Multipotent Hematopoietic Progenitor Cells Research Articles

Monoallelic KRAS (G13C) mutation triggers dysregulated expansion in induced pluripotent stem cell-derived hematopoietic progenitor cells.

Although oncogenic RAS mutants are thought to exert mutagenic effects upon blood cells, it remains uncertain how a single oncogenic RAS impacts non-transformed multipotent hematopoietic stem or progenitor cells (HPCs). Such potential pre-malignant status may characterize HPCs in patients with RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). This study sought to elucidate the biological and molecular alterations in human HPCs carrying monoallelic mutant KRAS (G13C) with no other oncogene mutations. We utilized induced pluripotent stem cells (iPSCs) derived from two unrelated RALD patients. Isogenic HPC pairs harboring either wild-type KRAS or monoallelic KRAS (G13C) alone obtained following differentiation enabled reliable comparative analyses. The compound screening was conducted with an established platform using KRAS (G13C) iPSCs and differentiated HPCs. Cell culture assays revealed that monoallelic KRAS (G13C) impacted both myeloid differentiation and expansion characteristics of iPSC-derived HPCs. Comprehensive RNA-sequencing analysis depicted close clustering of HPC samples within the isogenic group, warranting that comparative studies should be performed within the same genetic background. When compared with no stimulation, iPSC-derived KRAS (G13C)-HPCs showed marked similarity with the wild-type isogenic control in transcriptomic profiles. After stimulation with cytokines, however, KRAS (G13C)-HPCs exhibited obvious aberrant cell-cycle and apoptosis responses, compatible with "dysregulated expansion," demonstrated by molecular and biological assessment. Increased BCL-xL expression was identified amongst other molecular changes unique to mutant HPCs. With screening platforms established for therapeutic intervention, we observed selective activity against KRAS (G13C)-HPC expansion in several candidate compounds, most notably in a MEK- and a BCL-2/BCL-xL-inhibitor. These two compounds demonstrated selective inhibitory effects on KRAS (G13C)-HPCs even with primary patient samples when combined. Our findings indicate that a monoallelic oncogenic KRAS can confer dysregulated expansion characteristics to non-transformed HPCs, which may constitute a pathological condition in RALD hematopoiesis. The use of iPSC-based screening platforms will lead to discovering treatments that enable selective inhibition of RAS-mutated HPC clones.

Abstract P03: Splicing factor mutations induce stress granule upregulation in myeloid malignancies

Abstract Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are aggressive blood cancers with a 5-year relative survival rate of only 37% and 28%, respectively [Sekeres MA, Taylor J. JAMA. 2022; https://www.cancer.net/cancer-types/leukemia-acute-myeloid-aml/statistics]. This highlights the need to better characterize the crucial oncogenic mechanisms sustaining cancer stem cell fitness in order to develop more effective therapies. Somatic mutations affecting splicing factor genes represent a hallmark of myeloid blood cancers [Saez B, et al. Blood. 2017]. Splicing factor mutations alter the splicing process of thousands of RNA molecules, but how they provide clonal advantage with consequent ineffective hematopoiesis remains an unanswered question. We integrated fractionated enhanced UV-crosslinking and immunoprecipitation (freCLIP-seq), bulk RNA-seq and immunofluorescence imaging demonstrating that U2AF1 mutations in myeloid malignancies alter the biology of stress granules (SGs), whose upregulation has been linked to tumorigenesis [Redding A, Grabocka E. Trends Cancer. 2023]. U2AF1 mutations, through RNA binding and splicing alterations, increase the availability of SG components leading to enhanced stress adaptation. Crucially, U2AF1-mutant cells show improved cell fitness that can be reverted by a SG inhibitor [Biancon G, et al. Mol Cell. 2022]. These observations have been recently validated at single-cell level in MDS patients (n=10) without splicing factor mutations, with U2AF1 S34F mutation, or with SRSF2 P95H mutation that carries an equally poor prognosis as U2AF1-mutant disease. We isolated CD34+ blasts from bone marrow samples and performed 10x Genomics 5' scRNA-seq. Integrating cell type annotation (https://github.com/TebaldiLab/TheCellMarkerAccordion) with mutational analysis (VarTrix v1.1.19), we clearly distinguished an enrichment of U2AF1 mutant cells and of SRSF2 mutant cells in hematopoietic multipotent progenitor cells. The enrichment is observed also in monocytic cells that are also characterized by high SG score. Of note, it has been recently showed that leukemic stem cells with monocytic phenotype are associated to relapse in AML patients treated with BCL2-inh [Pei S, et al. Cancer Discov. 2023]. We then identified the top influential SG genes in each annotated mutant population and conducted functional enrichment analysis. In hematopoietic multipotent progenitor cells, the biological processes mainly affected by the SG upregulation are regulation of translation, of microtubule, of transcription and of cell growth. Changes in SGs may represent the collective result of splicing aberrancies that drive ineffective hematopoiesis in MDS and AML, or other potential tumorigenic phenotypes. Splicing factor mutations are in fact reported in several cancer types [Stanley RF, Abdel-Wahab O. Nat Cancer. 2022] and SGs are involved in several cancer hallmarks [Boija A, et al. Cancer Cell. 2021]. Alterations in SGs provide therefore novel therapeutic opportunities in blood cancers and potentially in other cancers. Citation Format: Giulia Biancon, Emma Busarello, Matthew Cheng, Domitilla Baccon, Jennifer VanOudenhove, Fabio Ciceri, Toma Tebaldi, Stephanie Halene. Splicing factor mutations induce stress granule upregulation in myeloid malignancies [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P03.

