RESUMEN

Oncogenic mutations are abundant in the tissues of healthy individuals, but rarely form tumours1-3. Yet, the underlying protection mechanisms are largely unknown. To resolve these mechanisms in mouse mammary tissue, we use lineage tracing to map the fate of wild-type and Brca1-/-;Trp53-/- cells, and find that both follow a similar pattern of loss and spread within ducts. Clonal analysis reveals that ducts consist of small repetitive units of self-renewing cells that give rise to short-lived descendants. This offers a first layer of protection as any descendants, including oncogenic mutant cells, are constantly lost, thereby limiting the spread of mutations to a single stem cell-descendant unit. Local tissue remodelling during consecutive oestrous cycles leads to the cooperative and stochastic loss and replacement of self-renewing cells. This process provides a second layer of protection, leading to the elimination of most mutant clones while enabling the minority that by chance survive to expand beyond the stem cell-descendant unit. This leads to fields of mutant cells spanning large parts of the epithelial network, predisposing it for transformation. Eventually, clone expansion becomes restrained by the geometry of the ducts, providing a third layer of protection. Together, these mechanisms act to eliminate most cells that acquire somatic mutations at the expense of driving the accelerated expansion of a minority of cells, which can colonize large areas, leading to field cancerization.

Asunto(s)

Proteína BRCA1 , Linaje de la Célula , Transformación Celular Neoplásica , Glándulas Mamarias Animales , Mutación , Proteína p53 Supresora de Tumor , Animales , Ratones , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linaje de la Célula/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Transformación Celular Neoplásica/genética , Células Clonales/metabolismo , Células Clonales/citología , Carcinogénesis/genética , Carcinogénesis/patología , Autorrenovación de las Células/genética

RESUMEN

A major next step in hematopoietic stem cell (HSC) biology is to enhance our quantitative understanding of cellular and evolutionary dynamics involved in undisturbed hematopoiesis. Mathematical models have been and continue to be key in this respect, and are most powerful when parameterized experimentally and containing sufficient biological complexity. In this paper, we use data from label propagation experiments in mice to parameterize a mathematical model of hematopoiesis that includes homeostatic control mechanisms as well as clonal evolution. We find that nonlinear feedback control can drastically change the interpretation of kinetic estimates at homeostasis. This suggests that short-term HSC and multipotent progenitors can dynamically adjust to sustain themselves temporarily in the absence of long-term HSCs, even if they differentiate more often than they self-renew in undisturbed homeostasis. Additionally, the presence of feedback control in the model renders the system resilient against mutant invasion. Invasion barriers, however, can be overcome by a combination of age-related changes in stem cell differentiation and evolutionary niche construction dynamics based on a mutant-associated inflammatory environment. This helps us understand the evolution of e.g., TET2 or DNMT3A mutants, and how to potentially reduce mutant burden.

Asunto(s)

Diferenciación Celular , Hematopoyesis , Células Madre Hematopoyéticas , Mutación , Animales , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ratones , Hematopoyesis/genética , Hematopoyesis/fisiología , ADN Metiltransferasa 3A/metabolismo , Homeostasis , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Modelos Biológicos , Linaje de la Célula , Dioxigenasas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Evolución Clonal , Modelos Teóricos

RESUMEN

BACKGROUND: Understanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models, lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate, and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule. METHODS: To establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells, we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways, and construct cell lines carrying an inducible NKX3-1 expressing cassette, together with three-dimensional culture system. Unpaired t test was applied for statistical analyses. RESULTS: We first successfully generate the definitive endoderm, hindgut, and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1, but fail to express NKX3-1. Therefore, we construct NKX3-1-inducible cell line by homologous recombination, which is eventually able to yield AR, FOXA1, and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally, combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations. CONCLUSIONS: This study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line, as well as provides a stepwise differentiation protocol to generate human prostate-like organoids, which should facilitate the studies on prostate development and disease pathogenesis.

