RESUMEN
OBJECTIVE: Hirschsprung disease (HSCR) is a severe congenital disorder affecting 1:5000 live births. HSCR results from the failure of enteric nervous system (ENS) progenitors to fully colonise the gastrointestinal tract during embryonic development. This leads to aganglionosis in the distal bowel, resulting in disrupted motor activity and impaired peristalsis. Currently, the only viable treatment option is surgical resection of the aganglionic bowel. However, patients frequently suffer debilitating, lifelong symptoms, with multiple surgical procedures often necessary. Hence, alternative treatment options are crucial. An attractive strategy involves the transplantation of ENS progenitors generated from human pluripotent stem cells (hPSCs). DESIGN: ENS progenitors were generated from hPSCs using an accelerated protocol and characterised, in detail, through a combination of single-cell RNA sequencing, protein expression analysis and calcium imaging. We tested ENS progenitors' capacity to integrate and affect functional responses in HSCR colon, after ex vivo transplantation to organotypically cultured patient-derived colonic tissue, using organ bath contractility. RESULTS: We found that our protocol consistently gives rise to high yields of a cell population exhibiting transcriptional and functional hallmarks of early ENS progenitors. Following transplantation, hPSC-derived ENS progenitors integrate, migrate and form neurons/glia within explanted human HSCR colon samples. Importantly, the transplanted HSCR tissue displayed significantly increased basal contractile activity and increased responses to electrical stimulation compared with control tissue. CONCLUSION: Our findings demonstrate, for the first time, the potential of hPSC-derived ENS progenitors to repopulate and increase functional responses in human HSCR patient colonic tissue.
Asunto(s)
Colon , Sistema Nervioso Entérico , Enfermedad de Hirschsprung , Enfermedad de Hirschsprung/cirugía , Enfermedad de Hirschsprung/terapia , Humanos , Células Madre Pluripotentes , Trasplante de Células Madre/métodos , Diferenciación CelularRESUMEN
The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are essential for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. Although the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non-cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative-feedback loop with FGF signalling.
Asunto(s)
Tipificación del Cuerpo , Genes Homeobox , Animales , Embrión de Pollo , Humanos , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Mesodermo/metabolismo , Médula Espinal , Expresión Génica , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Spinal muscular atrophy with lower extremity dominant (SMALED) is a hereditary neuromuscular disorder characterized by degeneration of spinal cord motor neurons resulting in lower limbs muscle weakness and paralysis. Mutations in DYNC1H1, which encodes BICD2, a multifunctional adaptor for microtubule motor proteins, cause the disorder. Here, we generated four induced pluripotent stem cell (iPSC) lines from patients with SMALED. Dermal fibroblasts were obtained from the MRC neuromuscular disease biobank and reprogrammed using non-integrating mRNA-based protocol. Characterization of the four iPSC lines included karyotyping and Sanger sequencing, while the expression of associated markers confirmed pluripotency and differentiation potential.
Asunto(s)
Células Madre Pluripotentes Inducidas , Atrofia Muscular Espinal , Humanos , Atrofia Muscular Espinal/genética , Atrofia MuscularRESUMEN
The production of the tissues that make up the mammalian embryonic trunk takes place in a head-tail direction, via the differentiation of posteriorly-located axial progenitor populations. These include bipotent neuromesodermal progenitors (NMPs), which generate both spinal cord neurectoderm and presomitic mesoderm, the precursor of the musculoskeleton. Over the past few years, a number of studies have described the derivation of NMP-like cells from mouse and human pluripotent stem cells (PSCs). In turn, these have greatly facilitated the establishment of PSC differentiation protocols aiming to give rise efficiently to posterior mesodermal and neural cell types, which have been particularly challenging to produce using previous approaches. Moreover, the advent of 3-dimensional-based culture systems incorporating distinct axial progenitor-derived cell lineages has opened new avenues toward the functional dissection of early patterning events and cell vs non-cell autonomous effects. Here, we provide a brief overview of the applications of these cell types in disease modelling and cell therapy and speculate on their potential uses in the future.
Asunto(s)
Tipificación del Cuerpo , Células-Madre Neurales , Animales , Tipificación del Cuerpo/fisiología , Diferenciación Celular/fisiología , Linaje de la Célula , Humanos , Mamíferos , Mesodermo , RatonesRESUMEN
The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by WNT/fibroblast growth factor (FGF) signals at the posterior end of the embryo. NMPs can be generated from human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, NMPs are transient, which compromises production of the full range of rostrocaudal spinal cord identities in vitro. Here we report the generation of NMP-derived pre-neural progenitors (PNPs) with stem cell-like self-renewal capacity. PNPs maintain pre-spinal cord identity for 7-10 passages, dividing to self-renew and to make neural crest progenitors, while gradually adopting a more posterior identity by activating colinear HOX gene expression. The HOX clock can be halted through GDF11-mediated signal inhibition to produce a PNP and NC population with a thoracic identity that can be maintained for up to 30 passages.
Asunto(s)
Cresta Neural , Células Madre Pluripotentes , Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Humanos , Cresta Neural/metabolismo , Células Madre Pluripotentes/metabolismo , Médula Espinal/metabolismoRESUMEN
The neural crest (NC) is a multipotent cell population which can give rise to a vast array of derivatives including neurons and glia of the peripheral nervous system, cartilage, cardiac smooth muscle, melanocytes and sympathoadrenal cells. An attractive strategy to model human NC development and associated birth defects as well as produce clinically relevant cell populations for regenerative medicine applications involves the in vitro generation of NC from human pluripotent stem cells (hPSCs). However, in vivo, the potential of NC cells to generate distinct cell types is determined by their position along the anteroposterior (A-P) axis and, therefore the axial identity of hPSC-derived NC cells is an important aspect to consider. Recent advances in understanding the developmental origins of NC and the signalling pathways involved in its specification have aided the in vitro generation of human NC cells which are representative of various A-P positions. Here, we explore recent advances in methodologies of in vitro NC specification and axis patterning using hPSCs.
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Cresta Neural , Células Madre Pluripotentes , Diferenciación Celular , Humanos , Cresta Neural/metabolismo , NeuronasRESUMEN
The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co-localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.
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Caseína Quinasa Ialfa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Vía de Señalización Wnt , Animales , Proteína Axina/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular Tumoral , Núcleo Celular , Expresión Génica Ectópica , Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Complejos Multiproteicos/metabolismo , Fosforilación , Unión Proteica , Transporte de Proteínas , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismoRESUMEN
DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.