Breast cancer remotely imposes a myeloid bias on haematopoietic stem cells by reprogramming the bone marrow niche.

Myeloid cell infiltration of solid tumours generally associates with poor patient prognosis and disease severity1-13. Therefore, understanding the regulation of myeloid cell differentiation during cancer is crucial to counteract their pro-tumourigenic role. Bone marrow (BM) haematopoiesis is a tightly regulated process for the production of all immune cells in accordance to tissue needs14. Myeloid cells differentiate during haematopoiesis from multipotent haematopoietic stem and progenitor cells (HSPCs)15-17. HSPCs can sense inflammatory signals from the periphery during infections18-21 or inflammatory disorders22-27. In these settings, HSPC expansion is associated with increased myeloid differentiation28,29. During carcinogenesis, the elevation of haematopoietic growth factors supports the expansion and differentiation of committed myeloid progenitors5,30. However, it is unclear whether cancer-related inflammation also triggers demand-adapted haematopoiesis at the level of multipotent HSPCs. In the BM, HSPCs reside within the haematopoietic niche which delivers HSC maintenance and differentiation cues31-35. Mesenchymal stem cells (MSCs) are a major cellular component of the BM niche and contribute to HSC homeostasis36-41. Modifications of MSCs in systemic disorders have been associated with HSC differentiation towards myeloid cells22,42. It is unknown if MSCs are regulated in the context of solid tumours and if their myeloid supportive activity is impacted by cancer-induced systemic changes. Here, using unbiased transcriptomic analysis and in situ imaging of HSCs and the BM niche during breast cancer, we show that both HSCs and MSCs are transcriptionally and spatially modified. We demonstrate that breast tumour can distantly remodel the cellular cross-talks in the BM niche leading to increased myelopoiesis.

Single-Cell Transcriptomics Reveal Altered Hematopoietic Mechanisms Driven By T21 and GATA1s

Introduction Trisomy 21 (T21) is associated with baseline erythrocytosis and thrombocytopenia and risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia of Down syndrome (ML-DS). TAM and ML-DS are characterized by mutations in the transcription factor GATA1, resulting in the truncated isoform GATA1s (G1S). We previously found that T21 increases erythropoiesis, while G1S severely impairs erythroid development but enhances megakaryocyte (MK) proliferation (Byrska-Bishop et al, JCI 2015). To better understand how T21 and G1S each impact hematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) of multipotent and late hematopoietic progenitor cells (HPCs) differing only by chromosome 21 and/or G1S status. Methods Four isogenic human induced pluripotent stem cell (iPSC) lines differing only by chromosome 21 (euploid vs. T21) and/or GATA1 (WT vs. G1S) status underwent hematopoietic differentiation by embryoid body formation. Cells were collected at 3 timepoints: on day 7 (D7), CD41 +235 + multipotent HPCs were purified by flow cytometry and on days 9 (D9) and 11 (D11), late HPCs biased to a single lineage were collected. Cells were sequenced by scRNA-seq and underwent quality control followed by clustering and analysis using Seurat. Clusters were manually annotated based on expression of 141 key hematopoietic genes, including aggregated expression of lineage markers. Early vs. committed lineage clusters were determined based on the relative expression of lineage-specific markers. Within each lineage, differentially expressed genes (DEGs) associated with a genetic background were assessed using the Wilcoxon rank-sum test. The χ² test was used to compare categorical data. A 2-tailed p or adjusted p <0.05 was considered statistically significant. Results Cluster annotation revealed HPC, early and committed erythroid and MK, and myeloid cells (Figure 1). D7 T21/G1S HPCs surprisingly showed an erythroid bias, with upregulation of genes such as GYPA and AHSP, while euploid/G1S HPCs showed a MK bias, with upregulation of genes including PF4V1 and PF4. With wtGATA1, neither euploid nor T21 D7 HPCs showed a significant lineage skew. On D9 and D11, the euploid/WT, T21/WT, and T21/G1S cells developed subpopulations committing to the erythroid lineage, while the euploid/G1S cells demonstrated minimal erythroid potential (Figure 1). Across all 3 timepoints, early and committed erythrocytes from T21 and euploid cells with G1S had a greater number of combined DEGs than their wtGATA1 counterparts (G1S vs. WT, T21: 692 vs. 378; euploid: 1235 vs. 599). On D9, T21 was associated with a greater erythroid skew (38.6% of T21/WT and 32.2% of T21/G1S cells vs. 27.8% of euploid/WT cells; p <0.00001). By D11, this erythroid drive was no longer apparent, and the T21/G1S erythrocytes were significantly less mature (Table 1; p <0.00001). These data suggest that T21 initially enhances erythropoietic drive, while G1S results in a near-complete erythroid block in euploid cells but an incomplete developmental block with T21. All 4 genotypes yielded MK subpopulations on D9 and D11 (Figure 1). With wtGATA1, euploid and T21 MKs were predominantly early MKs by D11 (Table 1). Interestingly, G1S was associated with enhanced MK commitment and maturation in the euploid context but arrest at the early stage with T21 (Table 1; p <0.00001). G1S was again associated with increased DEGs compared to wtGATA1 (G1S vs. WT, T21: 594 vs. 454; euploid: 1689 vs. 1310). These data suggest that G1S in a euploid background promotes MK differentiation, but T21 and G1S cooperate to generate immature, abnormal MKs. Conclusions Although the hematopoietic abnormalities and leukemic risk of T21 have been well-described, the underlying developmental events remain unclear. This scRNA-seq timecourse of HPCs differentiated from isogenic iPSCs reveals the individual and synergistic effects of T21 and G1S. Overall, T21 enhances erythropoietic drive while G1S alone suppresses erythropoiesis. G1S in euploid cells enhances megakaryopoiesis, but with T21, megakaryopoietic commitment and maturation are uncoupled with a strong bias to early MKs. Together, the competing effects of T21 and G1S appear to result in abnormal, immature erythrocytes and MKs with impaired lineage commitment, consistent with a pro-leukemic phenotype.

