HSC Populations Research Articles

Abstract 3044: Novel agents with efficacy against germline mutant RUNX1 familial platelet disorder with myeloid malignancy (FPD-MM)

Abstract RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Germline, mono-allelic, missense mutations in RUNX1 cause large deletions or truncations that are mostly loss-of-function mutations. They cause familial platelet disorder (RUNX1-FPD), which evolves into myeloid malignancy (FPD-MM), e.g., MDS or AML. Although curative in some patients, allogeneic stem cell transplantation from matched, un-related donors carries the risk of GVHD and relapse in FPD-MM. Thus, there is a need for novel therapies to revert FPD-MM back to FPD. LINCS1000-CMap analysis, of the RUNX1 knockdown RNA-Seq signature revealed homoharringtonine (HHT or omacetaxine) and the anthelmintic fenbendazole (analog of mebendazole (MB)) as the top expression mimickers. In our current studies, we demonstrate that treatment with HHT or MB induced significantly greater loss of viability in seven patient-derived samples of FPD-MM vs RUNX1-FPD. Analyses conducted on BMA cells harvested longitudinally from the same patient at the RUNX1-FPD versus FPD-MM stage demonstrated greater chromatin accessibility in FPD-MM cells along with increased mRNA expressions of MYB, MECOM and BCL2. Following HHT treatment, scRNA-Seq analysis demonstrated a marked reduction in the HSC population, but concomitant increase in the macrophage population in FPD-MM. This was associated with positive enrichment of apoptosis signaling and TP53 targets gene-sets, while negatively enriching those of c-Myc targets. CyTOF analysis demonstrated that HHT treatment reduced c-Myc, EVI1, MCL1, BFL1 and CDK6 expression in phenotypically characterized FPD-MM stem-progenitor cells. Utilizing an FPD-MM sample harboring Runx1 K194N, also detected in other pedigree members with FPD or FPD-MM, we established the first ever cell line (GMR-AML1) expressing germline mtRUNX1. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL-3R) and CD33. GMR-AML1 cells in which Runx1 was knocked-out by CRISPR-gRNA were outcompeted in vitro and in vivo, by cells that retained Runx1. Treatment with HHT or MB induced loss of viability in GMR-AML1 cells (LD50: 40 or 330 nM, respectively). HHT also caused concordant decline in ATAC-Seq and RNA-Seq peaks in ribosomal genes involved in protein translation. Treatment with MB inhibited cell cycle and protein translation. CyTOF analysis revealed that MB treatment also depleted CDK6, RUNX1, c-Myc and EVI1 in phenotypically defined FPD-MM stem cells. Finally, in the GMR-AML1 cell xenograft, treatment with omacetaxine or MB significantly reduced AML burden and improved overall survival of NSG mice. These preclinical findings highlight the molecular features associated with progression of RUNX1-FPD to FPD-MM and highlight HHT or MB as effective against cellular models of FPD-MM. Citation Format: Christopher P. Mill, Warren C. Fiskus, Courtney D. DiNardo, Patrick K. Reville, John A. Davis, Christine E. Birdwell, Kaberi Das, Hanxi Hou, Koichi Takahashi, Sanam Loghavi, Joseph D. Khoury, Kapil Bhalla. Novel agents with efficacy against germline mutant RUNX1 familial platelet disorder with myeloid malignancy (FPD-MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3044.

Understanding the Functional Differences between PRC2.1 and PRC2.2 in Hematopoiesis

Epigenetic mechanisms play an important role in the determination of hematopoietic stem and progenitor cell (HSPC) fate through the modification of chromatin architecture. Polycomb repressive complex 2 (PRC2) is an indispensable epigenetic modifier that catalyzes histone 3 lysine 27 tri-methylation (H3K27me3) repressive mark through its catalytic subunit EZH2. PRC2 is divided into two independent complexes, PRC2.1 and PRC2.2, based on the specific interacting accessory proteins. PRC2.1 comprises one of the three polycomb-like proteins (PCL1/PHF1, PCL2/MTF2, or PCL3/PHF19), as well as one of the two recently identified accessory proteins, EPOP or PALI1. However, PRC2.2 contains either JARID2 or AEBP2 as co-factors. These accessory proteins aid in the recruitment of the PRC2 complexes to different sites in the genome to catalyze H3K27me3. Recent studies performed on mouse embryonic stem cells showed that PRC2.1 and PRC2.2 are involved in distinct lineage specification and commitment through their specific co-factors. However, the functional differences between PRC2.1 and PRC2.2 in HSPC differentiation and lineage commitment has not been thoroughly characterized. To compare the complete loss of PRC2 versus PRC2.2-specific loss of function in normal hematopoiesis, we generated Mx1-Cre: Jarid2fl/fl and Mx1-Cre: Ezh2fl/fl mouse strains. Two-month old Mx1-Cre(+) control, Mx1-Cre(+): Jarid2fl/fl, and Mx1-Cre(+): Ezh2fl/fl mice were injected with six doses of polyinosinic:polycytidylic acid (pIpC) to promote conditional deletion of Jarid2 and Ezh2. Two months post-pIpC injection, whole bone marrow (WBM) cells were used to perform competitive transplantation (5x10 5 CD45.2 test WBM plus 5x10 5 CD45.1 competitor WBM cells). Jarid2-KO primary recipients showed the highest CD45.2 peripheral blood (PB) engraftment (~80%), and the highest tri-lineage (myeloid 80%, B cell 70%, T cell 80%) chimerism compared to the control (myeloid 50%, B cell 45%, T cell 25%) and Ezh2-KO (myeloid 65%, B cell 5%, T cell 5%) recipients. Ezh2-KO recipients showed the lowest CD45.2 PB engraftment (~20%), and were deficient for PB lymphoid cell production. Jarid2-KO recipients showed the highest bone marrow (BM) engraftment (~ 90%), while Ezh2-KO recipients showed significant BM lymphoid engraftment (B cell 55% and T cell 40%) inverse to what was observed in the PB (B cell 5% and T cell 5%). This suggested that complete loss of PRC2 function promotes blockage in the migration of mature lymphoid cells from BM to the PB. Jarid2-KO and Ezh2-KO recipients showed significant reductions in phenotypically defined HSC cell number and frequency compared to control recipients suggesting that PRC2.2 function through its co-factor Jarid2 to maintain HSC pool. To further investigate the functional effect of both genes' loss of function at the molecular level, we performed single cell transcriptomic analysis from KSL (lineage-, c-Kit+, Sca1+, CD45.2+) cells sorted from the aforementioned recipients. Based on transcriptomic analysis, Ezh2 loss of function promoted an increase in MPP4 and precursor B cell populations in the BM of recipient mice. However, no significant changes was observed between control and Jarid2-KO clusters. Analysis showed that the Mtf2 co-factor has the highest expression level throughout the whole KSL clusters compared to other PRC2.1 and PRC2.2 accessory proteins. These results suggested that Mtf2-associated PRC2.1 is necessary to restrict MPP4 self-renewal and to repress B cell priming in HSPCs. Although no significant change was observed in the HSC cluster between Jarid2-KO and Ezh2-KO genotypes, Jarid2 and Ezh2 loss of functions showed significant differential gene expression in the HSC population when compared to each other and to the control suggesting that Jarid2-associated PRC2.2 regulates a unique molecular program in HSCs independently from PRC2.1. Combined with our functional data, these results suggested that PRC2.2 restricts HSC-specific gene expression in non-self-renewing progenitor cells through its co-factor Jarid2; while PRC2.1 restricts MPP4 fate and B cell priming through its co-factor Mtf2. Ongoing studies are aimed to delineate the distinct molecular programs regulated by PRC2.1 and PRC2.2 in HSPCs using CRISPR-Cas9 sgRNA lentiviral system targeting each of the PRC2.1 and PRC2.2 accessory proteins independently and validation in lineage-specific functional assays.