Asunto(s)

Diferenciación Celular , Linaje de la Célula , Proteínas de Homeodominio , Células Madre Embrionarias Humanas , Próstata , Factores de Transcripción , Humanos , Próstata/citología , Próstata/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Masculino , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Organoides/metabolismo , Organoides/citología , Ratones , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Animales , Línea Celular

RESUMEN

The lymphatic system is formed during embryonic development by the commitment of specialized lymphatic endothelial cells (LECs) and their subsequent assembly in primary lymphatic vessels. Although lymphatic cells are in continuous contact with mesenchymal cells during development and in adult tissues, the role of mesenchymal cells in lymphatic vasculature development remains poorly characterized. Here, we show that a subpopulation of mesenchymal cells expressing the transcription factor Osr1 are in close association with migrating LECs and established lymphatic vessels in mice. Lineage tracing experiments revealed that Osr1+ cells precede LEC arrival during lymphatic vasculature assembly in the back of the embryo. Using Osr1-deficient embryos and functional in vitro assays, we show that Osr1 acts in a non-cell-autonomous manner controlling proliferation and early migration of LECs to peripheral tissues. Thereby, mesenchymal Osr1+ cells control, in a bimodal manner, the production of extracellular matrix scaffold components and signal ligands crucial for lymphatic vessel formation.

Asunto(s)

Células Endoteliales , Linfangiogénesis , Vasos Linfáticos , Factores de Transcripción , Animales , Vasos Linfáticos/embriología , Vasos Linfáticos/metabolismo , Vasos Linfáticos/citología , Ratones , Linfangiogénesis/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Células Endoteliales/metabolismo , Células Endoteliales/citología , Movimiento Celular/genética , Proliferación Celular , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Mesodermo/metabolismo , Mesodermo/citología , Regulación del Desarrollo de la Expresión Génica , Linaje de la Célula

RESUMEN

Lineage plasticity in small cell lung carcinoma (SCLC) causes therapeutic difficulties. This study aimed to investigate the pathological findings of plasticity in SCLC, focusing on combined SCLC, and elucidate the involvement of YAP1 and other transcription factors. We analysed 100 surgically resected SCLCs through detailed morphological observations and immunohistochemistry for YAP1 and other transcription factors. Component-by-component next-generation sequencing (n = 15 pairs) and immunohistochemistry (n = 35 pairs) were performed on the combined SCLCs. Compared with pure SCLCs (n = 65), combined SCLCs (n = 35) showed a significantly larger size, higher expression of NEUROD1, and higher frequency of double-positive transcription factors (p = 0.0009, 0.04, and 0.019, respectively). Notably, 34% of the combined SCLCs showed morphological mosaic patterns with unclear boundaries between the SCLC and its partner. Combined SCLCs not only had unique histotypes as partners but also represented different lineage plasticity within the partner. NEUROD1-dominant combined SCLCs had a significantly higher proportion of adenocarcinomas as partners, whereas POU2F3-dominant combined SCLCs had a significantly higher proportion of squamous cell carcinomas as partners (p = 0.006 and p = 0.0006, respectively). YAP1 expression in SCLC components was found in 80% of combined SCLCs and 62% of pure SCLCs, often showing mosaic-like expression. Among the combined SCLCs with component-specific analysis, the identical TP53 mutation was found in 10 pairs, and the identical Rb1 abnormality was found in 2 pairs. On immunohistochemistry, the same abnormal p53 pattern was found in 34 pairs, and Rb1 loss was found in 24 pairs. In conclusion, combined SCLC shows a variety of pathological plasticity. Although combined SCLC is more plastic than pure SCLC, pure SCLC is also a phenotypically plastic tumour. The morphological mosaic pattern and YAP1 mosaic-like expression may represent ongoing lineage plasticity. This study also identified the relationship between transcription factors and partners in combined SCLC. Transcription factors may be involved in differentiating specific cell lineages beyond just 'neuroendocrine'.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Factores de Transcripción , Proteínas Señalizadoras YAP , Humanos , Proteínas Señalizadoras YAP/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Masculino , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Femenino , Persona de Mediana Edad , Anciano , Inmunohistoquímica , Linaje de la Célula , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Mutación , Plasticidad de la Célula , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética

