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The bone marrow supports and regulates hematopoiesis, responding to physiological requirements for blood cell production over ontogeny and during pathological challenges. Interactions between hematopoietic cells and niche components are challenging to study mechanistically in the human context, but are important to delineate in order to explore the pathobiology of blood and bone marrow disorders. Organoids are proving transformative in many research settings, but an accurate human bone marrow model incorporating multiple hematopoietic and stromal elements has been lacking. This protocol describes a method to generate three-dimensional, multilineage bone marrow organoids from human induced pluripotent stem cells (hiPSCs), detailing the steps for the directed differentiation of hiPSCs using a series of cytokine cocktails and hydrogel embedding. Over 18 days of differentiation, hiPSCs yield the key lineages that are present in central myelopoietic bone marrow, organized in a well-vascularized architecture that resembles native hematopoietic tissues. This presents a robust, in vitro system that can model healthy and perturbed hematopoiesis in a scalable three-dimensional microenvironment. Bone marrow organoids also support the growth of immortalized cell lines and primary cells from healthy donors and patients with myeloid and lymphoid cancers, including cell types that are poorly viable in standard culture systems. Moreover, we discuss assays for the characterization of organoids, including interrogation of pathogenic remodeling using recombinant TGF-ß treatment, and methods for organoid engraftment with exogenous cells. This protocol can be readily adapted to specific experimental requirements, can be easily implemented by users with tissue culture experience and does not require access to specialist equipment.
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Descoberta de Drogas , Células-Tronco Pluripotentes Induzidas , Organoides , Humanos , Organoides/citologia , Descoberta de Drogas/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Hematopoese , Células da Medula Óssea/citologiaRESUMO
ABSTRACT: Nonmelanoma skin cancers (NMSCs) in ruxolitinib-treated patients with myeloproliferative neoplasms behave aggressively, with adverse features and high recurrence. In our cohort, mortality from metastatic NMSC exceeded that from myelofibrosis. Vigilant skin assessment, counseling on NMSC risks, and prospective ruxolitinib-NMSC studies are crucial.
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Transtornos Mieloproliferativos , Pirazóis , Pirimidinas , Neoplasias Cutâneas , Humanos , Estudos Prospectivos , Transtornos Mieloproliferativos/tratamento farmacológico , Nitrilas , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
Understanding the genetic and nongenetic determinants of tumor protein 53 (TP53)-mutation-driven clonal evolution and subsequent transformation is a crucial step toward the design of rational therapeutic strategies. Here we carry out allelic resolution single-cell multi-omic analysis of hematopoietic stem/progenitor cells (HSPCs) from patients with a myeloproliferative neoplasm who transform to TP53-mutant secondary acute myeloid leukemia (sAML). All patients showed dominant TP53 'multihit' HSPC clones at transformation, with a leukemia stem cell transcriptional signature strongly predictive of adverse outcomes in independent cohorts, across both TP53-mutant and wild-type (WT) AML. Through analysis of serial samples, antecedent TP53-heterozygous clones and in vivo perturbations, we demonstrate a hitherto unrecognized effect of chronic inflammation, which suppressed TP53 WT HSPCs while enhancing the fitness advantage of TP53-mutant cells and promoted genetic evolution. Our findings will facilitate the development of risk-stratification, early detection and treatment strategies for TP53-mutant leukemia, and are of broad relevance to other cancer types.
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Leucemia , Multiômica , Humanos , Proteínas de Neoplasias , Inflamação/genética , Alelos , Leucemia/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Introduction: Recent advances in human cardiac 3D approaches have yielded progressively more complex and physiologically relevant culture systems. However, their application in the study of complex pathological processes, such as inflammation and fibrosis, and their utility as models for drug development have been thus far limited. Methods: In this work, we report the development of chamber-specific, vascularised human induced pluripotent stem cell-derived cardiac microtissues, which allow for the multi-parametric assessment of cardiac fibrosis. Results: We demonstrate the generation of a robust vascular system in the microtissues composed of endothelial cells, fibroblasts and atrial or ventricular cardiomyocytes that exhibit gene expression signatures, architectural, and electrophysiological resemblance to in vivo-derived anatomical cardiac tissues. Following pro-fibrotic stimulation using TGFß, cardiac microtissues recapitulated hallmarks of cardiac fibrosis, including myofibroblast activation and collagen deposition. A study of Ca2+ dynamics in fibrotic microtissues using optical mapping revealed prolonged Ca2+ decay, reflecting cardiomyocyte dysfunction, which is linked to the severity of fibrosis. This phenotype could be reversed by TGFß receptor inhibition or by using the BET bromodomain inhibitor, JQ1. Discussion: In conclusion, we present a novel methodology for the generation of chamber-specific cardiac microtissues that is highly scalable and allows for the multi-parametric assessment of cardiac remodelling and pharmacological screening.