Chronic Injury of Mice Bone Marrow Multipotent Hematopoietic Progenitor Cells Induced by Ionizing Radiation

To explore the chronic injury and its possible mechanism of ionizing radiation on multipotent hematopoietic progenitor cells (MPPs) by determining the related indicators of MPPs in bone marrow of mice post-radiation. Sixteen C57BL/6 adult mice were randomly divided into normal control and irradiation groups, 8 mice in each group. The mice in irradiation group were exposed to 6 Gy X-ray. The proportion of bone marrow MPPs, their apoptosis and proliferation 2 months after irradiation were detected by flow cytometry. Mitochondrial activity and levels of reactive oxygen species (ROS) in each MPPs population were detected by Mitotracker Red and DCFDA probes, and the senescent state of MPPs in the bone marrow was analyzed. Ionizing radiation could reduce the proportion of MPPs in mouse bone marrow. The proportions and numbers of MPP1, MPP3 and MPP4 in the bone marrow were significantly decreased after whole-body irradiation with 6 Gy X-ray (P<0.05). In addition, radiation significantly reduced the colony-forming capacity of MPPs in bone marrow (P<0.05), the proportions of apoptotic cells in the MPP1 and MPP4 cell populations increased significantly in the bone marrow (P<0.05). The activity of mitochondria was significantly reduced in the bone marrow MPP2, MPP3 and MPP4 cell populations compared with that of the control group (P<0.05). It was also found that the radiation could significantly increase the ROS levels of MPPs in bone marrow, and the content of ROS in the MPP2, MPP3 and MPP4 cell population of the bone marrow was significantly increased(P<0.05). The senescent cells ratios of MPP1, MPP3 and MPP4 cells in the bone marrow after irradiation were significantly higher than those in the control group (P<0.05). Ionizing radiation can cause chronic MPPs damage in mice, which is closely associated with persistent oxidative stress, cells apoptosis, and cellular senescence.

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.

Skewed fate and hematopoiesis of CD34+ HSPCs in umbilical cord blood amid the COVID-19 pandemic.

Umbilical cord blood (UCB) is an irreplaceable source for hematopoietic stem progenitor cells (HSPCs). However, the effects of SARS-CoV-2 infection and COVID-19 vaccination on UCB phenotype, specifically the HSPCs therein, are currently unknown. We thus evaluated any effects of SARS-CoV-2 infection and/or COVID-19 vaccination from the mother on the fate and functionalities of HSPCs in the UCB. The numbers and frequencies of HSPCs in the UCB decreased significantly in donors with previous SARS-CoV-2 infection and more so with COVID-19 vaccination via the induction of apoptosis, likely mediated by IFN-γ-dependent pathways. Two independent hematopoiesis assays, a colony forming unit assay and a mouse humanization assay, revealed skewed hematopoiesis of HSPCs obtained from donors delivered from mothers with SARS-CoV-2 infection history. These results indicate that SARS-CoV-2 infection and COVID-19 vaccination impair the functionalities and survivability of HSPCs in the UCB, which would make unprecedented concerns on the future of HSPC-based therapies.

