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1.
Genome Biol ; 25(1): 122, 2024 May 13.
Article En | MEDLINE | ID: mdl-38741214

BACKGROUND: Pluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive. RESULTS: By single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes. CONCLUSIONS: Our data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity.


Pluripotent Stem Cells , Single-Cell Analysis , Humans , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Genome, Human , Euchromatin/genetics , Euchromatin/metabolism , Chromatin/metabolism , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/cytology , Heterochromatin/metabolism , Embryonic Stem Cells/metabolism , Chromatin Assembly and Disassembly
2.
Sci Rep ; 14(1): 10420, 2024 05 07.
Article En | MEDLINE | ID: mdl-38710730

In the mouse embryo, the transition from the preimplantation to the postimplantation epiblast is governed by changes in the gene regulatory network (GRN) that lead to transcriptional, epigenetic, and functional changes. This transition can be faithfully recapitulated in vitro by the differentiation of mouse embryonic stem cells (mESCs) to epiblast-like cells (EpiLCs), that reside in naïve and formative states of pluripotency, respectively. However, the GRN that drives this conversion is not fully elucidated. Here we demonstrate that the transcription factor OCT6 is a key driver of this process. Firstly, we show that Oct6 is not expressed in mESCs but is rapidly induced as cells exit the naïve pluripotent state. By deleting Oct6 in mESCs, we find that knockout cells fail to acquire the typical morphological changes associated with the formative state when induced to differentiate. Additionally, the key naïve pluripotency TFs Nanog, Klf2, Nr5a2, Prdm14, and Esrrb were expressed at higher levels than in wild-type cells, indicating an incomplete dismantling of the naïve pluripotency GRN. Conversely, premature expression of Oct6 in naïve cells triggered a rapid morphological transformation mirroring differentiation, that was accompanied by the upregulation of the endogenous Oct6 as well as the formative genes Sox3, Zic2/3, Foxp1, Dnmt3A and FGF5. Strikingly, we found that OCT6 represses Nanog in a bistable manner and that this regulation is at the transcriptional level. Moreover, our findings also reveal that Oct6 is repressed by NANOG. Collectively, our results establish OCT6 as a key TF in the dissolution of the naïve pluripotent state and support a model where Oct6 and Nanog form a double negative feedback loop which could act as an important toggle mediating the transition to the formative state.


Cell Differentiation , Gene Regulatory Networks , Mouse Embryonic Stem Cells , Nanog Homeobox Protein , Animals , Mice , Nanog Homeobox Protein/metabolism , Nanog Homeobox Protein/genetics , Cell Differentiation/genetics , Mouse Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Gene Expression Regulation, Developmental , Octamer Transcription Factor-3/metabolism , Octamer Transcription Factor-3/genetics , Germ Layers/metabolism , Germ Layers/cytology , Mice, Knockout
3.
Stem Cell Res Ther ; 15(1): 139, 2024 May 12.
Article En | MEDLINE | ID: mdl-38735988

The concept of "stemness" incorporates the molecular mechanisms that regulate the unlimited self-regenerative potential typical of undifferentiated primitive cells. These cells possess the unique ability to navigate the cell cycle, transitioning in and out of the quiescent G0 phase, and hold the capacity to generate diverse cell phenotypes. Stem cells, as undifferentiated precursors endow with extraordinary regenerative capabilities, exhibit a heterogeneous and tissue-specific distribution throughout the human body. The identification and characterization of distinct stem cell populations across various tissues have revolutionized our understanding of tissue homeostasis and regeneration. From the hematopoietic to the nervous and musculoskeletal systems, the presence of tissue-specific stem cells underlines the complex adaptability of multicellular organisms. Recent investigations have revealed a diverse cohort of non-hematopoietic stem cells (non-HSC), primarily within bone marrow and other stromal tissue, alongside established hematopoietic stem cells (HSC). Among these non-HSC, a rare subset exhibits pluripotent characteristics. In vitro and in vivo studies have demonstrated the remarkable differentiation potential of these putative stem cells, known by various names including multipotent adult progenitor cells (MAPC), marrow-isolated adult multilineage inducible cells (MIAMI), small blood stem cells (SBSC), very small embryonic-like stem cells (VSELs), and multilineage differentiating stress enduring cells (MUSE). The diverse nomenclatures assigned to these primitive stem cell populations may arise from different origins or varied experimental methodologies. This review aims to present a comprehensive comparison of various subpopulations of multipotent/pluripotent stem cells derived from stromal tissues. By analysing isolation techniques and surface marker expression associated with these populations, we aim to delineate the similarities and distinctions among stromal tissue-derived stem cells. Understanding the nuances of these tissue-specific stem cells is critical for unlocking their therapeutic potential and advancing regenerative medicine. The future of stem cells research should prioritize the standardization of methodologies and collaborative investigations in shared laboratory environments. This approach could mitigate variability in research outcomes and foster scientific partnerships to fully exploit the therapeutic potential of pluripotent stem cells.