Hematopoietic Stem and Progenitor Cells Acquire an Inflammatory Memory of the Acute Myeloid Leukemia Niche

Recent studies suggest that acute myeloid leukemia (AML) generates inflammation in the bone marrow (BM) niche to an extent that compromises the function of healthy hematopoietic stem and progenitor cells (HSPC) and impacts patient prognosis. AML xenograft studies have identified several pro-inflammatory mediators (IL-6, IL-1b) that are secreted into the BM niche by leukemic blasts. Prior work by our laboratory also showed contributions involving other AML-secreted factors, in regulating HSPCs in the leukemic niche. HSPC are cellular components of the innate immune system, but the full extent of their inflammatory adaptation by AML is unknown. To overcome some of the experimental constraints inherent in the xenograft approach, we first established non-conditioned congenic grafts of C1498 murine AML cells in C57B/6 recipients. Assessment of the BM revealed that, even at low AML burden, these animals contained a range of elevated pro-inflammatory factors. Single cell RNA-Seq studies of healthy HSPCs, obtained concurrently at sacrifice confirmed an inflammatory transcriptional signature in HSC and MPP populations, with GSEA enrichment for interferon-alpha and -gamma signaling pathways. These observations led us to hypothesize that AML-experienced (inflammation-primed) healthy HSPCs may acquire a durable long-term inflammatory memory that shapes their response to subsequent stimulation during relapse or infection. To test this hypothesis, we adoptively transferred FACS-sorted healthy AML-experienced HSPCs from C1498-engrafted mice (HSPC AML) into secondary recipients. HSPCs transplanted from PBS-injected recipients (HSPC PBS) serve as controls. To assess whether HSPC AML would respond to immune challenge more aggressively compared to HSPC PBS, we exposed these animals with a single-injection of lipopolysaccharide (LPS), followed by tissue harvest and analysis after 24 hours. Bulk RNA-Seq analysis of HSPC AML from the LPS-challenged recipients showed amplified GSEA signatures for tumor necrosis factor- and NF-kB signaling pathways compared to HSPC PBS. In addition, LPS-challenged HSPC AML showed amplified expression by several krüppel-like factors (KLF) genes. Analysis for transposase-accessible chromatin loci (ATAC-Seq), concurrently obtained from LPS-challenged HSPC AML echoed the enrichment of KLF motifs as well. Collectively, the C1498 model suggests that initial AML exposure programs HSPC AML for an amplified inflammatory and differentiation program. To further ascertain disease relevance and reproducibility we turned to a doxycycline-inducible (i)MLL-AF9 AML model. Given the low leukemic burden in the BM of C1498-grafted animals, we wanted to test the hypothesis that secreted factors accounted, at least in part, for the inflammatory memory. Indeed, injection of AML secreted factors from iMLL-AF9 models revealed a strong inflammatory signature in bulk RNA-Seq studies. To determine if innate immune priming in HSPC can be directly mediated by AML crosstalk, we exposed ex vivo-expanded HSPCs to purified secreted factors (SF AML) from (i)MLL-AF9 AML blasts, followed by a secondary inflammatory challenge with LPS seven days later. Gene expression analysis of SF AML-experienced LPS-challenged HSPCs confirmed myeloid-skewed progenitor differentiation and increased expression of inflammatory targets. Together, our data show for the first time that phenotypically normal, AML-experienced HSPCs carry a durable inflammatory legacy whereby primary exposure to AML secreted factors alters their response to secondary stimuli. Our experiments phenocopy observations of trained immunity in other experimental systems, and suggest the acquisition of an innate immune memory in the AML niche. Immune priming may impact the role of these HSPC in the context of inflammation associated with aging, or clones with predisposing molecular lesions such as TET2 or DNMT3A.

IPSC-Derived Mesenchymal Stem Cells Supports the Ex Vivo expansion of Human Hematopoietic Stem and Progenitor Cells Via Sterile Inflammation Pathway

Ex-vivo expansion of human hematopoietic stem/progenitor cells (HSPCs) represents a promising technology for investigating HSPCs' function and overcoming donor shortage for transplantation. HSPCs appropriately regulate self-renewal and differentiation, relying on hematopoietic supporting cells within the bone marrow microenvironment. Notably, various strategies employing primary mesenchymal stem cells (MSCs) as hematopoietic supporting cells have demonstrated successful HSPC expansion (De Lima, et al, N. Engl. J. Med. 2012). However, the preparation of primary MSCs includes harmful procedures, hindering the stable supply of high-quality hematopoietic supporting cells, thereby limited expansion capability and enhancement of clone variation. To overcome these issues, we have established the high-quality type of an immortalized MSCs (imMSCs) cell line derived from human induced pluripotent cells (iPSCs) with knockdown of p53 gene. The imMSCs exhibited exponential proliferation over 2 months, while retaining their MSC phenotype and functionality. To examine the supporting capability of imMSCs on HSPC expansion, we performed coculture experiments with cord blood-derived CD34 + cells in the presence or absence of combination of small compounds StemRegenin-1 and UM171 (SU). We compared four distinct culture conditions, i.e., i) the basic culture with cytokines (SCF, THPO, and FLT3L), as a control; ii) cocultured with imMSCs (imMSC condition); iii) SU alone (SU condition); and iv) cocultured with imMSC and SU (imMSC+SU condition). After a 7-day culture period, we observed a 2 to 3-fold expansion of the CD34 +CD45RA -CD90 + (phenotypic HSC) population in the imMSC condition compared to control. Moreover, the addition of SU (imMSC+SU condition) demonstrated a synergistical effect in promoting HSC expansion (up to 9-fold). Meanwhile, to evaluate the functional potential of expanded HSPCs in vivo, we conducted a xenotransplantation assay using NOG mice as recipients. The expanded HSPCs obtained from the initial 1,000 CD34 + cells were transplanted into the femoral bone of sub-lethally irradiated mice (2Gy). After 12 weeks, mice were sacrificed, and the proportion of human CD45 + cells in the murine bone marrow were analyzed with flow cytometer. All experimental conditions (ii, iii, and iv in the above) enabled to increase chimerism levels compared to the control (i). Notably, the imMSC+SU condition showed a significantly higher chimerism level compared to that of the freshly isolated CD34 + cells, highlighting the efficacy of our expanded HSPCs. Furthermore, a limiting dilution assay using immunodeficient mice revealed that the imMSC, SU, and imMSC+SU conditions showed 3.5-fold, 7.4-fold, and 20-fold expansion of functional HSCs, respectively, in comparison to the control. To elucidate the underlying mechanism how intervention promote HSPC expansion, we employed RNA sequencing analysis. We found that interferon signaling genes (ISGs) were significantly elevated in HSPCs from either imMSC or imMSC+SU compared to other conditions. This finding of elevated ISGs was recapitulated by freshly isolated CB-derived HSCs (uncultured), indicating that ISGs may contribute to the self-renewal ability of both uncultured HSC and those cocultured with imMSCs. While multiple roles of inflammatory signaling, including ISGs, have been identified in hematopoiesis, its role in the fate decision of cord blood-derived HSCs remains poorly understood. To gain further insights, we examined the role of the IFN pathway through shRNA-mediated knockdown of IRF3/7 in CD34 + HSPCs. Our results revealed that the knockdown of IRF3/7 in HSPCs led to a significant reduction in the phenotypic HSC population in all conditions, suggesting the critical role of interferon signaling in HSC expansion. In this context, we attempted to recapitulate the effects of imMSCs by administering recombinant IFN-2α with various doses. However, IFN-2α administration at any doses failed to promote HSC expansion, suggesting transcript of ISGs-dependent but not IFN protein-dependent mechanism might be crucial for the HSC self-renewal. Taken together, our new co-culture system using imMSCs should contribute to a better understanding of the regulatory networks governing HSPC biology as well as the future clinical application of HSPC expansion.