Hematological Response to Avatrombopag Switch in Thrombopoietin Receptor Agonist-Refractory Aplastic Anemia

ATG combined with CsA is the first-line treatment for patients with SAA who cannot choose HLA-matched HSCT because of various conditions. The HR rate is only approximately 60-80%, and the CR is not high. The lack of residual HSC in patients is an important reason for this unsatisfactory effect. RhTPO/TPO-RA combine with HSC membrane receptors to promote their proliferation and differentiation, expand multipotent hematopoietic progenitor cells in vivo. Studies have found that TPO-RA eltrombopag (EPAG) combined with IST can significantly increase the HR rate and improve the quality of the HR. From 40-50% hematologic improvement can also be obtained with refractory SAA. However, TPO-RA treatment is ineffective in some patients. Avatrombopag (APAG) acts on the transmembrane region of the TPO receptor, has a stronger effect than EPAG, and has no obvious liver toxicity. We analyzed the efficacy and safety of IST combined with rhTPO and/or EPAG in patients with SAA of switching to APAG. We retrospectively analyzed the data of 10 patients who were hospitalized between January and December 2021. All patients have been diagnosed with V/SAA. They received IST combined with rhTPO/TPO-RA treatment, failed to obtain a HR, and were converted to APAG. The patients were treated with IST for at least 6 months, combined with rhTPO and/or EPAG for at least 3 months, and did not get HR. None of the patients included in this study was suitable for undergo allogeneic HSCT. APAG was administered orally at a dose of 40 mg/day once a day for at least 6 months.The efficacy criteria for refractory SAA were defined as follows: one-line response included platelet response (HI-P), neutrophil response (HI-N), and erythroid response (HI-E). The 2-line HR could be a combination of any two of the aforementioned one-line responses. A trilineage response was defined as a patient with simultaneous platelet, erythroid, and neutrophil responses, and the quality of the trilineage response was the same as that of the initial treatment with SAA/TDNSAA. Ten patients were included in the study, with a median age of 54 (14-68) years, including five males and five females. All patients received IST for a median of 8.5 (6-40) months. Four patients received rhTPO for 1-2 months followed by EPAG for 2-8 months. Six patients received only EPAG for 3-4 months. All the patients received rhTPO/TP-RA for a median of 3.5 (3-10) months. Five patients with PNH clones were positive by flow cytometry, and there was no hemolytic or biochemical evidence of hemolysis. Two patients had clonal chromosomal abnormalities: one with 47,XY; +8 [2/20] and the other with 45,XY; -Y [10/20]. Ten patients were converted to APAG treatment for a median of 6 (6-10) months. After 3 months of follow-up , seven patients obtained a HR (7/10, 70%), of which two patients obtained a HI-P and HI-N, and five patients (5/10, 50%) obtained a three-line HR (1-CR, 3-GPR, 1-PR). Seven patients (7/10, 70%) obtained HR after 6 months of follow-up, of which one patient obtained HI-P/N and six patients (6/10, 60%) obtained three-line HR (3-CR, t2-GPR, 1-PR). The patient with two-line HR further improved after 8 months of APAG, reaching a three-line blood cell response to PR. At the end of the last follow-up, the HR rate of all patients with APAG conversion therapy was 70%, and all of them achieved three-line blood cell responses (4-CR, 1-GPR, 2-PR). During the follow-up period, no APAG-related adverse events of grade 2 or above were found in any patient, no thrombotic events occurred, and no fibrous tissue hyperplasia was found in the bone marrow pathology review after 6 months of treatment. Among the five patients with PNH clones before APAG conversion therapy, three patients did not change significantly. Otherwise one patient's granulocyte PNH clone decreased from 16.2% at baseline to 6.3%, and the other patient's PNH clone increased from 9.8% at baseline to 19.6%, whereas there was no symptomatic hemolysis in all patient. One patient (1/10, 10%) with normal chromosomal before therapy get a new clonal chromosomal abnormality after 3 months. It was 46XX, t(X;4 )(p11.4;q21) [4/20]; the HR was assessed as GPR. In conclusion, patients with SAA refractory to TPO-RA, drug conversion therapy can be based on the safety, pharmacological, and pharmacodynamic characteristics of TPO-RA drugs. Therefore, APAG is a better choice for switch therapy in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Development of a Real-Time qPCR Assay for Detection of Common MPL Mutations in Myeloproliferative Neoplasms (MPNS).

Myeloproliferative neoplasms (MPNs) are blood cell disorders, characterized by overproduction of abnormal cells in bone marrow due to stem cell mutation. The proliferations of blood cell are controlled by many genes particularly MPL gene which encodes thrombopoietin receptor, a hematopoietic growth factor involved in the production and regulation of the platelets and multipotent hematopoietic progenitor cells. Acquired mutations including (W515L and W515K) in this gene have been observed in patients with primary myelofibrosis or essential thrombocythemia lacking JAK2 (V617F) mutations. MPL mutation detection is important for MPNs diagnosis, but due to low frequency of mutant allele burden (< 15%) may be missed by already available common assays such as Sanger sequencing. Furthermore, these techniques are costly, time-consuming, and less sensitive. In present study, we aimed to develop sensitive, less time-consuming, and cost-effective real-time PCR assay for the detection of MPL mutations that is based on TaqMan fluorescent probes. DNA was extracted from blood sample of 128 MPNs patients collected and further analysis was performed on TaqMan RT-PCR. Reference curve was obtained for amplified product of MPL gene containing mutated sequence. The predicted sensitivity level was at least 5% mutant allele burden by our developed assay that is much higher than sequencing output. Out of 128, 2 (1.56%) patients harbored W515L mutation and 1 (0.78%) harbored W515K mutation. It was concluded that TaqMan qRT-PCR assay is an efficient, sensitive, cost-effective, and less time-consuming method capable of detecting MPL mutation in MPNs patients. We suggested that this assay might be helpful in investigating mutant allele load in MPNs patients.

