Cord Blood-derived Hematopoietic Progenitor Research Articles

Combinatorial gene targeting in primary human hematopoietic stem and progenitor cells

The CRISPR/Cas9 system offers enormous versatility for functional genomics but many applications have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, to establish a paradigm for multiplexed gene editing in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs), we used co-delivery of lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The fluorescent markers allow for tracking of either single- or double-edited cells, and we could achieve robust double knockout of the cell surface molecules CD45 and CD44 with an efficiency of ~ 70%. As a functional proof of concept, we demonstrate that this system can be used to model gene dependencies for cell survival, by simultaneously targeting the cohesin genes STAG1 and STAG2. Moreover, we show combinatorial effects with potential synergy for HSPC expansion by targeting the Aryl Hydrocarbon Receptor (AHR) in conjunction with members of the CoREST complex. Taken together, our traceable multiplexed CRISPR/Cas9 system enables studies of genetic dependencies and cooperation in primary HSPCs, and has important implications for modelling polygenic diseases, as well as investigation of the underlying mechanisms of gene interactions.

3029 – COMBINATORIAL GENE TARGETING IN PRIMARY HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

The CRISPR/Cas9 system offers enormous versatility for functional genomics. However, many applications, such as combinatorial perturbations for disease modelling and studies of gene interactions, have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, we addressed the challenge of multiplexed gene perturbations in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs) by co-delivering lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The use of fluorescent markers allows for tracing of single- or double-edited cells and we achieved robust double knockout of the cell surface markers CD45 and CD44 with an efficiency of around 70%. As a functional proof of concept, we demonstrated the use of this system to study gene dependencies for cell survival by simultaneously targeting the cohesin genes STAG1 and STAG2, as a model of synthetic lethality. Furthermore, we showed that this system can be used to study cooperative genetic events, and found a combinatorial effect with potential synergy for HSPC expansion when targeting the Aryl Hydrocarbon Receptor (AHR) in combination with members of the epigenetic modifier CoREST. Taken together, we established a paradigm for multiplexed CRISPR/Cas9 editing in primary human HSPCs, which enables studies of genetic dependencies and cooperation. This traceable multiplexed system has important implications for modelling polygenic diseases and for investigating gene interactions. The CRISPR/Cas9 system offers enormous versatility for functional genomics. However, many applications, such as combinatorial perturbations for disease modelling and studies of gene interactions, have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, we addressed the challenge of multiplexed gene perturbations in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs) by co-delivering lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The use of fluorescent markers allows for tracing of single- or double-edited cells and we achieved robust double knockout of the cell surface markers CD45 and CD44 with an efficiency of around 70%. As a functional proof of concept, we demonstrated the use of this system to study gene dependencies for cell survival by simultaneously targeting the cohesin genes STAG1 and STAG2, as a model of synthetic lethality. Furthermore, we showed that this system can be used to study cooperative genetic events, and found a combinatorial effect with potential synergy for HSPC expansion when targeting the Aryl Hydrocarbon Receptor (AHR) in combination with members of the epigenetic modifier CoREST. Taken together, we established a paradigm for multiplexed CRISPR/Cas9 editing in primary human HSPCs, which enables studies of genetic dependencies and cooperation. This traceable multiplexed system has important implications for modelling polygenic diseases and for investigating gene interactions.

Alginate-Chitosan Microencapsulated Cells for Improving CD34+ Progenitor Maintenance and Expansion

Protocols for isolation, characterization, and transplantation of hematopoietic stem cells (HSCs) have been well established. However, difficulty in finding human leucocyte antigens (HLA)-matched donors and scarcity of HSCs are still the major obstacles of allogeneic transplantation. In this study, we developed a double-layered microcapsule to deliver paracrine factors from non-matched or low-matched HSCs to other cells. The umbilical cord blood-derived hematopoietic progenitor cells, identified as CD34+ cells, were entrapped in alginate polymer and further protected by chitosan coating. The microcapsules showed no toxicity for surrounding CD34+ cells. When CD34+ cells-loaded microcapsules were co-cultured with bare CD34+ cells that have been collected from unrelated donors, the microcapsules affected surrounding cells and increased the percentage of CD34+ cell population. This study is the first to report the potency of alginate-chitosan microcapsules containing non-HLA-matched cells for improving proliferation and progenitor maintenance of CD34+ cells.

