Number Of Hematopoietic Stem And Progenitor Cells Research Articles

Novel Small Molecule VLA-4 Inhibitors Optimized for Extended Hematopoietic and Progenitor Cell Mobilization

Background: Hematopoietic stem and progenitor cell (HSPC) transplant is an essential treatment for a variety of blood disorders and malignancies, and more recently, for gene editing therapies such as sickle cell anemia. A key step in this procedure is the effective mobilization of a large quantity of primitive donor HSPCs to ensure a durable and long lasting engraftment capable of developing into all blood cell lineages. The most commonly used regimen for donor mobilization is a 5-day course of Granulocyte Colony Stimulating Factor (G-CSF). However, G-CSF does not effectively mobilize a sufficient number or quality of stem cells in all donors, can have unwanted side effects, and is contraindicated in sickle cell disease. Therefore, it is desirable to identify an alternative treatment that is safe, rapid and cost effective. The cell surface integrin receptor VLA-4 (a4b1) and the chemokine receptor CXCR4 are essential for HSPC homing and retention in the bone marrow. Inhibition of these receptors rapidly mobilizes HSPCs within an hour, as opposed to the multi-day process using G-CSF. Small molecule inhibitors of VLA-4 have been reported previously, but most have poor pharmacokinetic properties and/or weak inhibition of VLA4. These inhibitors rapidly mobilize HSPCs into the peripheral blood of mice after a single s.c. injection, but their effect is short lived, yielding an inadequate number of HSPCs necessary for stem cell transplantation or for gene therapy. Therefore, VLA4 inhibitors with improved pharmacokinetic properties imparting extended and greater HSPC mobilization after a single dose is desirable. Methods: We designed and synthesized multiple novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. Inhibitors determined to be most potent with improved aqueous solubility were then tested in vivo in DBA mice for extended HSPC mobilization beyond 4 hours after a single SC injection, alone and in combination with the CXCR4 inhibitors. Results: We developed two main core molecules that each incorporate key VLA4 receptor binding motifs critical for potent inhibition of VLA4 binding to VCAM-1. Our first generation of inhibitors were quite potent and rapidly mobilized HSPCs, but their effect was short lived and yielded insufficient numbers of HSPCs for gene editing and engraftment studies. Most had poor solubility and very short plasma half-lives after s.c. injection in mice. We designed later generation analogues that strategically exploited a region of each optimized core, allowing for covalent addition of polyethylene glycol (PEG) chains of increasing length without decreasing potency. We determined that a minimum PEG length of 24 PEG units was required for extended HSPC mobilization in mice. WU-106, one of our VLA-4 inhibitors containing 24 PEG units, exhibited excellent aqueous solubility, longer plasma half-life after s.c. dosing, and mobilized 2-fold more murine HSPCs for a longer time than previously published best-in-class VLA-4 inhibitors or our first generation VLA4 inhibitor leads. Importantly, a synergistic effect on HSPC mobilization was achieved when WU-106 was co-administered with CXCR4 inhibitors (Plerixafor or BL-8040). Further optimization by the addition of longer PEG chains, even up to 40KD, retained sub nM potency and extended mobilization beyond 24 hours after a single injection. Our 20kD PEG analogues attached to our two different core molecules (WU-125 and WU-133) allowed for dosing of the drug in the evening, with maximum mobilization still present the next morning, thereby enabling HSPC collection to initiate soon (2-4 hours) after CXCR4 inhibitor dosing in the morning. The quantity of HSPCs obtained using this staggered dosing regimen was greater than simultaneous administration of the VLA-4 and CXCR4 inhibitors ( Fig. 1). Furthermore, our novel, long-acting PEG analogues demonstrated superior HSPC mobilization compared to an alpha 4-targeted antibody (anti-CD49d) in mice ( Fig.2). Conclusion: We have developed novel, potent PEGylated small molecule VLA-4 inhibitors that have excellent aqueous solubility and pharmacokinetics. The kinetics and magnitude of HSPC mobilization by our PEGylated inhibitors are superior to other previously described VLA-4 inhibitors and an antibody to alpha-4(anti-CD49d).

Polycom Group Protein Mel18 Inhibits Hematopoietic Stem Cell Self-Renewal through Repressing the Expression of Genes Important for Cellular Senescence and Proliferation

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cell (HSC) and leukemia stem cell (LSC) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. To further determine the role of Mel18 in hematopoiesis, we have generated Mel18 conditional knockout mice ( Mel18 f/f-Mx1Cre +). Acute deletion of Mel18 in the hematopoietic compartment did not affect the frequency and survival of hematopoietic stem and progenitor cells (HSPCs). To determine the impact of Mel18 deficiency on HSPC proliferation, we performed serial replating assays and found that loss of Mel18 increases the replating potential of HSPCs in vitro. To determine the role of Mel18 in HSC self-renewal, we first performed serial bone marrow (BM) transplantation assays and found that Mel18 null BM cells show increased engraftment in both primary and secondary transplantation assays. Limiting dilution transplantation showed that there were more functional HSCs in the BM of Mel18 knockout mice. We then performed HSC transplantation assays and found that loss Mel18 enhances HSC self-renewal. Thus, we demonstrated that Mel18 inhibits HSC self-renewal and decreases the number of functional HSCs in vivo. To understand the mechanism by which Mel18 inhibits HSC self-renewal and proliferation, we performed RNA-seq using Mel18 +/+ and Mel18 -/- HSCs. We then performed gene set enrichment analysis (GSEA) to identify potential Mel18 target genes important for HSC behavior. We found that HSC gene signatures are enriched in Mel18 -/- HSCs. We also found that leukemia stem cell gene signatures and CBF-mutant-AML genes are significantly enriched in Mel18 -/- HSCs, suggesting that loss of Mel18 may prime normal HSCs for leukemic transformation. Notably, our RNA-seq assay showed that senescence-related genes, including S100a9 and S100a8, were down-regulated in Mel18 -/- HSPCs. Importantly, loss of Mel18 significantly decreased the number of senescent HSPCs. To understand how Mel18 regulates gene transcription, we performed ATAC-seq assays in Mel18 +/+ and Mel18 -/- HSPCs. Mel18 null HSPCs showed decreased chromatin accessibility near both S100a8 and S100a9 genes. We confirmed that both S100a8 and S100a9 are downregulated in Mel18 -/- HSPCs compared to Mel18 +/+ HSPCs. Given that cell cycle arrest is a common feature of cellular senescence, we also examined the expression of cell cycle related genes in Mel18 -/- HSPCs. The expression of cell cycle regulators, including CDK4 and CCND2, is increased in Mel18 -/- HSPCs. Mel18 can bind to a specific DNA sequence, 5‘-GACTNGACT-3‘, thereby repressing the expression of target genes. Mel18 directly binds to the CCND2 gene locus as revealed by ChIP-seq assays in embryonic stem cells, suggesting that Mel18 may directly repress CCND2 expression in HSPCs. In summary, we demonstrate that Mel18 inhibits HSC self-renewal via repressing the expression of genes that are important for cellular senescence and proliferation.

