RESUMEN
Physical processes ultimately shape tissue during development. Two emerging proposals are that cells migrate toward stiffer tissue (durotaxis) and that the extent of cell rearrangements reflects tissue phase, but it is unclear whether and how these concepts are related. Here, we identify fibronectin-dependent tissue stiffness as a control variable that underlies and unifies these phenomena in vivo. In murine limb bud mesoderm, cells are either caged, move directionally, or intercalate as a function of their location along a stiffness gradient. A modified Landau phase equation that incorporates tissue stiffness accurately predicts cell diffusivity upon loss or gain of fibronectin. Fibronectin is regulated by WNT5A-YAP feedback that controls cell movements, tissue shape, and skeletal pattern. The results identify a key determinant of phase transition and show how fibronectin-dependent directional cell movement emerges in a mixed-phase environment in vivo.
Asunto(s)
Movimiento Celular , Fibronectinas , Mesodermo , Fibronectinas/metabolismo , Animales , Mesodermo/metabolismo , Mesodermo/citología , Ratones , Proteína Wnt-5a/metabolismoRESUMEN
MECP2 duplication syndrome (MDS) is a neurodevelopmental disorder caused by tandem duplication of the MECP2 locus and its surrounding genes, including IRAK1. Current MDS mouse models involve transgenic expression of MECP2 only, limiting their applicability to the study of the disease. Herein, we show that an efficient and precise CRISPR/Cas9 fusion proximity-based approach can be utilized to generate an Irak1-Mecp2 tandem duplication mouse model ('Mecp2 Dup'). The Mecp2 Dup mouse model recapitulates the genomic landscape of human MDS by harboring a 160â kb tandem duplication encompassing Mecp2 and Irak1, representing the minimal disease-causing duplication, and the neighboring genes Opn1mw and Tex28. The Mecp2 Dup model exhibits neuro-behavioral abnormalities, and an abnormal immune response to infection not previously observed in other mouse models, possibly owing to Irak1 overexpression. The Mecp2 Dup model thus provides a tool to investigate MDS disease mechanisms and develop potential therapies applicable to patients.
Asunto(s)
Modelos Animales de Enfermedad , Duplicación de Gen , Quinasas Asociadas a Receptores de Interleucina-1 , Discapacidad Intelectual Ligada al Cromosoma X , Proteína 2 de Unión a Metil-CpG , Animales , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Discapacidad Intelectual Ligada al Cromosoma X/genética , Discapacidad Intelectual Ligada al Cromosoma X/patología , Humanos , Ratones Endogámicos C57BL , Ratones , Sistemas CRISPR-Cas/genética , Conducta Animal , MasculinoRESUMEN
In the rapidly moving field of stem cell and embryo research, research questions often sit at the intersection of scientific inquiry and ethical considerations. The International Society for Stem Cell Research (ISSCR) produces guidelines to help navigate decisions in this area. For Cell's 50th Anniversary Focus on Developmental Biology, scientific editor Sarah Geisler discussed the importance of the ISSCR guidelines on stem cell and embryo research for both the stem cell community and the broader public with Amander Clark, Robin Lovell-Badge, and Janet Rossant, who have been involved in the ongoing evolution of the guidelines. A lightly edited transcript of their conversation is shared here.
Asunto(s)
Investigaciones con Embriones , Sociedades Científicas , Investigación con Células Madre , Humanos , Investigación con Células Madre/ética , Investigaciones con Embriones/ética , Guías como AsuntoRESUMEN
Understanding the processes and mechanisms underlying early human embryo development has become an increasingly active and important area of research. It has potential for insights into important clinical issues such as early pregnancy loss, origins of congenital anomalies and developmental origins of adult disease, as well as fundamental insights into human biology. Improved culture systems for preimplantation embryos, combined with the new tools of single cell genomics and live imaging, are providing new insights into the similarities and differences between human and mouse development. However, access to human embryo material is still restricted and extended culture of early embryos has regulatory and ethical concerns. Stem cell-derived models of different phases of human development can potentially overcome these limitations and provide a scalable source of material to explore the early postimplantation stages of human development. To date, such models are clearly incomplete replicas of normal development but future technological improvements can be envisaged. The ethical and regulatory environment for such studies remains to be fully resolved.
