Human CD34+ hematopoietic stem cell hierarchy: how far are we with its delineation at the most primitive level?

The ability to isolate and characterize different hematopoietic stem cell (HSC) or progenitor cell populations opens avenues to understand how hematopoiesis is regulated during development, homeostasis, and regeneration as well as in age-related conditions such as clonal hematopoiesis and leukemogenesis. Significant progress has been made in the past few decades in determining the composition of the cell types that exist in this system, but the most significant advances have come from mouse studies. However, recent breakthroughs have made significant strides that have enhanced the resolution of the human primitive hematopoietic compartment. Therefore, we aim to review this subject not only from a historical perspective but also to discuss the progress made in the characterization of the human postnatal CD34+ HSC-enriched populations. This approach will enable us to shed light on the potential future translational applicability of human HSCs.

Perturbed hematopoiesis in mice lacking ATMIN.

The ataxia telangiectasia mutated (ATM)-interacting protein ATMIN mediates noncanonical ATM signaling in response to oxidative and replicative stress conditions. Like ATM, ATMIN can function as a tumor suppressor in the hematopoietic system: deletion of Atmin under the control of CD19-Cre results in B-cell lymphomas in aging mice. ATM signaling is essential for lymphopoiesis and hematopoietic stem cell (HSC) function; however, little is known about the role of ATMIN in hematopoiesis. We thus sought to investigate whether the absence of ATMIN would affect primitive hematopoietic cells in an ATM-dependent or -independent manner. Apart from its role in B-cell development, we show that ATMIN has an ATM-independent function in the common myeloid progenitors (CMPs) by deletion of Atmin in the entire hematopoietic system using Vav-Cre. Despite the lack of lymphoma formation, ATMIN-deficient mice developed chronic leukopenia as a result of high levels of apoptosis in B cells and CMPs and induced a compensatory mechanism in which HSCs displayed enhanced cycling. Consequently, ATMIN-deficient HSCs showed impaired regeneration ability with the induction of the DNA oxidative stress response, especially when aged. ATMIN, therefore, has multiple roles in different cell types, and its absence results in perturbed hematopoiesis, especially during stress conditions and aging.

MRTF-SRF signaling is required for seeding of HSC/Ps in bone marrow during development.

Chemokine signaling is important for the seeding of different sites by hematopoietic stem cells (HSCs) during development. Serum response factor (SRF) controls multiple genes governing adhesion and migration, mainly by recruiting members of the myocardin-related transcription factor (MRTF) family of G-actin-regulated cofactors. We used vav-iCre to inactivate MRTF-SRF signaling early during hematopoietic development. In both Srf- and Mrtf-deleted animals, hematopoiesis in fetal liver and spleen is intact but does not become established in fetal bone marrow. Srf-null HSC progenitor cells (HSC/Ps) fail to effectively engraft in transplantation experiments, exhibiting normal proximal signaling responses to SDF-1, but reduced adhesiveness, F-actin assembly, and reduced motility. Srf-null HSC/Ps fail to polarize in response to SDF-1 and cannot migrate through restrictive membrane pores to SDF-1 or Scf in vitro. Mrtf-null HSC/Ps were also defective in chemotactic responses to SDF-1. Srf-null HSC/Ps exhibit substantial deficits in cytoskeletal gene expression. MRTF-SRF signaling is thus critical for expression of genes required for the response to chemokine signaling during hematopoietic development.

Arginine deprivation using pegylated arginine deiminase has activity against primary acute myeloid leukemia cells in vivo.

The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia. Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials.

Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation.

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC-progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34(+)CD38(-) stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34(+)CD38(+) progenitors were reduced. Residual normal CD34(+) cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC-progenitor transition.

Influence of donor-recipient sex on engraftment of normal and leukemia stem cells in xenotransplantation.

Immunodeficient mouse models are widely used for the assessment of human normal and leukemic stem cells. Despite the advancements over the years, reproducibility, as well as the differences in the engraftment of human cells in recipient mice remains to be fully resolved. Here, we used various immunodeficient mouse models to characterize the effect of donor-recipient sex on the engraftment of the human leukemic and healthy cells. Donor human cells and recipient immunodeficient mice demonstrate sex-specific engraftment levels with significant differences observed in the lineage output of normal CD34+ hematopoietic stem and progenitor cells upon xenotransplantation. Intriguingly, human female donor cells display heightened sensitivity to the recipient mice's gender, influencing their proliferation and resulting in significantly increased engraftment in female recipient mice. Our study underscores the intricate interplay taking place between donor and recipient characteristics, shedding light on important considerations for future studies, particularly in the context of pre-clinical research.

Active RHOA favors retention of human hematopoietic stem/progenitor cells in their niche.