Kdm6a Modulates Hematopoiesis and Leukemogenesis Via Demethylase-Dependent Epigenetic Programming

Hematopoietic stem and progenitor cells (HSPCs) maintain their differentiation by precisely regulating gene expression patterns at the transcriptional and epigenetic levels. KDM6A encodes an epigenetic regulator of the repressive chromatin mark histone 3 lysine 27 tri-methylation (H3K27me3). Growing evidence suggested the involvement of KDM6A in hematological malignancies. KDM6A mutations in leukemia frequently occur in males, reflecting that KDM6A escapes X chromosome inactivation. Therefore, female patients with single-allele loss-of-function KDM6A mutations will partially retain KDM6A expression, which is called haploinsufficiency. Furthermore, KDM6A haploinsufficiency was reported to be associated with refractory and/or relapsed leukemia, while the role of KDM6A haploinsufficiency in hematopoiesis remains unexplored. We established kdm6a-null zebrafish using CRISPR-Cas9 gene editing. We found that kdm6a loss impaired HSPCs formation. kdm6a-null embryos showed reduced expression of the hemogenic endothelial and HSPC markers runx1 at 28 hours post-fertilization (hpf). Using Tg(kdrl:mCherry;cmyb:en-GFP) embryos, we also found that the number of hemogenic endothelium and emerging HSPCs in kdm6a-null was significantly decreased. To study the importance of Kdm6a-mediated demethylase activity, we ectopically expressed kdm6a constructs (wild-type kdm6a, or catalytically-dead kdm6a) in kdm6a-null embryos. The result showed that, unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for decreased HSPC numbers. Then, we aimed to understand the potential role of kdm6a haploinsufficiency in hematopoiesis and leukemogenic mechanisms. We found that zebrafish mutants that are haploinsufficient in kdm6a ( kdm6a +/-) exhibited uncontrolled myeloid differentiation. The expression of myeloid-specific markers substantially increased in kdm6a +/- mutants, compared with WT siblings at 72 hpf. Confocal analysis showed that the ratio of myeloid-primed HSPCs was markedly increased in kdm6a +/- embryos compared to WT siblings. Consistently, a significant upregulation in transcripts encoding key myeloerythorid TFs including cebps, irf8 and gata1a was observed in kdm6a +/- embryonic HSPCs. Furthermore, kdm6a +/- adults exhibited the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or exhibited the phenotype of increased progenitors, which mimics acute myeloid leukemia. We next sorted HSPCs from WT or kdm6a +/- at 72 hpf for RNA-sequencing and ATAC-sequencing. Enrichment analysis showed transcriptional dysregulation of genes involving “Purine metabolism”, “Cysteine and methionine metabolism”, “TGF-beta signaling pathway”, and “Cell cycle”. GSEA further uncovered positive enrichment of gene sets in kdm6a +/- embryonic HSPCs, including “Oxidative phosphorylation”, “mTORC1 signaling” and “DNA repair”. These dysregulated pathways in kdm6a +/- HSPCs are highly involved in cellular homeostasis and hallmarks of aged hematopoietic system. ATAC analysis also revealed robust changes in chromatin accessibility, especially including genes related to aged hematopoietic system. Integrated RNA-seq and ATAC-seq analyses showed that the chromatin accessibility at promoters and transcription of 63 genes were decreased in kdm6a +/- HSPCs. And we found that the negative regulator of Jak-Stat signaling- socs3a was on the list. Ectopic expression of socs3a in kdm6a +/- could partially rescue the uncontrolled myeloid differentiation of HSPCs. Consistently, abnormal myeloid differentiation from kdm6a +/- mutations was rescued by Jak or Stat3 inhibitors. We also tested the importance of Kdm6a-mediated demethylase activity. Unlike restoring effect by wild-type kdm6a, the ectopic expression of catalytically-dead kdm6a constructs ( kdm6a H1134A) showed no rescue effect for dysregulated expression of socs3a and key myeloerythorid genes including cebps. In conclusion, our data demonstrate the multiple roles of kdm6a in regulating HSPC homeostasis and provide new insight into the mechanism of kdm6a haploinsufficiency in uncontrolled myeloid differentiation through demethylase-dependent epigenetic programming.

Infusion of UM171-Expanded Cord Blood Led to Excellent Survival in Patients with High-Risk Leukemias: Results from Two Independent Phase II Studies

Background: Cord blood transplant (CBT) recipients experience delayed hematopoietic recovery and an increased risk of non-relapse mortality (NRM). UM171 is a small molecule that expands hematopoietic stem and progenitor cells (HSPC). An initial phase I-II clinical trial of a single UM171-expanded CBT was associated with low NRM and excellent long-term outcomes. (S Cohen et al. Lancet Haematology 2020). With the primary goal of generating increased numbers of HSPC to supplement a conventional single cord blood (CB) graft, we tested the same approach exclusively in high and very high-risk patients with acute leukemias/myelodysplastic syndromes (MDS). Methods: Between 2019 and 2022, two single independent prospective phase II studies were conducted in Montreal, at the Maisonneuve Rosemont Hospital and in Seattle, at the Fred Hutchinson Cancer Center. Primary objectives were safety/NRM and progression free survival at 2-years post-transplant. Only patients with high and very-high risk acute myeloid leukemia (AML), acute lymphoid leukemia (ALL) and MDS were eligible. Patients were required to have adequate organ function and comorbidity index as defined by the transplant center. Patients received a single UM171 7-day expanded and cryopreserved CB after either a myeloablative or mid-intensity conditioning regimen. All patients received tacrolimus and mycophenolate mofetil for GVHD prophylaxis. Results: A total of 52 patients were enrolled (31 in Montreal and 21 in Seattle) with 2 patients not receiving the UM171 expanded product and therefore removed from the final analysis. Patient's characteristics are shown in Table 1. The median age was 44 years (range 19-66) and weight 78 kg (range, 44-145). Twenty-eight patients (54%) were either in a no complete response (CR) status or in ≥CR2, ten (19%) were p53+ and 17 (33%) were second or third transplants. Forty patients (77%) received a 5/8 HLA-matched CB unit and the median of CD34+ cells infused was 4.7 10 6/Kg (0.9-7.5). All patients engrafted (ANC >500 10^ 9/L for 2 consecutive days) at a median of 17 days (range, 9-39 days). The median time to platelet recovery (>20 x 10^ 9/L) was 37 days (range 24-116). With a median follow-up of 24 months (range 0.7 - 39), the probability of 2-year overall survival (OS) and progression-free survival (PFS) was 67% (95% CI, 54-82) and 63% (95% CI, 50-79). OS for second/third transplants and p53+ patients was 64% (95% CI, 43-96) and 40 (95% CI, 19-86), PFS was 51 % (95% CI, 29-87) and 40 (95% CI, 19-86), respectively. At 2 years, the cumulative incidence of NRM was 19% (95% CI, 8-31) while the incidence of relapse was 18% (95% CI, 6-29). The incidence of grade II-IV and III-IV acute GVHD was 69% (95% CI, 56−82%) and 16% (95% CI, 6−27%). The rate of moderate/severe chronic GVHD was 7% (95% CI,1−15%) and translated in an incidence of a chronic-relapse-free-survival of 55% (95% CI, 42−72%). Of note, Kaplan-Meier analysis of all clinical outcomes showed no difference for patients treated in Montreal and Seattle, and results were similar for both centers (Figure 1). Conclusions: The results of 2 independent Phase II clinical studies demonstrated that infusion of a UM171 single CBT led to excellent clinical outcomes in patients with high and very-high risk malignancies including patients with p53+ mutation. We also observed encouraging results for patients undergoing second/third transplants (33% of the entire cohort). Clinical outcomes were similar between the two Transplant Centers. Based on these positive results, a multicenter randomized clinical trial is currently in the planning stage.

A Scalable, Off-the-Shelf, and Cost-Effective NK Cell Manufacturing Platform for Developing NK Cell-Based Immunotherapy

Natural killer (NK) cells are innate lymphocytes that play a key role in the control of infection and malignancy. NK cells are potent killers of target cells and act in an MHC-independent manner, enabling their application as allogenic therapy for cancer. Human iPSCs are pluripotent with the ability to self-renew, thus providing an inexhaustible source for deriving NK cells. With a two-tiered cell bank system, we can manufacture NK cells with more homogeneity between batches. In principle, one single iPSC clone-derived master cell bank and working cell banks can support the life cycle of an affordable, off-the-shelf NK cell product. We developed a robust stepwise protocol for large-scale generation of high-purity NK cells from human iPSCs without the cell isolation procedure. First, human iPSCs formed embryoid bodies and then were differentiated into highly purified (>98.0%) CD34 + CD43 + hematopoietic stem and progenitor cells (HSPCs) using a combination of cytokines and small molecles such as BMP-4, VEGF, BFGF, SCF, FLT-3 and ascorbic acid. During this period, we observed up to an 180-fold cell increase in the number of HSPCs compared with human iPSCs. Second, these HSPCs were differentiated into NK cells with FLT3, SCF, IL-3, IL-7, and IL-15 etc., with cell number increased approximately 9000-fold compared with iPSCs. In the final step, iPSC-derived NK cells were further expanded up to 8000-fold which enable us to produce 1E12 NK cells from less than 1 million iPSCs. After expansion, iNK cells became more homogenous and exhibited a more mature phenotype. More than 99.0% of cells expressed both CD45 and CD56, and more than 70% expressed CD16. The expression of activating and co-stimulatory receptors (NKG2D, NKp46,NKp44, NKp30, CD244 and CD226) of NK cells was also high in these iNK cells. Intrestingly, the cells exhibited robust anti-tumor efficacy against K562 (chronic myelogenous leukemia), RPMI 8226 (multiple myeloma). Furthermore, the cryopreserved iNK cells demonstrated dramatic cytotoxicity to U-87 MG cells (malignant gliomas) in 2D culture and the tumor organoid, indicating that iNK cells derived from our protocol can be used as off-the-shelf therapy for both hematological and solid tumors. We also observed that these iNK cells had variable cytotoxicity in other tumors. For example, our iNK were not obviously cytotoxic to the NCI-H929 cell line, derived from a plasmacytoma myeloma patient and expressing BCMA at a high level. To improve the specific cytotoxicity of NK cells to NCI-H929 cells, we transduced a anti-BCMA CAR to the donor iPSC. Following differentiation, the CAR-transduced iNK cells showed approximately 6-fold cytotoxicity compared with WT iNK. (Figure 1) Collectively, our serum-free and strom cell-free NK cell generation produre from human iPSCs provides a scalable, consistent, off-the-shelf and cost-effective NK/CAR-NK cell manufacturing platform for clinical application and NK cell-based immunotherapies.