Human Adult Long-Term Repopulating Hematopoietic Stem Cells Can be Purified By Surface cMPL Expression

Simple combinations of markers that reliably demarcate human long-term repopulating hematopoietic stem cells (LT-HSCs) would facilitate our understanding of their unique molecular state and the development of stem cell-based therapeutics. The complex CD34+CD38-CD45RA-CD90+CD49f+ phenotype is now routinely accepted to define a subset of cord blood CD34+ cells enriched in LT-HSCs (Notta, Science 2011). The same phenotype with the addition of rhodamine can enrich human LT-HSCs within mobilized peripheral blood (MPB) CD34+ cells, but lower stem cell enrichment is observed (Huntsman, Blood 2015). The hematopoietic cytokine thrombopoietin (TPO) and its receptor cMPL act as primary regulators of HSC function. In recent clinical studies, the synthetic TPO agonist eltrombopag was shown to improve long-term trilineage hematopoiesis in patients with immune aplastic anemia, providing evidence that activation of cMPL receptor stimulates residual multipotent LT-HSCs within the bone marrow (BM). Building on these observations, we hypothesized that cMPL might also be a relevant marker to delineate LT-HSC activity within human adult CD34+ cell populations and provide the basis for a novel LT-HSC purification scheme. To assess the utility of surface cMPL expression for purification of human adult LT-HSCs, we first performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) of human MPB CD34+ cells from a healthy volunteer. CITE-seq was conducted with antibodies designed to react with human cMPL receptor and previously described HSC epitopes (CD34, CD38, CD90, CD45RA, and CD49f). We identified 7 distinct clusters in dimension-reduction (UMAP) analysis. By confirming expression levels of HSPC gene signatures and HSC surface markers, such as CD38, we assigned each CD34+ cell cluster to a distinct HSPC subpopulation, including a single LT-HSC population (cluster 2) (Fig A). Notably, CD34+ cell subsets expressing higher levels of surface cMPL expression were highly enriched in transcriptionally defined HSC population (cluster 2) and consistently co-expressing the known human LT-HSC phenotypic markers, including lower levels of surface CD38 and CD45RA expression and higher levels of surface CD90 and CD49f expression (Fig B). To determine whether surface cMPL expression can enrich functional LT-HSCs, we next performed serial xenotransplantation assays. We used fluorescence-activated cell sorting to partition human MPB CD34+ cells into cMPL high (top 10%) and cMPL low (bottom 10%) populations, and transplanted these cells into immunodeficient NBSGW mice. Serial peripheral blood (PB) sampling of primary xenografted mice revealed a striking difference in patterns of hematopoietic reconstitution over time between the two groups. In mice transplanted with cMPL high cells, human cell chimerism increased gradually throughout the 16-week engraftment period, while a progressive decline in engraftment was observed in the cMPL low group. Notably, mean human cell engraftment within the PB, BM and spleen of mice transplanted with cMPL high cells was 209-fold (p < 0.01), 37-fold (p < 0.0001) and 283-fold (p < 0.001) higher than mean human cell chimerism in the cMPL low group at 16 weeks post-transplantation, respectively. These data suggest that cMPL high and cMPL low populations are distinctly enriched in LT-HSCs and hematopoietic progenitors, respectively. Next, to quantitatively compare the frequency of self-renewing LT-HSCs within the cMPL high and cMPL low HSPC subsets, human CD45+ cells obtained from the BM of primary mice were injected into secondary NBSGW mice at limiting dilution and BM engraftment was measured at 16 weeks post-transplantation (total period of engraftment: 32 weeks). After accounting for engraftment parameters (i.e., cell dose and engraftment levels) in both primary and secondary mice, extreme limiting dilution analysis computed self-renewing LT-HSC frequencies of 1 in 1,267 within the cMPL high CD34+ cell population, and 1 in 149,010 in the cMPL low CD34+ cell fraction, representing a 116-fold enrichment of LT-HSCs using cMPL as a single marker purification strategy. Collectively, these data suggest that cMPL expression can effectively delineate LT-HSCs in human adult CD34+ HSPCs and thus serve as an additional relevant marker for the development of human adult HSC targeting therapeutics.

The Ultrastructure of Hepatic Stellate Cell-Macrophage Intercellular Crosstalk as a New Morphological Insight into Phenomenon of Fibrogenesis in Pediatric Autoimmune Hepatitis.

Ultrastructural assessment of the HSC population and NPCs was performed in transmission electron microscopy (TEM) using pretreatment liver biopsies from 25 children (8 boys and 17 girls) aged 4-17 with clinic-pathologically diagnosed untreated AIH. Submicroscopic evaluation allowed easy identification of numerous HSCs in the form of transitory cells, i.e., T-HSCs, accompanied by signs of fibrosis. T-HSCs included cells with features of activation initiation (iHSCs) and activation perpetuation (pHSCs), indicating high HSC activation plasticity. The pHSCs were markedly elongated and mainly showed a distinct loss of lipid cytoplasmic material, expanded and dilated channels of granular endoplasmic reticulum, and linear bundles of microfilaments beneath the cell membrane. They were surrounded by usually mature collagen fibers. Frequently activated KCs/MPs adhered directly to T-HSCs. Between them, tight intercellular junctions were formed by means of point desmosomes. Our qualitative TEM observations indicate a key role of T-HSCs in liver fibrogenesis in pediatric AIH, with the essential involvement of activated KCs/MPs that directly adhere to them. Tight intercellular junctions, being the ultrastructural exponent of the specific cellular mechanisms of the crosstalk between NPCs, can play a vital role in hepatic collagen fibroplasia. A better understanding of HSC population morphology at the ultrastructural level in AIH seems important not only to improve the disease morphological diagnostics but to also provide new insights into therapeutic interventions for the phenomenon of liver fibrogenesis.