BACKGROUND: Hematopoietic stem/progenitor cells (HSPCs) maintain the hematopoietic system by balancing their self-renewal and differentiation events. Hematopoietic stem cells also migrate to various sites and interact with their specific microenvironment to maintain the integrity of the system. Rho GTPases have been found to control the migration of hematopoietic cells and other cell types. Although the role of RAC1, RAC2 and CDC42 has been studied, the role of RHOA in human hematopoietic stem cells is unclear. RESULTS: By utilizing constitutively active and dominant negative RHOA, we show that RHOA negatively regulates both in vitro and in vivo migration and dominant negative RHOA significantly increased the migration potential of human HSC/HPCs. Active RHOA expression favors the retention of hematopoietic stem/progenitor cells in the niche rather than migration and was found to lock the cells in the G0 cell cycle phase thereby affecting their long-term self-renewal potential. CONCLUSION: The current study demonstrates that down-regulation of RHOA might be used to facilitate the migration and homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.

Engraftment defect of cytokine-cultured adult human mobilized CD34(+) cells is related to reduced adhesion to bone marrow niche elements.

In vitro exposure of haematopoietic stem and progenitor cells (HSPC) to cytokines in expansion or gene therapy protocols reduces homing and engraftment in vivo. We have previously reported that this is related in part to altered tissue specificity of short-term homing, leading to loss of cells in non-haematopoietic tissues. Here we demonstrate that defective engraftment persists when cultured HSPC are transplanted by intrabone injection. Changes in engraftment function occur within 24 h of cytokine exposure, and are evident when engraftment is analysed solely in the injected bone. A novel ex vivo model of the bone marrow was developed, in which the attachment of infused HSPC in rodent long bones is reduced following culture with cytokines. Finally, cultured HSPC demonstrated reduced adhesion to N-cadherin, osteopontin and vascular cell-adhesion molecule-1, ligands present in bone marrow niches. These changes in adhesive function occur rapidly, and are not related to downregulation of the relevant receptors. Our findings suggest that cytokine exposure of adult human HSPC results in altered adhesion within bone marrow niches, further leading to reduced engraftment potential in vivo.

Single cell analyses identify a highly regenerative and homogenous human CD34+ hematopoietic stem cell population.

The heterogeneous nature of human CD34+ hematopoietic stem cells (HSCs) has hampered our understanding of the cellular and molecular trajectories that HSCs navigate during lineage commitment. Using various platforms including single cell RNA-sequencing and extensive xenotransplantation, we have uncovered an uncharacterized human CD34+ HSC population. These CD34+EPCR+(CD38/CD45RA)- (simply as EPCR+) HSCs have a high repopulating and self-renewal abilities, reaching a stem cell frequency of ~1 in 3 cells, the highest described to date. Their unique transcriptomic wiring in which many gene modules associated with differentiated cell lineages confers their multilineage lineage output both in vivo and in vitro. At the single cell level, EPCR+ HSCs are the most transcriptomically and functionally homogenous human HSC population defined to date and can also be easily identified in post-natal tissues. Therefore, this EPCR+ population not only offers a high human HSC resolution but also a well-structured human hematopoietic hierarchical organization at the most primitive level.

Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells.

Immunodeficient mice are increasingly used to assay human hematopoietic repopulating cells as well as leukemia-initiating cells. One method commonly used to isolate these rare cells is to sort cells stained with fluorochrome-conjugated antibodies into fractions, then transplant the different fractions into immunodeficient mice to test their repopulating ability. The antibodies are generally treated as being neutral in terms of their effects on the experiment. Human repopulating cells are thought to express CD34 and lack CD38. Here we present evidence that anti-CD38 antibodies have a profound inhibitory effect on engraftment of cord blood and leukemia cells. We show that this effect is Fc-mediated and can be overcome by treating mice with immunosuppressive antibodies. When this inhibitory effect is prevented, we demonstrate that the CD34(+)CD38(+) fraction of certain acute myeloid leukemia samples contains all, or at least most, leukemia-initiating cell capacity. This study highlights the potential pitfall of antibody-mediated clearance of repopulating cells and is important for any groups working with this model. More importantly, the work suggests that there is greater variation in the phenotypes of leukemia-initiating cells than previously suggested.

Advances in Human Immune System Mouse Models for Studying Human Hematopoiesis and Cancer Immunotherapy.

Immunotherapy has established itself as a promising tool for cancer treatment. There are many challenges that remain including lack of targets and some patients across various cancers who have not shown robust clinical response. One of the major problems that have hindered the progress in the field is the dearth of appropriate mouse models that can reliably recapitulate the complexity of human immune-microenvironment as well as the malignancy itself. Immunodeficient mice reconstituted with human immune cells offer a unique opportunity to comprehensively evaluate immunotherapeutic strategies. These immunosuppressed and genetically modified mice, with some overexpressing human growth factors, have improved human hematopoietic engraftment as well as created more functional immune cell development in primary and secondary lymphoid tissues in these mice. In addition, several new approaches to modify or to add human niche elements to further humanize these immunodeficient mice have allowed a more precise characterization of human hematopoiesis. These important refinements have opened the possibility to evaluate not only human immune responses to different tumor cells but also to investigate how malignant cells interact with their niche and most importantly to test immunotherapies in a more preclinically relevant setting, which can ultimately lead to better success of these drugs in clinical trials.

Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment.

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches.

Correction: Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gata2 as a Crucial Regulator of Stem Cells in Adult Hematopoiesis and Acute Myeloid Leukemia.

Subversion of transcription factor (TF) activity in hematopoietic stem/progenitor cells (HSPCs) leads to the development of therapy-resistant leukemic stem cells (LSCs) that drive fulminant acute myeloid leukemia (AML). Using a conditional mouse model where zinc-finger TF Gata2 was deleted specifically in hematopoietic cells, we show that knockout of Gata2 leads to rapid and complete cell-autonomous loss of adult hematopoietic stem cells. By using short hairpin RNAi to target GATA2, we also identify a requirement for GATA2 in human HSPCs. In Meis1a/Hoxa9-driven AML, deletion of Gata2 impedes maintenance and self-renewal of LSCs. Ablation of Gata2 enforces an LSC-specific program of enhanced apoptosis, exemplified by attenuation of anti-apoptotic factor BCL2, and re-instigation of myeloid differentiation--which is characteristically blocked in AML. Thus, GATA2 acts as a critical regulator of normal and leukemic stem cells and mediates transcriptional networks that may be exploited therapeutically to target key facets of LSC behavior in AML.

IMiDs mobilize acute myeloid leukemia blasts to peripheral blood through downregulation of CXCR4 but fail to potentiate AraC/Idarubicin activity in preclinical models of non del5q/5q- AML.

Treatment for acute myeloid leukemia (AML) remains suboptimal and many patients remain refractory or relapse upon standard chemotherapy based on nucleoside analogs plus anthracyclines. The crosstalk between AML cells and the BM stroma is a major mechanism underlying therapy resistance in AML. Lenalidomide and pomalidomide, a new generation immunomodulatory drugs (IMiDs), possess pleiotropic anti-leukemic properties including potent immune-modulating effects and are commonly used in hematological malignances associated with intrinsic dysfunctional BM such as myelodysplastic syndromes and multiple myeloma. Whether IMiDs may improve the efficacy of current standard treatment in AML remains understudied. Here, we have exploited in vitro and in vivo preclinical AML models to analyze whether IMiDs potentiate the efficacy of AraC/Idarubicin-based standard AML chemotherapy by interfering with the BM stroma-mediated chemoresistance. We report that IMiDs do not exert cytotoxic effects on either non-del5q/5q- AML cells nor BM-MSCs, but they enhance the immunomodulatory properties of BM-MSCs. When combined with AraC/Idarubicin, IMiDs fail to circumvent BM stroma-mediated resistance of non-del5q/5q- AML cells in vitro and in vivo but induce robust extramedullary mobilization of AML cells. When administered as a single agent, lenalidomide specifically mobilizes non-del5q/5q- AML cells, but not healthy CD34+ cells, to peripheral blood (PB) through specific downregulation of CXCR4 in AML blasts. Global gene expression profiling supports a migratory/mobilization gene signature in lenalidomide-treated non-del5q/5q- AML blasts but not in CD34+ cells. Collectively, IMiDs mobilize non-del5q/5q- AML blasts to PB through CXCR4 downregulation, but fail to potentiate AraC/Idarubicin activity in preclinical models of non-del5q/5q- AML.

Notch Signaling in the Regulation of Hematopoietic Stem Cell.

PURPOSE OF REVIEW: Understanding the signaling pathways that govern hematopoietic stem and progenitor cells (HSPCs) is fundamental to uncover their regulation and how this is skewed in hematological malignancies. Whether Notch is necessary for the regulation of mammalian HSPCs is still unclear. We therefore critically review the current literature on the role of Notch in HSPCs. RECENT FINDINGS: HSPCs have shown different requirements for Notch signals in vitro and in vivo and at different stages of differentiation. Additionally, bone marrow niche cells activate Notch signaling in HSPCs enhancing their regenerative and self-renewal capacity. SUMMARY: Despite the controversy, adequate levels of Notch signaling appear necessary to avoid the development of hematological malignancies. Contrary to early studies, recent research suggests that Notch signaling may play a role in homeostatic and regenerative hematopoiesis but further investigation is necessary to understand how it is regulated by the different ligand/receptor pairings and the molecular mechanisms that are triggered.

Dendritic Cell Lineage Potential in Human Early Hematopoietic Progenitors.