Combination of Propranolol and a Novel CXCR4 Antagonist Burixafor (GPC-100) for Enhanced Hematopoietic Cell Mobilization

Burixafor (GPC-100), a novel CXCR4 antagonist, has shown safe and effective mobilization of hematopoietic stem and progenitor cells (HSPC) in a phase I clinical study. In multiple myeloma patients, GPC-100 combined with granulocyte-colony stimulating factor (G-CSF) elicited a significant increase in circulating HSPCs that was comparable with the historical results from G-CSF plus the CXCR4 antagonist plerixafor. GPC-100 is currently being investigated for HSPC mobilization in combination with the FDA-approved ß-adrenergic receptor (ß-AR) blocker propranolol in a phase II clinical trial for autologous stem cell transplant (ASCT) in multiple myeloma. Here, we present preclinical findings indicating that the addition of propranolol to GPC-100 alone or GPC-100 and G-CSF combination can address the unmet needs of patients undergoing mobilization. Additionally, we show an enhanced and unique pattern of lymphoid cell mobilization that may have applications in adoptive cell therapies. Compared to chemotherapy, ASCT significantly improves survival in multiple myeloma patients. Because HSPCs constitute a very small fraction (~ 0.1%) of circulating mononuclear cells, it is critical to mobilize a sufficient number of HSPC into peripheral blood during the ASCT process. Current therapies for HSPC mobilization include G-CSF as a monotherapy or combined with plerixafor. However, mobilization failure is experienced by 15-50% patients, leading to potential loss of ASCT as a treatment option and significant toxicity from repeated mobilization attempts. Moreover, G-CSF is contraindicated in sickle cell disease and may exacerbate autoimmune diseases. Therefore, our goal is to identify novel strategies that will not only address the mobilization failure, but also explore non-G-CSF options for adequate mobilization. G protein-coupled receptors like CXCR4 can form heteromers with other receptors and result in altered signaling pathways. Using Proximity Ligation Assay in Namalwa (lymphoma) cells, we show that endogenously expressed CXCR4 and ß 2AR form heteromers, which are absent in CXCR4 knockout cells. In cell-based assays, co-activation of both receptors with their respective ligands, CXCL12 and epinephrine, leads to a synergistic increase in calcium flux (Figure 1) as well as ß-arrestin recruitment, indicating functional consequences of the heteromerization. Complete blockade of this enhanced signaling is only achieved with co-inhibition by both antagonists. Like CXCR4, ß 2AR is also expressed by HSPCs, and in lymph nodes was shown to heteromerize with CXCR4 to increase lymphocyte retention. In multiple myeloma patients, propranolol upregulated stem-cell like gene signature. Therefore, we sought to understand the effects of propranolol on HSPC mobilization by GPC-100. In mice, single injection of GPC-100 effectively mobilized hematopoietic stem cells characterized as Lin negSca1 +cKit +(LSK) cells as well as mature Lin negCD34 + progenitor cells by 2-4-fold, respectively. Seven-day pretreatment with propranolol enhanced LSK cell mobilization by GPC-100 resulting in a 4-fold increase in circulating HSPCs (Figure 2). Addition of propranolol to G-CSF and GPC-100 led to 8-17-fold increases in mobilization of long-term repopulating HSCs (CD34 negLSK and CD150 +LSK), which were greater than the 4-7-fold increases elicited by G-CSF and G-CSF with plerixafor. Additionally, propranolol enhanced GPC-100 induced mobilization of white blood cells, neutrophils, and lymphocytes (Figure 2). Immune cell subset analyses revealed that GPC-100 induced a 3-fold increase in circulating CD8 +T cells, which was increased to 7-fold when combined with propranolol (Figure 2). For comparison, plerixafor increased CD8 + T cell mobilization by only 2-fold. GPC-100 induced mobilization of NK and B cells was also improved by propranolol. These pre-clinical data support our phase II clinical trial currently in progress (NCT05561751). In the first arm of this two-arm trial, GPC-100 is combined with orally administered propranolol, and in the second arm, G-CSF is added to this combination. We anticipate that our clinical studies can fill the gap in the current standard of care without adding a significant financial burden or co-morbidities. Furthermore, enhanced mobilization of CD8+ T cells can provide extended opportunities for improved adoptive cell therapies.

Differential roles of prostaglandin E2 EP4 receptor on stromal cell populations for hematopoietic stem and progenitor cell function

Prostaglandin E2 (PGE2) signaling through its EP4 receptor regulates hematopoietic stem and progenitor cell (HSPC) functions. Here we generated mouse strains with conditional and inducible deletion of EP4 in stromal cell populations, including osteolineage cells, mesenchymal progenitor cells, perivascular stromal cells, and endothelial cells, to evaluate the role of EP4 in HSPC regulation through signaling in each of these niche cell populations. We found that EP4 deletion in different stromal cells had distinct effects on HSPC proliferation, long-term repopulating capacity, and the peripheral blood stem cell mobilization response. Lack of EP4 signaling in osteolineage cells increased HSPC number but impaired their long-term engraftment and mobilization. EP4 deletion in mesenchymal progenitor cells and endothelial cells reduced HSPC number and function, while EP4 deletion in perivascular stromal cells had sex-specific effects on HSPC engraftment. Our results demonstrate that PGE2/EP4 signaling in bone marrow stromal cells plays a significant and complex role in HSPC regulation, with both positive and negative effects depending on the stromal cell type.