Chimeric Antigen Receptor (CAR) T Cell Therapy for CD7-Positive Acute Myeloid Leukemia

Expression of CD7 is an adverse prognostic factor in both AML and myelodysplastic syndromes. Because CD7 is not constitutively expressed by mature myeloid cells or early myeloid progenitor cells, we explored the feasibility of targeting these malignancies using T cells expressing a CD7-directed chimeric antigen receptor (CAR7). Due to high expression of CD7 on normal T cells, expression of CAR7 induced substantial fratricide of T cells, preventing their expansion and long term survival. We therefore disrupted the CD7 gene in T cells using CRISPR/Cas9 prior to transduction with the CAR. Electroporation of the Cas9 protein with CD7-specific gRNA produced little toxicity to T cells, and the resulting CD7KOCAR7 T cells (henceforth CAR7 T cells) had no evident fratricide, expanding >300-fold within two weeks.Upon coculture with primary AML blasts, CAR7 T cells eliminated a mean 85% of malignant cells within 48h (Fig. A) and expanded 5-fold. Furthermore, CAR7 T cells effectively targeted primitive AML progenitor cells resulting in a mean >25-fold decrease in the number of leukemic colonies after a 5h coculture (Fig. B). CAR7 T cells demonstrated robust cytotoxicity against CD7+ AML cell lines eliminating >95% of malignant cells after 72h of coculture at a 1:40 effector-to-target ratio but had no activity against CD7-negative tumor cells. To test the protective effect in vivo, we established a mouse xenograft model of disseminated AML by intravenous injection of the CD7+ AML cell line KG-1a. A single injection of 2x10^6 CAR7 T cells produced a >2-log reduction in tumor burden and significantly extended median survival (65 days in control group; median survival not reached at 88 days for the CAR7 T treated animals, P=0.0025). In a second, more aggressive AML model using Kasumi-3 cells, we observed a 2-log reduction in tumor volume following administration of CAR7 T cells.CD7 may also be transiently expressed in normal early myeloid progenitors. To assess the off-tumor hematotoxicity of CAR7 T cells, we cocultured them with cord blood-derived hematopoietic progenitor cells and measured colony formation on a methylcellulose medium. We observed no differences in the numbers of granulocytic, monocytic and erythrocytic colonies after exposure to CAR7 T or control non-transduced T cells indicating a lack of toxicity to normal myeloid and erythroid progenitor cells (Fig. C). Similarly, we observed no reactivity of CAR7 T cells against peripheral monocytes. However, CAR7 T cells were cytotoxic to normal peripheral T- and NK-cells suggesting potential aplasia of these lymphocyte subsets subsequent to in vivo administration of the CAR7 T cells. We may be able to circumvent this toxicity, by infusing pathogen-directed CD7KO T cells from the same donor to repopulate the host, since CD7KO cells appear to retain TCR-mediated activity against a range of pathogens. Hence, CD7KO T cells expressing a CD7-directed CAR can expand without fratricide and have high activity against AML while sparing normal myeloid cells. These data support the feasibility of this approach for the targeted therapy of AML. [Display omitted] DisclosuresLulla:ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding; Leukemia Texas: Membership on an entity's Board of Directors or advisory committees.

Extracellular vesicles from human iPSCs enhance reconstitution capacity of cord blood-derived hematopoietic stem and progenitor cells

Cord blood (CB) represents a source of hematopoietic stem and progenitor cells (CB-HSPCs) for bone marrow (BM) reconstitution, but clinical CB application is limited in adult patients due to the insufficient number of CB-HSCPCs and the lack of effective ex vivo approaches to increase CB-HSPC functionality. Since human-induced pluripotent stem cells (hiPSCs) have been indicated as donor cells for bioactive extracellular vesicles (EVs) modulating properties of other cells, we are the first to employ hiPSC-derived EVs (hiPSC-EVs) to enhance the hematopoietic potential of CB-derived CD45dimLin-CD34+ cell fraction enriched in CB-HSPCs. We demonstrated that hiPSC-EVs improved functional properties of CB-HSPCs critical for their hematopoietic capacity including metabolic, hematopoietic and clonogenic potential as well as survival, chemotactic response to stromal cell-derived factor 1 and adhesion to the model components of hematopoietic niche in vitro. Moreover, hiPSC-EVs enhanced homing and engraftment of CB-HSPCs in vivo. This phenomenon might be related to activation of signaling pathways in CB-HSPCs following hiPSC-EV treatment, as shown on both gene expression and the protein kinases activity levels. In conclusion, hiPSC-EVs might be used as ex vivo modulators of CB-HSPCs capacity to enhance their functional properties and augment future practical applications of CB-derived cells in BM reconstitution.

Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

2020 – ACTIVATION OF THE RECEPTOR TYROSINE KINASE (RET) BY GDNF/GFRa1 IMPROVES CORD BLOOD-DERIVED HSC IN VITRO EXPANSION AND IN VIVO ENGRAFTMENT

Allogeneic hematopoietic stem cell (HSC) transplantation is the gold standard therapy for numerous hematological disorders, but limitations exist in HSC number available per donor and our relative inability to successfully expand transplantable HSCs in vitro. In recent years, promising new molecules have been discovered, which are able to expand HSCs in vitro and improve transplantation in vivo in immunodeficient mouse models and non-human primates (e.g. SR1 and UM171). Here we demonstrate that acute activation of the receptor tyrosine kinase, RET, by its key ligand and co-receptor (GDNF/GFRa1), significantly improves the outgrowth of HSCs in vitro and long-term engraftment in immunodeficient Kit-mutant (KitW41/W41) mice. The expression of RET at the cell surface of cord blood-derived hematopoietic stem and progenitor cells (HSPCs, CD34+CD38-REThi) enriches for stem cell frequency in limiting dilution transplantation assays (∼1 in 135), with CD34+CD38-RETlow cells showing low stem cell frequency with limited multilineage engraftment (∼1 in 531). Activation of RET in vitro with a single dose of GDNF/GFRa1 reduces apoptosis at early time points, leading to increased HSC expansion over a 7-day period (>4-fold increase vs control). Transplantation of GDNF/GFRa1 treated HSPCs significantly improves engraftment of primary mice, comparable to the previously published SR1/UM171 treatment, and combination treatment of GDNF/GFRa1 with SR1/UM171 improves engraftment compared to individual GDNF/GFRa1 or SR1/UM171 treatments alone. RET is a single pass transmembrane receptor tyrosine kinase, governing a variety of downstream signalling pathways. Interrogation of the dynamic kinome changes governed by RET in HSPCs, using PAMGene kinome array kinetic technology and single cell mass cytometry revealed key phosphorylation cascades responsive to GDNF/GFRa1 treatment. Key cascades including anti-apoptotic p53 phosphorylation, NFkB activation and reduced reactive oxygen species, revealed important cellular programs for HSC survival, outgrowth and engraftment activated by GDNF/GFRa1. Our results provide a promising new target to improve the outgrowth of transplantable HSCs for clinical use, with multiple downstream functions activated for successful transplantation.

HCMV Infection in a Mesenchymal Stem Cell Niche: Differential Impact on the Development of NK Cells versus ILC3.

Human cytomegalovirus (HCMV) is highly prevalent in most populations worldwide and has a major influence on shaping the human immune system. Natural killer (NK) cells are important antiviral effectors that adapt to HCMV infection by expansion of virus-specific effector/memory cells. The impact of HCMV infection on the development of NK cells and innate lymphoid cells (ILC) in general is less well understood. In this context, we have recently established a novel in vitro platform to study human NK cell development in a stem cell niche based on human bone marrow-derived mesenchymal stem cells (MSC). Here, the system was modified by infecting MSC with HCMV to study the influence of virus infection on NK/ILC development. We show that cord blood-derived hematopoietic progenitor cells are successfully differentiated into mature CD56+CD94+NKG2A+ NK cells on HCMV-infected MSC with significant higher anti-viral cytokine production compared to NK cells developing on non-infected MSC. Furthermore, the generation of ILC3, characterized by expression of the signature transcription factor RAR-related orphan receptor gamma (RORγt) and the production of IL-22, was strongly impaired by HCMV infection. These observations are clinically relevant, given that ILC3 are associated with protection from graft-versus-host disease (GvHD) following stem cell transplantation and HCMV reactivation in turn is associated with increased incidence of GvHD.