Human Schwann Cells in vitro II. Passaging, Purification, Banking, and Labeling of Established Cultures.

This manuscript describes step-by-step procedures to establish and manage fresh and cryopreserved cultures of nerve-derived human Schwann cells (hSCs) at the desired scale. Adaptable protocols are provided to propagate hSC cultures through serial passaging and perform routine manipulations such as enzymatic dissociation, purification, cryogenic preservation, live-cell labeling, and gene delivery. Expanded hSCs cultures are metabolically active, proliferative, and phenotypically stable for at least three consecutive passages. Cell yields are expected to be variable as determined by the rate of growth of individual batches and the rounds of subculture. The purity, however, can be maintained high at >95% hSC regardless of passage. The cells obtained in this manner are suitable for various applications, including small drug screens, in vitro modeling of neurodevelopmental processes, and cell transplantation. One caveat of this protocol is that continued expansion of same-batch hSC populations is eventually restricted due to senescence-linked growth arrest.

The Second Generation Type II JAK2 Inhibitor, AJ1-10502, Demonstrates Enhanced Selectivity, Improved Therapeutic Efficacy and Reduced Mutant Cell Fraction Compared to Type I JAK2 Inhibitors in Models of Myeloproliferative Neoplasms (MPNs)

Type I JAK inhibition (JAKi) represents a mainstay therapy for myelofibrosis and hydroxyurea-resistant high-risk polycythemia vera. Current type I JAKi, including ruxolitinib (RUX) and fedratinib, improve symptoms and outcomes in MPNs; however, JAK2VF allele burden remains essentially unchanged, and efficacy wanes over time. Sustained JAK/STAT signaling plays a critical role in MPN cell persistence in the setting of type I inhibition. CHZ868, a type II JAKi that binds the inactive conformation of the kinase domain, overcomes RUX persistence in vitro and reduces Jak2VF allele fraction in vivo suggesting improved JAK2 targeting might enhance clinical efficacy. However, current type II JAKi, including CHZ868, are limited by lack of kinome specificity and off-target toxicity. We therefore sought to develop novel type II JAK2i with improved potency and selectivity. Computational free energy perturbation and structure-activity relationship-based methods were used to identify lead type II inhibitor compounds. This work, followed by Absorption, Distribution, Metabolism and Excretion modeling, led to the development of AJ1-10502. Kinome selectivity profiling of AJ1-10502 revealed potent JAK2 selectivity with minimal kinase cross-reactivity compared to CHZ868, including among other JAK family tyrosine kinases (Figure 1A). Assessment of in vitro efficacy of AJ1-10502 in SET2 inhibitor naïve and SET2 RUX persistence (RUXper) cells revealed concentration-dependent inhibition of proliferation of RUXper cells comparable to that of CHZ868. The IC50 for AJ1-10502 in RUXper cells was similar to the IC50 observed in inhibitor naïve cells. We next evaluated AJ1-10502 in vivo using a novel dual Dre/Cre-recombinase Jak2VF knock-in/knock-out model allowing for the comparison of JAKi to Jak2VF genetic deletion. AJ1-10502 demonstrated reductions in leukocytosis comparable to that of RUX (K/uL: VEH 18.7 vs. RUX 10.1 vs. AJ1-10502 9.3) but dose-dependent improvements in hematocrit, platelet levels, and spleen weights superior to RUX, with reductions in spleen weights on par to that of Jak2VF deletion (mg: VEH 480 vs. RUX 255 vs. AJ1-10502 145 vs. del 107, p<0.05). A greater degree of restoration of splenic architecture was also observed with AJ1-10502 compared to type I JAKi. Critically, we observed reductions in peripheral blood (PB) and bone marrow (BM) mutant allele fraction with AJ1-10502 not observed with RUX, including within Mac1+Gr1+ myeloid cell fractions (VEH 94% vs. RUX 92% vs. AJ1-10502 78.5%, p<0.05), consistent with a mutant-biased reduction in myeloid output. In a separate series of competitive transplants using a Cre-inducible human JAK2VF transgenic mouse line, we validated the phenotypic changes in regard to leukocytosis, hematocrit, and spleen weight reduction seen with the DreCre model. Most importantly, we confirmed significant reductions in mutant cell fraction within the hematopoietic stem (HSC) compartment of BM and spleen, including among granulocytic-monocytic progenitor (GMP) and long-term HSC (LT-HSC) populations (Figure 1B). Finally, a separate study comparing AJ1-10502 to CHZ868 revealed no significant weight loss with AJ1-10502 (-0.5g) compared to CHZ868 (-2.2g, p<0.05) despite similar reductions in Jak2VF allele fraction suggesting comparable efficacy without systemic toxicity. In sum, AJ1-10502 is a potent, selective type II JAK2i with improved efficacy compared to RUX and an enhanced safety profile compared to previous non-selective type II JAKi. Most importantly, AJ1-10502 results in superior reductions in PB and BM mutant cell fraction in vivo not observed with type I JAKi. These data demonstrate the preclinical utility of type II JAKi with AJ1-10502 and inform a path to clinical development of type II JAKi for MPN patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase 2, Open-Label Study to Evaluate the Efficacy and Safety of Mgta-145 in Combination with Plerixafor for the Mobilization of Hematopoietic Stem Cells in Patients with Sickle Cell Anemia

