Expansion Of Hematopoietic Progenitor Cells Research Articles

A Transient Induction of Blood-Circulating Bile Acid Is a Therapeutic Marker and Target of Chemotherapy-Oriented Myelosuppression

Rapid recovery of normal blood production is essential for the completion of chemotherapy for different types of solid tumors and leukemia's. However, chemotherapeutic agents can damage bone marrow cells and cause myelosuppression, which is one of the serious side effects of the cancer treatment. It is hard to predict in which patients this will be problematic but this results in a delay in the completion of the treatment protocol. An early, reliable, marker of successful recovery of blood production during cycles of chemotherapy would thus be highly beneficial.Under normal steady state conditions concentrations of bile acids (BA) in peripheral blood are low. However, in several liver diseases and critically ill patients circulating BA levels rise substantially. Some studies even argue that the levels of BA are correlated to clinical outcome and mortality. In contrast, we have previously reported that a specific BA, tauroursodeoxycholic acid (TUDCA), has a beneficial role as a molecular chaperone in maintenance and expansion of hematopoietic stem and progenitor cells (HSPC). More recently, we have also seen that BA in mouse PB are increased during hematopoietic stress (irradiation, LPS injections and 5-FU treatment). Circulating BA levels may thus have a more clinical relevance in hematopoiesis than previously has been recognized. At present the cause and precise role of increased BA levels during hematopoietic stress are unknown. In light of these findings we hypothesized that in the rebuild of the blood system after stress, circulating BA levels may be an indicator of recovery and they may have an unknown role in the blood recovery process.Initially, we measured levels of BA in pediatric patients in treatment for different types of childhood cancers. These measurements showed clearly that circulating BA levels are upregulalted during recovery after chemotherapy. This was especially seen during the recovery phase from myelosuppression. Furthermore, there was a significant, positive, correlation between increased BA levels and favorable clinical outcome in these patients. Mass spectrometry analyses showed that the main component of the increased BA was glycochenodeoxycholic acid (GCDCA), glycoursodeoxycholic acid (GUDCA) and GUDCA-3S. To replicate these results in a mouse setting we used 5-FU injections and examined PB, plasma and BM cells at different time points. In the nadir (day 3 to day 6) in the mouse, BA levels were increased moderately but during BM recovery (day 6 to day 12) the levels were highly and significantly increased.To assess if ursodeoxycholic acid (UDCA) plays a role during hematopoietic recovery, we injected TUDCA into 5-FU treated mice. PB and BM analyses revealed that TUDCA treated mice showed more rapid and better recovery. Since T/G/UDCA are known for their ability to suppress an induction of endoplasmic reticulum (ER) stress, 5-FU treated mice were also treated with an ER stress inhibitor, Salubrinal, and the mice showed similar or better degree of recovery. These findings strongly indicate that the induction of specific types of BA can serve as a supportive factor in blood recovery possibly through chaperone function and suppression of ER stress.We also tested if the induced BA upon 5-FU treatment has a biological meaning in mice, 5-FU treated mice were injected with a chemical compound, GW4064, which suppresses BA production. However, the suppression of BA levels did not impact PB and BM profiles. This might be due to the fact that the UDCA is rarely detected in rodents even under stress and compositions of BA are largely different between human and rodents.During critical illness glucocorticoids (GC) are known to increase substantially and the induction of circulating BA might be reflecting the GC levels. We have seen that the concentration of GC is increased at similar timing with BA induction after 5-FU treatment. In support of this, injection of corticosterone to mice also increased the level of circulating BA. Our data suggests that the BA increase upon chemotherapy is a consequence of the elevation of GC levels, possibly through the alteration of production and/or reabsorption of BA.To conclude, BA in the circulating blood is a potential marker to assess the recovery timing of chemotherapy-treated patients. Moreover, medication of TUDCA/GUDCA may be a novel optional treatment to boost-up the recovery from myelosuppresive conditions. DisclosuresNo relevant conflicts of interest to declare.