Activation of the Receptor Tyrosine Kinase (RET) By GDNF/GFRα1 Improves Cord Blood-Derived HSC in Vitro expansion and In Vivo engraftment

Allogeneic hematopoietic stem cell (HSC) transplantation is the gold standard therapy for numerous hematological disorders, but limitations exist in HSC number available per donor and our relative inability to successfully expand transplantable HSCs in vitro. In recent years, promising new molecules have been discovered, which are able to expand HSCs in vitro and improve transplantation in vivo in immunodeficient mouse models and non-human primates (e.g. SR1 and UM171). Here we demonstrate that acute activation of the receptor tyrosine kinase, RET, by its key ligand and co-receptor (GDNF/GFRα1), significantly improves the outgrowth of HSCs invitro and long-term engraftment in immunodeficient Kit-mutant (KitW41/W41) mice. The expression of RET at the cell surface of cord blood-derived hematopoietic stem and progenitor cells (HSPCs, CD34+CD38-REThi) enriches for stem cell frequency in limiting dilution transplantation assays (~1 in 135), with CD34+CD38-RETlow cells showing low stem cell frequency with limited multilineage engraftment (~1 in 531). Activation of RET in vitro with a single dose of GDNF/GFRα1 reduces apoptosis at early time points, leading to increased HSC expansion over a 7-day period (>4-fold increase vs control). Transplantation of GDNF/GFRα1 treated HSPCs significantly improves engraftment of primary mice, comparable to the previously published SR1/UM171 treatment, and combination treatment of GDNF/GFRα1 with SR1/UM171 improves engraftment compared to individual GDNF/GFRα1 or SR1/UM171 treatments alone. RET is a single pass transmembrane receptor tyrosine kinase, governing a variety of downstream signalling pathways. Interrogation of the dynamic kinome changes governed by RET in HSPCs, using PAMGene kinome array kinetic technology and single cell mass cytometry revealed key phosphorylation cascades responsive to GDNF/GFRα1 treatment. Key cascades including anti-apoptotic p53 phosphorylation, NFκB activation and increased CD44 cell surface expression, revealed important cellular programs for HSC survival, outgrowth and engraftment activated by GDNF/GFRα1. Our results provide a promising new target to improve the outgrowth of transplantable HSCs for clinical use, with multiple downstream functions activated for successful transplantation. Disclosures No relevant conflicts of interest to declare.

3105 - Functional Integrity and Gene Expression Profiles of Human Cord Blood-Derived Hematopoietic Stem and Progenitor Cells Generated in Vitro

3105 - Functional Integrity and Gene Expression Profiles of Human Cord Blood-Derived Hematopoietic Stem and Progenitor Cells Generated in Vitro

BMS 493 Modulates Retinoic Acid-Induced Differentiation During Expansion of Human Hematopoietic Progenitor Cells for Islet Regeneration.

Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cord blood-derived hematopoietic progenitor cells (HPCs), isolated by high aldehyde dehydrogenase activity (ALDHhi), have previously been shown to reduce hyperglycemia after intrapancreatic (iPan) transplantation into streptozotocin (STZ)-treated nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. However, these cells are rare and require ex vivo expansion to reach clinically applicable numbers for human therapy. Therefore, we investigated whether BMS 493, an inverse retinoic acid receptor agonist, could prevent retinoic acid-induced differentiation and preserve islet regenerative functions during expansion. After 6-day expansion, BMS 493-treated cells showed a twofold increase in the number of ALDHhi cells available for transplantation compared with untreated controls. Newly expanded ALDHhi cells showed increased numbers of CD34 and CD133-positive cells, as well as a reduction in CD38 expression, a marker of hematopoietic cell differentiation. BMS 493-treated cells showed similar hematopoietic colony-forming capacity compared with untreated cells, with ALDHhi subpopulations producing more colonies than low aldehyde dehydrogenase activity subpopulations for expanded cells. To determine if the secreted proteins of these cells could augment the survival and/or proliferation of β-cells in vitro, conditioned media (CM) from cells expanded with or without BMS 493 was added to human islet cultures. The total number of proliferating β-cells was increased after 3- or 7-day culture with CM generated from BMS 493-treated cells. In contrast to freshly isolated ALDHhi cells, 6-day expansion with or without BMS 493 generated progeny that were unable to reduce hyperglycemia after iPan transplantation into STZ-treated NOD/SCID mice. Further strategies to reduce retinoic acid differentiation during HPC expansion is required to expand ALDHhi cells without the loss of islet regenerative functions.

Functional Integrity and Gene Expression Profiles of Human Cord Blood-Derived Hematopoietic Stem and Progenitor Cells Generated In Vitro.