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The grading of fibrosis in myeloproliferative neoplasms (MPN) is an important component of disease classification, prognostication and monitoring. However, current fibrosis grading systems are only semi-quantitative and fail to fully capture sample heterogeneity. To improve the quantitation of reticulin fibrosis, we developed a machine learning approach using bone marrow trephine (BMT) samples (n = 107) from patients diagnosed with MPN or a reactive marrow. The resulting Continuous Indexing of Fibrosis (CIF) enhances the detection and monitoring of fibrosis within BMTs, and aids MPN subtyping. When combined with megakaryocyte feature analysis, CIF discriminates between the frequently challenging differential diagnosis of essential thrombocythemia (ET) and pre-fibrotic myelofibrosis with high predictive accuracy [area under the curve = 0.94]. CIF also shows promise in the identification of MPN patients at risk of disease progression; analysis of samples from 35 patients diagnosed with ET and enrolled in the Primary Thrombocythemia-1 trial identified features predictive of post-ET myelofibrosis (area under the curve = 0.77). In addition to these clinical applications, automated analysis of fibrosis has clear potential to further refine disease classification boundaries and inform future studies of the micro-environmental factors driving disease initiation and progression in MPN and other stem cell disorders.
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Transtornos Mieloproliferativos , Policitemia Vera , Mielofibrose Primária , Trombocitemia Essencial , Humanos , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/patologia , Policitemia Vera/patologia , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/patologia , Medula Óssea/patologia , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/patologia , FibroseRESUMO
Myeloproliferative neoplasms (MPN) are a group of clonal stem cell-derived hematopoietic malignancies driven by aberrant Janus kinase-signal transducer and activator of transcription proteins (JAK/STAT) signaling. Although these are genetically simple diseases, MPNs are phenotypically heterogeneous, reflecting underlying intratumoral heterogeneity driven by the interplay of genetic and nongenetic factors. Their evolution is determined by factors that enable certain cellular subsets to outcompete others. Therefore, techniques that resolve cellular heterogeneity at the single-cell level are ideally placed to provide new insights into MPN biology. With these insights comes the potential to uncover new approaches to predict the clinical course and treat these cancers, ultimately improving outcomes for patients. MPNs present a particularly tractable model of cancer evolution, because most patients present in an early disease phase and only a small proportion progress to aggressive disease. Therefore, it is not surprising that many groundbreaking technological advances in single-cell omics have been pioneered by their application in MPNs. In this review article, we explore how single-cell approaches have provided transformative insights into MPN disease biology, which are broadly applicable across human cancers, and discuss how these studies might be swiftly translated into clinical pathways and may eventually underpin precision medicine.
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Neoplasias da Medula Óssea , Neoplasias Hematológicas , Transtornos Mieloproliferativos , Neoplasias , Humanos , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/terapia , Transtornos Mieloproliferativos/metabolismo , Janus Quinases/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/metabolismo , Transdução de Sinais , Janus Quinase 2/genética , MutaçãoRESUMO
Chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) are rare myeloid disorders that are challenging with regard to diagnosis and clinical management. To study the similarities and differences between these disorders, we undertook a multicenter international study of one of the largest case series (CNL, n = 24; aCML, n = 37 cases, respectively), focusing on the clinical and mutational profiles (n = 53 with molecular data) of these diseases. We found no differences in clinical presentations or outcomes of both entities. As previously described, both CNL and aCML share a complex mutational profile with mutations in genes involved in epigenetic regulation, splicing, and signaling pathways. Apart from CSF3R, only EZH2 and TET2 were differentially mutated between them. The molecular profiles support the notion of CNL and aCML being a continuum of the same disease that may fit best within the myelodysplastic/myeloproliferative neoplasms. We identified 4 high-risk mutated genes, specifically CEBPA (ß = 2.26, hazard ratio [HR] = 9.54, P = .003), EZH2 (ß = 1.12, HR = 3.062, P = .009), NRAS (ß = 1.29, HR = 3.63, P = .048), and U2AF1 (ß = 1.75, HR = 5.74, P = .013) using multivariate analysis. Our findings underscore the relevance of molecular-risk classification in CNL/aCML as well as the importance of CSF3R mutations in these diseases.
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Leucemia Mieloide Crônica Atípica BCR-ABL Negativa , Leucemia Neutrofílica Crônica , Doenças Mieloproliferativas-Mielodisplásicas , Humanos , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/diagnóstico , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Leucemia Neutrofílica Crônica/diagnóstico , Leucemia Neutrofílica Crônica/genética , Epigênese Genética , Doenças Mieloproliferativas-Mielodisplásicas/genética , MutaçãoRESUMO
A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow-stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFß stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers. SIGNIFICANCE: We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed ex vivo tool for the prioritization of new therapeutics. See related commentary by Derecka and Crispino, p. 263. This article is highlighted in the In This Issue feature, p. 247.
