the chemotactic SDF-1 gradient across the BM–peripheral blood (PB) barrier determines whether cells home from PB into the BM microenvironment. However, this simple explanation has been challenged by several observations supporting the existence of SDF-1–CXCR4-independent homing mechanisms. For example, i) CXCR4−/− fetal liver HSPCs can home to BM in an SDF-1-independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by preincubation and co-injection with a CXCR4 receptor antagonist (AMD3100) is normal or only mildly reduced, iii) HSPCs in which CXCR4 expression is reduced by a SDF-1 intrakine strategy remain able to engraft in lethally irradiated recipients, and, as we recently reported, iv) myeloablative conditioning for transplantation induces a highly proteolytic microenvironment in BM that leads to proteolytic degradation of SDF-1 (Cancer Res. 2010;70:3402, Leukemia. 2012;26:106). Aim of the study.Based on these observations strongly suggesting the involvement of other factors and/or supportive mechanisms in the SDF-1-mediated homing of HSPCs, we became interested in identifying these unknown factors, which support homing of HSPCs when SDF-1 is degraded in the proteolytic microenvironment of BM or even if it is completely absent. Experimental approach.We tested several growth factors, cytokines, bioactive lipids, extracellular nucleotides, and antimicrobial cationic peptides for their potential involvement in homing by employing i) Transwell migration assays and ii) signaling studies in the presence or absence of specific inhibitors. We studied the chemotactic responsiveness of these factors against BM, umbilical cord blood (UCB), and mobilized peripheral blood (mPB) cells. We also focused on the molecular mechanisms responsible for the observed phenomena. Salient observations.Out of >50 different factors tested in addition to SDF-1, only sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), ATP, UTP, and GTP, which are released by cells after BM microenvironment damaged by radio/chemotherapy conditioning for transplantation, were able to chemoattract BM-purified HSPCs. The responsiveness of HSPCs to these factors was inhibited after exposure to pertussis toxin (PTX) and after inhibiting MAPKp42/44 and AKT. Interestingly, in contrast to BM-isolated HSPCs, the chemotactic responsiveness of UCB and mPB HSPCs to these factors was significantly weaker, which suggests desensitization of the corresponding receptors by factors already present in peripheral blood plasma. We also found that BM stroma exposed to myeloablative doses of radio-chemotherapy secretes two antimicrobial cationic peptides, LL-37 and β2-defensin, that, while not direct chemottractants for HSPCs, strongly enhance the responsiveness of HSPCs to an SDF-1 gradient. This phenomenon plays a crucial role in situations in which the SDF-1 homing gradient is impaired by a highly proteolytic BM microenvironment after myeloablative conditioning for transplantation. Conclusions.Since all these direct chemottractants and priming factors are upregulated in BM after myeloblative conditioning for transplantation, a more complex picture of homing emerges that involves several factors that support, and in some situations even replace, the SDF-1–CXCR4 axis. We also conclude that the priming effects by LL-37 and β2-defensin play a critical role in homing of UCB- and mPB-derived HSPCs, which respond robustly to an SDF-1 gradient. Moreover, data in an animal model lend further support to this concept. Disclosures:No relevant conflicts of interest to declare.

Differential Responsiveness of HSPCs Harvested From Bone Marrow (BM), Mobilized Peripheral Blood (mPB) and Umbilical Cord Blood (UCB) to the BM Homing Factors Stromal-Derived Factor-1 (SDF-1), Sphingosine-1 Phosphate (S1P) and Ceramide-1 Phosphate (C1P) Is Related to the Desensitization of mPB and UCB HSPCs by S1P and C1P Present in mPB and UCB Plasma