To date, different experimental strategies have been developed for the ex vivo expansion of human hematopoietic stem (HSCs) and progenitor (HPCs) cells. This has resulted in significant advances on the use of such expanded cells in transplantation settings. To this day, however, it is still unclear to what extent those stem and progenitor cells generated in vitro retain the functional and genomic integrity of their freshly isolated counterparts. In trying to contribute to the solving of this issue, in the present study we have selected and purified three different hematopoietic cell populations: HSCs (CD34+ CD38‐ CD45RA‐ CD71‐ Lin‐ cells), myeloid progenitor cells (CD34+ CD38+ CD45RA+ CD71‐ Lin‐ cells), and erythroid progenitor cells (CD34+ CD38+ CD45RA‐ CD71+ Lin‐ cells), obtained directly from fresh human umbilical cord blood (UCB) units or generated in vitro under particular culture conditions. We, then, compared their functional integrity in vitro and their gene expression profiles. Our results indicate that in spite of being immunophenotipically similar, fresh and in vitro generated cells showed significant differences, both in functional and genetic terms. As compared to their fresh counterparts, those HSCs generated in our culture system showed a deficient content of long‐term culture‐initiating cells, and a marked differentiation bias toward the myeloid lineage. In addition, in vitro generated HSCs and HPCs showed a limited expansion potential. Such functional alterations correlated with differences in their gene expression profiles. These observations are relevant in terms of HSC biology and may have implications in UCB expansion and transplantation. Stem Cells Translational Medicine 2018;7:602–614

Small Molecule Inhibition of LARG/RhoA Signaling Blocks Migration of ETV6-RUNX1 Positive B-Cell Precursor Acute Lymphoblastic Leukemia

Background and ObjectivesTranslocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the favorable prognosis associated with ETV6-RUNX1 positive ALL, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. Leukemic cells reside in the bone marrow microenvironment, where they are nurtured and protected against chemotherapy. In this study, we investigated novel ways to disrupt this leukemic niche by targeting signaling pathways contributing to the migration of ETV6-RUNX1 positive leukemic cells.ResultsGene expression profiling and subsequent pathway analysis of leukemic blasts of 654 ALL patients revealed a significant enrichment of genes involved in regulation of cellular movement and cell morphology in ETV6-RUNX1 positive BCP-ALL patients compared with ETV6-RUNX1 negative BCP-ALL patients (p < 0.001). In correspondence, the same pathways were significantly upregulated in cord blood-derived hematopoietic progenitor cells (CB-CD34+) ectopically expressing ETV6-RUNX1 (p < 10E-06). LARG (ARHGEF12) was identified to be the most important regulator of this pro-migratory signature. This gene encodes for the G-protein-regulated Rho Guanine Exchange Factor 12, a specific activator of the GTPase RhoA. LARG expression was 5.7-fold higher in ETV6-RUNX1 positive BCP-ALL cells than in ETV6-RUNX1 negative BCP-ALL cells (p < 10E-06). Similarly, LARG was upregulated 5.4-fold in CB-CD34+ cells expressing ETV6-RUNX1 compared with empty vector controls (p = 0.03).To determine the importance of the LARG/RhoA pathway in the induction of this migratory phenotype, we used two recently identified small molecule inhibitors of the LARG/RhoA pathway: Y16, a specific LARG inhibitor, and G04 (Rhosin), a specific RhoA inhibitor. Both inhibitors significantly reduced the migration of ETV6-RUNX1 positive leukemic cells towards a gradient of CXCL12 (75%, p < 0.01) whereas the migration potential of ETV6-RUNX1 negative leukemic cells remained unaffected. Migration of ETV6-RUNX1 positive leukemic cells towards patient derived mesenchymal stromal cells was inhibited to a similar extent (75-85%, p < 0.01). In contrast, LARG/RhoA inhibition in ETV6-RUNX1 negative cells resulted in an induction of migration towards MSCs. While LARG/RhoA inhibitors reduced migration of ETV6-RUNX1 positive cells in a targeted manner, inhibition of CXCR4 by the CXCR4 antagonist AMD3100 (Plerixafor) reduced the migration of both ETV6-RUNX1 positive cells and ETV6-RUNX1 negative BCP-ALL cells.Next, we studied the additional cellular effects of LARG/RhoA inhibition on leukemic cells. shRNA-mediated silencing of LARG modestly reduced the proliferation rate of both ETV6-RUNX1 positive and ETV6-RUNX1 negative cell lines (p<0.05). This reduced proliferation rate was accompanied by a moderate induction of apoptosis (p < 0.05). Using a multiplexed cell signalling assay, we further show that silencing of LARG moderately reduced the phosphorylation level of CREB (S133, 25%, p<0.05), while the phosphorylation state of other key signaling proteins was unaffected (including PI3K/PKB, MAPK/ERK, JAK/STAT and NFκB). In line with these data, inhibition of LARG/RhoA signaling with small molecule inhibitors did not affect cell survival of primary ETV6-RUNX1 positive BCP-ALL cells or resistance of these cells to Prednisolone or L-Asparaginase.ConclusionTogether, our data indicate that small molecule inhibition of LARG/RhoA signaling affects the migration potential of ETV6-RUNX1 positive cells. In contrast to inhibition of the CXCR4/CXCL12 axis that reduces the migration of both leukemic and healthy hematopoietic cells, inhibition of the LARG/RhoA signaling pathway appears to specifically inhibit ETV6-RUNX1 positive cell migration. Our results warrant further studies to establish the clinical benefit of disrupting the leukemic niche in ETV6-RUNX1 positive BCP-ALL patients using small molecule inhibitors of LARG/RhoA signaling. DisclosuresNo relevant conflicts of interest to declare.