Background: In sickle cell anemia (SCA), a point mutation in the β-globin gene results in abnormal hemoglobin prone to polymerization under hypoxic conditions. Erythrocytes become sickle-shaped and rigid, leading to painful vaso-occlusion, ischemic tissue damage, and endothelial dysfunction. Despite significant, life-threatening risks, allogeneic hematopoietic cell transplant (allo-HCT) is the only cure for SCA. Results of autologous HCT, using the addition of a non-sickling, post-transcriptional gene silencing or gene editing to increase fetal hemoglobin, are promising and suggest an alternative option for those who lack a suitable donor. Procuring hematopoietic stem and progenitor cells (HSPCs) is challenging in patients with SCA, as granulocyte colony-stimulating factor is contraindicated due to severe adverse effects. Peripheral blood mobilization with Plerixafor (Mozobil®, Genzyme) has recently been shown to be safe and effective in SCA. While mobilization with plerixafor allows collection of an HSPC product, many SCA patients require ≥2 cycles of multiday plerixafor doses and apheresis to collect sufficient quantities of HSCs for genetic manipulation. Reports suggest 30% of patients experience vaso-occlusive events (VOEs) of grade ≥3 during and after apheresis; therefore, a considerable unmet need exists for agents that augment stem cell (SC) yields and reduce apheresis time. MGTA-145 is an agonist of cell-surface chemokine receptor 2 (CXCR2) under development for the mobilization of CD34+ cells in combination with plerixafor. MGTA-145 binds to CXCR2, resulting in HSC release, which when given with plerixafor, increases mobilization of HSCs. Preclinical studies in animal models and clinical trials in healthy subjects and patients with multiple myeloma demonstrated that MGTA-145 with plerixafor results in more rapid and robust mobilization of CD34+ cells, including primitive HSC populations, compared to plerixafor alone. Objective: To evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of 1 or 2 doses of MGTA-145 combined with plerixafor for the mobilization of HSCs in individuals with SCA. Study Design and Methods: This phase 2, open-label, 2-part study (NCT05445128) will enroll approximately 10-14 adults ages 18-35 years with SCA, at 3 sites in the US. Participants must have a documented diagnosis of SCA, have a hydroxyurea washout period, and have adequate white blood cell counts and cardiopulmonary, renal, and liver function. Key exclusion criteria include history of VOE requiring a visit to a healthcare facility within 30 days of screening, poorly controlled healthcare conditions, including but not limited to cardiopulmonary or hepatorenal events ≤6 months prior to screening, and cerebrovascular accident or retinal infarct ≤2 years prior to screening. In the 1st cohort of Part A, subjects will receive a single 0.24 mg/kg subcutaneous dose of plerixafor followed by a single 0.03 mg/kg IV dose of MGTA-145, with apheresis occurring within approximately 30-45 minutes of MGTA-145 dosing. After a 1-week follow-up visit and an Independent Data Monitoring Committee (DMC) review, the 2nd cohort with additional subjects may be enrolled and given plerixafor and a 0.03 or 0.015 mg/kg or lower dose of MGTA-145 (FIGURE). Following a 1-week follow-up visit and a 2nd DMC review, patients will be enrolled in Part B and will receive, on 2 consecutive days, plerixafor and the same dose of MGTA-145 as the 2nd Part A cohort, followed by apheresis on each dosing day. The primary endpoint is total yield of CD34+ cells (CD34+ cells/kg). Safety outcomes include incidence of treatment-emergent adverse events (TEAEs), drug-related TEAEs, Grade ≥3 TEAEs, treatment-emergent serious adverse events, and TEAEs leading to study drug discontinuation. Core exploratory endpoints include characterization of the phenotype and function of cells collected by apheresis and assessment of the gene-modifying potential of mobilized CD34+ cells. The results of this trial will establish if MGTA-145 with plerixafor will rapidly and safely mobilize robust numbers of high-quality SCs for HSCT in SCA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mitochondrial Membrane Potential Defines the HSC Population with Serial Repopulating Potential in Young but Not Aged Animals

Mitochondrial Membrane Potential Defines the HSC Population with Serial Repopulating Potential in Young but Not Aged Animals

S121: CD123-CD33 COMPOUND CAR-T CELLS WITH NOVEL ANTIGEN BINDING DOMAINS PROVIDE A NEW HOPE FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA

Background: CAR-T therapy needs to be optimized to treat myeloid malignancies. Antigen-escape mediated relapse after CAR-T treatment is the vital mechanism responsible for the nondurable response. Targeting CD33 and CD123 simultaneously could cover almost all patients with AML. The off-target cytotoxicity of CD123 CAR-T and CD33 CAR-T, especially on HSPCs, has always been concerned. Several researchers have explored this issue and the conclusions are inconsistent in different studies. Aims: To improve the outcome of relapse and/or refractory acute myeloid leukemia and explore the influence of CD123-CD33 CAR-T on hematopoiesis. Methods: We develpoed a panel of anti-CD123 monoclonal antibodies (mAbs) using hybridoma technology, named 6E11, 8D7, 12H7 and 13C3 and incorporated each of the four scFvs into 4-1BB/CD3ζ signaling domain to generate 4 second generation CARs. T cells were infected with lentiviral supernatants to generate CAR-T cells. Comparative analysis were made from the expression property of CAR protein, in vitro and in vivo anti-leukemia efficacy to screen out the best candidate CD123 CAR. Next, we generated a CD123-CD33 dual targeting CAR in a compound way. We established an escape-simulation model to explore whether the compound CAR-T could reduce the risk of relapse associated with antigen loss. Flowcytometry analysis, CFU test and single cell RNA sequecing methods were jointly used to explore the influence of our CD123-CD33 CAR-T on hematopoiesis. Results: CD123 CAR constructs with four different novel scFvs differed in CAR protein expression ratio. A changing trend of CAR proteins from dropping to rising after encountering target cells was observed in 3 CAR-T cell group except in 8D7 CAR-T. 13C3 CAR was the most suitable construct from the perspective of basic and dynamic expression. 123CAR-T cells with different scFvs mediated variable anti-leukemia effect in vitro and in vitro. 123CAR and 33CAR proteins expressed efficiently on the compound CAR-T cell surface without codon optimization. The compound CAR-T showed superiority in the treatment of antigen-escape mouse model. FCM analysis showed no significant difference of residual CD34+ cells proportion between NCT cell group and compound CAR-T group. scRNA-seq analysis showed that the most undifferentiated HSC population barely changed among each group. GSEA revealed that the HSC population in CAR-T groups had no difference in the enrichment of quiescence, cell cycle, G2M checkpoint related genes compared with that of NCT group. In addition, no downregulation/enrichment of TGF-beta signaling pathway/ PI3K-AKT-mTOR signaling pathway was observed in compound CAR-T group compared with NCT group.The CFU assay demonstrated that there were still quite numbers of hematopoietic colonies formed in compound CAR-T treatment groups. Image:Summary/Conclusion: Taken together, in this study we developed a panel of second generation CD123 CARs with novel scFvs on the basis of 4 novel anti-CD123 mAbs. We proved that scFv sequence has a significantly impact on CAR expression both originally and dynamically and further affects CAR-T function, addressing the importance of scFv development in CAR-T therapy refining. The compound CAR-T in our study minimize the risk of antigen-loss mediated escape. Notably, wildtype DNA sequence of cCAR without codon optimization brings no potential risk such as immunogenic complications. scRNA-seq and CFU assay convincingly demonstrated that the CD123-CD33 compound CAR-T had limited toxicity on hematopoiesis. Thus, the CD123-CD33 compound CAR-T with novel scFvs provides new hope for R/R AML patients.

