Expansion Of Human Hematopoietic Stem Cells Research Articles

Ex vivo expansion of human hematopoietic stem cells and clinical applications.

Hematopoietic stem cells (HSCs) are a rare population of cells found in the bone marrow that play a critical role in lifelong hematopoiesis and the reconstitution of the hematopoietic system after hematopoietic stem cell transplantation. Hematopoietic stem cell transplantation remains the only curative treatment for patients with refractory hematologic disorders, and umbilical cord blood (CB) serves as an alternative stem cell source due to its several advantageous characteristics, including human leukocyte antigen flexibility and reduced donor burden. However, CB also has the disadvantage of containing a small number of cells, resulting in limited donor selection and a longer time for engraftment. Therefore, the development of techniques to expand HSCs exvivo, particularly umbilical CB, is a goal in hematology. While various combinations of cytokines were once the mainstream approach, these protocols had limited expansion rates and did not lead to clinical application. However, in recent years, the development of a technique in which small molecules are added to cytokines has enabled the stable, long-term exvivo expansion of human HSCs. Clinical trials of expanded umbilical CB using these techniques have been undertaken and have confirmed their efficacy and safety. In addition, we have successfully developed a recombinant-cytokine-free and albumin-free culture system for the long-term expansion of human HSCs. This approach could offer the potential for more selective expansion of human HSCs compared to previous protocols. This review discusses exvivo culture protocols for expanding human HSCs and presents the results of clinical trials using these techniques, along with future perspectives.

Expansion of human megakaryocyte-biased hematopoietic stem cells by biomimetic Microniche

Limited numbers of available hematopoietic stem cells (HSCs) limit the widespread use of HSC-based therapies. Expansion systems for functional heterogenous HSCs remain to be optimized. Here, we present a convenient strategy for human HSC expansion based on a biomimetic Microniche. After demonstrating the expansion of HSC from different sources, we find that our Microniche-based system expands the therapeutically attractive megakaryocyte-biased HSC. We demonstrate scalable HSC expansion by applying this strategy in a stirred bioreactor. Moreover, we identify that the functional human megakaryocyte-biased HSCs are enriched in the CD34+CD38-CD45RA-CD90+CD49f lowCD62L-CD133+ subpopulation. Specifically, the expansion of megakaryocyte-biased HSCs is supported by a biomimetic niche-like microenvironment, which generates a suitable cytokine milieu and supplies the appropriate physical scaffolding. Thus, beyond clarifying the existence and immuno-phenotype of human megakaryocyte-biased HSC, our study demonstrates a flexible human HSC expansion strategy that could help realize the strong clinical promise of HSC-based therapies.

Aberrant activation of TCL1A promotes stem cell expansion in clonal haematopoiesis.

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis1. These lesions are precursors for blood cancers2-6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, butthis effect was not seen inclones withdriver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimentalknockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

Chemically defined cytokine-free expansion of human haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases1,2. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation3. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo4. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.

Recent advances in ex vivo expansion of human hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are a rare cell population present in the bone marrow. They possess self-renewal and multipotent differentiation capacities and play a crucial role in lifelong hematopoiesis and reconstitution of the hematopoietic system after hematopoietic stem cell transplantation (HSCT). HSCT remains the only curative treatment for refractory hematologic disorders. Umbilical cord blood (CB) has several advantages as an alternative donor for HSCT, including HLA flexibility and lack of donor burden. However, CB has limitations in terms of cell dose, restricted donor options, and prolonged time to engraftment. Development of techniques for expanding HSCs ex vivo, especially those contained in CB, has become a goal in the field of hematology. Attempts have been made to use various combinations of cytokines for this purpose, but these protocols showed limited expansion rates and did not progress to clinical applications. Recent advances that include the addition of small molecules to cytokines have enabled long-term and stable ex vivo expansion of human HSCs. Clinical trials have been conducted with HSCs expanded in CB using these techniques, confirming their efficacy and safety. Furthermore, we recently developed a recombinant cytokine-free, albumin-free culture system for long-term expansion of human HSCs. This approach has the potential to selectively expand human HSCs more effectively than the previous protocols. We herein present an overview of ex vivo culture protocols for expanding human HSCs together with the results of clinical trials that utilized these techniques.