Conventional dendritic cells (cDCs) are thought to descend from a DC precursor downstream of the common myeloid progenitor (CMP). However, a mouse lymphoid-primed multipotent progenitor has been shown to generate cDCs following a DC-specific developmental pathway independent of monocyte and granulocyte poiesis. Similarly, here we show that, in humans, a large fraction of multipotent lymphoid early progenitors (MLPs) gives rise to cDCs, in particular the subset known as cDC1, identified by co-expression of DNGR-1 (CLEC9A) and CD141 (BDCA-3). Single-cell analysis indicates that over one-third of MLPs have the potential to efficiently generate cDCs. cDC1s generated from CMPs or MLPs do not exhibit differences in transcriptome or phenotype. These results demonstrate an early imprinting of the cDC lineage in human hematopoiesis and highlight the plasticity of developmental pathways giving rise to human DCs.

Frequency and Dynamics of Leukemia-Initiating Cells during Short-term Ex Vivo Culture Informs Outcomes in Acute Myeloid Leukemia Patients.

Acute myeloid leukemia (AML) is sustained by a subpopulation of rare leukemia-initiating cells (LIC) detected in the xenograft assay by their capacity to self-renew and to generate non-LICs in vivo The xenotransplantation model captures functional properties of LICs that have clinical prognostic value. However, the long duration of this in vivo assay has hampered its use as a prognostic tool. Here, we show, using an ex vivo coculture system, that intermediate and poor risk AML patient samples at diagnosis have a 5 to 7 times higher frequency of leukemic long-term culture-initiating cells (L-LTC-IC) compared with the good risk group. We defined a fluorescence dilution factor (FDF) parameter that monitors sample proliferation over 1 week and established a strong correlation of this parameter with the L-LTC-IC frequency. A higher FDF was found for poor prognostic AMLs or for samples capable of engrafting NSG mice compared with good risk AMLs or nonengrafters. Importantly, FDF could classify normal karyotype intermediate risk patients into two groups with a significant difference in their overall survival, thus making this nongenetic and non-in vivo approach a new clinically relevant tool for better diagnosis of AML patients. Cancer Res; 76(8); 2082-6. ©2016 AACR.

Different Motile Behaviors of Human Hematopoietic Stem versus Progenitor Cells at the Osteoblastic Niche.

Despite advances in our understanding of interactions between mouse hematopoietic stem cells (HSCs) and their niche, little is known about communication between human HSCs and the microenvironment. Using a xenotransplantation model and intravital imaging, we demonstrate that human HSCs display distinct motile behaviors to their hematopoietic progenitor cell (HPC) counterparts, and the same pattern can be found between mouse HSCs and HPCs. HSCs become significantly less motile after transplantation, while progenitor cells remain motile. We show that human HSCs take longer to find their niche than previously expected and suggest that the niche be defined as the position where HSCs stop moving. Intravital imaging is the only technique to determine where in the bone marrow stem cells stop moving, and future analyses should focus on the environment surrounding the HSC at this point.

APOBEC3A is implicated in a novel class of G-to-A mRNA editing in WT1 transcripts.

Classic deamination mRNA changes, including cytidine to uridine (C-to-U) and adenosine to inosine (A-to-I), are important exceptions to the central dogma and lead to significant alterations in gene transcripts and products. Although there are a few reports of non-classic mRNA alterations, as yet there is no molecular explanation for these alternative changes. Wilms Tumor 1 (WT1) mutations and variants are implicated in several diseases, including Wilms tumor and acute myeloid leukemia (AML). We observed two alternative G-to-A changes, namely c.1303G>A and c.1586G>A in cDNA clones and found them to be recurrent in a series of 21 umbilical cord blood mononuclear cell (CBMC) samples studied. Two less conserved U-to-C changes were also observed. These alternative changes were found to be significantly higher in non-progenitor as compared to progenitor CBMCs, while they were found to be absent in a series of AML samples studied, indicating they are targeted, cell type-specific mRNA editing modifications. Since APOBEC/ADAR family members are implicated in RNA/DNA editing, we screened them by RNA-interference (RNAi) for WT1-mRNA changes and observed near complete reversal of WT1 c.1303G>A alteration upon APOBEC3A (A3A) knockdown. The role of A3A in mediating this change was confirmed by A3A overexpression in Fujioka cells, which led to a significant increase in WT1 c.1303G>A mRNA editing. Non-progenitor CBMCs showed correspondingly higher levels of A3A-mRNA and protein as compared to the progenitor ones. To our knowledge, this is the first report of mRNA modifying activity for an APOBEC3 protein and implicates A3A in a novel G-to-A form of editing. These findings open the way to further investigations into the mechanisms of other potential mRNA changes, which will help to redefine the RNA editing paradigm in both health and disease.