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Medula Óssea , Neoplasias Hematológicas , Humanos , Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Organoides , Microambiente TumoralRESUMO
The latency between acquisition of an initiating somatic driver mutation by a single-cell and clinical presentation with cancer is largely unknown. We describe a remarkable case of monozygotic twins presenting with CALR mutation-positive myeloproliferative neoplasms (MPNs) (aged 37 and 38 years), with a clinical phenotype of primary myelofibrosis. The CALR mutation was absent in T cells and dermal fibroblasts, confirming somatic acquisition. Whole-genome sequencing lineage tracing revealed a common clonal origin of the CALR-mutant MPN clone, which occurred in utero followed by twin-to-twin transplacental transmission and subsequent similar disease latency. Index sorting and single-colony genotyping revealed phenotypic hematopoietic stem cells (HSCs) as the likely MPN-propagating cell. Furthermore, neonatal blood spot analysis confirmed in utero origin of the JAK2V617F mutation in a patient presenting with polycythemia vera (aged 34 years). These findings provide a unique window into the prolonged evolutionary dynamics of MPNs and fitness advantage exerted by MPN-associated driver mutations in HSCs.
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Transtornos Mieloproliferativos , Mielofibrose Primária , Calreticulina , Humanos , Janus Quinase 2/genética , Mutação/genética , Transtornos Mieloproliferativos/genética , Mielofibrose Primária/genética , Gêmeos Monozigóticos/genéticaRESUMO
Single-cell RNA sequencing has led to unprecedented levels of data complexity. Although several computational platforms are available, performing data analyses for multiple datasets remains a significant challenge. Here, we provide a comprehensive analytical protocol to interrogate multiple datasets on SingCellaR, an analysis package in R. This tool can be applied to general single-cell transcriptome analyses. We demonstrate steps for data analyses and visualization using bespoke pipelines, in conjunction with existing analysis tools to study human hematopoietic stem and progenitor cells. For complete details on the use and execution of this protocol, please refer to Roy et al. (2021).
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Análise de Dados , Análise de Célula Única , Humanos , RNA-Seq , Análise de Célula Única/métodos , Transcriptoma/genética , Sequenciamento do ExomaRESUMO
CD19-directed immunotherapies have revolutionized the treatment of advanced B-cell acute lymphoblastic leukemia (B-ALL). Despite initial impressive rates of complete remission (CR) many patients ultimately relapse. Patients with B-ALL successfully treated with CD19-directed T cells eventually relapse, which, coupled with the early onset of CD22 expression during B-cell development, suggests that preexisting CD34+CD22+CD19- (pre)-leukemic cells represent an "early progenitor origin-related" mechanism underlying phenotypic escape to CD19-directed immunotherapies. We demonstrate that CD22 expression precedes CD19 expression during B-cell development. CD34+CD19-CD22+ cells are found in diagnostic and relapsed bone marrow samples of â¼70% of patients with B-ALL, and their frequency increases twofold in patients with B-ALL in CR after CD19 CAR T-cell therapy. The median of CD34+CD19-CD22+ cells before treatment was threefold higher in patients in whom B-ALL relapsed after CD19-directed immunotherapy (median follow-up, 24 months). Fluorescence in situ hybridization analysis in flow-sorted cell populations and xenograft modeling revealed that CD34+CD19-CD22+ cells harbor the genetic abnormalities present at diagnosis and initiate leukemogenesis in vivo. Our data suggest that preleukemic CD34+CD19-CD22+ progenitors underlie phenotypic escape after CD19-directed immunotherapies and reinforce ongoing clinical studies aimed at CD19/CD22 dual targeting as a strategy for reducing CD19- relapses. The implementation of CD34/CD19/CD22 immunophenotyping in clinical laboratories for initial diagnosis and subsequent monitoring of patients with B-ALL during CD19-targeted therapy is encouraged.
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Antígenos CD19 , Linfoma de Burkitt , Antígenos CD34 , Linfócitos B , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Recidiva , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
Myeloproliferative neoplasms (MPNs) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although the actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the high mobility group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is upregulated in MPN, with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and BM. RNA-sequencing and chromatin immunoprecipitation sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion, whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and a promising therapeutic target to treat or prevent disease progression.