Manipulating Osteogenic/Adipogenic Balance in Mesenchymal Stromal Cells (MSCs) for Hematopoietic Applications

Aims: Bone marrow-derived mesenchymal stromal cells (BM-MSCs) have the potential to improve hematopoietic transplantation by supporting hematopoietic stem and progenitor cell (HSPC) expansion as well as by promoting HSPC engraftment to the BM. However, culture expanded MSCs becomes a mixture of progenitor cells committed to different cell lineages, each with different potencies for any one desired application. Clinically, the accumulation of adipogenic MSCs in the BM is believed to contribute towards age-related impairment of regeneration and hematopoiesis; this observation, together with our understanding of the inverse relationship between MSC osteogenic and adipogenic differentiation prompted a more in depth study of the ability of these different MSC lineages to support hematopoietic clinical applications. Clinically relevant methods for engineering a pure cell population from heterogeneous MSC culture will also be discussed.Methods: MSCs were derived from the healthy BM of patients aged 30-50 years. At various stages of osteogenic and adipogenic differentiation in vitro , cultures were evaluated for their ability to support cord blood (CB) CD34+ HSC expansion and stimulate regenerative processes such as angiogenesis and cell proliferation. qPCR, coupled with secretome analysis was performed to discover gene expression markers for MSCs with potent hematopoietic supportive capabilities. Finally, due to the lack of unique surface markers for identification of MSC subpopulations, we utilized multivariate biophysical analysis (microfluidic sorting, atomic force microscopy, etc.), correlating these quantitative measures with biomolecular markers as well as in vitro and in vivo functionality. Novel label-free cell separation strategies that take advantage of unique biophysical traits in a potent MSC subpopulation for HSPC transplantation was also developed to enable clinical translation.Results: Differentiated osteoblasts (OB) as well as pre-OB demonstrated a higher ability to support CB CD34+ HSC expansion compared to undifferentiated MSCs or MSCs in the adipogenic state. Over a period of 10 days, the total HSPC fold expansion was 44.4 ± 4.83, 57.8 ± 10.67, 40.2 ± 4.65, 34.2 ± 6.18 and 13.8 ± 5.50 for OB, pre-OB, MSCs, pre-AD and AD, respectively (n = 5). Absolute expanded CD34+ HSCs was highest with pre-OB cultures (~1.7 fold, n = 5) compared to undifferentiated MSCs. Adipogenic MSCs had much reduced capabilities for supporting HSPC expansion. In vitro angiogenic and proliferation assays showed the greatest degree of HUVEC sprouting and proliferation (~ 2.6 fold greater than undifferentiated MSCs) when exposed to a pre-OB secretome. Pre-OBs are distinct in their transcriptome (up-regulated osteopontin, runx2, angiopoietin-1, FGF1, etc) but no surface marker reliably distinguishes them from other MSCs. We subsequently found a minimal set of three biophysical markers: cell diameter, membrane stiffness and nuclear membrane fluctuations, which collectively are predictive of MSC subpopulations (multipotent vs committed progenitors). We hypothesized and experimentally verified that a biophysically derived pre-OB subpopulation, defined biophysically as MSCs with >20 μm diameter, >375Pa stiffness and <1.2 % nuclear fluctuation, exhibits significantly greater efficacy over other MSC subpopulations for promoting bone marrow repair (~2-3 fold improvements in medial survival following BM injury) and expansion of HSPCs (~1.5-2 fold increase in HSPC numbers). Following co-transplantation with HSPCs, biophysically derived pre-OB promoted faster engraftment of donor cells in the peripheral blood by week 4 (~ 9% vs < 5% in other MSC groups, P<0.05).Conclusion: These results demonstrate a correlation between MSCs with high osteogenic activity and the capabilities for BM repair as well as hematopoietic support, making such cells ideal for augmentation of HSC transplantations. In the absence of reliable surface markers for isolation of these pre-OBs, we developed a clinically relevant biophysical isolation strategy for derivation of pre-OBs that can markedly improve the efficacy of MSC-based approaches for augmentation of hematopoietic transplants. DisclosuresNo relevant conflicts of interest to declare.

Inhibitors of Chemoresistance Pathways in Combination with Ara-C to Overcome Multidrug Resistance in AML. A Mini Review.