PRC2 inhibition counteracts the culture-associated loss of engraftment potential of human cord blood-derived hematopoietic stem and progenitor cells.

Cord blood hematopoietic stem cells (CB-HSCs) are an outstanding source for transplantation approaches. However, the amount of cells per donor is limited and culture expansion of CB-HSCs is accompanied by a loss of engraftment potential. In order to analyze the molecular mechanisms leading to this impaired potential we profiled global and local epigenotypes during the expansion of human CB hematopoietic stem and progenitor cells (HPSCs). Human CB-derived CD34+ cells were cultured in serum-free medium together with SCF, TPO, FGF, with or without Igfbp2 and Angptl5 (STF/STFIA cocktails). As compared to the STF cocktail, the STFIA cocktail maintains in vivo repopulation capacity of cultured CD34+ cells. Upon expansion, CD34+ cells genome-wide remodel their epigenotype and depending on the cytokine cocktail, cells show different H3K4me3 and H3K27me3 levels. Expanding cells without Igfbp2 and Angptl5 leads to higher global H3K27me3 levels. ChIPseq analyses reveal a cytokine cocktail-dependent redistribution of H3K27me3 profiles. Inhibition of the PRC2 component EZH2 counteracts the culture-associated loss of NOD scid gamma (NSG) engraftment potential. Collectively, our data reveal chromatin dynamics that underlie the culture-associated loss of engraftment potential. We identify PRC2 component EZH2 as being involved in the loss of engraftment potential during the in vitro expansion of HPSCs.

Targeted Therapy for ETV6-RUNX1-Driven B-Cell Precursor Acute Lymphoblastic Leukemia