UM171 cooperates with PIM1 inhibitors to restrict HSC expansion markers and suppress leukemia progression

The pyrimido-indole derivative UM171 promotes human Hematopoietic Stem Cells Expansion (HSCE), but its impact on leukemia is not known. Herein, we show in a mouse model of erythroleukemia that UM171 strongly suppresses leukemia progression. UM171 inhibits cell cycle progression and apoptosis of leukemic cells in culture. The effect of UM171 on leukemia differentiation was accompanied by increased expression of HSCE markers. RNAseq analysis combined with Q-RT-PCR and western blotting revealed that the PIM1 protein kinase is highly elevated in response to UM171 treatment. Moreover, docking analysis combined with immunoprecipitation assays revealed high binding affinity of UM171 to PIM1. Interestingly, pan-PIM kinase inhibitors counteracted the effect of UM171 on HSCE marker expression and PIM1 transcription, but not its suppression of leukemic cell growth. Moreover, combination treatment with UM171 and a pan-PIM inhibitor further suppressed leukemic cell proliferation compared to each drug alone. To uncover the mechanism of growth inhibition, we showed strong upregulation of the cyclin-dependent kinase inhibitor P21CIP1 and the transcription factor KLF2 by UM171. In accordance, KLF2 knockdown attenuated growth inhibition by UM171. KLF2 upregulation by UM171 is also responsible for the activation of P21CIP1 in leukemic cells leading to a G1/S arrest and suppression of leukemogenesis. Thus, suppression of leukemic growth by UM171 through KLF2 and P21CIP1 is thwarted by PIM-mediated expansion of leukemic stemness, uncovering a novel therapeutic modality involving combined UM171 plus PIM inhibitors.

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.

S249: EX VIVO HEMATOPOIETIC STEM CELL (HSC) EXPANSION USING BONE-LINING REINVIGORATING MESENCHYMAL STROMAL CELLS (RMSCS)

Background: Bone marrow transplants (BMTs) have highlighted the Hematopoietic Stem Cell (HSC) potential to restore a new functional hematopoietic system in diseased recipients. However, a major roadblock for the clinical application and translational research on HSCs is our limited potential for ex vivo HSC expansion. Aims: To develop an approach for improved ex vivo HSC expansion using novel bone-lining reinvigorating Mesenchymal Stromal Cells (rMSC)-based culture system. Methods: Using a functional approach, we developed a robust pipeline for the fluorescence-activated cell sorting (FACS)-based isolation and ex vivo expansion of rMSCs from both murine and patient-individualized human samples. We tested the long-term HSC expansion potential in the rMSC-based co-culture system, phenotypically using FACS based proliferation analysis and functionally using limiting dilution and colony forming assays. Further, we performed transplantation experiments to study the hematopoietic reconstitution ability of the ex vivo expanded HSCs. Moreover, we expanded a single HSC over multiple cell divisions using our culture system to demonstrate bona fide HSC self-renewal potential of the rMSC-based system. Results: We propose a potent ex vivo HSC expansion system based on novel bone lining-derived reinvigorating Mesenchymal Stromal Cells (rMSCs). Both bulk- and single-HSCs expanded long-term using the rMSC co-culture system maintained phenotypic stemness over multiple cell differentiation cycles and possessed functional bone marrow reconstitution capabilities upon transplant. Notably, our rMSC co-culture system outperformed existing alternatives for HSC expansion including systems using stromal cells, non-cellular coating factors, or different medium compositions. Further, our results highlight the reliable isolation and robust culture of human rMSCs using our experimental strategy, and the potential to also utilize the rMSC co-culture system for the ex vivo expansion of human HSCs. Summary/Conclusion: We could demonstrate that our rMSC-based system for HSC expansion can play a pivotal role in research to reduce the number of mice used for ex vivo experiments. Moreover, our data also shows that the rMSC-based co-culture can be used for human HSC expansion, opening possibilities of applying this potent system for research on numerous diseases including immunodeficiencies and leukaemia.

Lamin B1 deletion in myeloid neoplasms causes nuclear anomaly and altered hematopoietic stem cell function.

Abnormal nuclear morphology is a hallmark of malignant cells widely used in cancer diagnosis. Pelger-Huët anomaly (PHA) is a common abnormality of neutrophil nuclear morphology of unknown molecular etiology in myeloid neoplasms (MNs). We show that loss of nuclear lamin B1 (LMNB1) encoded on chromosome 5q, which is frequently deleted in MNs, induces defects in nuclear morphology and human hematopoietic stem cell (HSC) function associated with malignancy. LMNB1 deficiency alters genome organization inducing invitro and invivo expansion of HSCs, myeloid-biased differentiation with impaired lymphoid commitment, and genome instability due to defective DNA damage repair. Nuclear dysmorphology of neutrophils in patients with MNs is associated with 5q deletions spanning the LMNB1 locus, and lamin B1 loss is both necessary and sufficient to cause PHA in normal and 5q-deleted neutrophils. LMNB1 loss thus causes acquired PHA and links abnormal nuclear morphology with HSCs and progenitor cell fate determination via genome organization.

