RESUMO
Hematopoietic stem cells (HSCs) reconstitute multilineage human hematopoiesis after clinical bone marrow (BM) transplantation and are the cells of origin of some hematological malignancies. Although HSCs provide multilineage engraftment, individual murine HSCs are lineage biased and contribute unequally to blood cell lineages. Here, we performed high-throughput single-cell RNA sequencing in mice after xenograft with molecularly barcoded adult human BM HSCs. We demonstrated that human individual BM HSCs are also functionally and transcriptionally lineage biased. Specifically, we identified platelet-biased and multilineage human HSCs. Quantitative comparison of transcriptomes from single HSCs from young and aged BM showed that both the proportion of platelet-biased HSCs and their level of transcriptional platelet priming increase with age. Therefore, platelet-biased HSCs and their increased prevalence and transcriptional platelet priming during aging are conserved features of mammalian evolution.
Assuntos
Plaquetas , Células-Tronco Hematopoéticas , Animais , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Plaquetas/imunologia , Envelhecimento/imunologia , Linhagem da Célula/imunologia , Evolução Biológica , Análise de Célula ÚnicaRESUMO
Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.
Assuntos
Plaquetas , Diferenciação Celular , Células-Tronco Hematopoéticas , Megacariócitos , Plaquetas/imunologia , Plaquetas/metabolismo , Animais , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Diferenciação Celular/imunologia , Megacariócitos/citologia , Linhagem da Célula , Camundongos Endogâmicos C57BL , Hematopoese , Trombopoese , Camundongos Knockout , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/imunologiaRESUMO
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.
Assuntos
Leucemia , Multiômica , Humanos , Proteínas de Neoplasias , Inflamação/genética , Alelos , Leucemia/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Haematopoietic stem cells (HSCs) are multipotent, but individual HSCs can show restricted lineage output in vivo. Currently, the molecular mechanisms and physiological role of HSC fate restriction remain unknown. Here we show that lymphoid fate is epigenetically but not transcriptionally primed in HSCs. In multi-lineage HSCs that produce lymphocytes, lymphoid-specific upstream regulatory elements (LymUREs) but not promoters are preferentially accessible compared with platelet-biased HSCs that do not produce lymphoid cell types, providing transcriptionally silent lymphoid lineage priming. Runx3 is preferentially expressed in multi-lineage HSCs, and reinstating Runx3 expression increases LymURE accessibility and lymphoid-primed multipotent progenitor 4 (MPP4) output in old, platelet-biased HSCs. In contrast, platelet-biased HSCs show elevated levels of epigenetic platelet-lineage priming and give rise to MPP2 progenitors with molecular platelet bias. These MPP2 progenitors generate platelets with faster kinetics and through a more direct cellular pathway compared with MPP2s derived from multi-lineage HSCs. Epigenetic programming therefore predicts both fate restriction and differentiation kinetics in HSCs.
Assuntos
Células-Tronco Hematopoéticas , Linfócitos , Linhagem da Célula/genética , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular/genética , Linfócitos/metabolismo , Epigênese Genética , Células-Tronco Multipotentes/metabolismoRESUMO
Alternative splicing is an important source of heterogeneity underlying gene expression between individual cells but remains an understudied area due to the paucity of computational tools to analyze splicing dynamics at single-cell resolution. Here, we present MARVEL, a comprehensive R package for single-cell splicing analysis applicable to RNA sequencing generated from the plate- and droplet-based methods. We performed extensive benchmarking of MARVEL against available tools and demonstrated its utility by analyzing multiple publicly available datasets in diverse cell types, including in disease. MARVEL enables systematic and integrated splicing and gene expression analysis of single cells to characterize the splicing landscape and reveal biological insights.
Assuntos
Processamento Alternativo , Software , Biologia Computacional , Splicing de RNA , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3' ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.
RESUMO
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).
Assuntos
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.
Assuntos
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
Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta-gonad-mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Células Eritroides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/metabolismo , Células Progenitoras Mieloides/metabolismo , Proteínas de Transporte Vesicular/genética , Saco Vitelino/metabolismo , Animais , Diferenciação Celular , Embrião de Mamíferos , Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Epigênese Genética , Células Eritroides/citologia , Feminino , Feto , Genes Reporter , Hematopoese/genética , Fígado/citologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células Progenitoras Mieloides/patologia , Cultura Primária de Células , Proteínas de Transporte Vesicular/metabolismo , Via de Sinalização Wnt , Saco Vitelino/citologia , Saco Vitelino/crescimento & desenvolvimento , Proteína Vermelha FluorescenteRESUMO
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.