Distinct roles of core autophagy-related genes in zebrafish definitive hematopoiesis

ABSTRACT Despite the well-described discrepancy between ATG (macroautophagy/autophagy-related) genes in the regulation of hematopoiesis, varying essentiality of core ATG proteins in vertebrate definitive hematopoiesis remains largely unclear. Here, we employed zebrafish (Danio rerio) to compare the functions of six core atg genes, including atg13, becn1 (beclin1), atg9a, atg2a, atg5, and atg3, in vertebrate definitive hematopoiesis via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein and morpholino targeting. Zebrafish with various atg mutations showed autophagic deficiency and presented partially consistent hematopoietic abnormalities during early development. All six atg mutations led to a declined number of spi1b + (Spi-1 proto-oncogene b) myeloid progenitor cells. However, only becn1 mutation resulted in the expansion of myb + (v-myb avian myeloblastosis viral oncogene homolog) hematopoietic stem and progenitor cells (HSPCs) and transiently increased coro1a + (coronin, actin binding protein, 1A) leukocytes, whereas atg3 mutation decreased the number of HSPCs and leukocytes. Proteomic analysis of caudal hematopoietic tissue identified sin3aa (SIN3 transcription regulator family member Aa) as a potential modulator of atg13- and becn1-regulated definitive hematopoiesis. Disruption of sin3aa rescued the expansion of HSPCs and leukocytes in becn1 mutants and exacerbated the decrease of HSPCs in atg13 mutants. Double mutations were also performed to examine alternative functions of various atg genes in definitive hematopoiesis. Notably, becn1 mutation failed to induce HSPCs expansion with one of the other five atg mutations. These findings demonstrated the distinct roles of atg genes and their interplays in zebrafish definitive hematopoiesis, thereby suggesting that the vertebrate definitive hematopoiesis is regulated in an atg gene-dependent manner. Abbreviations: AGM: aorta-gonad-mesonephros; AO: acridine orange; atg: autophagy related; becn1: beclin 1, autophagy related; CHT: caudal hematopoietic tissue; CKO: conditional knockout; coro1a: coronin, actin binding protein, 1A; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; dpf: days post fertilization; FACS: fluorescence-activated cell sorting; hbae1.1: hemoglobin, alpha embryonic 1.1; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; KD: knockdown; KO: knockout; map1lc3/lc3: microtubule-associated protein 1 light chain 3; MO: morpholino; mpeg1.1: macrophage expressed 1, tandem duplicate 1; mpx: myeloid-specific peroxidase; myb: v-myb avian myeloblastosis viral oncogene homolog; PE: phosphatidylethanolamine; p-H3: phospho-H3 histone; PtdIns3K: class 3 phosphatidylinositol 3-kinase; rag1: recombination activating 1; rb1cc1/fip200: RB1-inducible coiled-coil 1; RFLP: restriction fragment length polymorphism; RNP: ribonucleoprotein; sin3aa: SIN3 transcription regulator family member Aa; spi1b: Spi-1 proto-oncogene b; ulk: unc-51 like autophagy activating kinase; vtg1: vitellogenin 1; WISH: whole-mount in situ hybridization.

Modeling Gilteritinib Resistance By Enforced cxcl8a Expression in Zebrafish HSPCs

In Acute Myeloid Leukemia (AML), the FMS-like tyrosine kinase III Internal Tandem Duplication (FLT3-ITD) is common and increases the risk of relapse which is only partially mitigated by tyrosine kinase inhibitor (TKI) therapy. Gilteritinib is the current mainstay for treating relapsed FLT3-ITD AML, but therapy is non-curative, and the duration of remission is transient. Mutations in the RAS pathway are known to promote gilteritinib resistance, however non-mutational mechanisms of resistance remain under-explored and could provide new avenues for combination therapy. Our previous work has associated AML cell-autonomous expression of chemokines such as CXCL8 (IL-8) as a mechanism of resistance to gilteritinib therapy. Here we enforce expression of cxcl8a ( cxcl8, ENSDARG00000104795) by hematopoietic stem and progenitor cells (HSPCs) in transgenic zebrafish embryos using a defined Runx1 enhancer element. In the absence of gilteritinib, Runx1:cxcl8(+) animals had greater numbers of HPSCs compared to Runx1:cxcl8(-) animals (4.0 vs 2.1, p = 0.04). When treated with gilteritinib at 100 nM from 48-72 hours post-fertilization (hpf), HSPC numbers were decreased in both groups; however, Runx1:cxcl8(+) animals still had more HSPCs compared to Runx1:cxcl8(-) animals (1.6 vs 0.4, p = 0.02). This suggests that cxcl8 may act as a resistance factor for gilteritinib in vivo and that understanding regulators of cxcl8 expression may provide insight into gilteritinib resistance. Reanalysis of published single cell RNA-seq data in AML patients revealed an association between expression of CXCL8 and BTK and BMX but not other Tec-family kinases such as ITK and TEC itself. Zebrafish do not have an annotated BMX gene; BTK is expressed in HSPCs and neutrophils but not the erythroid lineage. CHMFL is a specific and potent inhibitor of human BMX with an unknown spectrum of activity in zebrafish. In Runx1:cxcl8(-) Runx1:GFP zebrafish, CHMFL treatment reduced HSPC numbers, but this was not rescued in Runx1:cxcl8(+) animals (untreated vs treated, cxcl8(-): 2.9 vs 1.5, p = 0.02, untreated vs treated, cxcl8(+): 4.0 vs 1.4, p = 0.0006). This contrasts with our prior work with the non-specific Tec family kinase inhibitor, ibrutinib, where HSPC numbers were rescued in Runx1:cxcl8(+) animals. Ongoing studies are focused on understanding the targets of ibrutinib and CHMFL in the zebrafish and how these pathways may affect gilteritinib resistance.

GATA1 controls numbers of hematopoietic progenitors and their response to autoimmune neuroinflammation

Abstract GATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b +myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge. This work was supported by a grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (T32 training grant T32AI134646).

Glia maturation factor-γ is required for initiation and maintenance of hematopoietic stem and progenitor cells