Leukemia Stem Cells as a Potential Target to Achieve Therapy-Free Remission in Chronic Myeloid Leukemia.

Simple SummaryLeukemic stem cells represent a rare subpopulation of leukemic cells, which not only drive leukemia initiation and progression, but also contribute to drug resistance and/or disease relapse. To achieve permanent cures and prevent relapse, eradication of leukemia stem cells is essential. Chronic myeloid leukemia is a myeloproliferative disorder, and tyrosine kinase inhibitors have dramatically improved long-term outcomes and quality of life for patients. Point mutations of the kinase domain of BCR-ABL1 lead to drug resistance, and as a result, several new generations of tyrosine kinase inhibitor have been introduced to the clinic. Some patients develop drug resistance without known mutations, however, and the presence of leukemia stem cells is believed to be at least partially associated with resistance development and disease relapse. The identification of specific markers distinguishing leukemia stem cells from healthy hematopoietic stem cells, and the potential contributions of the bone marrow microenvironment to leukemia pathogenesis, have also been explored. In this review, we revisit the current knowledge regarding the roles of leukemia stem cells in response to pharmacological treatment and explore how durable treatment-free remission may be achieved after discontinuing tyrosine kinase inhibitor treatment.Leukemia stem cells (LSCs, also known as leukemia-initiating cells) not only drive leukemia initiation and progression, but also contribute to drug resistance and/or disease relapse. Therefore, eradication of every last LSC is critical for a patient’s long-term cure. Chronic myeloid leukemia (CML) is a myeloproliferative disorder that arises from multipotent hematopoietic stem and progenitor cells. Tyrosine kinase inhibitors (TKIs) have dramatically improved long-term outcomes and quality of life for patients with CML in the chronic phase. Point mutations of the kinase domain of BCR-ABL1 lead to TKI resistance through a reduction in drug binding, and as a result, several new generations of TKIs have been introduced to the clinic. Some patients develop TKI resistance without known mutations, however, and the presence of LSCs is believed to be at least partially associated with resistance development and CML relapse. We previously proposed targeting quiescent LSCs as a therapeutic approach to CML, and a number of potential strategies for targeting insensitive LSCs have been presented over the last decade. The identification of specific markers distinguishing CML-LSCs from healthy HSCs, and the potential contributions of the bone marrow microenvironment to CML pathogenesis, have also been explored. Nonetheless, 25% of CML patients are still expected to switch TKIs at least once, and various TKI discontinuation studies have shown a wide range in the incidence of molecular relapse (from 30% to 60%). In this review, we revisit the current knowledge regarding the role(s) of LSCs in CML leukemogenesis and response to pharmacological treatment and explore how durable treatment-free remission may be achieved and maintained after discontinuing TKI treatment.

Guardians of the eye: new tales about retinal microglia and other ocular macrophages.

Guardians of the eye: new tales about retinal microglia and other ocular macrophages.

Genetically Determined Obesity and Adipose Distribution Impact Human Blood Trait Variation across Cell Lineages

IntroductionGenome wide association studies (GWAS) have catalogued thousands of loci that influence blood traits, but genetic mechanisms and impacted cell types are often unknown. While some regulatory factors are blood cell-autonomous, other extrinsic regulatory factors respond to systemic physiology. Epidemiologic studies have correlated generalized obesity (increased body mass index, BMI) and high cholesterol with anemia, but cell types and mechanisms that mediate these effects are incompletely understood, and these studies do not provide evidence for causality. Thus, we wanted to use genetic effects of BMI and cholesterol on hemoglobin level (HGB) and other blood traits to infer causal relationships, identify relevant cell types, and propose genetic mechanisms.MethodsMendelian randomization (MR), akin to a “genetic randomized controlled trial”, uses variants linked to an exposure trait to estimate causal effects of that exposure on a given outcome. 1 Random variant allele allocation at meiosis enables this approach to address confounding and reverse causality that can otherwise preclude causal inference from epidemiologic and cohort studies. Further, effects of multiple factors can be parsed using multivariable MR and causal mediation analyses to help explain genetic associations. 2 Our study used gender- and age-adjusted GWAS summary statistics from European individuals.ResultsWe hypothesized that increased genetically determined BMI would decrease HGB. Using a MR framework, we found that a 5 kg/m 2 (1 standard deviation unit) increase in BMI decreased hemoglobin by 0.06±0.01 g/dL (p=1x10 -5, Fig). Increased BMI also decreased erythrocyte count, and unexpectedly also decreased platelet and white blood cell counts (all with p<5x10 -3). These data showed that obesity-related mechanisms extended beyond the erythroid lineage, perhaps impacting multipotent hematopoietic progenitor cells (HPCs).Similar to BMI, a 1 SD unit increase in total cholesterol decreased HGB (0.10±0.03 g/dL, p=2x10 -3, Fig). However, genetic effects of cholesterol were restricted to erythroid traits (HGB and hematocrit). Multivariable and mediation analyses confirmed that effects of BMI and cholesterol functioned through distinct genetic mechanisms.We speculated that multilineage effects from BMI could reflect a genetic predisposition to accumulate bone marrow adipose tissue, which can impact HPCs and hematopoiesis. 3 A tendency for ‘central’ adiposity increases one's waist-hip ratio (WHR), and increases cardiovascular disease risk concordant with BMI. Unexpectedly, we found that increased WHR, in contrast to BMI, increased HGB (0.08±0.02 g/dL, p=9x10 -6) as well as erythrocyte, platelet, and white blood cell counts (all with p<4x10 -3, Fig). In multivariable experiments, the effects of WHR were exacerbated after accounting for BMI at the individual or population level. Thus, obesity impacts blood traits through genetically determined adipose distribution.ConclusionsOur results confirm that BMI and cholesterol negatively impact HGB at a genetic level, consistent with clinical observations. The unexpected multilineage effects of genetically determined BMI most likely reflects a tendency to accumulate bone marrow adipose tissue, which in turn impacts HPCs and downstream blood cell production. Our findings suggest that adjustment for BMI and adiposity traits may be considered in blood trait GWAS analyses and illuminate opportunities to functionally dissect related genes and molecular pathways.