Assuntos
Linhagem da Célula/genética , Redes Reguladoras de Genes/genética , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , Hematopoese , Células-Tronco Hematopoéticas/citologia , Humanos , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , TranscriptomaRESUMO
Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Here, we investigate how mutations in either ribosomal protein large (RPL) or ribosomal protein small (RPS) subunit genes selectively affect erythroid progenitor development and clinical phenotypes in Diamond-Blackfan anemia (DBA), a rare ribosomopathy with limited therapeutic options. Using single-cell assays of patient-derived bone marrow, we delineated two distinct cellular trajectories segregating with ribosomal protein genotypes. Almost complete loss of erythroid specification was observed in RPS-DBA. In contrast, we observed relative preservation of qualitatively abnormal erythroid progenitors and precursors in RPL-DBA. Although both DBA genotypes exhibited a proinflammatory bone marrow milieu, RPS-DBA was characterized by erythroid differentiation arrest, whereas RPL-DBA was characterized by preserved GATA1 expression and activity. Compensatory stress erythropoiesis in RPL-DBA exhibited disordered differentiation underpinned by an altered glucocorticoid molecular signature, including reduced ZFP36L2 expression, leading to milder anemia and improved corticosteroid response. This integrative analysis approach identified distinct pathways of erythroid failure and defined genotype-phenotype correlations in DBA. These findings may help facilitate therapeutic target discovery.
Assuntos
Anemia de Diamond-Blackfan , Medula Óssea , Eritropoese , Humanos , Proteínas RibossômicasRESUMO
Corticotrophinomas represent 10% of all surgically removed pituitary adenomas, however, current treatment options are often not effective, and there is a need for improved pharmacological treatments. Recently, JQ1+, a bromodomain inhibitor that promotes gene transcription by binding acetylated histone residues and recruiting transcriptional machinery, has been shown to reduce proliferation in a murine corticotroph cell line, AtT20. RNA-Seq analysis of AtT20 cells following treatment with JQ1+ identified the calcium-sensing receptor (CaSR) gene as significantly downregulated, which was subsequently confirmed using real-time PCR and Western blot analysis. CaSR is a G protein-coupled receptor that plays a central role in calcium homeostasis but can elicit non-calcitropic effects in multiple tissues, including the anterior pituitary where it helps regulate hormone secretion. However, in AtT20 cells, CaSR activates a tumour-specific cAMP pathway that promotes ACTH and PTHrP hypersecretion. We hypothesised that the Casr promoter may harbour binding sites for BET proteins, and using chromatin immunoprecipitation (ChIP)-sequencing demonstrated that the BET protein Brd3 binds to the promoter of the Casr gene. Assessment of CaSR signalling showed that JQ1+ significantly reduced Ca2+e-mediated increases in intracellular calcium (Ca2+i) mobilisation and cAMP signalling. However, the CaSR-negative allosteric modulator, NPS-2143, was unable to reduce AtT20 cell proliferation, indicating that reducing CaSR expression rather than activity is likely required to reduce pituitary cell proliferation. Thus, these studies demonstrate that reducing CaSR expression may be a viable option in the treatment of pituitary tumours. Moreover, current strategies to reduce CaSR activity, rather than protein expression for cancer treatments, may be ineffective.
Assuntos
Azepinas/farmacologia , Hipófise/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Triazóis/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Camundongos , Receptores de Detecção de Cálcio/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin-CD34+CD38-CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a "first hit," (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.
Assuntos
Células-Tronco Hematopoéticas/patologia , Leucemia Mielomonocítica Juvenil/patologia , Animais , Biomarcadores Tumorais/genética , Linhagem Celular , Feminino , Humanos , Leucemia Mielomonocítica Juvenil/genética , Masculino , Camundongos , Mutação/genética , Células-Tronco Neoplásicas/patologia , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Células-Tronco Neoplásicas , Proteína Morfogenética Óssea 4 , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases , Fator de Transcrição STAT3/genética , Microambiente TumoralRESUMO
We present VALERIE (Visualising alternative splicing events from single-cell ribonucleic acid-sequencing experiments), an R package for visualising alternative splicing events at single-cell resolution. To explore any given specified genomic region, corresponding to an alternative splicing event, VALERIE generates an ensemble of informative plots to visualise cell-to-cell heterogeneity of alternative splicing profiles across single cells and performs statistical tests to compare percent spliced-in (PSI) values across the user-defined groups of cells. Among the features available, VALERIE displays PSI values, in lieu of read coverage, which is more suitable for representing alternative splicing profiles for a large number of samples typically generated by single-cell RNA-sequencing experiments. VALERIE is available on the Comprehensive R Archive Network (CRAN): https://cran.r-project.org/web/packages/VALERIE/index.html.