BackgroundIn vertebrates, hematopoietic stem and progenitor cells (HSPCs) emerge from hemogenic endothelium in the floor of the dorsal aorta and subsequently migrate to secondary niches where they expand and differentiate into committed lineages. Glia maturation factor γ (gmfg) is a key regulator of actin dynamics that was shown to be highly expressed in hematopoietic tissue. Our goal is to investigate the role and mechanism of gmfg in embryonic HSPC development.MethodsIn-depth bioinformatics analysis of our published RNA-seq data identified gmfg as a cogent candidate gene implicated in HSPC development. Loss and gain-of-function strategies were applied to study the biological function of gmfg. Whole-mount in situ hybridization, confocal microscopy, flow cytometry, and western blotting were used to evaluate changes in the number of various hematopoietic cells and expression levels of cell proliferation, cell apoptosis and hematopoietic-related markers. RNA-seq was performed to screen signaling pathways responsible for gmfg deficiency-induced defects in HSPC initiation. The effect of gmfg on YAP sublocalization was assessed in vitro by utilizing HUVEC cell line.ResultsWe took advantage of zebrafish embryos to illustrate that loss of gmfg impaired HSPC initiation and maintenance. In gmfg-deficient embryos, the number of hemogenic endothelium and HSPCs was significantly reduced, with the accompanying decreased number of erythrocytes, myelocytes and lymphocytes. We found that blood flow modulates gmfg expression and gmfg overexpression could partially rescue the reduction of HSPCs in the absence of blood flow. Assays in zebrafish and HUVEC showed that gmfg deficiency suppressed the activity of YAP, a well-established blood flow mediator, by preventing its shuttling from cytoplasm to nucleus. During HSPC initiation, loss of gmfg resulted in Notch inactivation and the induction of Notch intracellular domain could partially restore the HSPC loss in gmfg-deficient embryos.ConclusionsWe conclude that gmfg mediates blood flow-induced HSPC maintenance via regulation of YAP, and contributes to HSPC initiation through the modulation of Notch signaling. Our findings reveal a brand-new aspect of gmfg function and highlight a novel mechanism for embryonic HSPC development.

DRAG in situ barcoding reveals an increased number of HSPCs contributing to myelopoiesis with age

Ageing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity. However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche. To address this issue, we present an in situ single cell lineage tracing technology to quantify the clonal composition and cell production of single cells in their native niche. Our results demonstrate that a pool of HSPCs with unequal output maintains myelopoiesis through overlapping waves of cell production throughout adult life. During ageing, the increased frequency of myeloid cells is explained by greater numbers of HSPCs contributing to myelopoiesis rather than the increased myeloid output of individual HSPCs. Strikingly, the myeloid output of HSPCs remains constant over time despite accumulating significant transcriptomic changes throughout adulthood. Together, these results show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs in their native microenvironment do not functionally decline in their regenerative capacity.

Motixafortide and G-CSF to mobilize hematopoietic stem cells for autologous transplantation in multiple myeloma: a randomized phase 3 trial

Autologous hematopoietic stem cell transplantation (ASCT) improves survival in multiple myeloma (MM). However, many individuals are unable to collect optimal CD34+ hematopoietic stem and progenitor cell (HSPC) numbers with granulocyte colony-stimulating factor (G-CSF) mobilization. Motixafortide is a novel cyclic-peptide CXCR4 inhibitor with extended in vivo activity. The GENESIS trial was a prospective, phase 3, double-blind, placebo-controlled, multicenter study with the objective of assessing the superiority of motixafortide + G-CSF over placebo + G-CSF to mobilize HSPCs for ASCT in MM. The primary endpoint was the proportion of patients collecting ≥6 × 106 CD34+ cells kg–1 within two apheresis procedures; the secondary endpoint was to achieve this goal in one apheresis. A total of 122 adult patients with MM undergoing ASCT were enrolled at 18 sites across five countries and randomized (2:1) to motixafortide + G-CSF or placebo + G-CSF for HSPC mobilization. Motixafortide + G-CSF enabled 92.5% to successfully meet the primary endpoint versus 26.2% with placebo + G-CSF (odds ratio (OR) 53.3, 95% confidence interval (CI) 14.12–201.33, P < 0.0001). Motixafortide + G-CSF also enabled 88.8% to meet the secondary endpoint versus 9.5% with placebo + G-CSF (OR 118.0, 95% CI 25.36–549.35, P < 0.0001). Motixafortide + G-CSF was safe and well tolerated, with the most common treatment-emergent adverse events observed being transient, grade 1/2 injection site reactions (pain, 50%; erythema, 27.5%; pruritis, 21.3%). In conclusion, motixafortide + G-CSF mobilized significantly greater CD34+ HSPC numbers within two apheresis procedures versus placebo + G-CSF while preferentially mobilizing increased numbers of immunophenotypically and transcriptionally primitive HSPCs. Trial Registration: ClinicalTrials.gov, NCT03246529

Loss of Shp1 impairs myeloid cell function and causes lethal inflammation in zebrafish larvae.

PTPN6 encodes SHP1, a protein tyrosine phosphatase with an essential role in immune cell function. SHP1 mutations are associated with neutrophilic dermatoses and emphysema in humans, which resembles the phenotype seen in motheaten mice that lack functional SHP1. To investigate the function of Shp1 in developing zebrafish embryos, we generated a ptpn6 knockout zebrafish line lacking functional Shp1. Shp1 knockout caused severe inflammation and lethality around 17 days post fertilization (dpf). During early development, the myeloid lineage was affected, resulting in a decrease in the number of neutrophils and a concomitant increase in the number of macrophages. The number of emerging hematopoietic stem and progenitor cells (HSPCs) was decreased, but due to hyperproliferation, the number of HSPCs was higher in ptpn6 mutants than in siblings at 5 dpf. Finally, the directional migration of neutrophils and macrophages was decreased in response to wounding, and fewer macrophages were recruited to the wound site. Yet, regeneration of the caudal fin fold was normal. We conclude that loss of Shp1 impaired neutrophil and macrophage function, and caused severe inflammation and lethality at the larval stage.

Innovations in hematopoietic stem-cell mobilization: a review of the novel CXCR4 inhibitor motixafortide.