Abstract 2958 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches in stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis-dependent manner. While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady state conditions is undisputed, recent evidence confirms that due to induction of proteolytic microenvironment in BM after myeloablative radio-chemotherapy, SDF-1 level in BM decreases (Leukemia 2011, doi: 10.1038/leu.2011.185) which supports the potential role of other non-SDF-1-mediated homing mechanisms. The cumulative evidence indicates that gradients of bioactive lipids, such as sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are important homing factors. To support this further we demonstrated recently that S1P and C1P are upregulated in BM after conditioning for transplantation (Leukemia 2011, doi: 10.1038/leu.2011.185). Hypothesis. Based on the fact that SDF-1, S1P and C1P are present at relatively high concentrations in umbilical cord blood (UCB) and mobilized peripheral blood (mPB) plasma, they may desensitize responsiveness of HSPCs to BM homing gradients of SDF-1, S1P and C1P. Experimental approach. Based on this we i) measured concentration of SDF-1, S1P and C1P in BM aspirates, mPB and UCB, ii) evaluated chemotactic responsiveness of BM-, UCB- and mPB-derived HSPCs to homing gradients of SDF-1, S1P and C1P and iii) investigated molecular mechanisms potentially involved in this phenomenon by examining the surface expression of CXCR4 and S1P receptors 1–5 (S1PR1-5) at baseline and following exposure to appropriate ligands. Finally, we modulated the expression of S1PR1-5 on HSPCs and their responsiveness to chemotactic gradients by removal of S1PR1-5 ligands from the culture medium. Results. The highest expression of S1PR1-5 was detected on the surface of BM-derived CD34+/Lin− cells as compared to mPB and UCB counterparts. The downregulation of S1PR1-5 on mPB- and UCB-derived BM CD34+Lin− cells correlated with elevated level of circulating S1P in UCB and mPB plasma. Next, we found that BM-derived HSPCs responded more robustly to S1P and C1P as compared to SDF-1 when these chemoattractants were employed at physiologically relevant concentrations. Addition of S1P to the BM-derived HSPCs or incubation of BM HSPCs in S1P and C1P rich UCB or mPB plasma lead to downregulation of S1PR1-5 and desensitized their responsiveness to S1P and C1P, but not SDF-1 gradient. The expression of S1PR1-5 and responsiveness to S1P gradient by UCB- and mPB-derived HSPCs, however, could be re-established after incubating HSPCs in S1P-free medium. At the same time, since SDF-1 concentration in mPB and UCB is very low, the responsiveness of mPB- and UCB-derived HSPCs to SDF-1 gradient was not affected. Conclusions. We demonstrate, for the first time, significant differences in responsiveness of HSPCs from different sources to homing gradients of bioactive lipids. The relatively high concentration of S1P and C1P in mPB and UCB plasma may potentially desensitize responsiveness of HSPCs to BM homing gradients of bioactive lipids and interfere with their homing. These observations will have substantial clinical implications in HSPCs’ transplantation. Disclosures:No relevant conflicts of interest to declare.

A Novel Perspective on Stem Cell Homing – Emerging Interplay Between Bioactive Lipids As Potent Chemoattractants and Cationic Peptides As Underappreciated Modulators of Responsiveness to SDF-1 Gradients

Abstract 2957 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4 or a4b1 integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady state conditions is undisputed, its role in stem cell homing needs further clarification. For many years, it has been proposed that concentration differences between the chemotactic SDF-1 gradient across the BM–peripheral blood (PB) barrier determines whether cells will home from PB into the BM microenvironment. However, this simple explanation has been challenged by several observations supporting the existence of SDF-1–CXCR4-independent homing mechanisms. For example, i) CXCR4−/– fetal liver HSPCs can home to BM in an SDF-1-independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by preincubation and co-injection with a CXCR4 receptor antagonist (AMD3100) is normal or only mildly reduced, iii) HSPCs in which CXCR4 expression is reduced by a SDF-1 intrakine strategy remain able to engraft in lethally irradiated recipients, and as we recently reported, iv) myeloablative conditioning for transplantation induces a highly proteolytic microenvironment in BM that leads to proteolytic degradation of SDF-1 (Leukemia 2011, doi: 10.1038/leu.2011.185). Aim of the study. Based on these observations that strongly suggest the involvement of other factors and/or supportive mechanisms in the SDF-1-mediated homing of HSPCs we become interested in a role of bioactive lipids (sphingosine-1 phosphate; S1P and ceramide-1 phosphate; C1P) as well as priming phenomenon of SDF-1-CXCR4 axis by cationic antimicrobial peptides (CAMPs) in HSPCs homing. Results. We present evidence that gradients of bioactive lipids, such as S1P and C1P, which are products of membrane lipid metabolism, are involved in stem cell lodgment into BM. In addition, we present another important mechanism that enhances stem cell response to an low SDF-1 concentration gradient, namely, the “priming effect”. This mechanism is based on a phenomenon in which CAMPs such as complement cascade cleavage fragment C3a and stroma fibroblast- and granulocytes-derived antimicrobial peptide LL-37 significantly increase (up to 25 times) the chemotactic responsiveness of HSPCs to very low “tissue concentration-relevant” SDF-1 gradients (1–3 ng/ml). To support this further murine HSPCs primed ex vivo with CAMPs engrafted by 3–5 days faster in lethally irradiated littermates. At the molecular level, this sensitization of the responsiveness to SDF-1 after exposure to CAMPs depends on the incorporation of the CXCR4 receptor into membrane lipid rafts. This phenomenon is specific for SDF-CXCR4 axis as we did not observe that this phenomenon influences S1P- and C1P-mediated in vitro chemotaxis. Overall, these changes of SDF-1, S1P, and C1P chemotactic gradients and the appearance of priming factors in the BM microenvironment and PB plasma are triggered during conditioning for transplantation by radio-chemotherapy by the induction of a proteolytic microenvironment in BM and the activation of the complement cascade (CC). Conclusions. We provide in vivo evidence that C3a and LL-37, which have primarily antimicrobial functions and are harmless to mammalian cells, could be clinically applied to accelerate engraftment as ex vivo priming agent for transplanted human HSPCs. This novel approach based on a short ex vivo incubation of HSPCs in the graft before transplantation by CAMPs (C3a and LL-37) is of particular important in umbilical cord blood transplantations, where the number of available HSPCs in single unit for transplant is limited. This strategy is currently tested in the clinic. Disclosures:No relevant conflicts of interest to declare.