Assuntos
Processamento Alternativo/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Software , Animais , Células Cultivadas , Biologia Computacional , CamundongosRESUMO
Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage- hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.
Assuntos
Hematopoese/fisiologia , Megacariócitos/patologia , Mielofibrose Primária/sangue , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Feminino , Regulação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Megacariócitos/fisiologia , Pessoa de Meia-Idade , Mutação , Receptores Imunológicos/genética , Análise de Célula Única/métodosRESUMO
Acute erythroid leukemia (AEL) commonly involves both myeloid and erythroid lineage transformation. However, the mutations that cause AEL and the cell(s) that sustain the bilineage leukemia phenotype remain unknown. We here show that combined biallelic Cebpa and Gata2 zinc finger-1 (ZnF1) mutations cooperatively induce bilineage AEL, and that the major leukemia-initiating cell (LIC) population has a neutrophil-monocyte progenitor (NMP) phenotype. In pre-leukemic NMPs Cebpa and Gata2 mutations synergize by increasing erythroid transcription factor (TF) expression and erythroid TF chromatin access, respectively, thereby installing ectopic erythroid potential. This erythroid-permissive chromatin conformation is retained in bilineage LICs. These results demonstrate that synergistic transcriptional and epigenetic reprogramming by leukemia-initiating mutations can generate neomorphic pre-leukemic progenitors, defining the lineage identity of the resulting leukemia.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Linhagem da Célula , Transformação Celular Neoplásica/patologia , Células Precursoras Eritroides/patologia , Fator de Transcrição GATA2/genética , Leucemia Eritroblástica Aguda/patologia , Mutação , Neutrófilos/patologia , Idoso , Alelos , Animais , Diferenciação Celular , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Células Precursoras Eritroides/metabolismo , Feminino , Fator de Transcrição GATA1/genética , Humanos , Leucemia Eritroblástica Aguda/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Dedos de ZincoRESUMO
Alternative splicing of RNAs generates isoform diversity, resulting in different proteins that are necessary for maintaining cellular function and identity. The discovery of alternative splicing has been revolutionized by next-generation transcriptomic sequencing mainly using bulk RNA-sequencing, which has unravelled RNA splicing and mis-splicing of normal cells under steady-state and stress conditions. Single-cell RNA-sequencing studies have focused on gene-level expression analysis and revealed gene expression signatures distinguishable between different cellular types. Single-cell alternative splicing is an emerging area of research with the promise to reveal transcriptomic dynamics invisible to bulk- and gene-level analysis. In this review, we will discuss the technological advances for single-cell alternative splicing analysis, computational strategies for isoform detection and quantitation in single cells, and current applications of single-cell alternative splicing analysis and its potential future contributions to personalized medicine.
Assuntos
Anemia Falciforme , Antígenos CD34 , Células da Medula Óssea , Regulação da Expressão Gênica , Análise de Sequência de RNA , Análise de Célula Única , Talassemia , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Criança , Feminino , Humanos , Masculino , Talassemia/genética , Talassemia/metabolismo , Talassemia/patologiaRESUMO
Human myelopoiesis has been proposed to occur through oligopotent common myeloid progenitor (CMP) and lymphoid-primed multipotent progenitor (LMPP) populations. However, other studies have proposed direct commitment of multipotent cells to unilineage fates, without specific intermediary lineage cosegregation patterns. We here show that distinct human myeloid progenitor populations generate the neutrophil/monocyte and mast cell/basophil/eosinophil lineages as previously shown in mouse. Moreover, we find that neutrophil/monocyte potential selectively cosegregates with lymphoid lineage and mast cell/basophil/eosinophil potentials with megakaryocyte/erythroid potential early during lineage commitment. Furthermore, after this initial commitment step, mast cell/basophil/eosinophil and megakaryocyte/erythroid potentials colocalize at the single-cell level in restricted oligopotent progenitors. These results show that human myeloid lineages are generated through two distinct cellular pathways defined by complementary oligopotent cell populations.