Hematopoietic stem-cell transplantation (HCT) and stem-cell-based gene therapies rely on the ability to collect sufficient CD34+ hematopoietic stem and progenitor cells (HSPCs), typically via peripheral blood mobilization. Commonly used HSPC mobilization regimens include single-agent granulocyte colony-stimulating factor (G-CSF), plerixafor, chemotherapy, or a combination of these agents. These regimens, however, frequently require multiple days of injections and leukapheresis procedures to collect adequate HSPCs for HCT (minimum = >2 × 106 CD34+ cells/kg; optimal = 5-6 × 106 CD34+ cells/kg). In addition, these regimens frequently yield suboptimal CD34+ HSPC numbers for HSPC-based gene-edited therapies, given the significantly higher HSPC number needed for successful gene-editing and manufacturing. Meanwhile, G-CSF is associated with common adverse events such as bone pain as well as an increased risk of rare but potentially life-threatening splenic rupture. Moreover, G-CSF is unsafe in patients with sickle-cell disease, a key patient population that may benefit from autologous HSPC-based gene-edited therapies, where it has been associated with unacceptable rates of serious vaso-occlusive and thrombotic events. Motixafortide is a novel CXCR4 inhibitor with extended in vivo activity (>48 h) that has been shown in preclinical and clinical trials to rapidly mobilize robust numbers of HSPCs in preparation for HCT, while preferentially mobilizing increased numbers of more primitive HSPCs by immunophenotyping and single-cell RNA expression profiling. In this review, we present a history of stem-cell mobilization and update of recent innovations in novel mobilization strategies with a specific focus on the development of motixafortide, a long-acting CXCR4 inhibitor, as a novel HSPC mobilizing agent.

Bnip3lb Regulated Mitophagy Maintains the Embryonic Pool of Hematopoietic Stem Cells By Protecting Them from ROS Induced Apoptosis

We have previously shown that Reactive Oxygen Species (ROS) promote Hematopoietic Stem and Progenitor Cell (HSPC) specification in the dorsal aorta, however, in the adult marrow it is established that HSPCs must maintain low levels of ROS to protect the stem cell pool against excessive cycling, differentiation, loss of stemness and apoptosis. We aim to elucidate the developmental timing and the mechanism by which HSCs transition from their initial dependence on ROS, to their precise regulation of it in the adult marrow. Since adult HSPCs regulate ROS by maintaining high levels of mitophagy, a process in which damaged mitochondria, which disproportionately generate ROS, are removed from the cell and recycled, we sought to define the initiation, regulation and impact of mitophagy in embryonic HSPC development. Our recent data shows that oxidative stress limits HSPC numbers as early as their colonization of the secondary hematopoietic niche, the Caudal Hematopoietic Tissue (CHT). This is the zebrafish analog of the mammalian fetal liver in that it is the tissue to which the HSPCs migrate after emerging from the hemogenic endothelium of the dorsal aorta. Once in the CHT they mature and proliferate before migrating again to their terminal niche in the marrow. In contrast to published data showing that ROS promotes HSPC specification in the dorsal aorta, when ROS levels were increased in the embryo using a morpholino against PRDX1 we saw a decrease in runx1/c-myb expression in the CHT. Conversely, when ROS was decreased using the antioxidant N-acetyl cystine (NAC), there was an increase in HSPC marker expression. By live imaging of zebrafish embryos expressing a recently developed fluorescent transgenic mitophagy-reporter, Tg(ubi:mitoGR), we observed that mitophagy is low in the hemogenic endothelial tissue which gives rise to HSPCs, but is upregulated as HSPCs emerge and begin to migrate to the CHT, and is occurring at high levels at the developmental stage when ROS begins to become damaging to the HSPC population. Our scRNAseq analysis and in situ-hybridization experiments allowed us to examine which genes and pathways were regulating the induction of mitophagy in these cells. These data show that the increase in mitophagy induction in the HSPC population at this timepoint coincides precisely with the initialization of expression of the bnip3lb mitophagy receptor gene. Fluorescent whole-mount in situ hybridization experiments demonstrated that bnip3lb is co-expressed with runx1 (a HSPC marker), in the CHT (figure 1), and, by examining our Tg(ubi:mitoGR) mitophagy reporter line after morpholino knock down of bnip3lb we confirmed that it is this gene which is driving mitophagy in HSPCs at this developmental stage. NIX, the murine homolog of bnip3lb, is responsible for developmentally regulated mitophagy in reticulocytes, lens fibers and macrophages, but has, thus far, had no reported role in maintaining HSPCs. Functionally, mitophagy appears to be essential for the survival of the HSPC population in the CHT. Reducing mitophagy via morpholino directed knockdown of bnip3lb has no effect on the initial specification of HSPCs in the dorsal aorta, but significantly decreases HSPC marker expression in the CHT, as measured by runx1/c-myb in-situ hybridization, and quantified by CD41:GFP HSPC reporter expression. In contrast upregulating mitophagy, which can be done via a novel heat-shock driven ΔOTC transgenic line or via small molecule mitophagy induction, elevates HSPC numbers in the CHT. Mechanistically, we found that bnip3lb knockdown increases detection of CellROX, a fluorescent dye which reports ROS levels, and increases the number of acridine orange+ apoptotic runx1+ HSPCs. Further, reducing ROS with antioxidant NAC reduces apoptosis and rescues the HSPC population (figure 2). We therefore propose that developmentally programmed mitophagy directed by bnip3lb is responsible for protecting proliferative embryonic HSPCs from the harmful effects of oxidative stress while the HSPC pool is expanding to seed lifelong hematopoiesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Link between Blood Flow and the Inflammatory Networks That Govern Hematopoietic Stem Cell Specification