Acute myeloid leukemia (AML), the most common type of leukemia in older adults, is a heterogeneous disease that originates from the clonal expansion of undifferentiated hematopoietic progenitor cells. These cells present a remarkable variety of genes and proteins with altered expression and function. Despite significant advances in understanding the molecular panorama of AML and the development of therapies that target mutations, survival has not improved significantly, and the therapy standard is still based on highly toxic chemotherapy, which includes cytarabine (Ara-C) and allogeneic hematopoietic cell transplantation. Approximately 60% of AML patients respond favorably to these treatments and go into complete remission; however, most eventually relapse, develop refractory disease or chemoresistance, and do not survive for more than five years. Therefore, drug resistance that initially occurs in leukemic cells (primary resistance) or that develops during or after treatment (acquired resistance) has become the main obstacle to AML treatment. In this work, the main molecules responsible for generating chemoresistance to Ara-C in AML are discussed, as well as some of the newer strategies to overcome it, such as the inclusion of molecules that can induce synergistic cytotoxicity with Ara-C (MNKI-8e, emodin, metformin and niclosamide), subtoxic concentrations of chemotherapy (PD0332991), and potently antineoplastic treatments that do not damage nonmalignant cells (heteronemin or hydroxyurea + azidothymidine).

Mutation of Gemin5 Causes Defective Hematopoietic Stem/Progenitor Cells Proliferation in Zebrafish Embryonic Hematopoiesis.

Fate determination and expansion of Hematopoietic Stem and Progenitor Cells (HSPCs) is tightly regulated on both transcriptional and post-transcriptional level. Although transcriptional regulation of HSPCs have achieved a lot of advances, its post-transcriptional regulation remains largely underexplored. The small size and high fecundity of zebrafish makes it extraordinarily suitable to explore novel genes playing key roles in definitive hematopoiesis by large-scale forward genetics screening. Here, we reported a novel zebrafish mutant line gemin5cas008 with a point mutation in gemin5 gene obtained by ENU mutagenesis and genetic screening, causing an earlier stop codon next to the fifth WD repeat. Gemin5 is an RNA-binding protein with multifunction in post-transcriptional regulation, such as regulating the biogenesis of snRNPs, alternative splicing, stress response, and translation control. The mutants displayed specific deficiency in definitive hematopoiesis without obvious defects during primitive hematopoiesis. Further analysis showed the impaired definitive hematopoiesis was due to defective proliferation of HSPCs. Overall, our results indicate that Gemin5 performs an essential role in regulating HSPCs proliferation.

A single-cell resolution developmental atlas of hematopoietic stem and progenitor cell expansion in zebrafish

During vertebrate embryogenesis, fetal hematopoietic stem and progenitor cells (HSPCs) exhibit expansion and differentiation properties in a supportive hematopoietic niche. To profile the developmental landscape of fetal HSPCs and their local niche, here, using single-cell RNA-sequencing, we deciphered a dynamic atlas covering 28,777 cells and 9 major cell types (23 clusters) of zebrafish caudal hematopoietic tissue (CHT). We characterized four heterogeneous HSPCs with distinct lineage priming and metabolic gene signatures. Furthermore, we investigated the regulatory mechanism of CHT niche components for HSPC development, with a focus on the transcription factors and ligand-receptor networks involved in HSPC expansion. Importantly, we identified an endothelial cell-specific G protein-coupled receptor 182, followed by in vivo and in vitro functional validation of its evolutionally conserved role in supporting HSPC expansion in zebrafish and mice. Finally, comparison between zebrafish CHT and human fetal liver highlighted the conservation and divergence across evolution. These findings enhance our understanding of the regulatory mechanism underlying hematopoietic niche for HSPC expansion in vivo and provide insights into improving protocols for HSPC expansion in vitro.

Extracellular Vesicles Derived From Adult and Fetal Bone Marrow Mesenchymal Stromal Cells Differentially Promote ex vivo Expansion of Hematopoietic Stem and Progenitor Cells.

Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV vs. the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24 h treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

Comparative engraftment and clonality of macaque HSPCs expanded on human umbilical vein endothelial cells versus non-expanded cells

Ex vivo hematopoietic stem and progenitor cell (HSPC) expansion platforms are under active development, designed to increase HSPC numbers and thus engraftment ability of allogeneic cord blood grafts or autologous HSPCs for gene therapies. Murine and in vitro models have not correlated well with clinical outcomes of HSPC expansion, emphasizing the need for relevant pre-clinical models. Our rhesus macaque HSPC competitive autologous transplantation model utilizing genetically barcoded HSPC allows direct analysis of the relative short and long-term engraftment ability of lentivirally transduced HSPCs, along with additional critical characteristics such as HSPC clonal diversity and lineage bias. We investigated the impact of ex vivo expansion of macaque HSPCs on the engineered endothelial cell line (E-HUVECs) platform regarding safety, engraftment of transduced and E-HUVEC-expanded HSPC over time compared to non-expanded HSPC for up to 51 months post-transplantation, and both clonal diversity and lineage distribution of output from each engrafted cell source. Short and long-term engraftment were comparable for E-HUVEC expanded and the non-expanded HSPCs in both animals, despite extensive proliferation of CD34+ cells during 8 days of ex vivo culture for the E-HUVEC HSPCs, and optimization of harvesting and infusion of HSPCs co-cultured on E-HUVEC in the second animal. Long-term hematopoietic output from both E-HUVEC expanded and unexpanded HSPCs was highly polyclonal and multilineage. Overall, the comparable HSPC kinetics of macaques to humans, the ability to study post-transplant clonal patterns, and simultaneous multi-arm comparisons of grafts without the complication of interpreting allogeneic effects makes our model ideal to test ex vivo HSPC expansion platforms, particularly for gene therapy applications.

Embryonic Expression of NrasG 12 D Leads to Embryonic Lethality and Cardiac Defects.

Ras proteins control a complex intracellular signaling network. Gain-of-function mutations in RAS genes lead to RASopathy disorders in humans, including Noonan syndrome (NS). NS is the second most common syndromic cause of congenital heart disease. Although conditional expression of the NrasG12D/+ mutation in adult hematopoietic system is leukemogenic, its effects on embryonic development remain unclear. Here, we report that pan-embryonic expression of endogenous NrasG12D/+ by Mox2-Cre in mice caused embryonic lethality from embryonic day (E) 15.5 and developmental defects predominantly in the heart. At E13.5, NrasG12D/+; Mox2Cre/+ embryos displayed a moderate expansion of hematopoietic stem and progenitor cells without a significant impact on erythroid differentiation in the fetal liver. Importantly, the mutant embryos exhibited cardiac malformations resembling human congenital cardiac defects seen in NS patients, including ventricular septal defects, double outlet right ventricle, the hypertrabeculation/thin myocardium, and pulmonary valve stenosis. The mutant heart showed dysregulation of ERK, BMP, and Wnt pathways, crucial signaling pathways for cardiac development. Endothelial/endocardial-specific expression of NrasG12D/+ caused the cardiac morphological defects and embryonic lethality as observed in NrasG12D/+; Mox2Cre/+ mutants, but myocardial-specific expression of NrasG12D/+ did not. Thus, oncogenic NrasG12D mutation may not be compatible with embryonic survival.

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.

Сord blood hematopoietic stem cells ex vivo enhance the bipotential commitment of adipose mesenchymal stromal progenitors

AimsStroma-dependent ex vivo expansion of hematopoietic stem progenitor cells (HSPCs) is a valid approach for cell therapy needs. Our goal was to verify whether HSPCs can affect stromal cells to optimize their functions during ex vivo expansion. Main methodsHSPCs from cord blood (cb) were cocultured with growth-arrested adipose mesenchymal stromal cells (MSCs). Commitment-related transcriptional and secretory profiles as well as hematopoiesis-supportive activity of intact and osteo-induced MSCs were examined. Key findingsDuring expansion, cbHSPCs affected the functional state of MSCs, contributing to the formation of early stromal progenitors with a bipotential osteo-adipogenic profile. This was evidenced by the upregulation of certain MSC genes of osteo- and adipodifferentiation (ALPL, RUNX2, BGLAP, CEBPA, ADIPOQ), as well as by elevated alkaline phosphatase activity and altered osteoprotein patterns. Joint paracrine profiles upon coculture were characterized by a balance of “positive” (GM-SCF) and “negative” (IP-10, MIP-1α, MCP-3) myeloid regulators, effectively supporting expansion of both committed and primitive cbHSPCs. Short-term (72 h) osteoinduction prior to coculture resulted in more pronounced shift of the bipotential transcriptomic and osteoprotein profiles. The increased proportions of late primitive CD133−/CD34+cbHSPCs and unipotent CFUs suggested that cbHSPCs after expansion on osteo-MSCs were more committed versus cbHSPCs from coculture with non-differentiated MSCs. SignificanceDuring ex vivo expansion, cbHSPCs can drive the bipotential osteo-adipogenic commitment of MSCs, providing a specific hematopoiesis-supportive milieu. Short-term preliminary osteo-induction enhanced the development of the bipotential profile, leading to more pronounced functional polarization of cbHSPCs, which may be of interest in an applied context.