RESUMEN

While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Combined deletion of the histone methyl transferases Prdm3 and Prdm16 in early lung endoderm causes perinatal lethality due to respiratory failure from loss of AT2 cells and the accumulation of partially differentiated AT1 cells. Combination of single-cell RNA-seq, bulk ATAC-seq, and CUT&RUN data demonstrate that PRDM3 and PRDM16 regulate chromatin accessibility at NKX2-1 transcriptional targets critical for perinatal AT2 cell differentiation and surfactant homeostasis. Lineage specific deletion of PRDM3/16 in AT2 cells leads to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16.

Asunto(s)

Células Epiteliales Alveolares , Diferenciación Celular , Cromatina , Proteínas de Unión al ADN , Factor Nuclear Tiroideo 1 , Factores de Transcripción , Animales , Factor Nuclear Tiroideo 1/metabolismo , Factor Nuclear Tiroideo 1/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Cromatina/metabolismo , Ratones , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/citología , Ratones Noqueados , Pulmón/citología , Pulmón/metabolismo , Linaje de la Célula , Femenino

RESUMEN

Age is a risk factor for hematologic malignancies. Attributes of the aging hematopoietic system include increased myelopoiesis, impaired adaptive immunity, and a functional decline of the hematopoietic stem cells (HSCs) that maintain hematopoiesis. Changes in the composition of diverse HSC subsets have been suggested to be responsible for age-related alterations, however, the underlying regulatory mechanisms are incompletely understood in the context of HSC heterogeneity. In this study, we investigated how distinct HSC subsets, separated by CD49b, functionally and molecularly change their behavior with age. We demonstrate that the lineage differentiation of both lymphoid-biased and myeloid-biased HSC subsets progressively shifts to a higher myeloid cellular output during aging. In parallel, we show that HSCs selectively undergo age-dependent gene expression and gene regulatory changes in a progressive manner, which is initiated already in the juvenile stage. Overall, our studies suggest that aging intrinsically alters both cellular and molecular properties of HSCs.

Asunto(s)

Envejecimiento , Células Madre Hematopoyéticas , Ratones Endogámicos C57BL , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Animales , Envejecimiento/genética , Envejecimiento/fisiología , Ratones , Diferenciación Celular , Linaje de la Célula/genética , Hematopoyesis/genética , Células Mieloides/metabolismo , Células Mieloides/citología , Masculino , Regulación de la Expresión Génica , Femenino

RESUMEN

Cancer is a highly heterogeneous disease, where phenotypically distinct subpopulations coexist and can be primed to different fates. Both genetic and epigenetic factors may drive cancer evolution, however little is known about whether and how such a process is pre-encoded in cancer clones. Using single-cell multi-omic lineage tracing and phenotypic assays, we investigate the predictive features of either tumour initiation or drug tolerance within the same cancer population. Clones primed to tumour initiation in vivo display two distinct transcriptional states at baseline. Remarkably, these states share a distinctive DNA accessibility profile, highlighting an epigenetic basis for tumour initiation. The drug tolerant niche is also largely pre-encoded, but only partially overlaps the tumour-initiating one and evolves following two genetically and transcriptionally distinct trajectories. Our study highlights coexisting genetic, epigenetic and transcriptional determinants of cancer evolution, unravelling the molecular complexity of pre-encoded tumour phenotypes.

