Direct conversion of human fibroblasts to pancreatic epithelial cells through transient progenitor states is controlled by temporal activation of defined factors
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Abstract Cell fate can be reprogrammed by ectopic expression of lineage-specific transcription factors (TF). For example, few specialized cell types like neurons, hepatocytes and cardiomyocytes have been generated from fibroblasts by defined factors (Wang et al , 2021). However, the exact cell state transitions and their control mechanisms during cell fate conversion are still poorly understood. Moreover, the defined TFs for generating vast majority of the human cell types are still elusive. Here, we report a novel protocol for reprogramming human fibroblasts to pancreatic exocrine cells with phenotypic and functional characteristics of ductal epithelial cells using a minimal set of six TFs. We mapped the molecular determinants of lineage dynamics at single-cell resolution using a novel factor-indexing method based on single-nuclei multiome sequencing (FI-snMultiome-seq) that enables dissecting the role of each individual TF and pool of TFs in cell fate conversion. We show that transdifferentiation – although being considered a direct cell fate conversion method – occurs through transient progenitor states orchestrated by stepwise activation of distinct TFs. Specifically, transition from mesenchymal fibroblast identity to epithelial pancreatic exocrine fate involves two deterministic steps: first, an endodermal progenitor state defined by activation of HHEX concurrently with FOXA2 and SOX17, and second, temporal GATA4 activation essential for maintenance of pancreatic cell fate program. Collectively, our data provide a high-resolution temporal map of the epigenome and transcriptome remodeling events that facilitate cell fate conversion, suggesting that direct transdifferentiation process occurs through transient dedifferentiation to progenitor cell states controlled by defined TFs.Keywords:
Transdifferentiation
Cell fate determination
Reprogramming
Fate mapping
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Progenitor
Single-Cell Analysis
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Natural interconversions between distinct somatic cell types have been reported in species as diverse as jellyfish and mice. The efficiency and reproducibility of some reprogramming events represent unexploited avenues in which to probe mechanisms that ensure robust cell conversion. We report that a conserved H3K27me3/me2 demethylase, JMJD-3.1, and the H3K4 methyltransferase Set1 complex cooperate to ensure invariant transdifferentiation (Td) of postmitotic Caenorhabditis elegans hindgut cells into motor neurons. At single-cell resolution, robust conversion requires stepwise histone-modifying activities, functionally partitioned into discrete phases of Td through nuclear degradation of JMJD-3.1 and phase-specific interactions with transcription factors that have conserved roles in cell plasticity and terminal fate selection. Our results draw parallels between epigenetic mechanisms underlying robust Td in nature and efficient cell reprogramming in vitro.
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Although fate maps of early embryos exist for nearly all model organisms, a fate map of the gastrulating human embryo remains elusive. Here we use human gastruloids to piece together a rudimentary fate map for the human primitive streak (PS). This is possible because differing levels of BMP, WNT, and NODAL leads to self-organization of gastruloids into homogenous subpopulations of endoderm and mesoderm, and comparative analysis of these gastruloids, together with the fate map of the mouse embryo, allows the organization of these subpopulations along an anterior-posterior axis. We also developed a novel cell tracking technique that detected robust fate-dependent cell migrations in our gastruloids comparable to those found in the mouse embryo. Taken together, our fate map and recording of cell migrations provides a first coarse view of what the human PS may resemble in vivo.
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Over the last decade significant advances have been made toward reprogramming the fate of somatic cells, typically by overexpression of cell lineage-determinant transcription factors. As key regulators of cell fate, the SOX family of transcription factors has emerged as potent drivers of direct somatic cell reprogramming into multiple lineages, in some cases as the sole overexpressed factor. The vast capacity of SOX factors, especially those of the SOXB1, E and F subclasses, to reprogram cell fate is enlightening our understanding of organismal development, cancer and disease, and offers tremendous potential for regenerative medicine and cell-based therapies. Understanding the molecular mechanisms through which SOX factors reprogram cell fate is essential to optimize the development of novel somatic cell transdifferentiation strategies.