Loss of FGFR3 Accelerates Bone Marrow Suppression-Induced Hematopoietic Stem and Progenitor Cell Expansion by Activating FGFR1-ELK1-Cyclin D1 Signaling

Patients with chemotherapy or radiation therapy often generate anemia and low immunity due to the therapy-induced bone marrow (BM) suppression. To enhance hematopoietic regeneration during the therapy-induced BM suppression urgently need to be solved. Fibroblast growth factors (FGFs) play important regulatory roles in hematopoietic stem and progenitor cell (HSPC) expansion in vitro and in vivo by the FGF receptor (FGFR1-4)-mediated signaling pathway. FGFR3 is an important member of the FGFR family, and its regulatory function in hematopoiesis is largely unknown. Using knockout (KO) mice of FGFR3, we found that loss of FGFR3 does not affect HSPC functions or lineage differentiation during steady-state hematopoiesis, but FGFR3 deletion accelerates HSPC expansion and hematopoiesis recovery via a cell-autonomous manner under 5-fluorouracil-induced BM suppression. Our results showed that FGFR3 inactivation accelerates BM suppression-induced HSPC expansion by upregulating FGFR1 and its downstream transcriptional factor, ELK, which regulates the expression of the cyclin D1 gene attheleveloftranscription. Further studies confirmed that loss of FGFR3 in hematopoietic cells inhibits in vivo leukemogenesis under BM suppression. Our data found a novel hematopoietic regulatory mechanism by which FGFR3 deletion promotes HSPC expansion under BM suppression and also provided a promising approach to enhance antileukemia and hematopoietic regeneration by inhibiting FGFR3 functions in HSPCs combined with leukemic chemotherapy.

Ex Vivo Expansion of Adult Hematopoietic Stem and Progenitor Cells with Valproic Acid.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic stem cell transplantation but lack a matched donor. However, the limited number of HSCs within each UCB unit remains a major challenge for their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of human HSCs in ex vivo cultures initiated with CD34+ cells isolated from UCBs can overcome this limitation. The method described here utilizes a deacetylase inhibitor, valproic acid (VPA), to rapidly expand to a high degree the numbers of functional HSCs and committed progenitors (HPCs). The expanded HSCs are capable of establishing both short-term and long-term multilineage hematopoietic reconstitution. This highly reproducible and simple protocol can be also applied to expansion of both HSCs and HPCs from different sources including the bone marrow and peripheral blood.

