Cord Blood Hematopoietic Progenitor Cells Research Articles

In vivo and in vitro effects of cord blood hematopoietic stem and progenitor cell (HSPC) expansion using valproic acid and/or nicotinamide

BackgroundHigh self-renewal capacity and most permissive nature of umbilical cord blood (CB) results with successful transplant outcomes but low hematopoietic stem and progenitor cell (HSPC) counts limits wider use. In order to overcome this problem ex vivo expansion with small molecules such as Valproic acid (VPA) or Nicotinamide (NAM) have been shown to be effective. To the best of our knowledge, the combinatory effects of VPA and NAM on HSPC expansion has not been studied earlier. The aim of this study was to analyze ex vivo and in vivo efficacy of VPA and NAM either alone or in combination in terms of expansion and engraftment. MethodsA total of 44 CB units were included in this study. To determine the ex vivo and in vivo efficacy, human CB CD34+ cells were expanded with VPA and/or NAM and colony forming unit (CFU) assay was performed on expanded HSPC. Xenotransplantation was performed simultaneously by intravenous injection of expanded HSPC to NOD-SCID gamma (NSG) mice (n = 22). Significance of the difference between the expansion groups or xenotransplantation models was analyzed using t-test, Mann-Whitney, ANOVA or Kruskal-Wallis tests as appropriate considering the normality of distributions and the number of groups analyzed. ResultsIn vitro CD34+ HSPC expansion fold relative to cytokines-only was significantly higher with VPA compared to NAM [2.23 (1.07–5.59) vs 1.48 (1.00–4.40); p < 0.05]. Synergistic effect of VPA+NAM has achieved a maximum relative expansion fold at 21 days (D21) of incubation [2.95 (1.00–11.94)]. There was no significant difference between VPA and VPA+NAM D21 (p = 0.44). Fold number of colony-forming unit granulocyte-macrophage (CFU-GM) colonies relative to the cytokine-only group was in favor of NAM compared to VPA [1.87 (1.00–3.59) vs 1.00 (1.00–1.81); p < 0.01]. VPA+NAM D21 [1.62 (1.00–2.77)] was also superior against VPA (p < 0.05). There was no significant difference between NAM and VPA+NAM D21. Following human CB34+ CB transplantation (CBT) in the mouse model, fastest in vivo leukocyte recovery was observed with VPA+NAM expanded cells (6 ± 2 days) and the highest levels of human CD45 chimerism was detectable with VPA-expanded CBT (VPA: 5.42 % at day 28; NAM: 2.45 % at day 31; VPA+NAM 1.8 % at day 31). ConclusionOur study results suggest using VPA alone, rather than in combination with NAM or NAM alone, to achieve better and faster expansion and engraftment of CB HSPC.

Investigation of CD83 As a Novel Human Hematopoietic Stem Cell Inflammatory Activation Marker