Background: Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the favorable prognostic parameters of this B-ALL subgroup, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. The molecular mechanisms underlying ETV6-RUNX1-driven leukemia are largely unknown. Increased knowledge of these mechanisms is essential to develop novel therapeutic strategies to selectively target ETV6-RUNX1-positive leukemia.Objectives: This study aims to identify and target the molecular drivers behind ETV6-RUNX1-positive BCP-ALL.Results: Gene expression profiling of leukemic blasts of 654 ALL patients revealed that the class III PI3-kinase Vps34, an important regulator of autophagy, was exclusively up-regulated in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (2.7-fold; p ≤ 10-30). In addition, ectopic expression of ETV6-RUNX1 in cord blood-derived hematopoietic progenitor cells (CB-HPCs) significantly induced expression of Vps34 1.3-fold already 40 hours after transduction (p ≤ 0.05). This suggests that the Vps34-autophagy pathway is activated by ETV6-RUNX1, which may mechanistically explain the leukemogenic and pro-survival properties ascribed to ETV6-RUNX1. In correspondence, Ingenuity Pathway Analysis (IPA) predicted a pro-survival and pro-proliferative phenotype in ETV6-RUNX1 transduced CB-HPCs and highlighted a network of up-regulated transcription factors, including HEY1, EGR1, GATA1 and GATA2 (2 – 25-fold up-regulation; p ≤ 0.05). Luciferase reporter assays revealed that not only the ETV6-RUNX1 fusion protein, but also the ETV6-RUNX1-induced target genes HEY1, EGR1 and GATA1 positively regulate Vps34 promoter activity (5 – 13-fold up-regulation; p ≤ 0.01).Lentiviral knockdown experiments were performed to elucidate the importance of Vps34 expression in ETV6-RUNX1-positive BCP-ALL cells. Knockdown of all Vps34 transcript variants, with two independent constructs, led to complete growth arrest of the ETV6-RUNX1-positive cell lines REH and AT2, while this only led to a decrease in proliferation of the ETV6-RUNX1-negative cell line NALM6. This growth arrest was caused by a significant induction of apoptosis (more than 4-fold 7 days after transduction; p ≤ 0.001) and a significantly reduced percentage of cycling cells (1.3-fold 7 days after transduction; p ≤ 0.05). Analysis of p62 protein expression by western blot and reverse phase protein arrays revealed that the levels of autophagy were significantly higher in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (p ≤ 0.001). In addition, knockdown of ETV6-RUNX1 and Vps34 significantly reduced autophagy, quantified with confocal microscopy, in ETV6-RUNX1-positive cells with 50% and 84%, respectively (p ≤ 0.01). Furthermore, pharmacological inhibition of autophagy with hydroxychloroquine (HCQ) significantly reduced cell viability of BCP-ALL cell lines and primary patient-derived BCP-ALL cells (p ≤ 0.001). Treatment of the ETV6-RUNX1-positive BCP-ALL cell lines REH and AT2 with 20 µg/mL HCQ resulted in a 82% and 95% reduced cell viability, while the viability of ETV6-RUNX1-negative BCP-ALL cell lines and T-ALL cell lines were reduced to a lesser extent (NALM6: 43%; TOM-1: 50%; Loucy: 40%; Jurkat: 0%). Importantly, HCQ selectively sensitized ETV6-RUNX1-positive leukemic cells to L-asparaginase treatment in clinically relevant concentrations. Treatment of primary ETV6-RUNX1-positive patient cells with 10 µg/mL HCQ resulted in a 70% reduction in cell survival during L-asparaginase exposure (p ≤ 0.01). This sensitization was not observed in ETV6-RUNX1-negative BCP-ALL cells.Conclusion: The ETV6-RUNX1 fusion protein activates autophagy via Vps34, which is essential for survival and proliferation of ETV6-RUNX1-positive cells. Inhibition of autophagy in primary ETV6-RUNX1-positive leukemic cells inhibited cell survival and sensitized these cells to L-asparaginase treatment. These results indicate that autophagy inhibition may provide a novel means to sensitize L-asparaginase-resistant ETV6-RUNX1-positive BCP-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

CAPE promotes the expansion of human umbilical cord blood-derived hematopoietic stem and progenitor cells in vitro

Due to the low number of collectable stem cells from single umbilical cord blood (UCB) unit, their initial uses were limited to pediatric therapies. Clinical applications of UCB hematopoietic stem and progenitor cells (HSPCs) would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity. In recent years, numerous attempts have been made to expand human UCB HSPCs in vitro. In this study, we report that caffeic acid phenethyl ester (CAPE), a small molecule from honeybee extract, can promote in vitro expansion of HSPCs. Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells. Importantly, culture of CD34(+) HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units. Burst-forming unit-erythroid was the mostly affected colony type, which increased more than 3.7-fold in 1 μg mL(-1) CAPE treatment group when compared to the controls. CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α. Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.

Vasoactive intestinal polypeptide suppresses proliferation of human cord blood-derived hematopoietic progenitor cells by increasing TNF-α and TGF-β production in the liver.

The physiology of hepatic hematopoiesis is largely unknown, although studies have indicated that vasoactive intestinal polypeptide (VIP) is involved in this disease. To validate this hypothesis, we assessed the effects of VIP on human cord blood CD34+ cells. We also measured VIP levels and the capacity of vasoactive intestinal polypeptide receptor (VIPR) to bind to VIP in the rat liver during different developmental phases. VIP inhibited the proliferation of cord blood-derived CD34(+) cells from concentrations of 10-7-10-12 M. The highest suppression was achieved with 10-8 M VIP at day 10. Intracellular levels of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 in CD34(+) cells treated with VIP were increased by 50.70 and 43.46%, respectively. Variations in VIP levels in the rat fetal liver generally increased rapidly with the stage of fetal development. In addition, the affinity of VIPR for VIP increased from relatively low levels in the rat fetal liver and peaked at birth, after which it gradually decreased. VIP had a suppressive effect on the proliferation of human cord blood-derived CD34(+) cells, partially by increasing the production of TNF-α and TGF-β. Low VIP levels in the fetal liver and gradually increasing levels after birth may in part be responsible for suppressing hematopoietic stem cell and progenitor proliferation in the liver.