A Novel Paradigm In Stem Cell Trafficking: The Ratio of Peripheral Blood Sphingosine-1 Phosphate (S1P) to Bone Marrow Ceramide-1 Phosphate (C1P) Regulates Mobilization and Homing of Hematopoietic Stem Cells

AbstractAbstract 554The stromal derived factor-1 (SDF-1)–CXCR4 axis plays an unquestioned role in developmental migration of hematopoietic stem cells (HSPCs) and their retention in the bone marrow (BM). However, changes in the SDF-1 gradient between BM and peripheral blood (PB) do not always support its having a crucial role as chemoattractant for mobilization or homing of HSPCs. As demonstrated by others (e.g., Bone Marrow Transplantation 2003; 31:651–654, and Transfus Apher Sci 2009;40:159) and us (Leukemia 2010;24:976–985) the plasma SDF-1 level does not correlate with mobilization of HSPCs. On the other hand, there is increasing doubt about an exclusive role for SDF-1 in homing of HSPCs in BM. This is based on evidence that i) CXCR4−/− fetal liver HSPCs may home to BM in an SDF-1–independent manner (Immunity 1999;10:463-471), ii) homing of murine HSPCs made refractory to SDF-1 by incubation and co-injection with a CXCR4 receptor antagonist is normal or only mildly reduced (Science 2004;305:1000), and finally iii) HSPCs in which CXCR4 has been knocked down by means of an SDF-1 intrakine strategy also engraft in lethally irradiated recipients (Blood 2000;96:2074–,2080). All this strongly suggests the existence of other factors involved in the mobilization and homing of HSPCs. Moreover, while SDF-1 is a potent chemoattractant for HSPCs when employed at supraphysiological concentrations in vitro, as a peptide it is highly susceptible to degradation by proteases that are elevated, for example, in PB during stem cell mobilization or in the BM microenvironment after myeloablative conditioning for transplantation. Employing ELISA for detection in the present study, we observed insignificant changes in SDF-1 level both in PB during mobilization and in BM after myeloablative conditioning. We also found that mobilized PB (mPB) plasma as well as conditioned media (CM) from lethally irradiated mice chemoattract HSPCs in an SDF-1–independent manner as demonstrated by i) normal chemotaxis of AMD3100 pre-treated cells and ii) preservation of chemotactic activity of plasma and BM-derived CM following heat inactivation. However, the chemotactic activity of mPB plasma and BM CM was inhibited after stripping by activated charcoal. This suggested the involvement of small molecule bioactive lipids. It is known that sphingolipids, which are important components of cell membranes, give rise to two bioactive derivatives, sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P), with S1P already identified as a chemoattractant for HSPCs (Ann N Y Acad Sci. 200;1044:84–89). To our surprise, we found that C1P is also a strong chemoattractant for human and murine HSPCs. In addition, we observed that at physiological concentrations both these bioactive lipids i) activate phosphorylation of MAPKp42/44 and AKT in HSPCs, ii) induce expression of matrix metalloproteinases (MMPs), and iii) modulate adhesion to stroma and endothelium. Interestingly, by employing ELISA and/or mass spectophotometry we found that, while the S1P level increases in PB during mobilization, the C1P level increases in BM after myeloablative conditioning for transplantation. Based on these findings, we propose a new paradigm in which the S1P:C1P ratio plays a role in mobilization and homing of HSPCs. While S1P is a major chemoattractant that directs egress of HSPCs from BM into PB, C1P released from damaged cells in BM after myeloablative conditioning creates a homing gradient for circulating HSPCs. We also postulate that the S1P:C1P ratio plays a more universal role and is involved in regulating migration of other types of stem cells, such as circulating mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like (VSEL) stem cells. Accordingly, while S1P plays a role in egress of stem cells into PB, C1P released from damaged cells (e.g., in infarcted myocardium or brain tissue after stroke) chemoattracts circulating stem cells for potential repair. Disclosures:No relevant conflicts of interest to declare.