Hematopoietic stem and progenitor cells (HSPCs) are multipotent, self-renewing cells that maintain the blood-forming system throughout the life of all individuals. The unique properties of these cells make HSPC transplantation an effective therapy for treatment of many blood disorders. The advent of induced-pluripotent stem cells (iPSCs) led to great optimism in the field as a potentially limitless source of immunologically-matched, clinical-grade hematopoietic cells. Despite success in producing in vitro hematopoietic progenitors and cells that are capable of limited in vivo reconstitution, there are no robust, reproducible protocols that can generate long-term reconstituting HSPCs. Having a more complete understanding of the signals and molecular pathways required to develop HSPCs in their native environment will move the field forward, leading to more robust protocols and the development of novel HSPC-based therapeutics. Years of investigation have revealed that HSPCs require multiple molecular inputs for proper specification, including activity of the Notch, Wnt, FGF, and BMP signaling pathways. In addition, we have reported inflammatory signaling (Tnfa and NF-kB) as a novel group of HSPC fate modulators. However, the spatial temporal requirement of NF-kB activity has been enigmatic due to: (1) lack of tools that faithfully report the dynamic activation of NF-kB; (2) difficulty on assessing NF-kB dynamics in vivo in a mammalian system during embryonic development due to artifactual inflammatory conditions triggered by invasive techniques required for in utero experimentation. Addressing this knowledge gap will be critical for optimizing in vitro protocols for the generation of patient-specific HSPCs. To date, all existing zebrafish NF-kB reporter lines fail to report dynamic cellular activation of NF-kB in real time due to the long half-life of their fluorescent reporter proteins. To overcome this limitation, we have generated a novel NF-kB reporter zebrafish line driving a destabilized version of eGFP (D2eGFP) containing a PEST domain. This modified eGFP has a short half-life of two hours, enabling us to pinpoint the precise developmental window these signals are required. Time-lapse confocal imaging, in conjunction with flow cytometry, of double transgenic kdrl:mCherry, NF-kB:D2eGFP embryos revealed that NF-kB was activated in vivo within the hemogenic endothelium (HE) just prior to the establishment of circulation (18-24 hpf) and was deactivated soon after its initiation (26 hpf). When this window of NF-kB activity was disrupted by inhibiting the translocation of the NF-kB p65 subunit to the nucleus using Caffeic acid phenethyl ester (CAPE), HSPCs failed to specify. qPCR analysis from FACS-isolated HE with activated NF-kB kdrl:mCherry+ ,NF-kB:D2eGFP+ and the surrounding endothelium kdrl:mCherry+ ,NF-kB:D2eGFP- showed a significant enrichment of the early HSPC markers gfi1 and runx1 in the NF-kB+ endothelial population at 22 hpf. In addition, mRNAseq of kdrl:mCherry+ HE of NF-kB+ showed hematopoietic GO terms enriched, demonstrating that NF-kB is a contributing activator of the endothelial to hematopoietic transition. Blood flow is a well-known conserved regulator of HSPC formation. To investigate the correlation between NF-kB deactivation and circulation, blood flow was inhibited by morpholino knockdown of tnnt2a in Tg(kdrl:mCherry, NF-kB:D2eGFP) embryos and assessed by confocal microscopy and flow cytometry for NF-kB activity. In the absence of circulation, NF-kB displayed sustained activity throughout the HE. To determine if this prolonged NF-kB activity was contributing to the loss of HSPCs observed in tnnt2a morphants, we blocked NF-kB at the developmental time circulation would normally occur using CAPE and observed a significant rescue of HSPC numbers. These data provide evidence of a link between the extrinsic biomechanical forces of blood flow and the intrinsic molecular regulation of the inflammatory networks that govern HSPC specification. Altogether, our NF-kB reporter line provides one of the earliest markers that can be utilized to isolate HE in vivo. These data pinpoint the early NF-kB requirement to form HSPCs and could be applied in vitro to improve current protocols of hematopoietic differentiation from human PSCs.

Jagged1+ Myeloid-Derived Suppressor Cells Are Required for Recovery of the Hematopoietic Bone Marrow Niche after Myelosuppression

Myelosuppressive treatments such as chemotherapy and radiotherapy are widely used to treat a variety of hematopoietic diseases and disorders, however current treatment regiments induce broad cytotoxicity illustrated by off-target tissue damage. In order for timely recovery of vascular and hematopoietic function, bone marrow (BM) endothelial cells must regenerate following myelosuppression. The identity and mechanism by which myelosuppressive-resistant cells that trigger endothelial regeneration in the damaged BM microenvironment are unknown. In the present study, we show that Jagged1 (Jag1) expression was significantly upregulated in various hematopoietic populations, particularly in myeloid-derived suppressor cells (MDSCs), following myelosuppression. By crossing Jag1conditional knockoutmice with Vav1-Cre driver mice, we generated the Jag1f/fVavCre+ strain that specific deletes Jag1 in hematopoietic cells. We demonstrate that loss of hematopoietic Jag1 does not affect numbers and function of hematopoietic stem and progenitor cells (HSPCs) under homeostatic condition, but Jag1f/fVavCre+ mice rapidly succumb to both chemo- and radio-therapeutic stress exhibited by severe pancytopenia, very limited endothelial regeneration, and rapid weight loss and morbidity. Full recovery can be achieved by adoptive transfer of WT bone marrow MDSCs, which we show can efficiently rescue the severe phenotypes of Jag1f/fVavCre+ by improving endothelial cell recovery and hematopoietic niche regeneration. Using reporter models, we show that robust Notch pathway activation in endothelial cells is mediated by Jag1+MDSCs. This leads to hematopoietic recovery due to proliferation and elevated numbers of HSPCs in Jag1f/fVavCre+ mice. Treatment with Jag1-coated nanoparticles similarly rescues viability of the BM endothelial cells. Our findings indicate that hematopoietic Jag1 is essential in accelerating niche regeneration and hematopoietic recovery and is a potential therapeutic target in ameliorating damage from multiple myelosuppressive treatments.

GATA1 controls numbers of hematopoietic progenitors and their response to autoimmune neuroinflammation

AbstractGATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b+ myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge.

Clinical Progress and Preclinical Insights Into Umbilical Cord Blood Transplantation Improvement.

The application of umbilical cord blood (UCB) as an important source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution in the clinical context has steadily grown worldwide in the past 30 years. UCB has advantages that include rapid availability of donors, less strict HLA-matching demands, and low rates of graft-versus-host disease (GVHD) versus bone marrow (BM) and mobilized peripheral blood (PB). However, the limited number of HSPCs within a single UCB unit often leads to delayed hematopoietic engraftment, increased risk of transplant-related infection and mortality, and proneness to graft failure, thus hindering wide clinical application. Many strategies have been developed to improve UCB engraftment, most of which are based on 2 approaches: increasing the HSPC number ex vivo before transplantation and enhancing HSPC homing to the recipient BM niche after transplantation. Recently, several methods have shown promising progress in UCB engraftment improvement. Here, we review the current situations of UCB manipulation in preclinical and clinical settings and discuss challenges and future directions.