Asunto(s)

Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias , Humanos , Neoplasias/genética , Animales , Análisis de la Célula Individual/métodos , Ratones , Linaje de la Célula/genética , Línea Celular Tumoral , Transcripción Genética , Fenotipo , Multiómica

RESUMEN

The periosteum contains skeletal stem/progenitor cells that contribute to bone fracture healing. However, the in vivo identity of periosteal skeletal stem cells (P-SSCs) remains unclear, and membrane protein markers of P-SSCs that facilitate tissue engineering are needed. Here, we identified integral membrane protein 2A (Itm2a) enriched in SSCs using single-cell transcriptomics. Itm2a+ P-SSCs displayed clonal multipotency and self-renewal and sat at the apex of their differentiation hierarchy. Lineage-tracing experiments showed that Itm2a selectively labeled the periosteum and that Itm2a+ cells were preferentially located in the outer fibrous layer of the periosteum. The Itm2a+ cells rarely expressed CD34 or Osx, but expressed periosteal markers such as Ctsk, CD51, PDGFRA, Sca1, and Gli1. Itm2a+ P-SSCs contributed to osteoblasts, chondrocytes, and marrow stromal cells upon injury. Genetic lineage tracing using dual recombinases showed that Itm2a and Prrx1 lineage cells generated spatially separated subsets of chondrocytes and osteoblasts during fracture healing. Bone morphogenetic protein 2 (Bmp2) deficiency or ablation of Itm2a+ P-SSCs resulted in defects in fracture healing. ITM2A+ P-SSCs were also present in the human periosteum. Thus, our study identified a membrane protein marker that labels P-SSCs, providing an attractive target for drug and cellular therapy for skeletal disorders.

Asunto(s)

Curación de Fractura , Proteínas de la Membrana , Periostio , Animales , Periostio/metabolismo , Periostio/citología , Ratones , Curación de Fractura/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Células Madre/metabolismo , Células Madre/citología , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Fracturas Óseas/patología , Fracturas Óseas/metabolismo , Fracturas Óseas/terapia , Fracturas Óseas/genética , Osteoblastos/metabolismo , Osteoblastos/citología , Diferenciación Celular , Condrocitos/metabolismo , Condrocitos/citología , Masculino , Linaje de la Célula

RESUMEN

Arterial endothelial cells (AECs) are the founder cells for intraembryonic haematopoiesis. Here, we report a method for the efficient generation of human haemogenic DLL4+ AECs from pluripotent stem cells (PSC). Time-series single-cell RNA-sequencing reveals the dynamic evolution of haematopoiesis and lymphopoiesis, generating cell types with counterparts present in early human embryos, including stages marked by the pre-haematopoietic stem cell genes MECOM/EVI1, MLLT3 and SPINK2. DLL4+ AECs robustly support lymphoid differentiation, without the requirement for exogenous NOTCH ligands. Using this system, we find IL7 acts as a morphogenic factor determining the fate choice between the T and innate lymphoid lineages and also plays a role in regulating the relative expression level of RAG1. Moreover, we document a developmental pathway by which human RAG1+ lymphoid precursors give rise to the natural killer cell lineage. Our study describes an efficient method for producing lymphoid progenitors, providing insights into their endothelial and haematopoietic ontogeny, and establishing a platform to investigate the development of the human blood system.

Asunto(s)

Hematopoyesis , Linfopoyesis , Humanos , Hematopoyesis/genética , Linfopoyesis/genética , Células Endoteliales/metabolismo , Células Endoteliales/citología , Diferenciación Celular , Linaje de la Célula/genética , Interleucina-7/metabolismo , Interleucina-7/genética , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/citología , Hemangioblastos/metabolismo , Hemangioblastos/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Análisis de la Célula Individual/métodos , Receptores Notch/metabolismo , Receptores Notch/genética

RESUMEN

Lymphocyte receptors independently evolved in both jawed and jawless vertebrates with similar adaptive immune responses. However, the diversity of functional subtypes and molecular architecture in jawless vertebrate lymphocytes, comparable to jawed species, is not well defined. Here, we profile the gills, intestines, and blood of the lamprey, Lampetra morii, with single-cell RNA sequencing, using a full-length transcriptome as a reference. Our findings reveal higher tissue-specific heterogeneity among T-like cells in contrast to B-like cells. Notably, we identify a unique T-like cell subtype expressing a homolog of the nonlymphoid hematopoietic growth factor receptor, MPL-like (MPL-L). These MPL-L+ T-like cells exhibit features distinct from T cells of jawed vertebrates, particularly in their elevated expression of hematopoietic genes. We further discovered that MPL-L+ VLRA+ T-like cells are widely present in the typhlosole, gill, liver, kidney, and skin of lamprey and they proliferate in response to both a T cell mitogen and recombinant human thrombopoietin. These findings provide new insights into the adaptive immune response in jawless vertebrates, shedding new light on the evolution of adaptive immunity.