Robust Autonomous Generation of Definitive Hematopoietic Stem Cells from Pre-Circulation Murine Embryos

Introduction:The ontogeny of mammalian hematopoiesis is organized in at least three embryonic hematopoietic waves. The first wave is characterized by the generation of primitive erythrocytes emerging from the yolk sac (YS); a second one produces bipotent erythro-myeloid progenitors also in the YS. Finally, multipotent definitive Hematopoietic Stem and Progenitor Cells (HSPCs) with the ability to reconstitute all mature adult blood lineages and sustain life long hematopoiesis emerge from a subset of endothelial cells named hemogenic endothelium (HE). The embryonic anatomical site of origin of these HSPCs is still under debate in the field. An intra-embryonic origin is well accepted, which primarily encompasses the Aorta-Gonad-Mesonephros region (AGM) and umbilical and vitelline arteries. In contrast, a YS contribution to adult hematopoiesis remains controversial. The same developmental origin (e.g. endothelial cells with a mesodermal ancestor) makes it difficult to identify specific markers to discern between an AGM- vs. YS-origin using a lineage trace approach. This together with the highly migratory nature of blood cells and the inability of cells isolated from pre-circulatory embryos to robustly engraft in transplantation assays, even after ex vivo culture, has precluded scientists from properly answering these questions. For example, cultured YS and para-aortic splanchnopleura (PSP, the precursor of the AGM) dissected from embryos prior to the onset of circulation resulted in the generation of HSPCs with multilineage engraftment potential only from the embryonic PSP and not the YS (Cumano A. et al., Immunity 2002). However, blood chimerism was low (<5%) and engraftment was achieved only after transplantation into severely immuno-compromised mice (i.e. Rag2gc-/-). This low engraftment baseline makes difficult any comparison between intraembryonic tissues (IET) and Yolk Sac because a smaller frequency of YS-derived transplantable HSPCs would be difficult to detect, yielding a false negative result.Thus, here we sought to interrogate if we could generate robust engraftment from pre-circulation embryos that would allow to rigorously compare the ability to derive definitive HSC in the IET and YS.Results:We have established ex vivo culture conditions to allow for the derivation of robust multi-lineage engraftment activity from pre-circulation murine embryos. CD45.2+ embryos at the 2-3 somite pairs (s.p.) stage and 5-7 s.p. embryos were dissected from their YS, cultured as explants in our optimized conditions and transplanted into lethally irradiated CD45.1+/CD45.2+ recipients. Here, we observed multilineage reconstitution of the peripheral blood (PB) for >16 weeks post-transplant in 5/7 and 9/11 recipients, respectively. Moreover these cells were serially transplantable, confirming the generation of self-renewing HSCs. In primary recipients, PB chimerism was >50% in 8/18 total recipients. This robust PB chimerism allowed us to rigorously interrogate the corresponding CD45.2+ YS (cultured under the same conditions). In these transplants, we observed PB engraftment in 0/7 and 0/11 recipients of cells isolated from 2-3 s.p. and 5-7 s.p. cultured YSs, respectively. These data suggest that either YS do not autonomously generate HSPCs or that they can not support their maturation ex vivo . Moreover we found that the engraftment ability from the IET is confined to an emerging CD31+CD45+c-Kit+CD41- population. Importantly, this population also develops in the YS explants, but lacks engraftment ability. We have repeated these studies with cultured 19-22 s.p. embryos and YSs, a post-circulation time point and still observed a failure of YS-derived cells to engraft irradiated recipients (0/9) while embryo-derived cells (9/9) engraft robustly. These data suggest that YS likely cannot support the maturation of HSPCs that might have migrated from other sites. Only when we transplanted 32-36 s.p. or 46-47 s.p. YS-derived cells we observed robust PB engraftment under our culture conditions (3/7 and 4/6 recipients engrafted, respectively).Conclusions:We observed robust generation of HSCs with long-term and serially transplantable multilineage potential from pre-circulation murine embryos. Our study demonstrates that the YS is not able to support the maturation of HSPCs. Further studies would be required to determine if the YS is an actual source of HSPCs. DisclosuresNo relevant conflicts of interest to declare.