Update on preclinical and clinical efforts on ex-vivo expansion of hematopoietic stem and progenitor cells.

Ex-vivo expansion of hematopoietic stem cells (HSCs) is one potential approach to enhance the clinical efficacy of hematopoietic cell transplantation-based therapy for malignant and nonmalignant blood diseases. Here, we discuss the major progress of preclinical and clinical studies on the ex-vivo expansion of human HSCs and progenitor cells (HPCs). Single-cell RNA sequencing identified ADGRG1 as a reliable marker of functional HSCs upon ex-vivo expansion-induced mitochondrial oxidative stress. Both SR1 and UM171 significantly promote ex-vivo expansion of human cord blood HSCs and HPCs, as determined in preclinical animal models. Encouraged by these findings from the bench, multiple phase I/II and phase II clinical trials have been conducted to evaluate the safety, feasibility and efficacy of SR1-expanded and UM171-expanded cord blood units in patients with hematological malignancy. Preliminary data from multiple phase I/II clinical trials regarding transplants of ex-vivo-expanded HSCs and HPCs have demonstrated that ex-vivo expansion may be used to overcome the limitation of the rarity of HSCs without compromising stemness.

Development of a novel and synthetic HematoMiR technology that broadly modulates quiescence of stem cells and enhances HSC expansion.

Hematopoietic stem cell (HSCs) transplantation is the primary therapeutic modality used to treat hematopoietic disorders. It centers on the capability of a small quantity of HSCs to repopulate whole blood lineages. Along with limited availability of suitable donors, the need for sufficient number of donor HSCs is still challenging in clinical relevance. This has been addressed by ex vivo HSC expansion albeit with partial success, and thus development of an alternative strategy that could improve HSC expansion is required. To that end, we aimed to build HematoMiR, an oligo-based technology that broadly targets HSC quiescence factors. Here, we show that HematoMiRs and their combinations targeting over 50 factors involved in HSC quiescence could induce robust ex vivo murine and human HSC expansion. In particular, HematoMiR-5 treatment enhanced cell cycle through down-regulation of negative cell cycle regulators in HSCs. HematoMiR-5 treated HSPCs had reduced DNA damage during the course of ex vivo expansion. Moreover, HematoMiR-5 treatment led to sustained HSC self-renewal ability and a low apoptosis rate. In addition, HematoMiR-5 expanded HSCs demonstrated successful engraftment and repopulation capacity in the recipient animals. Furthermore, combinatorial treatments of HematoMiR-2 and 5 allowed vigorous ex vivo HSC expansion. These findings demonstrate that novel and synthetic HematoMiR technology is feasible for HSC ex vivo expansion through the sequence-dependent modulation of numerous HSC quiescence modulators.

ADGRG1 enriches for functional human hematopoietic stem cells following ex vivo expansion-induced mitochondrial oxidative stress.

The heterogeneity of human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) under stress conditions such as ex vivo expansion is poorly understood. Here, we report that the frequencies of SCID-repopulating cells were greatly decreased in cord blood (CB) CD34+ HSCs and HPCs upon ex vivo culturing. Transcriptomic analysis and metabolic profiling demonstrated that mitochondrial oxidative stress of human CB HSCs and HPCs notably increased, along with loss of stemness. Limiting dilution analysis revealed that functional human HSCs were enriched in cell populations with low levels of mitochondrial ROS (mitoROS) during ex vivo culturing. Using single-cell RNA-Seq analysis of the mitoROS low cell population, we demonstrated that functional HSCs were substantially enriched in the adhesion GPCR G1-positive (ADGRG1+) population of CD34+CD133+ CB cells upon ex vivo expansion stress. Gene set enrichment analysis revealed that HSC signature genes including MSI2 and MLLT3 were enriched in CD34+CD133+ADGRG1+ CB HSCs. Our study reveals that ADGRG1 enriches for functional human HSCs under oxidative stress during ex vivo culturing, which can be a reliable target for drug screening of agonists of HSC expansion.