Concise Review: Ex Vivo Expansion of Cord Blood-Derived Hematopoietic Stem and Progenitor Cells: Basic Principles, Experimental Approaches, and Impact in Regenerative Medicine

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play key roles in the production of mature blood cells and in the biology and clinical outcomes of hematopoietic transplants. The numbers of these cells, however, are extremely low, particularly in umbilical cord blood (UCB); thus, ex vivo expansion of human UCB-derived HSCs and HPCs has become a priority in the biomedical field. Expansion of progenitor cells can be achieved by culturing such cells in the presence of different combinations of recombinant stimulatory cytokines; in contrast, expansion of actual HSCs has proved to be more difficult because, in addition to needing recombinant cytokines, HSCs seem to deeply depend on the presence of stromal cells and/or elements that promote the activation of particular self-renewal signaling pathways. Hence, there is still controversy regarding the optimal culture conditions that should be used to achieve this. To date, UCB transplants using ex vivo-expanded cells have already been performed for the treatment of different hematological disorders, and although results are still far from being optimal, the advances are encouraging. Recent studies suggest that HSCs may also give rise to nonhematopoietic cells, such as neural, cardiac, mesenchymal, and muscle cells. Such plasticity and the possibility of producing nonhematopoietic cells at the clinical scale could bring new alternatives for the treatment of neural, metabolic, orthopedic, cardiac, and neoplastic disorders. Once standardized, ex vivo expansion of human HSCs/HPCs will surely have a positive impact in regenerative medicine.

Ex vivo expanded human cord blood-derived hematopoietic progenitor cells induce lung growth and alveolarization in injured newborn lungs

BackgroundWe investigated the capacity of expanded cord blood-derived CD34+ hematopoietic progenitor cells to undergo respiratory epithelial differentiation ex vivo, and to engraft and attenuate alveolar disruption in injured newborn murine lungs in vivo.MethodsRespiratory epithelial differentiation was studied in CD34+ cells expanded in the presence of growth factors and cytokines (“basic” medium), in one group supplemented with dexamethasone (“DEX”). Expanded or freshly isolated CD34+ cells were inoculated intranasally in newborn mice with apoptosis-induced lung injury. Pulmonary engraftment, lung growth and alveolarization were studied at 8 weeks post-inoculation.ResultsSP-C mRNA expression was seen in 2/7 CD34+ cell isolates expanded in basic media and in 6/7 isolates expanded in DEX, associated with cytoplasmic SP-C immunoreactivity and ultrastructural features suggestive of type II cell-like differentiation. Administration of expanding CD34+ cells was associated with increased lung growth and, in animals treated with DEX-exposed cells, enhanced alveolar septation. Freshly isolated CD34+ cells had no effect of lung growth or remodeling. Lungs of animals treated with expanded CD34+ cells contained intraalveolar aggregates of replicating alu-FISH-positive mononuclear cells, whereas epithelial engraftment was extremely rare.ConclusionExpanded cord blood CD34+ cells can induce lung growth and alveolarization in injured newborn lungs. These growth-promoting effects may be linked to paracrine or immunomodulatory effects of persistent cord blood-derived mononuclear cells, as expanded cells showed limited respiratory epithelial transdifferentiation.

The AAA+ ATPase RUVBL2 is a critical mediator of MLL-AF9 oncogenesis

The most frequent chromosomal translocations in pediatric acute myeloid leukemia affect the 11q23 locus and give rise to mixed lineage leukemia (MLL) fusion genes, MLL-AF9 being the most prevalent. The MLL-AF9 fusion gene has been shown to induce leukemia in both mouse and human models. In this study, we demonstrate that leukemogenic activity of MLL-AF9 requires RUVBL2 (RuvB-like 2), an AAA+ ATPase family member that functions in a wide range of cellular processes, including chromatin remodeling and transcriptional regulation. Expression of RUVBL2 was dependent on MLL-AF9, as it increased upon immortalization of human cord blood-derived hematopoietic progenitor cells with the fusion gene and decreased following loss of fusion gene expression in conditionally immortalized mouse cells. Short hairpin RNA-mediated silencing experiments demonstrated that both the immortalized human cells and the MLL-AF9-expressing human leukemia cell line THP-1 required RUVBL2 expression for proliferation and survival. Furthermore, inhibition of RUVBL2 expression in THP-1 cells led to reduced telomerase activity and clonogenic potential. These data were confirmed with a dominant-negative Walker B-mutated RUVBL2 construct. Taken together, these data suggest the possibility of targeting RUVBL2 as a potential therapeutic strategy for MLL-AF9-associated leukemia.