Asunto(s)

Inmunidad Adaptativa , Linaje de la Célula , Lampreas , Animales , Lampreas/inmunología , Lampreas/genética , Inmunidad Adaptativa/genética , Linaje de la Célula/genética , Evolución Biológica , Transcriptoma , Linfocitos T/inmunología , Branquias/inmunología , Branquias/metabolismo , Linfocitos/inmunología , Análisis de la Célula Individual , Humanos

RESUMEN

Multiple embryonic origins give rise to forebrain oligodendrocytes (OLs), yet controversies and uncertainty exist regarding their differential contributions. We established intersectional and subtractional strategies to genetically fate map OLs produced by medial ganglionic eminence/preoptic area (MGE/POA), lateral/caudal ganglionic eminences (LGE/CGE), and dorsal pallium in the mouse brain. We found that, contrary to the canonical view, LGE/CGE-derived OLs make minimum contributions to the neocortex and corpus callosum, but dominate piriform cortex and anterior commissure. Additionally, MGE/POA-derived OLs, instead of being entirely eliminated, make small but sustained contribution to cortex with a distribution pattern distinctive from those derived from the dorsal origin. Our study provides a revised and more comprehensive view of cortical and white matter OL origins, and established valuable new tools and strategies for future OL studies.

Asunto(s)

Oligodendroglía , Prosencéfalo , Animales , Oligodendroglía/metabolismo , Oligodendroglía/citología , Prosencéfalo/embriología , Prosencéfalo/citología , Ratones , Linaje de la Célula/genética

RESUMEN

Long known as the site of ribosome biogenesis, the nucleolus is increasingly recognized for its role in shaping three-dimensional (3D) genome organization. Still, the mechanisms governing the targeting of selected regions of the genome to nucleolus-associated domains (NADs) remain enigmatic. Here, we reveal the essential role of ZNF274, a SCAN-bearing member of the Krüppel-associated box (KRAB)-containing zinc finger protein (KZFP) family, in sequestering lineage-specific gene clusters within NADs. Ablation of ZNF274 triggers transcriptional activation across entire genomic neighborhoods-encompassing, among others, protocadherin and KZFP-encoding genes-with loss of repressive chromatin marks, altered the 3D genome architecture and de novo CTCF binding. Mechanistically, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization via a previously uncharted function of the SCAN domain. Our findings illuminate the mechanisms underlying NAD organization and suggest that perinucleolar entrapment into repressive hubs constrains the activation of tandemly arrayed genes to enable selective expression and modulate cell differentiation programs during development.

Asunto(s)

Nucléolo Celular , Familia de Multigenes , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Animales , Humanos , Ratones , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Cromatina/metabolismo , Cromatina/genética , Linaje de la Célula/genética , Dedos de Zinc/genética , Diferenciación Celular/genética , Unión Proteica

RESUMEN

Pluripotent mouse embryonic stem cells (ESCs) can differentiate to all germ layers and serve as an in vitro model of embryonic development. To better understand the differentiation paths traversed by ESCs committing to different lineages, we track individual differentiating ESCs by timelapse imaging followed by multiplexed high-dimensional Imaging Mass Cytometry (IMC) protein quantification. This links continuous live single-cell molecular NANOG and cellular dynamics quantification over 5-6 generations to protein expression of 37 different molecular regulators in the same single cells at the observation endpoints. Using this unique data set including kinship history and live lineage marker detection, we show that NANOG downregulation occurs generations prior to, but is not sufficient for neuroectoderm marker Sox1 upregulation. We identify a developmental cell type co-expressing both the canonical Sox1 neuroectoderm and FoxA2 endoderm markers in vitro and confirm the presence of such a population in the post-implantation embryo. RNASeq reveals cells co-expressing SOX1 and FOXA2 to have a unique cell state characterized by expression of both endoderm as well as neuroectoderm genes suggesting lineage potential towards both germ layers.