Lead Optimization and Structure-Activity Relationship Studies on Myeloid Ecotropic Viral Integration Site 1 Inhibitor.

The pivotal role of the myeloid ecotropic viral integration site 1 (MEIS1) transcriptional factor was reported in cardiac regeneration and hematopoietic stem-cell (HSC) regulation with our previous findings. MEIS1 as a promising target in the context of pharmacological inhibition, we identified a potent myeloid ecotropic viral integration site (MEIS) inhibitor, MEISi-1, to induce murine and human HSC expansion ex vivo and in vivo. In this work, we performed lead optimization on MEISi-1 by synthesizing 45 novel analogues. Structure-activity relationship studies revealed the significance of a para-methoxy group on ring A and a hydrophobic moiety at the meta position of ring B. Obtained biological data were supported by inhibitor docking and molecular dynamics simulation studies. Eleven compounds were depicted as potent inhibitors demonstrating a better inhibitory profile on MEIS1 and target genes Meis1, Hif-1α, and p21. Among those, 4h, 4f, and 4b were the most potent inhibitors. The predicted pharmacokinetics properties fulfill drug-likeness criteria. In addition, compounds exerted neither cytotoxicity on human dermal fibroblasts nor mutagenicity.

Enhanced self-renewal of human long-term hematopoietic stem cells by a sulfamoyl benzoate derivative targeting p18INK4C

AbstractThe use of umbilical cord blood transplant has been substantially limited by the finite number of hematopoietic stem and progenitor cells in a single umbilical cord blood unit. Small molecules that not only quantitatively but also qualitatively stimulate enhancement of hematopoietic stem cell (HSC) self-renewal ex vivo should facilitate the clinical use of HSC transplantation and gene therapy. Recent evidence has suggested that the cyclin-dependent kinase inhibitor, p18INK4C (p18), is a critical regulator of mice HSC self-renewal. The role of p18 in human HSCs and the effect of p18 inhibitor on human HSC expansion ex vivo need further studies. Here we report that knockdown of p18 allowed for an increase in long-term colony-forming cells in vitro. We then identified an optimized small molecule inhibitor of p18, 005A, to induce ex vivo expansion of HSCs that was capable of reconstituting human hematopoiesis for at least 4 months in immunocompromised mice, and hence, similarly reconstituted secondary recipients for at least 4 more months, indicating that cells exposed to 005A were still competent in secondary recipients. Mechanistic studies showed that 005A might delay cell division and activate both the Notch signaling pathway and expression of transcription factor HoxB4, leading to enhancement of the self-renewal of long-term engrafting HSCs and the pool of progenitor cells. Taken together, these observations support a role for p18 in human HSC maintenance and that the p18 inhibitor 005A can enhance the self-renewal of long-term HSCs.

Polyvinyl alcohol hydrolysis rate and molecular weight influence human and murine HSC activity ex vivo

Ex vivo expansion of hematopoietic stem cells (HSCs) is one of the most promising strategies to increase the availability of transplantable HSCs and improve bone marrow transplantation outcomes. We recently demonstrated that mouse HSCs could be efficiently expanded in polyvinyl alcohol (PVA)-containing culture medium using only recombinant stem cell factor and thrombopoietin cytokines. However, the behavior of human HSCs in these simple PVA-based media was not fully elucidated. In this study, we analyzed the compatibility of PVA of different hydrolysis rates (HR) and molecular weights (MW) to support functional human and mouse HSCs ex vivo. Human and mouse HSCs proliferated more frequently in media containing PVA with lower HR than with higher HR, but both PVA types supported HSC multilineage reconstitution potential. Importantly, human HSCs cultured in PVA-containing media engrafted not only in irradiated recipients but also in non-irradiated recipients. Our results demonstrate that human HSCs can be maintained ex vivo using PVA-based culture systems and suggest approaches for future optimization of human HSC expansion.

CCL2 associated with CD38 expression during ex vivo expansion in human cord blood-derived hematopoietic stem cells.