The RNA Binding Protein IGF2BP3 Is Required for MLL-AF4 Mediated Leukemogenesis

Chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene are observed in acute lymphoblastic leukemias (ALL), acute myeloid leukemias (AML), and in rare mixed-lineage leukemia. Despite recent progress in therapeutic approaches, patients with MLL-rearranged (MLLr) leukemias still have very poor outcomes and a high risk of relapse. Of more than 90 fusion partner genes, MLL-AF4 is the most common MLL fusion protein in patients. Previously, we found that the RNA binding protein IGF2BP3 was specifically overexpressed in MLL-rearranged B-ALL, and enforced expression in vivo led to a pathologic expansion of hematopoietic stem and progenitor cells resulting in B and myeloid cell leukocytosis in the periphery. However, the requirement of IGF2BP3 in MLL-AF4 mediated leukemogenesis remains to be determined. Utilizing our previously generated list of differentially regulated targets with IGF2BP3 knockdown and a published dataset of MLL-Af4 targets, we determined that transcripts modulated by IGF2BP3 showed significant enrichment for MLL-Af4-bound genes. Furthermore, we observed that MLL-AF4 directly binds to and transcriptionally induces IGF2BP3. We performed ChIP-PCR assays on RS4;11 and SEM cell lines, human B-ALL cell lines that carry the MLL-AF4 translocation, and determined that the region in the first intron of IGF2BP3 is strongly bound by MLL-AF4. Furthermore, we observed a dose-dependent increase in luciferase reporter activity when we co-transfected a dual-luciferase reporter vector containing the promoter region of IGF2BP3 with increasing levels of MLL-AF4 expressing retroviral vector. To determine the role of Igf2bp3 in MLL-Af4 driven leukemogenesis, we generated the first Igf2bp3 KO murine model. Surprisingly, Igf2bp3 KO mice maintain normal, steady-state hematopoiesis. However, in striking contrast, deletion of Igf2bp3 in the MLL-Af4 leukemia model, significantly increases the survival of MLL-Af4 transplanted mice and greatly attenuates the disease. Furthermore, Igf2bp3 deficiency significantly reduced the tumor burden and disease severity. We observed significant decreases in WBC counts, spleen weights, and infiltrating leukemic cells visualized in histopathological analysis of hematopoietic tissues and quantified by FACS analysis. Moreover, deletion of Igf2bp3 led to a leukemia-initiating cell (LIC) disadvantage in vivo, demonstrated by significantly reduced engraftment in primary transplanted mice and reconstitution of secondary serially transplanted mice. To identify the transcripts directly regulated by Igf2bp3 in the context of MLL-Af4 driven leukemia, we carried out enhanced crosslinking and immunoprecipitation (eCLIP) transcriptome analysis of MLL-Af4 transformed early stem and progenitor cells and primary cells purified from splenic tumors of MLL-Af4 leukemic mice. We discovered an IGF2BP3-regulated post-transcriptional operon governing leukemic cell survival and proliferation, in which mRNA targets include the Hoxa locus and numerous genes within the Ras signaling pathway. In our study, we provide evidence that Igf2bp3 is required for the initiation of MLL-Af4 driven leukemia. We determined that Igf2bp3 is necessary for the development of and function of MLL-Af4 LICs. Mechanistically, we show that Igf2bp3 binds to and modulates the expression of hundreds of critical target transcripts. In summary, we demonstrate that Igf2bp3 is a positive regulator of MLLr leukemogenesis by targeting Hoxa transcripts such as Hoxa9 and numerous Ras signaling pathway transcripts, thereby controlling multiple downstream effector pathways required for disease initiation and aggressiveness. Together, our findings identify IGF2BP3 as an important, potential therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.

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.

Single-cell mutation analysis of clonal evolution in myeloid malignancies.

SummaryMyeloid malignancies, including acute myeloid leukemia (AML), arise from the expansion of hematopoietic stem/progenitor cells which acquire somatic mutations. Bulk molecular profiling suggests step-wise mutation acquisition, where mutant genes with high variant allele frequencies (VAFs) occur early in leukemogenesis and mutations with lower VAFs are thought to be acquired later1–3. Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate mutational order. To delineate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is dominated by a small number of clones, which frequently harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We next mapped clonal trajectories for each sample and uncovered mutation combinations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of single cell clonal architecture provides novel insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

Optimizing BIO feeding strategy promotes ex vivo expansion of human hematopoietic stem and progenitor cells

Exvivo expansion is critical in facilitating the application of hematopoietic/progenitor stem cells (HSPCs) for regenerative therapies. Wnt signaling is implicated in the expansion and self-renewal maintenance of HSPCs. However, a reasonable method to regulate Wnt signaling in exvivo cultures to achieve robust expansion of HSPCs has not yet been investigated. Here, cord blood-derived CD34+ cells were cultured with the activator of Wnt signaling 6-bromoindirubin-3'-oxime (BIO) under the following conditions: vehicle control (group A); BIO was added to the culture on days 0, 4, and 7 (group B); and BIO was added to the culture on days 0 and 7 (group C). Initial BIO treatment promoted the expansion of CD34+ cells on day 4. However, BIO supplementation on days 0 and 4 in group B attenuated HSPC expansion on day 7, while enhancing the multilineage commit potential and secondary expansion ability of expanded CD34+ cells. Based on this finding, an optimized BIO feeding strategy (group C) was proposed to support substantial expansion of HSPCs. After 10 days of culture, the expansion fold of CD34+ cells was 28.70 ± 0.46-folds, which was significantly higher than group A (16.20 ± 0.72-folds, p < 0.05). Moreover, the optimized BIO feeding strategy achieved increased primitive HSPC expansion without the loss of biological functions. Mechanistically, the optimized BIO feeding strategy avoided the excessive activation of Wnt observed in group B while maintaining a moderate level of intracellular β-catenin. These results provide an experimental and theoretical basis for Wnt regulation in exvivo culture process and a potential strategy to expand HSPCs for transplantation.

Metabolite Patterns in Human Myeloid Hematopoiesis Result from Lineage-Dependent Active Metabolic Pathways.

Assessment of hematotoxicity from environmental or xenobiotic compounds is of notable interest and is frequently assessed via the colony forming unit (CFU) assay. Identification of the mode of action of single compounds is of further interest, as this often enables transfer of results across different tissues and compounds. Metabolomics displays one promising approach for such identification, nevertheless, suitability with current protocols is restricted. Here, we combined a hematopoietic stem and progenitor cell (HSPC) expansion approach with distinct lineage differentiations, resulting in formation of erythrocytes, dendritic cells and neutrophils. We examined the unique combination of pathway activity in glycolysis, glutaminolysis, polyamine synthesis, fatty acid oxidation and synthesis, as well as glycerophospholipid and sphingolipid metabolism. We further assessed their interconnections and essentialness for each lineage formation. By this, we provide further insights into active metabolic pathways during the differentiation of HSPC into different lineages, enabling profound understanding of possible metabolic changes in each lineage caused by exogenous compounds.

Single-Cell Analysis of Neonatal HSC Ontogeny Reveals Gradual and Uncoordinated Transcriptional Reprogramming that Begins before Birth.

Fetal and adult hematopoietic stem cells (HSCs) have distinct proliferation rates, lineage biases, gene expression profiles, and gene dependencies. Although these differences are widely recognized, it is not clear how the transition from fetal to adult identity is coordinated. Here we show that murine HSCs and committed hematopoietic progenitor cells (HPCs) undergo a gradual, rather than precipitous, transition from fetal to adult transcriptional states. The transition begins prior to birth and is punctuated by a late prenatal spikein type I interferon signaling that promotes perinatal HPC expansion and sensitizes progenitors to the leukemogenic FLT3ITD mutation. Most other changes in gene expression and enhancer activation are imprecisely timed and poorly coordinated. Thus, heterochronic enhancer elements, and their associated transcripts, are activated independently of one another rather than as part of a robust network. This simplifies the regulatory programs that guide neonatal HSC/HPC ontogeny, but it creates heterogeneity within these populations.

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.

2012 – EXCESSIVE R-LOOPS TRIGGER AN INFLAMMATORY CASCADE LEADING TO ABERRANT HEMATOPOIETIC STEM AND PROGENITOR CELL EXPANSION

Aberrant expansion of hematopoietic stem and progenitor cells (HSPCs) is a major hallmark of myelodysplastic syndromes (MDS), although the underlying molecular regulation remains poorly understood. DEAD-box Helicase 41 (DDX41) is an understudied MDS-mutated factor whose function in hematopoiesis is unknown. Using an in vivo zebrafish model of Ddx41-deficiency, we unveiled a critical role for this helicase in regulating HSPCs. Mutants for ddx41 have more HSPCs than their siblings as measured by in situ hybridization and quantification by flow cytometry. We determined that in vivo Ddx41 suppresses the accumulation of R-loops, nucleic acid structures consisting of RNA:DNA hybrids and ssDNAs whose equilibrium is essential for cellular homeostasis. Using both global and hematovascular-restricted expression of RNASEH1-GFP to deplete R-loops, we revealed that excess R-loops in ddx41 mutants promote HSPC expansion. To uncover potential mechanisms driving HSPC expansion in ddx41 mutants, we identified elevated inflammatory target gene expression in HSPCs from ddx41 mutants compared to sibling controls using RNA-seq analysis. We established that excess R-loop levels induced by ddx41 deficiency triggered TBK1 phosphorylation and NFκB activation via the cGAS-STING pathway. Moreover, R-loop-triggered HSPC expansion was cGAS/STING-dependent. In human cells with diminished DDX41, blast counts, R-loop levels, and inflammatory signaling were elevated, suggesting a conservation of mechanism. These findings delineate a functional consequence for R-loop imbalance in HSPC biology and establish an in vivo link between R-loops, inflammation, and hematopoiesis.