Prostaglandin E2 Regulates Bipotent Monocyte-Dendritic Progenitor Cell Lineage-Commitment.

The factors/mechanisms regulating multipotent or bipotent hematopoietic progenitor cells lineage-commitment are not well understood. In this study, we found that prostaglandin E2 (PGE2) is a crucial physiological regulator of lineage choice for the bipotential monocyte-dendritic progenitor cell (MDP). Inhibition of endogenous PGE2 biosynthesis in mice by the dual cyclooxygenase inhibitor, indomethacin, enhances bone marrow and spleen monocyte (MO) differentiation and reduces dendritic cell (DC) differentiation. Ex vivo treatment of purified MDP with indomethacin preferentially increases MO development at the expense of DC generation, whereas addition of exogenous PGE2 reverses the indomethacin-mediated alteration in MDP differentiation potential. Treatment of MDP with selective EP receptor agonists demonstrated that EP1 signaling promotes MDP differentiation into DC at the expense of MO generation. Conversely, EP1 receptor knockout mice showed reduced DC and increased MO differentiation. Mechanistic studies revealed that PGE2 increases expression of the tyrosine kinase receptor Flt3 on MDP and increases the DC-lineage-related transcription factor PU.1, while reducing expression of M-CSFR and the MO-lineage-related transcription factor MafB. These data indicate that PGE2-EP1 signaling plays a critical role in MDP lineage commitment and DC and MO differentiation.

BRAFV600E-induced senescence drives Langerhans cell histiocytosis pathophysiology.

Langerhans cell histiocytosis (LCH) is a potentially fatal condition characterized by granulomatous lesions with characteristic clonal mononuclear phagocytes (MNPs) harboring activating somatic mutations in mitogen-activated protein kinase (MAPK) pathway genes, most notably BRAFV600E. We recently discovered that the BRAFV600E mutation can also affect multipotent hematopoietic progenitor cells (HPCs) in multisystem LCH disease. How the BRAFV600E mutation in HPCs leads to LCH is not known. Here we show that enforced expression of the BRAFV600E mutation in early mouse and human multipotent HPCs induced a senescence program that led to HPC growth arrest, apoptosis resistance and a senescence-associated secretory phenotype (SASP). SASP, in turn, promoted HPC skewing toward the MNP lineage, leading to the accumulation of senescent MNPs in tissue and the formation of LCH lesions. Accordingly, elimination of senescent cells using INK-ATTAC transgenic mice, as well as pharmacologic blockade of SASP, improved LCH disease in mice. These results identify senescent cells as a new target for the treatment of LCH.

Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin β1-ILK-β-catenin-JUN axis

Recombinant matrices have enabled feeder cell-free maintenance cultures of human pluripotent stem cells (hPSCs), with laminin 511-E8 fragment (LM511-E8) being widely used. However, we herein report that hPSCs maintained on LM511-E8 resist differentiating to multipotent hematopoietic progenitor cells (HPCs), unlike hPSCs maintained on LM421-E8 or LM121-E8. The latter two LM-E8s bound weakly to hPSCs compared with LM511-E8 and activated the canonical Wnt/β-catenin signaling pathway. Moreover, the extracellular LM-E8-dependent preferential hematopoiesis was associated with a higher expression of integrin β1 (ITGB1) and downstream integrin-linked protein kinase (ILK), β-catenin and phosphorylated JUN. Accordingly, the lower coating concentration of LM511-E8 or addition of a Wnt/β-catenin signaling activator, CHIR99021, facilitated higher HPC yield. In contrast, the inhibition of ILK, Wnt or JNK by inhibitors or mRNA knockdown suppressed the HPC yield. These findings suggest that extracellular laminin scaffolds modulate the hematopoietic differentiation potential of hPSCs by activating the ITGB1-ILK-β-catenin-JUN axis at the undifferentiated stage. Finally, the combination of low-concentrated LM511-E8 and a revised hPSC-sac method, which adds bFGF, SB431542 and heparin to the conventional method, enabled a higher yield of HPCs and higher rate for definitive hematopoiesis, suggesting a useful protocol for obtaining differentiated hematopoietic cells from hPSCs in general.

Calreticulin, a multi-faceted protein: thrombotic and bleeding risks in CALR mutation positive essential thrombocythemiaa

Essential thrombocythemia (ET) is a clonal disorder of a multipotent hematopoietic progenitor cell. In most patients, a driving mutation of Janus kinase 2 gene, calreticulin gene or myeloproliferative leukemia virus oncogene is detected. The occurrence of thrombotic and/or bleeding complications is very typical in manifestations of ET, with many cases of both occurring in the same patient. The thrombotic or bleeding phenotype can be a consequence of the coexistence of driving and non-driving molecular mutations and polymorphisms, affecting the platelet number and function. This paper discusses the nature of this disease, paying special attention to calreticulin gene function.