Asunto(s)

Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito , Células Madre Embrionarias de Ratones , Factores de Transcripción SOXB1 , Animales , Ratones , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Rastreo Celular/métodos , Proteína Homeótica Nanog/metabolismo , Proteína Homeótica Nanog/genética , Linaje de la Célula , Endodermo/metabolismo , Endodermo/citología , Análisis de la Célula Individual/métodos , Desarrollo Embrionario/genética , Placa Neural/metabolismo , Placa Neural/embriología , Placa Neural/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología

RESUMEN

BACKGROUND: Cardiovascular progenitor cells (CPCs) derived from human embryonic stem cells (hESCs) are considered valuable cell sources for investigating cardiovascular physiology in vitro. Meeting the diverse needs of this application requires the large-scale production of CPCs in an in vitro environment. This study aimed to use an effective culture system utilizing signaling factors for the large-scale expansion of hESC-derived CPCs with the potential to differentiate into functional cardiac lineage cells. METHODS AND RESULTS: Initially, CPCs were generated from hESCs using a 4-day differentiation protocol with a combination of four small molecules (CHIR99021, IWP2, SB-431542, and purmorphamine). These CPCs were then expanded and maintained in a medium containing three factors (bFGF, CHIR, and A83-01), resulting in a > 6,000-fold increase after 8 passages. These CPCs were successfully cryopreserved for an extended period in late passages. The expanded CPCs maintained their gene and protein expression signatures as well as their differentiation capacity through eight passages. Additionally, these CPCs could differentiate into four types of cardiac lineage cells: cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts, demonstrating appropriate functionality. Furthermore, the coculture of these CPC-derived cardiovascular lineage cells in rat tail collagen resulted in cardiac microtissue formation, highlighting the potential of this 3D platform for studying cardiovascular physiology in vitro. CONCLUSION: In conclusion, expandable hESC-derived CPCs demonstrated the ability to self-renewal and differentiation into functional cardiovascular lineage cells consistently across passages, which may apply as potential cell sources for in vitro cardiovascular studies.

Asunto(s)

Diferenciación Celular , Células Madre Embrionarias Humanas , Miocitos Cardíacos , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Animales , Ratas , Linaje de la Célula , Células Cultivadas

RESUMEN

We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a. These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL+ transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice.

Asunto(s)

Células Dendríticas , Animales , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ratones , Activación de Linfocitos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones Endogámicos C57BL , Perfilación de la Expresión Génica , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Linfoides/citología , Linaje de la Célula

RESUMEN

LMNA-related dilated cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C (LMNA) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. The molecular mechanisms of the disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA-related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA-mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (four from Patients and eight from Controls) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for cardiac progenitors to cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Data integration and comparative analyses of Patient and Control cells found cell type and lineage-specific differentially expressed genes (DEGs) with enrichment, supporting pathway dysregulation. Top DEGs and enriched pathways included 10 ZNF genes and RNA polymerase II transcription in pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CMs; LMNA and epigenetic regulation, as well as DDIT4 and mTORC1 signaling in EPDCs. Top DEGs also included XIST and other X-linked genes, six imprinted genes (SNRPN, PWAR6, NDN, PEG10, MEG3, MEG8), and enriched gene sets related to metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs, as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.