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Fator de Transcrição GATA2 , Proteína HMGA1a , Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Mielofibrose Primária , Animais , Proliferação de Células , Cromatina/genética , Fator de Transcrição GATA2/genética , Redes Reguladoras de Genes , Proteína HMGA1a/genética , Proteína HMGA1a/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/genética , Camundongos , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Mielofibrose Primária/genéticaRESUMO
Eltrombopag (ELT) is a thrombopoietic agent approved for immune thrombocytopenia and also a potent iron chelator. Here we found that ELT exhibited dose-dependent opposing effects on in vitro megakaryopoiesis: low concentrations (≤6 µM, ELT6) stimulated megakaryopoiesis, but high concentrations (30 µM, ELT30) suppressed megakaryocyte (MK) differentiation and proliferation. The suppressive effects of ELT30 were reproduced by other iron chelators, supporting iron chelation as a likely mechanism. During MK differentiation, committed MK progenitors (CD34+/CD41+ and CD34-/CD41+ cells) were significantly more sensitive than undifferentiated progenitors (CD34+/CD41- cells) to the suppressive effects of ELT30, which resulted from both decreased proliferation and increased apoptosis. The antiproliferative effects of ELT30 were reversed by increased iron in the culture, as were the proapoptotic effects when exposure to ELT30 was short. Because committed MK progenitors exhibited the highest proliferative rate and the highest sensitivity to iron chelation, we tested whether their iron status influenced their response to ELT during rapid cell expansion. In these studies, iron deficiency reduced the proliferation of CD41+ cells in response to all ELT concentrations. Severe iron deficiency also reduced the number of MKs generated in response to high thrombopoietin concentrations by â¼50%, compared with iron-replete cultures. Our findings support the hypothesis that although iron deficiency can stimulate certain cells and steps in megakaryopoiesis, it can also limit the proliferation of committed MK progenitors, with severity of iron deficiency and degree of thrombopoietic stimulation influencing the ultimate output. Further studies are needed to clarify how megakaryopoiesis, iron deficiency, and ELT stimulation are clinically interrelated.
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Sangue Fetal , Células Progenitoras de Megacariócitos , Benzoatos , Diferenciação Celular , Hidrazinas , Ferro/farmacologia , PirazóisRESUMO
Breakpoint cluster region-Abelson (BCR-ABL) negative myeloproliferative neoplasms (MPNs) are chronic myeloid neoplasms initiated by the acquisition of gene mutation(s) in a haematopoietic stem cell, leading to clonal expansion and over-production of blood cells and their progenitors. MPNs encompass a spectrum of disorders with overlapping but distinct molecular, laboratory and clinical features. This includes polycythaemia vera, essential thrombocythaemia and myelofibrosis. Dysregulation of the immune system is key to the pathology of MPNs, supporting clonal evolution, mediating symptoms and resulting in varying degrees of immunocompromise. Targeting immune dysfunction is an important treatment strategy. In the present review, we focus on the immune landscape in patients with MPNs - the role of inflammation in disease pathogenesis, susceptibility to infection and emerging strategies for therapeutic immune modulation. Further detailed work is required to delineate immune perturbation more precisely in MPNs to determine how and why vulnerability to infection differs between clinical subtypes and to better understand how inflammation results in a competitive advantage for the MPN clone. These studies may help shed light on new designs for disease-modifying therapies.
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Imunoterapia , Transtornos Mieloproliferativos/imunologia , Transtornos Mieloproliferativos/terapia , Animais , Proteínas de Fusão bcr-abl/análise , Humanos , Imunidade , Imunoterapia/métodos , Infecções/imunologia , Infecções/patologia , Infecções/terapia , Inflamação/imunologia , Inflamação/patologia , Inflamação/terapia , Transtornos Mieloproliferativos/patologia , Evasão TumoralRESUMO
Platelets are anucleate but contain a rich repertoire of mRNAs. In this issue of Cell Reports Medicine, Shen and colleagues1 present platelet transcriptomes from patients with myeloproliferative neoplasms to study disease mechanisms and generate a predictive algorithm for fibrotic progression.
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Transtornos Mieloproliferativos , Neoplasias , Plaquetas , Humanos , Transtornos Mieloproliferativos/genética , RNA Mensageiro , TranscriptomaRESUMO
Human hematopoiesis is a dynamic process that starts in utero 18-21 days post-conception. Understanding the site- and stage-specific variation in hematopoiesis is important if we are to understand the origin of hematological disorders, many of which occur at specific points in the human lifespan. To unravel how the hematopoietic stem/progenitor cell (HSPC) compartment changes during human ontogeny and the underlying gene regulatory mechanisms, we compare 57,489 HSPCs from 5 different tissues spanning 4 developmental stages through the human lifetime. Single-cell transcriptomic analysis identifies significant site- and developmental stage-specific transitions in cellular architecture and gene regulatory networks. Hematopoietic stem cells show progression from cycling to quiescence and increased inflammatory signaling during ontogeny. We demonstrate the utility of this dataset for understanding aberrant hematopoiesis through comparison to two cancers that present at distinct time points in postnatal life-juvenile myelomonocytic leukemia, a childhood cancer, and myelofibrosis, which classically presents in older adults.