Asunto(s)
Embrión de Mamíferos , Desarrollo Embrionario , Humanos , Embarazo , Adulto , Femenino , Animales , Ratones , Blastocisto , Células MadreAsunto(s)
Investigaciones con Embriones , Embrión de Mamíferos , Células Madre Embrionarias Humanas , Investigadores , Investigaciones con Embriones/ética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Células Madre Embrionarias Humanas/citología , Modelos Biológicos , Investigadores/ética , HumanosRESUMEN
Specification of the epiblast (EPI) and primitive endoderm (PE) in the mouse embryo involves fibroblast growth factor (FGF) signaling through the RAS/MAP kinase pathway. FGFR1 and FGFR2 are thought to mediate this signaling in the inner cell mass (ICM) of the mouse blastocyst and BMP signaling can also influence PE specification. In this study, we further explored the dynamics of FGFR2 expression through an enhanced green fluorescent protein (eGFP) reporter mouse line (FGFR2-eGFP). We observed that FGFR2-eGFP is present in the late 8-cell stage; however, it is absent or reduced in the ICM of early blastocysts. We then statistically correlated eGFP expression with PE and EPI markers GATA6 and NANOG, respectively. We detected that eGFP is weakly correlated with GATA6 in early blastocysts, but this correlation quickly increases as the blastocyst develops. The correlation between eGFP and NANOG decreases throughout blastocyst development. Treatment with FGF from the morula stage onwards did not affect FGFR2-eGFP presence in the ICM of early blastocysts; however, late blastocysts presented FGFR2-eGFP in all cells of the ICM. BMP treatment positively influenced FGFR2-eGFP expression and reduced the number of NANOG-positive cells in late blastocysts. In conclusion, FGFR2 is not strongly associated with PE precursors in the early blastocyst, but it is highly correlated with PE cells as blastocyst development progresses, consistent with the proposed role for FGFR2 in maintenance rather than initiating the PE lineage.
Asunto(s)
Endodermo , Estratos Germinativos , Animales , Ratones , Blastocisto/metabolismo , Diferenciación Celular , Linaje de la Célula , Embrión de Mamíferos/metabolismo , Endodermo/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/metabolismoRESUMEN
Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3-6 weeks in order to understand genetic determinants of disease and precision medicine.
Asunto(s)
Edición Génica , Células Madre Pluripotentes , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Células Madre Pluripotentes/metabolismo , Mutación , HeterocigotoRESUMEN
A hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue is formed only after gastrulation. Here, we discover that naive human pluripotent stem cells (hPSCs) are competent to differentiate into EXM cells (EXMCs). EXMCs are specified by inhibition of Nodal signaling and GSK3B, are maintained by mTOR and BMP4 signaling activity, and their transcriptome and epigenome closely resemble that of human and monkey embryo EXM. EXMCs are mesenchymal, can arise from an epiblast intermediate, and are capable of self-renewal. Thus, EXMCs arising via primate-specific specification between implantation and gastrulation can be modeled in vitro. We also find that most of the rare off-target cells within human blastoids formed by triple inhibition (Kagawa et al., 2021) correspond to EXMCs. Our study impacts our ability to model and study the molecular mechanisms of early human embryogenesis and related defects.
Asunto(s)
Células Madre Pluripotentes , Animales , Diferenciación Celular , Embrión de Mamíferos , Estratos Germinativos , Humanos , Mesodermo , PrimatesRESUMEN
This year, Cell Stem Cell and the International Society for Stem Cell Research (ISSCR) are celebrating their 15th and 20th anniversaries, respectively. We took the opportunity to ask the current and four former ISSCR presidents to reflect on major stem cell advances during this time, the evolution of policy, clinical translation and ethical aspects, and future challenges for the field.
Asunto(s)
Aniversarios y Eventos Especiales , Investigación con Células Madre , Políticas , Sociedades CientíficasRESUMEN
Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.
Asunto(s)
Ácidos Indolacéticos , Ubiquitina-Proteína Ligasas , Animales , Cromosomas/metabolismo , Ácidos Indolacéticos/metabolismo , Ligandos , Mamíferos/metabolismo , Ratones , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
From mRNA vaccines to community interventions in global child health, the 2022 Canada Gairdner awards demonstrate the importance of fundamental science and its translation into improved human health and well-being.
Asunto(s)
Distinciones y Premios , Investigación Biomédica , Canadá , Niño , Familia , Salud Global , HumanosRESUMEN
Greater transcultural and transdisciplinary engagement within Muslim contexts and deliberate inclusion of diverse Muslim voices in the development of international guidelines is required to improve understanding of the state of stem cell science, strengthen thinking about attendant ethical complexities, enhance compliance, deepen public deliberation, increase trust, and strengthen practice standards.