Asunto(s)

Cardiomiopatía Dilatada , Diferenciación Celular , Haploinsuficiencia , Células Madre Pluripotentes Inducidas , Lamina Tipo A , Miocitos Cardíacos , Transcriptoma , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Humanos , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Diferenciación Celular/genética , Haploinsuficiencia/genética , Femenino , Transcriptoma/genética , Pericardio/patología , Pericardio/metabolismo , Linaje de la Célula/genética , Análisis de la Célula Individual , Regulación de la Expresión Génica , Mutación/genética , Adulto

RESUMEN

B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), remain incurable, with MM particularly prone to relapse. Our study introduces a novel mouse model with active RANK signaling and the TCL1 oncogene, displaying both CLL and MM phenotypes. In younger mice, TCL1 and RANK expression expands CLL-like B1-lymphocytes, while MM originates from B2-cells, becoming predominant in later stages and leading to severe disease progression and mortality. The induced MM mimics human disease, exhibiting features like clonal plasma cell expansion, paraproteinemia, anemia, and kidney and bone failure, as well as critical immunosurveillance strategies that promote a tumor-supportive microenvironment. This research elucidates the differential impacts of RANK activation in B1- and B2-cells and underscores the distinct roles of single versus combined oncogenes in B-cell malignancies. We also demonstrate that human MM cells express RANK and that inhibiting RANK signaling can reduce MM progression in a xenotransplantation model. Our study provides a rationale for further investigating the effects of RANK signaling in B-cell transformation and the shaping of a tumor-promoting microenvironment.

Asunto(s)

Linfocitos B , Transformación Celular Neoplásica , Mieloma Múltiple , Proteínas Proto-Oncogénicas , Transducción de Señal , Animales , Humanos , Ratones , Linfocitos B/metabolismo , Linfocitos B/inmunología , Linaje de la Célula , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Microambiente Tumoral

RESUMEN

The contribution of endocardial cells (EdCs) to the hematopoietic lineages has been strongly debated. Here, we provide evidence that in zebrafish, the endocardium gives rise to and maintains a stable population of hematopoietic cells. Using single-cell sequencing, we identify an endocardial subpopulation expressing enriched levels of hematopoietic-promoting genes. High-resolution microscopy and photoconversion tracing experiments uncover hematopoietic cells, mainly hematopoietic stem and progenitor cells (HSPCs)/megakaryocyte-erythroid precursors (MEPs), derived from EdCs as well as the dorsal aorta stably attached to the endocardium. Emergence of HSPCs/MEPs in hearts cultured ex vivo without external hematopoietic sources, as well as longitudinal imaging of the beating heart using light sheet microscopy, support endocardial contribution to hematopoiesis. Maintenance of these hematopoietic cells depends on the adhesion factors Integrin α4 and Vcam1 but is at least partly independent of cardiac trabeculation or shear stress. Finally, blocking primitive erythropoiesis increases cardiac-residing hematopoietic cells, suggesting that the endocardium is a hematopoietic reservoir. Altogether, these studies uncover the endocardium as a resident tissue for HSPCs/MEPs and a de novo source of hematopoietic cells.

Asunto(s)

Endocardio , Células Madre Hematopoyéticas , Pez Cebra , Animales , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Endocardio/citología , Endocardio/metabolismo , Hematopoyesis/fisiología , Corazón/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Análisis de la Célula Individual , Linaje de la Célula , Eritropoyesis/fisiología , Animales Modificados Genéticamente

RESUMEN

Alternative cleavage and polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3' UTRs from the same genetic locus, potentially impacting mRNA translation, localization, and stability. Developmentally regulated APA can thus make major contributions to cell type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, ∼500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of cleavage factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knockdown of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell type-specific APA at selected genes.

Asunto(s)

Linaje de la Célula , Poliadenilación , Espermatocitos , Espermatogénesis , Animales , Poliadenilación/genética , Masculino , Espermatogénesis/genética , Espermatocitos/metabolismo , Espermatocitos/citología , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/genética , Células Madre Adultas/metabolismo , Células Madre Adultas/citología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Espermatogonias/citología , Espermatogonias/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética