Megakaryocytic-erythroid progenitors (MEPs) give rise to the cells that produce red blood cells and platelets. Although the mechanisms underlying megakaryocytic (MK) and erythroid (E) maturation have been described, those controlling their specification from MEPs are unknown. Single-cell RNA sequencing of primary human MEPs, common myeloid progenitors (CMPs), megakaryocyte progenitors, and E progenitors revealed a distinct transitional MEP signature. Inferred regulatory transcription factors (TFs) were associated with differential expression of cell cycle regulators. Genetic manipulation of selected TFs validated their role in lineage specification and demonstrated coincident modulation of the cell cycle. Genetic and pharmacologic modulation demonstrated that cell cycle activation is sufficient to promote E versus MK specification. These findings, obtained from healthy human cells, lay a foundation to study the mechanisms underlying benign and malignant disease states of the megakaryocytic and E lineages.

Infantile (fetal and neonatal) megakaryocytes (Mks) have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived Mks. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with the potential to affect MKL1 function and found that DYRK1A inhibition dramatically enhanced Mk morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal Mks. Mks derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss-of-function studies confirmed MKL1 involvement in this morphogenetic pathway. Expression of Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile Mks.

Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID-19 patients. Given that platelets are key regulators of thrombosis, inflammation, and immunity they represent prime candidates as mediators of COVID-19-associated pathogenesis. The objective of this study was to understand the contribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the platelet phenotype via phenotypic (activation, aggregation) and transcriptomic characterization.

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-β plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-β1 than TGF-β2 and TGF-β3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-β1/TGF-β3 protein trap, to block the excessive TGF-β signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-β1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-β1-induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-β1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

Single-cell assays have enriched our understanding of hematopoiesis and, more generally, stem and progenitor cell biology. However, these single-end-point approaches provide only a static snapshot of the state of a cell. To observe and measure dynamic changes that may instruct cell fate, we developed an approach for examining hematopoietic progenitor fate specification using long-term (> 7-day) single-cell time-lapse imaging for up to 13 generations with in situ fluorescence staining of primary human hematopoietic progenitors followed by algorithm-assisted lineage tracing. We analyzed progenitor cell dynamics, including the division rate, velocity, viability, and probability of lineage commitment at the single-cell level over time. We applied a Markov probabilistic model to predict progenitor division outcome over each generation in culture. We demonstrated the utility of this methodological pipeline by evaluating the effects of the cytokines thrombopoietin and erythropoietin on the dynamics of self-renewal and lineage specification in primary human bipotent megakaryocytic-erythroid progenitors (MEPs). Our data support the hypothesis that thrombopoietin and erythropoietin support the viability and self-renewal of MEPs, but do not affect fate specification. Thus, single-cell tracking of time-lapse imaged colony-forming unit assays provides a robust method for assessing the dynamics of progenitor self-renewal and lineage commitment.

Arhgap21 is a member of the Rho GTPase activating protein (RhoGAP) family, which function as negative regulators of Rho GTPases. Arhgap21 has been implicated in adhesion and migration of cancer cells. However, the role of Arhgap21 has never been investigated in hematopoietic cells. Herein, we evaluated functional aspects of hematopoietic stem and progenitor cells (HSPC) using a haploinsufficient (Arhgap21+/-) mouse. Our results show that Arhgap21+/- mice have an increased frequency of phenotypic HSC, impaired ability to form progenitor colonies in vitro and decreased hematopoietic engraftment in vivo, along with a decrease in LSK cell frequency during serial bone marrow transplantation. Arhgap21+/- hematopoietic progenitor cells have impaired adhesion and enhanced mobilization of immature LSK and myeloid progenitors. Arhgap21+/- mice also exhibit reduced erythroid commitment and differentiation, which was recapitulated in human primary cells, in which knockdown of ARHGAP21 in CMP and MEP resulted in decreased erythroid commitment. Finally, we observed enhanced RhoC activity in the bone marrow cells of Arhgap21+/- mice, indicating that Arhgap21 functions in hematopoiesis may be at least partially mediated by RhoC inactivation.