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Fate mapping experiments provide direct information on the differentiation pathways normally taken by cells or tissues during embryogenesis. Systematic analyses of the developmental fate of cell populations localized in different parts of the embryo enables the construction of fate maps. A comparison of the expression pattern of lineage-specific genes and the fate map allows the identification of precursor tissue for cell lineages well before definitive histogenesis takes place. The ability to trace the early lineage history of cells greatly facilitates the elucidation of the forces and processes which lead to the specification of cell lineages and the determination (or commitment) of cell fate. The knowledge of cell fate may also assist the interpretation of the phenotype of mutant embryos produced either by spontaneous mutation or by gene knockout experiments.
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Abstract Although fate maps of early gastrula embryos exist for nearly all model organisms, a fate map of the gastrulating human embryo remains elusive. Here we use human gastruloids to piece together part of a rudimentary fate map of the human primitive streak (PS). This is possible because stimulation with differing levels of BMP, WNT, and NODAL leads to self-organization of gastruloids into large and homogenous different subpopulations of endoderm and mesoderm, and comparative parallel analysis of these gastruloids, together with the fate map of the mouse embryo, allows the organization of these subpopulations along an anterior-posterior axis. We also developed a novel cell tracking technique that allowed the detection of robust fate-dependent cell migrations in our gastruloids comparable to those found in the mouse embryo. Taken together, our gastruloid derived fate map and recording of cell migrations provides a first coarse view of the embryonic human PS.
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Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.
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Abstract A fascinating aspect of developmental biology is how organs are assembled in three dimensions over time. Fundamental to understanding organogenesis is the ability to determine when and where specific cell types are generated, the lineage of each cell, and how cells move to reside in their final position. Numerous methods have been developed to mark and follow the fate of cells in various model organisms used by developmental biologists, but most are not readily applicable to mouse embryos in utero because they involve physical marking of cells through injection of tracers. The advent of sophisticated transgenic and gene targeting techniques, combined with the use of site‐specific recombinases, has revolutionized fate mapping studies in mouse. Furthermore, using genetic fate mapping to mark cells has opened up the possibility of addressing fundamental questions that cannot be studied with traditional methods of fate mapping in other organisms. Specifically, genetic fate mapping allows both the relationship between embryonic gene expression and cell fate (genetic lineage) to be determined, as well as the link between gene expression domains and anatomy (genetic anatomy) to be established. In this review, we present the ever‐evolving development of genetic fate mapping techniques in mouse, especially the recent advance of Genetic Inducible Fate Mapping. We then review recent studies in the nervous system (focusing on the anterior hindbrain) as well as in the limb and with adult stem cells to highlight fundamental developmental processes that can be discovered using genetic fate mapping approaches. We end with a look toward the future at a powerful new approach that combines genetic fate mapping with cellular phenotyping alleles to study cell morphology, physiology, and function using examples from the nervous system. Developmental Dynamics 235:2376–2385, 2006. © 2006 Wiley‐Liss, Inc.
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Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.
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AbstractThis chapter deals largely with the use of fluorescent dyes in the investigation of the development of the chick embryo. It covers three issues; generating fate maps, lineage (or clonal) analysis from single-cell injections, and axonal tracing techniques to uncover the neuronal organization of the early nervous system. The construction of fate maps in the early embryo is an important step in the process of understanding how an embryo is built. Fate maps tell us about the origin of particular cell groups, the morphogenetic movements that occur as the embryo takes shape, and can reveal the potential for signaling between cells whose proximity may be transient and obscured by subsequent cell rearrangements. Fate maps can be constructed by analyzing the fate of several neighboring cells labeled simultaneously (here the tracking dyes are usually applied to cell surfaces by extracellular injection) or more precisely by following the fate of individual cells (where the tracking dyes are usually injected intracellularly).KeywordsConcentration Of100Morphogenetic MovementInternal FilamentMicropipet PullerAxial DriveThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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