Clonal Dynamics of Normal Haematopoiesis with Human Ageing

The haematopoietic system manifests several age-associated phenotypes including anaemia; loss of regenerative capacity, especially in the face of insults such as infection, chemotherapy or blood loss; and increased risk of clonal haematopoiesis and blood cancers. The cellular alterations that underpin these age-related phenotypes, which typically manifest in individuals aged over 70, remain elusive. We aimed to investigate whether changes in HSC population structure with age might underlie any aspects of haematopoietic system ageing.We sequenced 3579 genomes from single-cell-derived colonies of haematopoietic stem cell/multipotent progenitors (HSC/MPPs) from 10 haematologically normal subjects aged 0-81 years. HSC/MPPs accumulated 17 somatic mutations/year after birth with no increased rate of mutation accumulation in the elderly. HSC/MPP telomere length declined by 30 bp/yr. In cord blood and adults aged <65, a small proportion of HSC/MPPs had unexpectedly long telomeres, as assessed using several criteria for outliers. The proportion of cells with unexpectedly long telomeres reduced in frequency with age. Given that telomeres shorten at cell division, these outlier cells have presumably undergone fewer historic cell divisions, supporting the existence of a rare population of dormant HSCs in humans that declines in frequency with age.To interrogate changes in HSC population structure with age, we used the pattern of unique and shared mutations between the sampled cells from each individual to reconstruct their phylogenetic relationships. The frequency of branch-points (known as coalescences) in phylogenetic trees in a neutrally evolving, well-mixed population of somatic cells is primarily determined by the product of population size and time between symmetric self-renewal cell divisions (Nt). Smaller populations and more frequent symmetric divisions both increase the density of coalescences. Specific clones can come to dominate either through neutral drift or positive selection.We found that haematopoiesis in adults aged <65 was polyclonal, with high indices of clonal diversity. The number and pattern of coalescent events in the phylogenies showed that a stable population of 20,000 to 200,000 HSC/MPPs was contributing evenly to blood production in young adult life. In contrast, haematopoiesis in individuals aged >75 showed profoundly decreased clonal diversity. In each elderly subject, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before age 40, but only 22% had known driver mutations.We used the ratio of non-synonymous to synonymous mutations (dN/dS) to identify any excess of non-synonymous (driver) mutations in the dataset. This genome-wide selection analysis estimated that 1/34 to 1/12 non-synonymous mutations were drivers, occurring at a constant rate throughout life, such that the set of 300 - 400 HSC/MPPs sampled from each adult individual harboured around 100 driver mutations, over 10-fold higher than the number of known drivers we could identify. Novel drivers affected a wider pool of genes than identified in blood cancers. The genes DNMT3A, ZNF318 and HIST2H3D were identified as being under significant positive selection in HSC/MPPs, despite ZNF318 and HIST2H3D not being enriched in the setting of myeloid malignancies. Loss of Y chromosome conferred selective benefits on HSC/MPPs in males.Simulations from a simple model of haematopoiesis, with constant HSC population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure observed in the elderly, which could not be explained by neutral models incorporating drift alone. Our data supports the view that dramatically decreased clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently known. By old age the majority of HSCs harbour at least one driver mutation. With such ubiquity of driver mutations, selected purely for their competitive advantage within the stem cell compartment, and with the wholesale rewiring of cellular pathways they induce, it is feasible that they may contribute to age-related phenotypes beyond the increased risk of blood cancer. DisclosuresSpencer: Wugen, Inc.: Consultancy, Other: Stock Options. Vassiliou: Kymab Ltd: Divested equity in a private or publicly-traded company in the past 24 months; STRM.BIO: Consultancy; Astrazeneca: Consultancy. Kent: STRM.bio: Research Funding. Campbell: Mu Genomics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Chromatin Regulator Mutations Are Drivers of Clonal Evolution in the Early Multipotent HSC Compartment of Primary Myelofibrosis

The definition of initiating genetic events remains up to date the main research quest in MPN disease biology. Identification of characteristic mutational clusters has shed more light on the complexity of various MPN genotypes, even though no specific genetic lesion or mutational order can be exclusively linked to the different MPN subtypes. Our aim is to determine the sequence of mutational clusters that orchestrate the expansion of aberrant clones in MPN with respect to the prime hematopoietic stem cell compartment.Genotyping for 54 MPN related gene mutations (100 MPN Patient cohort with myelofibrosis) indicated high incidence of mutations in chromatin regulator genes ASXL1 (40%) and EZH2 (14%). In 11% of the patients both ASXL1 and EZH2 gene mutations were detected, which correlated with significantly lower hemoglobin levels, higher leukocyte counts, higher numbers of additional mutations and higher risk score.In our former work we reported the identification of an expanded Lin-CD133+CD34+ neoplastic HSC population in patient peripheral blood that reproduces PMF parameters and fibrosis in vivo. Analysis of this aberrant HSC in the single cell level revealed the heterogeneity of the malignant clones and the mutational hierarchies that propagate myeloproliferation. From this analysis we report that ASXL1 and/or EZH2 mutations precede JAK2 signalling related genetic lesions (JAK2, CALR, MPL) or other epigenetic modifier mutational groups (DMT3A, TET2). Neoplastic stem cells bearing both ASXL1/EZH2 mutations exhibit higher levels of engraftment in vivo and limited erythroid colony output in vitro .In order to determine the exact contribution of chromatin regulator mutations with regard to clonal hematopoiesis determined by changes in prime HSC dynamics we established a sorting strategy for the isolation of early LT-HSC circulating in MF patient peripheral blood. By usage of 27 lineage specific and 6 additional HSC specific markers we sorted HSC on the apex of the hierarchy with the phenotype; 27xLin-CD34+CD38-CD90+CD93+CD49f+CD133+ and all classes of early and more committed progenitors from 11 patients with ASXL1 and/or EZH2 mutations. Our data indicate that both mutations emerge in the LT-HSC fraction (10/11 patients) dominate aberrant myelopoiesis and can be also detected in mature T cells (3/11 Patients). JAK2-V617F mutation was not detected in the early HSC fraction.The incidence of EZH2 mutations with or without the synergistic effect of ASXL1 mutations, is correlated with the expansion of morphologically aberrant erythroid progenitors in patient peripheral blood, which are found to be arrested in G0 phase of the cell cycle.Our results suggest that ASXL1 and EZH2 mutations are the drivers of clonal hematopoiesis in MPN and occur early at the multipotent HSC compartment. EZH2 mutations in PMF represent the first genetic cluster that correlates with aberrant erythropoiesis in these patients. DisclosuresNo relevant conflicts of interest to declare.

CCL3 Signaling Is Essential for Leukemia Progression but Dispensable for Hematopoietic Stem Cell Maintenance