To date, different experimental strategies have been developed for the ex vivo expansion of human hematopoietic stem cells (HSCs) for clinical applications. However, differences in the genomic function of expanded HSCs under different culture systems remain unclear. In this study, we compared the gene expression profiles of HSCs in ex vivo expanded serum (10% FBS, fetal bovine serum) and serum-free culture systems and analyzed the molecular functions of differentially expressed genes using microarray chips. We identified 839 differentially expressed genes between the two culture systems. These genes were enriched in the TNF -regulated inflammatory pathway in an FBS culture system. In addition, the mRNA expression of CCL2 (C-C motif chemokine ligand 2), TNF (tumor necrosis factor) and FOS (FBJ murine osteosarcoma viral oncogene homolog) was validated by RT-qPCR. Our data revealed that ex vivo expansion of HSCs using the FBS culture system induces an inflammatory response and high CD38 expression, indicating that this system might activate an inflammatory pathway and induce expression of the cancer marker CD38 during ex vivo expansion of HSCs. This study provides a transcriptional profile and new insights into the genomic functions of HSCs under different expanded cultures.

An Enhanced Zebrafish Xenograft Model to Study the Behavior of Human Hematopoietic Stem Cells and Patient Derived Leukemia In Vivo

Human hematopoietic stem cells (HSCs) are characterized by their unique ability to self-renew and differentiate into multiple cell types. Transplantation of human HSCs into immunocompromised mice is the gold standard for evaluating the in vivo repopulation capacity of these cells and has been adapted to evaluate the leukemic potential of leukemia-initiating cells (LICs) in acute leukemia. In spite of the popularity of this approach, certain cellular subsets do not survive post xenotransplantation as they are dependent on human factors that may be missing in the murine microenvironment. We pioneered human leukemia xenografts in the zebrafish model (Corkery et al., 2011; Bentley et al., 2015) as a parallel strategy, taking advantage of the immuno-permissiveness and transparency of zebrafish larvae to provide unprecedented continuous observation of leukemia evolution. However, as in mice, the addition of human cytokines and growth factors may further improve engraftment and optimize the utility of the zebrafish as a preclinical transplant model for medium to high-throughput therapeutic screening by creating a microenvironment more comparable to that found in patients.Using transgenic technology, we created novel zebrafish lines that express human stem cell factor (SCF/KITLG), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), and stromal cell-derived factor 1 alpha (SDF1α/CXCL12). All of these factors are essential for the survival and expansion of HSCs, progenitors and LICs. CMK, GRWi (Down Syndrome- acute myeloid leukemia (DS-AML)) and Jurkat (T-acute lymphoblastic leukemia) cell lines were injected into the yolk sac of these multi-cytokine zebrafish embryos. These cells engrafted and exhibited increased cell proliferation and physiological migration compared to control casper embryo transplants. Moreover, only in the multi-cytokine fish did these cells home to hematopoietic niches, including the caudal hematopoietic tissue (CHT) (equivalent to the fetal liver) at 7 days-post injection (dpi) and the kidney marrow at 11 dpi.The yolk sac is not the physiological site for hematopoietic cell growth and thus we injected patient-derived AML cells into the circulation of the multi-cytokine fish. We observed similar trends in proliferation and migration as seen with leukemia cell lines. Historically, human HSCs do not survive beyond 24h in zebrafish xenografts (Pruvot et al. 2011). Given our success with patient-derived leukemia samples, we wanted to see if our multi-cytokine fish would provide a more favorable environment for human HSC survival. Indeed, primary human umbilical cord blood-derived HSCs were injected into the circulation of zebrafish larvae and survived past 3 dpi in the multi-cytokine fish but not in casper control larvae.Transgenic zebrafish expressing human cytokines provide an enhanced model for studying human leukemia and stem cell biology in a visually tractable system that more closely recapitulates the human microenvironment. The inherent capacity of the zebrafish for rapid and efficient chemical screening will be exploited in this model for preclinical testing of anti-leukemic therapies and to evaluate HSC enhancing agents in vivo . DisclosuresBerman:AGADA Therapeutics: Research Funding.

Activation of the receptor tyrosine kinase RET improves long-term hematopoietic stem cell outgrowth and potency

AbstractExpansion of human hematopoietic stem cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche; how to apply this process to HSC maintenance and expansion has yet to be explored. We show a role for the GFL receptor, RET, at the cell surface of HSCs in mediating sustained cellular growth, resistance to stress, and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/coreceptor complex, glial-derived neurotrophic factor and its coreceptor, exhibit improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally, we show that RET drives a multifaceted intracellular signaling pathway, including key signaling intermediates protein kinase B, extracellular signal-regulated kinase 1/2, NF-κB, and p53, responsible for a wide range of cellular and genetic responses that improve cell growth and survival under culture conditions.

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.

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.