Hematopoietic stem cells (HSC) are responsible for life-long blood production and must balance homeostasis with sudden demand situations such as infections. However, the heterogeneity of molecular programs engaged by acute inflammatory stimuli to activate emergency hematopoiesis at the level of HSC are poorly understood. In a recent study linking human HSC heterogeneity to aging, recovery from severe COVID-19 infection, and clonal hematopoiesis (CH), we discovered two HSC subsets through multiomic analysis, denoted HSC-I and -II, in a xenograft model of inflammation-recovery. While HSC-I had few inflammation-driven molecular changes, HSC-II showed sustained epigenetic and transcriptional alterations in response to dual tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS) challenges despite a 2.5-month recovery period. Importantly, a signature of long-term memory T cells (Akondy et al 2017) was enriched in HSC-II (Zeng, Nagree, et al, in submission). We interrogated the human HSC multiomic signatures uncovered from this human xenograft model to identify potential regulators associated with HSC response to and recovery from acute inflammation. We identified CD83 as a candidate that is differentially expressed between our HSC subsets. Similarly, we found CD83 expression to be enriched in a subset of adult-derived HSCs from patients where CH status was established and enrichment of an HSC-II signature observed (Jakobsen et al, submitted). CD83, first identified as a dendritic cell marker, is expressed on many immune cells, with linkage to controlling murine T regulatory (T-Reg) vs memory cell fate (Doebbeler et al, 2018), but has not previously been shown to be relevant for human HSC biology. Flow cytometry analysis of cord blood (CB) hematopoietic stem and progenitor cells (HSPC) showed CD83 to be low or absent in HSC (Figure A) and their downstream progeny at steady state. However, robust CD83 expression was observed in multiple HSPC subpopulations including long-term (LT-) and short-term (ST-) HSC within 16 hours of ex vivo culture, with highest expression in LT-HSC (Figure A). Importantly, CD83 surface expression on LT-HSC was enhanced following culture with TNFα (Figure A). Bystander effects were excluded by demonstrating similar findings after culture of purified HSC populations. Acute in vivo LPS treatment of mice xenografted with CB-derived HSC was sufficient to upregulate CD83 surface expression after 16 hours on LT-HSC/HSPC. To elucidate the potential function of CD83-expressing HSC/multipotent progenitors (MPP), CD19-CD34+CD38-CD45RA- cells were sorted from xenografts following acute inflammatory challenge for low and high CD83 expression and subjected to bulk RNA sequencing. We found enrichment of a gene expression signature specific to activated human HSCs (Garcia-Prat et al, 2021) within CD83-positive HSC/MPP (Figure B). Strikingly, enrichment of a signature of stimulated vs resting T cells (Galetti et al, 2020) and of effector T cells (Akondy et al 2017) was found in CD83-positive compared to CD83-negative HSC/MPP (Figure B). T cell effector function is in part dictated by metabolic reprogramming. We thus examined reactive oxygen species (ROS) levels in LT-HSC following ex vivo culture with TNFα and found lower ROS levels with CD83 expression. We therefore hypothesize that CD83 may be an ‘effector’ marker defining a distinct HSC subset during homeostatic or inflammatory activation that is subsequently fated to become memory-like HSC-II. We are undertaking studies to determine the mechanistic role of CD83 in HSC function and HSC-II formation via Cas9-sgRNA-mediated knockout or lentivirus-mediated overexpression of CD83. In summary, CD83 represents a long-sought marker of inflammatory activation at the HSC level. CD83 may have significant clinical potential as a biomarker of aberrant activation in gene therapy manufacturing protocols or for HSC transplantation. Future studies will shed light on whether CD83 can functionally regulate the molecular programs underlying human HSC heterogeneity.

Vitamin C derivative/AA2P promotes erythroid differentiation by upregulating CA1

Abstract Vitamin C is used to treat anaemia; however, the mechanism through which vitamin C promotes erythroid differentiation is not comprehensively understood. The in vitro erythroid differentiation induction system can reveal the differentiation mechanism and provide erythrocytes for clinical transfusion and anaemia treatment. This process can be promoted by adding small-molecule compounds. In this study, we added l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P), a derivative of vitamin C, to an erythroid differentiation system induced from umbilical cord blood haematopoietic stem and progenitor cells in vitro and detected its effect on erythroid differentiation using single-cell transcription sequencing technology combined with non-targeted metabolism detection. AA2P increased the proportion of late basophilic erythroblasts, upregulating the expression of erythroid-related regulatory molecules GATA1, KLF1, ALAS2, and the globins HBG and HBB. CA1 is a target gene of AA2P, and CA1 knockdown affected the expression of globin-related genes. AA2P also increased glycolysis and decreased oxidative phosphorylation to facilitate terminal erythroid differentiation and enhanced the proliferation of early erythroid progenitors by altering the cell cycle. These results provide a reliable basis for using vitamin C to improve the efficiency of erythropoiesis in vitro and for the clinical treatment of anaemia.

Abstract 26 The Role of Oxygen in Cord Blood Hematopoietic Stem and Progenitor Cell Expansion and Engraftment