Attempts to expand ex vivo the numbers of human hematopoietic stem cells (HSCs) without compromising their marrow repopulating capacity and their ability to establish multilineage hematopoiesis has been the subject of intense investigation. Although most such efforts have focused on cord blood HSCs, few have been applied to adult HSCs, a more clinically relevant HSC source for gene modification. To date, the strategies that have been used to expand adult HSCs have resulted in modest effects or HSCs with lineage bias and a limited ability to generate T cells in vivo. We previously reported that culturing umbilical cord blood CD34+ cells in serum-free media supplemented with valproic acid (VPA), a histone deacetylase inhibitor, and a combination of cytokines led to the expansion of the numbers of fully functional HSCs. In the present study, we used this same approach to expand the numbers of adult human CD34+ cells isolated from mobilized peripheral blood and bone marrow. This approach resulted in a significant increase in the numbers of phenotypically defined HSCs (CD34+CD45RA-CD90+D49f+). Cells incubated with VPA also exhibited increased aldehyde dehydrogenase activity and decreased mitochondrial membrane potential, each functional markers of HSCs. Grafts harvested from VPA-treated cultures were able to engraft in immune-deficient mice and, importantly, to generate cellular progeny belonging to each hematopoietic lineage in similar proportion to that observed with unmanipulated CD34+ cells. These data support the utility of VPA-mediated ex vivo HSC expansion for gene modification of adult HSCs.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

Measuring multiple omics profiles from the same single cell opens up the opportunity to decode molecular regulation that underlies intercellular heterogeneity in development and disease. Here, we present co-sequencing of microRNAs and mRNAs in the same single cell using a half-cell genomics approach. This method demonstrates good robustness (~95% success rate) and reproducibility (R2 = 0.93 for both microRNAs and mRNAs), yielding paired half-cell microRNA and mRNA profiles, which we can independently validate. By linking the level of microRNAs to the expression of predicted target mRNAs across 19 single cells that are phenotypically identical, we observe that the predicted targets are significantly anti-correlated with the variation of abundantly expressed microRNAs. This suggests that microRNA expression variability alone may lead to non-genetic cell-to-cell heterogeneity. Genome-scale analysis of paired microRNA-mRNA co-profiles further allows us to derive and validate regulatory relationships of cellular pathways controlling microRNA expression and intercellular variability.

Myelofibrosis (MF) is a clonal stem cell neoplasm characterized by abnormal JAK-STAT signaling, chronic inflammation, cytopenias, and risk of transformation to acute leukemia. Despite improvements in the therapeutic options for patients with MF, allogeneic hematopoietic stem cell transplantation remains the only curative treatment. We previously demonstrated multiple immunosuppressive mechanisms in patients with MF, including increased expression of programmed cell death protein 1 (PD-1) on T cells compared with healthy controls. Therefore, we conducted a multicenter, open-label, phase 2, single-arm study of pembrolizumab in patients with Dynamic International Prognostic Scoring System category of intermediate-2 or greater primary, post-essential thrombocythemia or post-polycythemia vera myelofibrosis that were ineligible for or were previously treated with ruxolitinib. The study followed a Simon 2-stage design and enrolled a total of 10 patients, 5 of whom had JAK2V617mutation, 2 had CALR mutation, and 6 had additional mutations. Most patients were previously treated with ruxolitinib. Pembrolizumab treatment was well tolerated, but there were no objective clinical responses, so the study closed after the first stage was completed. However, immune profiling by flow cytometry, T-cell receptor sequencing, and plasma proteomics demonstrated changes in the immune milieu of patients, which suggested improved T-cell responses that can potentially favor antitumor immunity. The fact that these changes were not reflected in a clinical response strongly suggests that combination immunotherapeutic approaches rather than monotherapy may be necessary to reverse the multifactorial mechanisms of immune suppression in myeloproliferative neoplasms. This trial was registered at www.clinicaltrials.gov as #NCT03065400.

Chronic myeloproliferative neoplasms (MPN) frequently evolve to a blast phase (BP) that is almost uniformly resistant to induction chemotherapy or hypomethylating agents. We explored the functional properties, genomic architecture, and cell of origin of MPN-BP initiating cells (IC) using a serial NSG mouse xenograft transplantation model. Transplantation of peripheral blood mononuclear cells (MNC) from 7 of 18 patients resulted in a high degree of leukemic cell chimerism and recreated clinical characteristics of human MPN-BP. The function of MPN-BP ICs was not dependent on the presence of JAK2V617F, a driver mutation associated with the initial underlying MPN. By contrast, multiple MPN-BP IC subclones coexisted within MPN-BP MNCs characterized by different myeloid malignancy gene mutations and cytogenetic abnormalities. MPN-BP ICs in 4 patients exhibited extensive proliferative and self-renewal capacity, as demonstrated by their ability to recapitulate human MPN-BP in serial recipients. These MPN-BP IC subclones underwent extensive continuous clonal competition within individual xenografts and across multiple generations, and their subclonal dynamics were consistent with functional evolution of MPN-BP IC. Finally, we show that MPN-BP ICs originate from not only phenotypically identified hematopoietic stem cells, but also lymphoid-myeloid progenitor cells, which were each characterized by differences in MPN-BP initiating activity and self-renewal capacity.