Background: Hematologic malignancies remodel the bone marrow microenvironment (BMME), reducing support for normal hematopoiesis while increasing support for the malignancy. The chemokine CCL3 has been demonstrated to play a role in BMME dysfunction in multiple hematologic malignancies including myeloma, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and myelodysplastic syndrome. Therefore we used murine models to analyze the requirement for CCL3 expression in leukemia progression and the viability of CCL3 signaling as a target in myelogenous leukemias.Methods/Results: We utilized genetically altered mice with a global loss of CCL3 (CCL3KO) on a C57bl/6 background. We used sorted hematopoietic stem and progenitor cells (HSPC) (Lineage-sca1+ckit+) from CCL3KO mice or wild type controls to generate 2 models of AML, the BCR/ABL+Nup98/HoxA9 model of blast crisis CML (bcCML), and the MLL/AF9 model of AML. In the bcCML model, CCL3KO leukemic cells do not generate a leukemic disease when transplanted into naïve WT recipient mice in contrast to WT leukemic cells that will generate an acute myeloid leukemia with 100% penetrance over the course of 15-20 days. In the MLL/AF9 AML model, the disease progression of CCL3KO leukemic cells is significantly delayed as compared to WT controls (WT vs. CCL3KO p<0.05 n=15 mice/group). Therefore CCL3 modulates leukemogenesis in two genetically divergent AML models. The small molecule maraviroc is FDA approved and inhibits ligand binding to the chemokine receptor CCR5, one of the receptors for CCL3. Due to the poor pharmacokinetics of maraviroc in mice we utilized a novel micelle nanoparticle (NP) delivery platform that is targeted to the BMME through the use of a tartrate-resistant acid phosphatase (TRAP) binding peptide (TBP). Using this platform we can delay the release of maraviroc over 48 hours, and target that release to the bone marrow. To verify that CCL3 signaling is targetable in hematologic malignancies, and that the BMME targeted NP platform is a viable method of drug delivery we loaded maraviroc into TBP-NPs and treated the murine model of bcCML every 2 days over the course of 10 days. The bcCML model was used for these studies due to its exquisite sensitivity to CCL3 expression. In bcCML mice the BMME is altered including an increase in phenotypic MSC populations and HSPC populations. Following treatment with maraviroc TBP-NPs the increase in MSC and HSPC populations is reversed and leukemic burden was decreased >2 fold in the bone marrow (VEH vs Maraviroc TBP-NP treated p<0.01 n=10 mice/group). These data suggest that CCL3 can play a critical role in the progression of myelogenous leukemias and is a potential target for therapy.If CCL3 signaling is to be a viable therapeutic target in AML it must be specific to leukemic cells while sparing the normal hematopoietic system. Therefore we sorted Lineage-Sca1+Ckit+Flt3- (Flt3-LSK) bone marrow cells enriched for long term repopulating HSPCs (LT-HSCs) in order to establish stem cell activity on a per cell basis through competitive transplantation. Upon secondary transplantation, the true test of LT-HSC function, CCL3KO Flt3-LSK donor cells engrafted in recipient mice at a higher rate (2-way ANOVA, p<0.05 over 16 weeks, n=8 mice/group). These results confirm that CCL3KO mice maintain a functional LT-HSC population.Conclusions: These results demonstrate that in multiple mouse models of myelogenous leukemia CCL3 plays an important, and in some cases, indispensable role in leukemogenesis, likely involving dysruption of the normal BMME. Importantly however, a long-term engrafting normal HSC population is clearly maintained even in the complete absence of CCL3 suggesting that anti-CCL3 therapy would be well-tolerated by the normal hematopoietic system. DisclosuresNo relevant conflicts of interest to declare.

Reduction of Graft-versus-Host-Disease in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Mice by Cotransplantation of Syngeneic Human Umbilical Cord-Derived Mesenchymal Stromal Cells

Graft-versus-host disease (GVHD) is one of the major complications following hematopoietic stem cell transplantation, which remains the sole curative therapy for many malignant diseases of the hematopoietic system. The immunomodulatory potential of mesenchymal stromal cells (MSCs) to treat GVHD is currently being tested in various preclinical and clinical trials. Because the results of the preclinical and clinical trials on the use of MSCs to treat GVHD have not been consistent, we analyzed the potential beneficial effects of syngeneic versus allogenic treatment, culture expansion of MSCs, and various MSC cell doses and time points of MSC transplantation in a murine GVHD model. We established the murine GVHD model based on the transplantation of umbilical cord blood-derived hematopoietic stem cells (UC-HSCs) and used this model to assess the therapeutic potential of umbilical cord blood-derived MSCs (UC-MSCs). The use of HSC and MSC populations derived from the same donor allowed us to exclude third-party cells and test the UC-HSCs and UC-MSCs in a matched setting. Moreover, we were able to compare various doses, transplantation time points, and the influence of culture expansion of MSCs on the impact of treatment. This resulted in 16 different treatment groups. The most efficient setting for treatment of UC-HSC-induced GVHD reactions was based on the simultaneous administration of 1×106 culture-expanded, syngeneically matched UC-MSCs. This therapy effectively reduced the number of CD8+ T cells in the blood, protected the mice from weight loss, and prolonged their survival until the end of observation period. Taken together, our data show beneficial effects of (1) syngeneic over allogeneic UC-HSCs and UC-MSCs, (2) culture-expanded cells over freshly isolated primary cells, (3) simultaneous over sequential administration, and (4) high doses of UC-MSCs. The animal model of GVHD established here is now available for more detailed studies, including a comparative analysis of the efficacy of MSCs derived from alternative sources, such as adipose tissue and bone marrow.

THE PROTECTIVE ROLE OF N-ACETYL CYSTEINE (NAC) ON BROILERS EXPOSED TO CHRONIC HEAT STRESS

In hot areas, heat stress (HS) is one of the most prominent stressors in the broiler industry. To provide comprehensive information about the effect of N-acetyl cysteine (NAC) on hamartiology, biochemistry, and thyroid hormones in broilers under thermoneutral (TN) (22 4 ° C) and heat stress (HS) (34 2 ° C), The current study was carried out. A total of 80 one-day-old Cobb broiler chicks were used, with 20 birds in each group receiving either a control (basal diet) or a NAC (100 mg/Kg diet). Chronic HS at 42 days resulted in a considerable decrease in HGB, PCV, RBCs, total protein, Albumin, serum T3 and T4, while greatly increasing H/L, triglycerides, cholesterol, LDL-cholesterol, uric acid, AST, ALT, ALP, TBIL, DBIL, and creatinine. When compared to HSC broilers at 42 d old, NAC supplementation greatly increased PCV content with a substantial decrease in total leukocytic count, heterophils, and H/L ratio with an increase in lymphocytes and monocytes. When compared to HSC population, chickens given NAC had a significant decrease in serum AST, ALP, ALT and triglyceride levels, as well as a significant increase in serum T3 concentration.

The Negative Effect of Time and Air Exposure on HSC/HSPC Populations

Background: Hematopoietic stem and progenitor cells (HSC/HSPCs) inhabit niches within bone marrow that have significantly lower oxygen tension (1-4% O2, LowO2) than ambient air (21% O2) and provide signals regulating HSC signaling/function/self-renewal. Despite this knowledge, the majority of HSC/HSPC experiments are performed in ambient air, not replicating the endogenous environment. Our previous studies of HSC/HSPCs in LowO2 demonstrated enhanced phenotypic (CD150) marker expression, and frequency, of HSC/HSPC in live cells (~3fold LSK/LSKCD150 p=0.04) relative to historic data (fixed cells) as well as alterations in novel signaling/functional pathways. &#x0D; &#x0D; Methods: Our novel technology facilitates the isolation/sorting, and analysis of rare HSC populations, in live cells, continuously at their native LowO2 environments (3% O2). All assays have been adapted and validated in LowO2. &#x0D; &#x0D; Results: Time course (0 min, 15 min, 40 min, 60 min, 180 min) analysis comparing phenotypic markers of HSC/HSPC populations at LowO2 and ambient air, showed HSC/HSPC enhancement at baseline in LowO2 that were significantly blunted by exposure to air (p-value =.00007) and time beginning at 40 min (p-value =.00003). Phenotypic expression, as shown by the marker CD150, was also diminished by exposure to air (p-value =.0008) and via time (p-value =.05). Analysis via mRNA sequencing showed enhancement in 324 and 73 (LSK, LSKCD150) pathways including calcium channel complex and calcium ion binding when HSC/HSPC remained in LowO2 compared to air. Analysis of calcium regulation via FURA staining correlated with these data confirming increased calcium influx in LowO2 HSC/HSPC populations. &#x0D; &#x0D; Potential Impact: Our findings demonstrate significant negative impacts of ambient air, fixation, and time from harvest on HSC/HSPC phenotype and function. These findings suggest the critical importance of studying HSC/HSPC, live, quickly, and in their native environment, highlight potential caveats of current approaches, and rationale for lack of comparable results for studies of HSC/HSPC in the literature.