Asunto(s)
Islamismo , Células MadreRESUMEN
YAP protein is a critical regulator of mammalian embryonic development. By generating a near-infrared fusion YAP reporter mouse line, we have achieved high-resolution live imaging of YAP localization during mouse embryonic development. We have validated the reporter by demonstrating its predicted responses to blocking LATS kinase activity or blocking cell polarity. By time lapse imaging preimplantation embryos, we revealed a mitotic reset behaviour of YAP nuclear localization. We also demonstrated deep tissue live imaging in post-implantation embryos and revealed an intriguing nuclear YAP pattern in migrating cells. The YAP fusion reporter mice and imaging methods will open new opportunities for understanding dynamic YAP signalling in vivo in many different situations.
Asunto(s)
Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Proteínas Serina-Treonina Quinasas , Animales , Blastocisto/metabolismo , Proteínas de Ciclo Celular/metabolismo , Embrión de Mamíferos/embriología , Femenino , Ratones , Fosfoproteínas/metabolismo , Fosforilación , Embarazo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/fisiología , Proteínas Señalizadoras YAPRESUMEN
There has been recent renewed interest in studying human early embryonic development. The advent of improved culture conditions to maintain blastocysts in vitro for an extended period and the emerging stem-cell-based models of the blastocyst and peri-implantation embryos have provided new information that is relevant to early human embryogenesis. However, the mechanism of lineage development and embryonic patterning, and the molecular pathways involved in their regulation, are still not well understood. Interest in human embryonic development has been reinvigorated recently given numerous technical advances. In this review, Rossant and Tam discuss new insights into human embryogenesis gathered from successes in culturing human embryos in vitro and stem-cell-based embryo models. Then they outline what questions still need answering.
Asunto(s)
Blastocisto/fisiología , Desarrollo Embrionario/fisiología , Gastrulación/fisiología , Blastocisto/metabolismo , Tipificación del Cuerpo/fisiología , Linaje de la Célula , Técnicas de Cultivo de Embriones/métodos , Embrión de Mamíferos/fisiología , Células Madre Embrionarias/citología , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica/genética , HumanosRESUMEN
Pluripotent cells in the mammalian embryo undergo state transitions marked by changes in patterns of gene expression and developmental potential as they progress from pre-implantation through post-implantation stages of development. Recent studies of cultured mouse and human pluripotent stem cells (hPSCs) have identified cells representative of an intermediate stage (referred to as the formative state) between naive pluripotency (equivalent to pre-implantation epiblast) and primed pluripotency (equivalent to late post-implantation epiblast). We examine these recent findings in light of our knowledge of peri-implantation mouse and human development, and we consider the implications of this work for deriving human embryo models from pluripotent cells.
Asunto(s)
Células Madre Pluripotentes , Animales , Diferenciación Celular , Implantación del Embrión , Embrión de Mamíferos , Estratos Germinativos , Humanos , RatonesRESUMEN
While modulatory effects of gut microbes on neurological phenotypes have been reported, the mechanisms remain largely unknown. Here, we demonstrate that indole, a tryptophan metabolite produced by tryptophanase-expressing gut microbes, elicits neurogenic effects in the adult mouse hippocampus. Neurogenesis is reduced in germ-free (GF) mice and in GF mice monocolonized with a single-gene tnaA knockout (KO) mutant Escherichia coli unable to produce indole. External administration of systemic indole increases adult neurogenesis in the dentate gyrus in these mouse models and in specific pathogen-free (SPF) control mice. Indole-treated mice display elevated synaptic markers postsynaptic density protein 95 and synaptophysin, suggesting synaptic maturation effects in vivo. By contrast, neurogenesis is not induced by indole in aryl hydrocarbon receptor KO (AhR-/-) mice or in ex vivo neurospheres derived from them. Neural progenitor cells exposed to indole exit the cell cycle, terminally differentiate, and mature into neurons that display longer and more branched neurites. These effects are not observed with kynurenine, another AhR ligand. The indole-AhR-mediated signaling pathway elevated the expression of ß-catenin, Neurog2, and VEGF-α genes, thus identifying a molecular pathway connecting gut microbiota composition and their metabolic function to neurogenesis in the adult hippocampus. Our data have implications for the understanding of mechanisms of brain aging and for potential next-generation therapeutic opportunities.
Asunto(s)
Envejecimiento/metabolismo , Microbioma Gastrointestinal , Neurogénesis , Receptores de Hidrocarburo de Aril/metabolismo , Triptófano/metabolismo , Animales , Indoles/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Células-Madre Neurales/metabolismoRESUMEN
The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process.