Abstract Introduction Hematopoietic stem (HSC) and progenitor cells (HPCs) are exposed to differing oxygen tensions ranging from &amp;lt;1% to 21% as they reside in/move through different tissues or are harvested for clinical utility. Functional changes in HSCs/HPCs are induced by acute changes in oxygen tension (e.g., a change in percent of cells in cycle). Objectives We sought to determine if variable oxygen levels affect expansion and/or functional properties of cord blood (CB) HSCs/HPCs ex vivo and in vivo. Methods Human CB CD34+ cells were grown in expansion culture +/-UM171, an agonist of HSC self-renewal that expands transplantable CB HSCs, in five oxygen tensions: 1%O2, 3%O2, 5%O2, 14%O2, and 21%O2. HSCs/HPCs were enumerated by flow cytometry. Functional HPCs were enumerated by plating in semi solid media for colony forming unit assays (CFU). Cell cycle and reactive oxygen species (ROS) were measured by flow cytometry. Ability of expanded cells to engraft was determined by transplantation in non-lethally irradiated NSG mice. Results Immunophenotypic HPCs and functional HPC CFUs expanded significantly more after 7 days of growth in higher oxygen tensions (5%O2-21%O2) compared to lower (1%O2-3%O2), while immunophenotypic HSCs expanded best at 5% O2. HSCs/HPCs grown in low oxygen tensions had significantly lower ROS levels, significantly higher percentage of cells in G0, and were slightly but reproducibly smaller/less granular than those grown in high oxygen levels. HSC/HPC numbers were reduced in high oxygen tensions 1-2 days after plating but were better maintained in low, suggesting cells undergo a culture shock/stress after plating that is mitigated by reduced oxygen. In the presence of UM171, HSCs expanded significantly better at higher oxygen levels, but HPCs are better maintained in 5%O2. Ex vivo CD34+ expansions maintained under physiological O2 levels (1-14%O2) demonstrated significantly better/faster neutrophil recovery following transplantation compared to cells expanded at 21%O2 or input. Discussion HSCs/HPCs proliferate rapidly in high oxygen but have fewer quiescent cells, higher ROS, and are larger and more granular which are all characteristics associated with exhaustion. While high oxygen allows for faster growth, low tensions may mitigate cell stress and allow for prolonged growth (i.e., HSC/HPC expansion) while maintaining functional properties.

CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

BML-260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability.

Hematopoietic stem cells (HSCs), which are multipotent and have the ability to self-renew, are frequently used in the treatment of hematological diseases and cancer. Small molecules that target HSC quiescence regulators could be used for ex vivo expansion of both mobilized peripheral blood (mPB) and umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPC). We identified and investigated 35 small molecules that target HSC quiescence factors. We looked at how they affected HSC activity, such as expansion, quiescence, multilineage capacity, cycling ability, metabolism, cytotoxicity, and genotoxicity. A transplantation study was carried out on immunocompromised mice to assess the expanded cells' repopulation and engraftment abilities. 4-[(5Z)-5-benzylidene-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]benzoic acid (BML)-260 and tosyl-l-arginine methyl ester (TAME) significantly increased both mPB and UCB-HSPC content and activated HSC re-entry into the cell cycle. The improved multilineage capacity was confirmed by the colony forming unit (CFU) assay. Furthermore, gene expression analysis revealed that BML-260 and TAME molecules aided HSC expansion by modulating cell cycle kinetics, such as p27, SKP2, and CDH1. In addition to these in vitro findings, we discovered that BML-260-expanded HSCs had a high hematopoietic reconstitution capacity with increased immune cell content after xenotransplantation into immunocompromised mice. In addition to the BML-260 molecule, a comparison study of serum-containing and serum-free chemically defined media, including various supplements, was performed. These in vitro and xenotransplantation results show that BML-260 molecules can be used for human HSC expansion and regulation of function. Furthermore, the medium composition discovered may be a novel platform for human HSPC expansion that could be used in clinical trials.

Maternal lipid profiles vs. fetal growth and cord blood hematopoietic cells: weak associations in healthy Korean newborn-mother pairs.

We aimed to define the maternal lipid profiles that are associated with fetal growth and cord blood (CB) hematopoietic cells in healthy Korean full-term newborns. A total of 608 fetal-maternal pairs were enrolled; mothers voluntarily donated CB with informed consent. We analyzed birth weight (BW) as a marker of fetal growth, and we examined total nucleated cells (TNCs) and CD34+ cell concentrations of CB as markers of hematopoietic progenitor cell (HPC) contents. We also analyzed maternal lipid levels and investigated their associations with BW, TNCs and CD34+ cells. Maternal triglycerides (TG) showed a significant positive association with BW and CD34+ cells, and low-density lipoprotein (LDL) showed a negative association with BW and CD34+ cells. Though not statistically significant, higher maternal TG showed a tendency toward higher levels of TNCs. Maternal TG was independently and positively correlated with BW, and maternal LDL was independently and negatively correlated with CD34+ cells, although the impacts were not as strong, as indicated by small beta coefficients (0.157 and -0.226, respectively). We were able to investigate the association of maternal lipid profiles with BW and CB HPCs in healthy Korean newborn-mother pairs in this study. Both BW and the HPC contents showed independent associations with maternal TG and LDL, although the effect of maternal lipid levels on fetal growth and HPCs was not strong in the normal healthy population. Because maternal lipid levels were assessed once in the healthy fetal-maternal pairs, we could not investigate those associations across pregnancy.

Human Hematopoietic Progenitor Cells and Erythrocytes Generated from Human iPSCs Lacking CD55

CD55 (OMIM*125240; also known as Decay-Accelerating Factor for complement or DAF) is a plasma membrane protein that is widely distributed on all blood cells and many other cell types. A critical physiologic role of CD55 is to inhibit the complement cascade at the level of the central C3 convertase step. Variation in CD55 (GPI-anchored) forms the basis of the Cromer blood group system (CROM). CD55 also serves as a receptor for certain strains of E. coli and certain types of enteroviruses. More recently, CD55 was identified as a receptor for Plasmodium falciparum, a deadly malaria parasite that infects human erythrocytes. This finding was reached by experiments of gene knock-down using RNA interference in human cord blood hematopoietic progenitor cells that were subsequently differentiated into erythrocytes (1). In cells with lower expression of CD55, there were lower rates of infection. CD55 null erythrocytes from patient samples were refractory to infection, implying that CD55 is essential for invasion. We decided to use the erythrocytes generated from human induced pluripotent stem cells (iPSCs), which can be expanded without limit and efficiently genetically modified in order to obtain cell lines completely lacking CD55 expression. This novel approach is feasible because we previously established a methodology (2014 ASH meeting presentation) for generating human erythrocytes after terminal differentiation of iPSC-derived hematopoietic cells, in which 5-10% of the cells became enucleated (2). In this study, we found that a lab strain of P. falciparum parasites is able to invade and propagate in the iPSC-derived erythrocytes.To knock out the CD55 gene and generate large numbers of erythrocytes that are resistant to the infection of P. falciparum parasites, we used two different CRISPR guide RNAs as well as SpCas9 in human iPSCs. We successfully isolated and expanded human iPSC clones that have 85-bp deletion in CD55 exon 1 of both alleles, resulting in the frameshift and the disappearance of CD55 expression on cell surface. To assure that the CRISPR mediated the knock-out (KO) is specific to the CD55 gene, we used a doxycycline-inducible expression vector to deliver a CD55 transgene. Upon doxycycline induction, CD55 cell surface expression was restored in the selected CD55 KO iPSC lines. The complete CD55 KO had little effects in the growth of human iPSCs or their differentiation into hematopoietic progenitor cells that co-express CD45 and CD34. However, we observed that erythroid differentiation was reduced in the absence of CD55 expression. This observation contradicts the previous publication where CD55 RNA interference technology was likely incomplete (1). The inducible CD55 expression system we constructed will be able to overcome the requirement for CD55 before the formation of mature erythrocytes and invasion by P. falciparum parasites.The present study provides a novel genetic model for studying the infection of P. falciparum malaria parasites into human erythrocytes, based on the robust technologies of genome-edited human iPSCs and derived erythrocytes. The genetically modified erythrocytes such ones lacking CD55 expression may provide a novel source of P. falciparum resistant erythrocytes for blood transfusion in areas that malaria is epidemic and many people are anemic. This study will also provide a proof-of-principle that we can generate a large number of human erythrocytes with gain of function from a small number of genetically modified stem cells.

Targeting Mitochondrial Translation Overcomes Venetoclax Resistance in Acute Myeloid Leukemia (AML) through Activation of the Integrated Stress Response

Venetoclax is a highly specific BCL-2 inhibitor with promising activity against AML including leukemic stem cells (LSCs). However, venetoclax monotherapy in AML patients had limited clinical efficacy due to intrinsic or acquired drug resistance which has been attributed to the overexpression of other anti-apoptotic proteins including MCL-1 and BCL-xL. These findings highlight the importance of combination therapy to overcome resistance but the optimal strategy to achieving this goal is unclear.To address this issue, we generated a panel of highly resistant clones derived from the intrinsically sensitive MOLM-13 and MV-4-11 AML cell lines. Surprisingly, treatment with MCL-1 or BCL-xL inhibitors failed to overcome resistance despite higher expression of the anti-apoptotic proteins in resistant cells. To identify novel targets, we performed a genome-wide CRISPR knockout screen to find genes that upon inactivation, restore sensitivity to venetoclax. We transduced one of the resistant clones engineered to stably express Cas9 with a pooled lentiviral single-guide RNA (sgRNA) library that targets 17,661 protein-coding genes with 91,320 unique sgRNA sequences. Transduced cells were divided into 2 populations with one treated with venetoclax and the other untreated as control. The transduced cells were cultured for 29 days to negatively select sgRNAs that re-sensitized resistant cells to venetoclax. Cell samples were collected at regular intervals and subjected to next-generation sequencing of the sgRNA target region to quantify the abundance of each construct.We identified genes that were negatively selected in the presence of venetoclax using the MAGeCK algorithm. The top-ranked genes were highly enriched for gene ontology terms related to mitochondrial translation. We confirmed that RNAi-mediated knockdown of the top-ranked ribosomal subunit gene, DAP3, overcame venetoclax resistance. Pharmacologic inhibition of mitochondrial translation with a panel of protein synthesis inhibitor antibiotics similarly restored sensitivity in AML cell lines with intrinsic or acquired resistance. Tedizolid, a FDA-approved second generation oxazolidinone antibiotic, was the most effective in mediating this effect at clinically relevant concentrations. Furthermore, the combination of tedizolid and venetoclax targeted the LSC-enriched CD34+CD38- population in ex vivo -cultured primary AML samples. Importantly, normal cord blood hematopoietic stem and progenitor cells were more resistant to this combination suggestive of a wide therapeutic index .To decipher the mechanism by which tedizolid overcomes venetoclax resistance, we first profiled the expression of BCL-2 family members in total cellular and purified mitochondrial extracts. Antibiotic treatment did not affect total BCL-2, MCL-1, or BCL-xL protein levels but caused a substantial increase in BCL-2 levels in the mitochondrial fraction indicative of BCL-2 translocation to the organelle. Next, we confirmed that tedizolid treatment selectively reduced the expression of mitochondrial over nuclear-encoded proteins consistent with its inhibitory effect on mitochondrial translation. This mitonuclear protein imbalance has been shown to activate the integrated stress response (ISR). Tedizolid treatment activated this response as evidenced by an increase in eIF2-α phosphorylation and expression of ATF4, a master transcription factor regulator. To determine whether ISR activation is sufficient to overcome venetoclax resistance, we treated resistant cells with a small-molecule activator of PERK (CCT020312), which activates the ISR through direct eIF2-α phosphorylation, and observed re-sensitization to venetoclax.In summary, our results demonstrate that inhibition of mitochondrial translation, which activates ISR and triggers BCL-2 translocation to the mitochondria, is a novel approach to overcoming venetoclax resistance in AML cells. Our findings provide the rationale for exploring the use protein synthesis inhibitor antibiotics or direct ISR activators in combination with venetoclax for the treatment of AML or other malignancies. DisclosuresNo relevant conflicts of interest to declare.

The Bioactive Peptide SL-13R Expands Human Umbilical Cord Blood Hematopoietic Stem and Progenitor Cells In Vitro.

Hematopoietic stem and progenitor cell (HSPC) transplantation is a curative treatment of hematological disorders that has been utilized for several decades. Although umbilical cord blood (UCB) is a promising source of HSPCs, the low dose of HSPCs in these preparations limits their use, prompting need for ex vivo HSPC expansion. To establish a more efficient method to expand UCB HSPCs, we developed the bioactive peptide named SL-13R and cultured UCB HSPCs (CD34+ cells) with SL-13R in animal component-free medium containing a cytokine cocktail. Following 9 days of culture with SL-13R, the numbers of total cells, CD34+, CD38− cells, and hematopoietic stem cell (HSC)-enriched cells were significantly increased relative to control. Transplantation of cells cultured with SL-13R into immunodeficient NOD/Shi-scid/IL-2Rγ knockout mice confirmed that they possess long-term reconstitution and self-renewal ability. AHNAK, ANXA2, and PLEC all interact with SL-13R. Knockdown of these genes in UCB CD34+ cells resulted in reduced numbers of hematopoietic colonies relative to SL-13R-treated and non-knockdown controls. In summary, we have identified a novel bioactive peptide SL-13R promoting expansion of UCB CD34+ cells with long-term reconstitution and self-renewal ability, suggesting its clinical use in the future.

RXR Negatively Regulates Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem and Progenitor Cells.

Ex vivo expansion of human cord blood (CB) hematopoietic stem cells (HSCs) is one approach to overcome limited numbers of HSCs in single CB units. However, there is still no worldwide acceptable HSC ex vivo expansion system. A main reason is that we still have very limited knowldege regarding mechanisms underlying maintenance and expansion of CB HSCs. Here we report that retinoid X receptor (RXR) activity is of significance for CB HSC ex vivo expansion. RXR antagonist HX531 significantly promoted ex vivo expansion of CB HSCs and progenitor cells (HPCs). RXR agonist Bexarotene notably suppressed ex vivo expansion of CB HSCs. Activation of RXR by Bexarotene significantly blocked expansion of phenotypic HSCs and HPCs and expressed increased functional HPCs as assessed by colony formation induced by UM171 and SR1. In vivo transplantation experiments in immune-deficient mice demonstrated that HX531 expanded CB HSCs possess long-term reconstituting capacities, and Bexarotene treatment inhibited expansion of functional CB HSCs. RNA-seq analysis revealed that RXR regulates expression of FBP1 (a negative regulator of glucose metabolism) and many genes involved in differentation. ECAR analysis showed that HX531 significantly promoted glycolytic activity of CB CD34+ HSCs and HPCs. Our studies suggest that RXR is a negative regulator of ex vivo expansion of CB HSCs and HPCs.

Engineering of fully humanized and vascularized 3D bone marrow niches sustaining undifferentiated human cord blood hematopoietic stem and progenitor cells

Hematopoietic stem and progenitor cells (HSPCs) are frequently located around the bone marrow (BM) vasculature. These so-called perivascular niches regulate HSC function both in health and disease, but they have been poorly studied in humans due to the scarcity of models integrating complete human vascular structures. Herein, we propose the stromal vascular fraction (SVF) derived from human adipose tissue as a cell source to vascularize 3D osteoblastic BM niches engineered in perfusion bioreactors. We show that SVF cells form self-assembled capillary structures, composed by endothelial and perivascular cells, that add to the osteogenic matrix secreted by BM mesenchymal stromal cells in these engineered niches. In comparison to avascular osteoblastic niches, vascularized BM niches better maintain immunophenotypically-defined cord blood (CB) HSCs without affecting cell proliferation. In contrast, HSPCs cultured in vascularized BM niches showed increased CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) numbers. The vascularization also contributed to better preserve osteogenic gene expression in the niche, demonstrating that niche vascularization has an influence on both hematopoietic and stromal compartments. In summary, we have engineered a fully humanized and vascularized 3D BM tissue to model native human endosteal perivascular niches and revealed functional implications of this vascularization in sustaining undifferentiated CB HSPCs. This system provides a unique modular platform to explore hemato-vascular interactions in human healthy/pathological hematopoiesis.

The transcription factor ETS1 is an important regulator of human NK cell development and terminal differentiation

AbstractNatural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation–sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.

The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes

AbstractAlthough cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

Arterial endothelium creates a permissive niche for expansion of human cord blood hematopoietic stem and progenitor cells

BackgroundAlthough cord blood (CB) offers promise for treatment of patients with high-risk hematological malignancies and immune disorders, the limited numbers of hematopoietic stem cell (HSC)/progenitor cell in a CB unit and straitened circumstances in expanding ex vivo make it quite challenging to develop the successful cell therapies.MethodsIn this study, a novel strategy has been developed to support ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) by coculture with engineered human umbilical arterial endothelial cells (HuAECs-E4orf1-GFP), which expresses E4ORF1 stably by using a retroviral system.ResultsCoculture of CD34+ hCB cells with HuAECs-E4orf1-GFP resulted in generation of considerably more total nucleated cells, CD34+CD38−, and CD34+CD38−CD90+ HSPCs in comparison with that of cytokines alone or that of coculture with human umbilical vein endothelial cells (HuVECs) after 14-day amplification. The in vitro multilineage differentiation potential and in vivo repopulating capacity of the expanded hematopoietic cells cocultured with HuAECs-E4orf1-GFP were also markedly enhanced compared with the other two control groups. DLL4, a major determinant of arterial endothelial cell (EC) identity, was associated with CD34+ hCB cells amplified on HuAECs-E4orf1-GFP.ConclusionsCollectively, we demonstrated that HuAECs acted as a permissive niche in facilitating expansion of HSPCs. Our study further implicated that the crucial factors and related pathways presented in HuAECs may give a hint to maintain self-renewal of bona fide HSCs.

Gene editing using CRISPR enables FOXP3 gene repair in HSPCs and IPEX patient T cells

Background & Aim Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is the prototypical primary immune regulatory disorder caused by mutations in the forkhead box protein 3 (FOXP3) gene, a critical transcription factor required for regulatory T cells (Tregs). Patients typically present with severe early onset autoimmune manifestations that can be fatal within the first year of life. Currently, the only cure for IPEX is allogeneic hematopoietic stem cell transplant (HSCT), but this comes with risk of complications and the survival probability at 15 years is 73.2% (F. Barzaghi et al 2018). Moreover, a suitable donor is not always available. As a monogenic immune disease with limited treatment options, IPEX is an ideal candidate for a gene therapy approach whereby patient hematopoietic stem cells are gene edited and reinfused for autologous transplant. Methods, Results & Conclusion We developed a CRISPR/Cas9 approach combined with AAV delivery of a donor template to restore FOXP3 expression at the endogenous locus, permitting regulated expression of wild-type FOXP3 irrespective of downstream mutations. We were able to edit CD4+ effector T cells (Teff) and Tregs, showing that they maintain characteristic phenotypic markers and are functional in vitro. IPEX patient Tregs edited with the construct had partial restoration of both FOXP3 expression and functional suppression upon co-culture with responder T cells. Gene edited cord blood hematopoietic stem and progenitor Cells (HSPCs) are capable of long-term engraftment in humanized mice and differentiate into multiple hematopoietic subsets (CD13, CD19, CD56, CD3) and partially reconstitute Tregs (CD4+CD25+FOXP3+). These results demonstrate the feasibility of using gene edited HSPCs and hold promise for autologous transplant in genetic autoimmune diseases such as IPEX syndrome.

Human models of NUP98-KDM5A megakaryocytic leukemia in mice contribute to uncovering new biomarkers and therapeutic vulnerabilities

AbstractAcute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (<3 years of age). It remains plagued with extremely poor treatment outcomes (<40% cure rates), mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60% to 70% of cases and include nucleoporin 98 (NUP98) gene rearrangements, most commonly NUP98-KDM5A. Human models of NUP98-KDM5A–driven AMKL capable of faithfully recapitulating the disease have been lacking, and patient samples are rare, further limiting biomarkers and drug discovery. To overcome these impediments, we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest, sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A–transformed cells into immunodeficient mice led to multiple subtypes of leukemia, including AMKL, that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP, MPIG6B, and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib, a clinically approved JAK2 inhibitor. Overall, synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia, which lack effective and rationally designed treatments.

A stemness screen reveals C3orf54/INKA1 as a promoter of human leukemia stem cell latency

AbstractThere is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

MicroRNA-21 over expression in umbilical cord blood hematopoietic stem progenitor cells by leukemia microvesicles.

Microvesicles are able to induce the cell of origin’s phenotype in a target cell. MicroRNA-21, as an oncomir, is up-regulated in almost all cancer types such as leukemia which results in cell proliferation. In this study, we examine the ability of leukemia microvesicles to induce proliferation in hematopoietic stem progenitor cells (HSPCs) via microRNA-21 dysregulation. Herein, leukemia microvesicles were isolated from HL-60 and NB-4 cell lines by ultracentrifugation, and then their protein content was measured. Normal HSPCs were isolated from umbilical cord blood samples by a CD-34 antibody. These cells were treated with 20 and 40 μg/mL leukemia microvesicles for 5 and 10 days, respectively. Cell count, CD-34 analysis, and a microRNA-21 gene expression assay were done at days 5 and 10. HSPCs showed a significant increase in both microRNA-21 gene expression and cell count after treating with leukemia microvesicles compared with the control group. CD-34 analysis as stemness proof did not show any difference among the studied groups. This data suggests that HSPC proliferation followed by microRNA-21 gene over expression can be another evidence of a leukemia-like phenotype induction in a healthy target cell by leukemia microvesicles.

Identification of Gene Regulatory Networks Governing Stemness Properties of Human HSC and LSC

The gene regulatory networks (GRN) governing maintenance and expansion of normal and leukemic human hematopoietic stem-cells (HSC and LSC) are not well understood. Typically, GRNs are inferred from gene expression (GE) data of a limited subset of pre-selected genes implicated to be relevant to the cell types being studied. Such data are commonly derived from relatively homogeneous cell populations or cell lines, which do not reflect the heterogeneity of primary human samples. Importantly, there are currently no GRNs that directly interrogate the transcriptional circuitry controlling human HSC/LSC. To gain insight into the determinants of stem cell function in human HSC/LSC, we developed a unique method for building GRNs that employs GE and chromatin accessibility (ATAC-Seq) data derived from n=17 highly purified human umbilical cord blood hematopoietic stem and progenitor cell populations (hUCB-HSPC) and n=64 functionally-validated LSC-enriched and LSC-depleted cell fractions sorted from AML patient samples. Estimates of HSC/LSC frequencies based on limiting dilution xenotransplantation assays were also incorporated with statistical learning approaches to infer GRN models. Specifically, we determined transcription factor (TF) motif occurrence in HSC/LSC-enriched open chromatin regions near genes that are more highly expressed in stem versus non-stem profiles (P<0.05) to identify TF-target gene interactions in HSCs and LSCs. The effect of specific TF binding on target GE was modelled using statistical regression. A database comprising n=8,927 and n=7,916 HSC and LSC specific TF-target gene relationships, respectively, was constructed. Importantly, only a small set of n=95 TF-target gene interactions overlapped between HSC and LSC, suggesting divergent regulatory rules governing stemness maintenance, as well as differential downstream effects upon targeting of specific genes.Self-sustaining transcriptional loops between subsets of TFs were detected in HSC (ETS1, EGR1, RUNX2, FOSL1, ZNF274, ZNF683) and LSC (MEIS1, FOXK1) data, representing core regulatory hubs that are likely to be important to the maintenance of the HSC/LSC state. To determine how each gene in the transcriptome may interact with the core HSC and LSC networks, n=284,606 protein-protein interactions (PPI) between n=16,540 proteins were analyzed to define n=103,516 shortest PPI pathways connecting to the core HSC/LSC TFs. Statistical regression guided by functional data was used to identify likely HSC/LSC-relevant PPI pathway activity scores, defined as weighted combinations of constituent pathway component GE values, that were highly correlated to HSC/LSC frequency estimates from xenotransplantation assays. This generated 2 lists of n=9,948 and n=45,063 HSC- and LSC-relevant PPI pathways, respectively. We next analyzed these putative HSC/LSC-relevant pathways for points of perturbation (i.e. through gene knockdown (KD) or overexpression (OE)) that could lead to changes in stemness pathway activity scores and therefore potential HSC expansion or LSC eradication, resulting in a catalogue comprising n=976 and n=3,819 HSC and LSC targets, respectively. Prediction of several anti-LSC targets, including CDK6, XPO1, mir-126, CD47, and CD123, was supported by serial xenotransplantation data from our group and others. Furthermore, the HSC GRN correctly predicted increased HSC frequency as a consequence of mir-126 or CDK6 KD, or addition of a PROCR agonist to HSC-enriched hUCB or bone marrow. These functional validations of several GRN predictions support the overall validity of our model and accuracy of untested predictions.Collectively, we report a comprehensive resource for exploring the gene regulatory wiring and extended protein interactions that define the functional state of human HSC and LSC. The constructed GRNs can also serve as an in-silico screening platform for the systematic identification of gene/protein targets that can be exploited for clinical applications, including HSC expansion and LSC eradication. DisclosuresTakayama:Megakaryon co. Ltd.: Research Funding. Zandstra:ExCellThera: Equity Ownership.