Eltrombopag Specifically Expands Hematopoietic Multipotent Progenitors in Human Aplastic Anemia

Eltrombopag (EPAG), a thrombopoietin receptor agonist, has been recently added to first-line immunosuppressive therapy (IST) in immune aplastic anemia (AA), significantly improving hematologic recovery and survival (Townsley et al. NEJM 2017), corroborating preclinical data suggesting a direct effect of EPAG on hematopoietic precursors. Here, we analysed the in vivo effects of EPAG on the composition of the hematopoietic stem and progenitor cell (HSPC) compartment after hematologic recovery in human AA patients treated with IST combined with EPAG. Twelve patients with severe immune aplastic anemia who achieved hematologic response six months after initial treatment were invited to participate in the study. Six patients were treated with EPAG associated with cyclosporine and six were treated with rabbit antithymocyte globulin and cyclosporine, but treatment choice was not determined by this study. 25 mL of marrow aspirate was obtained from each patient for multiparametric analysis in an 8-color flow cytometry. Mononuclear cells were separated by gradient density centrifugation and CD34+ cells were purified using magnetic column beads. We used a 9-antibody panel to identify HSPC subpopulations, as follows: hematopoietic stem cells (HSC), CD34+ CD38- CD45RA- CD90+ CD49f+; multipotent progenitor cells (MPP), CD34+ CD38- CD45RA- CD90- CD49f-; multilymphoid progenitor cells (MLP), CD34+ CD38+ CD45RA+ CD10+ CD7-; common myeloid progenitor cells (CMP), CD34+ CD38+ CD45RA- CD135+ CD10- CD7-; granulocyte-monocyte progenitor cells (GMP), CD34+ CD38+ CD45RA+ CD135+ CD10- CD7-; and megakaryocytic-erythroid progenitor cells (MEP), CD34+ CD38+ CD45RA- CD135- CD10- CD7-. Purified marrow CD34+ cells were also assessed in methylcellulose medium and colonies were counted 14 days after triplicate plating. Cell populations and colony formation assay were compared between the two groups using a two-tailed non-parametric Mann-Whitney test. The percentage of CD34+ of total nucleated bone marrow cells was 1.2% (range, 0.28-2.9%) in patients who received IST/EPAG and 0.2% (0.11-0.65%) in those who received IST alone (P&amp;lt;0.005). The percentage of MPPs cells was the only one significantly expanded in patients who received IST/EPAG as compared to IST alone (P&amp;lt;0,05). There was no statistical difference in the number of colony-formation units between groups. Townsley et al. (NEJM 2017) reported on the CD34+ compartment reconstitution three and six months after initial therapy, but did not compare this recovery to patients not receiving EPAG. Additionally, they did not indicate which primitive cells are directly affected by EPAG. It was speculated that EPAG may act on HSPC proliferation via the THPO-MPL signalling pathway, supported by previously evidence that loss-of-function mutations in the THPO gene result in aplastic anemia (Dasouki et al. Blood 2013). Here, we compared for the first time the HSPC compartment reconstitution in AA patients treated with either IST alone or EPAG added to IST. We found that marrow CD34+ cell numbers were higher when EPAG was added to IST, indicating a positive effect on the HSPC compartment. More importantly, we found that MPPs were the only HSPC subset to be significantly increased when patients were treated with EPAG. The role of the THPO-MPL signalling pathway in HSPCs is not entirely clear, but our findings are in agreement with recent observations. In xenotransplant models, Matsuoka et al (Cell Transplantation 2017) found that human CD34+ MPL- cells are able to sustain long-term engraftment, whereas MPL+ cells showed deficient repopulation capacity. Likewise, Cocault et al (Experimental Hematology 2016) found that in human CD34+CD38- cells, MPL is preferentially expressed in CD90-CD49f- cells. Recent observations in refractory AA patients suggest that EPAG may expand quiescent preexisting abnormal clones in the marrow (Winkler et al. Blood 2019). However, in our cohort, we did not observe any aberrant HSPC immunophenotype suggesting the expansion of abnormal clones. In conclusion, we found that in patients with immune aplastic anemia, EPAG significantly expands CD34+ cells and specifically expands hematopoietic multipotent progenitor cells. Our findings support the hypothesis that in vivo the THPO-MPL signalling pathway plays a role in human MPP proliferation, but not in HSCs. Disclosures Scheinberg: Novartis Brasil S.A.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Calado:Novartis Brasil S.A.: Speakers Bureau.

A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

Nicotinamide Inhibits Self-renewal and Induces Granulocyte Differentiation of Multipotent Progenitor Cells.

Nicotinamide (NAM) a form of vitamin B3, is an essential precursor of NAD. This dinucleotide (pyridine nucleotide) participates in the regulation of fundamental processes including transcription, cell cycle progression and DNA repair. Here we assessed the effect of NAM on myeloid differentiation of the IL-3 dependent, multipotent hematopoietic progenitor cell line FDCP-Mix. We found that NAM reduces the pSTAT5 signaling response, cell cycling and self-renewal potential. It initiates an atypical program of myeloid differentiation that results in the emergence of granulocytic cells in the absence of added myeloid differentiation factors. NAM did not affect the expression the of cell surface granulocyte marker GR1 but led to a strong downregulation of MHC-II molecules. Taken together our data show that NAM induces a differentiation program in hematopoietic progenitors prompting them to undergo differentiation along the granulocyte path without reaching the status of fully developed granulocytes. Graphical abstract.