Notch-Mediated Expansion of Human Hematopoietic Stem and Progenitor Cells By Culture Under Hypoxia

Notch activation in human CD34+ hematopoietic stem/progenitor cells (HSPCs) by treatment with Delta1 ligand has enabled clinically relevant ex vivo expansion of short-term HSPCs. However, sustained engraftment of the expanded cells was not observed after transplantation, suggesting ineffective expansion of hematopoietic stem cells with long-term repopulating activity (LTR-HSCs). Recent studies have highlighted how increased proliferative demand in culture can trigger endoplasmic reticulum (ER) stress and impair HSC function. Here, we investigated whether ex vivo culture of HSPCs under hypoxia might limit cellular ER stress and thus offer a simple approach to preserve functional HSCs under high proliferative conditions, such as those promoted in culture with Delta1. Human adult mobilized CD34+ cells were cultured for 21 days under normoxia (21% O2) or hypoxia (2% O2) in vessels coated with optimized concentrations of Delta1. We observed enhanced progenitor cell activity within the CD34+ cell population treated with Delta1 in hypoxia, but the benefits provided by low-oxygen cultures were most notable in the primitive HSC compartment. At optimal coating densities of Delta1, the frequency of LTR-HSCs measured by limiting dilution analysis 16 weeks after transplantation into NSG mice was 4.9- and 4.2-fold higher in hypoxic cultures (1 in 1,586 CD34+ cells) compared with uncultured cells (1 in 7,706) and the normoxia group (1 in 5,090), respectively. Conversely, we observed no difference in expression of the homing CXCR4 receptor between cells cultured under normoxic and hypoxic conditions, indicating that hypoxia increased the absolute numbers of LTR-HSCs but not their homing potential after transplantation. To corroborate these findings molecularly, we performed transcriptomic analyses and found significant upregulation of a distinct HSC gene expression signature in cells cultured with Delta1 in hypoxia (Fig. A). Collectively, these data show that hypoxia supports a superior ex vivo expansion of human HSCs with LTR activity compared with normoxia at optimized densities of Delta1. To clarify how hypoxia improved Notch-mediated expansion of LTR-HSCs, we performed scRNA-seq of CD34+ cells treated with Delta1 under normoxic or hypoxic conditions. We identified 6 distinct clusters (clusters 0 to 5) in dimension-reduction (UMAP) analysis, with a comparable distribution of cells per cluster between normoxic and hypoxic cultures. Most clusters could be computationally assigned to a defined hematopoietic subpopulation, including progenitor cells (clusters 0 to 4) and a single transcriptionally defined HSC population (cluster 5). To assess the relative impact of normoxia and hypoxia on the HSC compartment, we performed gene set enrichment analysis (GSEA) of cells within HSC cluster 5 from each culture condition. A total of 32 genes were differentially expressed, and pathways indicative of cellular ER stress (unfolded protein response [UPR], heat shock protein [HSP] and chaperone) were significantly downregulated in hypoxia-treated cells relative to normoxic cultures (Fig. B). When examining expression of cluster 5 top differentially expressed genes across all cell clusters, we observed a more prominent upregulation of these genes within transcriptionally defined HSCs exposed to normoxia relative to more mature progenitors (Fig. C, red plots). Hypoxia lessened the cellular stress response in both progenitors and HSCs, but the mitigation was more apparent in the HSC population (Fig. C, grey plots), and decreased apoptosis was observed only within the HSC-enriched cluster 5 (Fig. D). These findings are consistent with several reports indicating that HSCs are more vulnerable to strong ER stress than downstream progenitors due to their lower protein folding capacity. In conclusion, we provide evidence that ex vivo culture of human adult CD34+ cells under hypoxic conditions enables a superior Delta1-mediated expansion of hematopoietic cells with LTR activity compared with normoxic cultures. Our data suggest a two-pronged mechanism by which optimal ectopic activation of Notch signaling in human HSCs promotes their self-renewal, and culture under hypoxia mitigates ER stress triggered by the increased proliferative demand, resulting in enhanced survival of expanding HSCs. This clinically feasible approach may be useful to improve outcomes of cellular therapeutics. Disclosures No relevant conflicts of interest to declare.

The IL-33 Receptor/ST2 acts as a positive regulator of functional mouse bone marrow hematopoietic stem and progenitor cells

There is a paucity of information on a potential role for the IL-33 receptor/ST2 in the regulation of mouse bone marrow (BM) hematopoietic stem (HSC) and progenitor (HPC) cells. Comparing the BM of st2−/− and wild type (WT) control mice using functional assays, it was found that st2−/− BM cells had poorer engrafting capacity than WT BM in a competitive repopulating assay using congenic mice, with no changes in reconstitution of B-, T- and myeloid cells following transplantation. The BM of st2−/− mice also had fewer granulocyte-macrophage, erythroid, and multipotential progenitors than that of WT BM and these st2−/− HPC were in a slow cycling state compared to that of the rapidly cycling HPC of the WT mice. While functional assessment of HSC and HPC demonstrated that ST2 has a positive influence on regulation of HSC, we could not pick up differences in st2−/− compared to WT BM using only phenotypic analysis of HSC and HPC populations prior to transplantation, again demonstrating that phenotypic analysis of HSC and HPC do not always recapitulate the functional assessments of these immature hematopoietic cells.

The properties of human Schwann cells: Lessons from in vitro culture and transplantation studies.

Human Schwann cells (hSCs) can be isolated directly from peripheral nerve and cultured using methods similar to those used for SCs from other species. Yet, important interspecies differences are revealed when the primary or expanded hSCs are compared to their nonhuman counterparts. This review addresses the special properties of nerve-derived hSCs that have resulted to date from both in vitro studies and in vivo research on cell transplantation in animal models and human subjects. A consensus has yet to emerge about the essential attributes of cultured normal hSCs. Thus, an emphasis is placed on the importance of validating hSC cultures by means of purity, identity, and biological activity to reliably use them as in vitro models of the SC phenotype and cell therapy products for injury repair. Combining traditional immunological methods, high-resolution omics approaches, and assorted cell-based assays is so far the best approach to unequivocally identify hSC populations obtained by direct isolation or derivation from stem cells. Special considerations are required to understand and manage the variability and heterogeneity proper of donor batches, as well as to evaluate risk factors. This is particularly important if the intended use of the hSCs is implantation in the human body, diagnosis of disease, or drug testing aimed at targeting any aspect of SC function in human patients. To conclude, in view of their unique properties, new concepts and methods are needed to better understand the biology of hSCs and exploit their full potential in basic science and regenerative medicine.