Multipotent Stem Cells , Pluripotent Stem Cells , Humans , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Cell Differentiation , Stromal Cells/cytology , Stromal Cells/metabolism , Animals
4.
Genome Res ; 34(4): 572-589, 2024 May 15.
Article En | MEDLINE | ID: mdl-38719471

Dormancy is a key feature of stem cell function in adult tissues as well as in embryonic cells in the context of diapause. The establishment of dormancy is an active process that involves extensive transcriptional, epigenetic, and metabolic rewiring. How these processes are coordinated to successfully transition cells to the resting dormant state remains unclear. Here we show that microRNA activity, which is otherwise dispensable for preimplantation development, is essential for the adaptation of early mouse embryos to the dormant state of diapause. In particular, the pluripotent epiblast depends on miRNA activity, the absence of which results in the loss of pluripotent cells. Through the integration of high-sensitivity small RNA expression profiling of individual embryos and protein expression of miRNA targets with public data of protein-protein interactions, we constructed the miRNA-mediated regulatory network of mouse early embryos specific to diapause. We find that individual miRNAs contribute to the combinatorial regulation by the network, and the perturbation of the network compromises embryo survival in diapause. We further identified the nutrient-sensitive transcription factor TFE3 as an upstream regulator of diapause-specific miRNAs, linking cytoplasmic MTOR activity to nuclear miRNA biogenesis. Our results place miRNAs as a critical regulatory layer for the molecular rewiring of early embryos to establish dormancy.


Cell Proliferation , MicroRNAs , Pluripotent Stem Cells , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Mice , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Embryonic Development/genetics , Germ Layers/metabolism , Germ Layers/cytology , Blastocyst/metabolism , Blastocyst/cytology , Female
5.
Int J Mol Sci ; 25(9)2024 Apr 29.
Article En | MEDLINE | ID: mdl-38732061

Embryonic stem-like cells (ES-like cells) are promising for medical research and clinical applications. Traditional methods involve "Yamanaka" transcription (OSKM) to derive these cells from somatic cells in vitro. Recently, a novel approach has emerged, obtaining ES-like cells from spermatogonia stem cells (SSCs) in a time-related process without adding artificial additives to cell cultures, like transcription factors or small molecules such as pten or p53 inhibitors. This study aims to investigate the role of the Nanog in the conversion of SSCs to pluripotent stem cells through both in silico analysis and in vitro experiments. We used bioinformatic methods and microarray data to find significant genes connected to this derivation path, to construct PPI networks, using enrichment analysis, and to construct miRNA-lncRNA networks, as well as in vitro experiments, immunostaining, and Fluidigm qPCR analysis to connect the dots of Nanog significance. We concluded that Nanog is one of the most crucial differentially expressed genes during SSC conversion, collaborating with critical regulators such as Sox2, Dazl, Pou5f1, Dnmt3, and Cdh1. This intricate protein network positions Nanog as a pivotal factor in pathway enrichment for generating ES-like cells, including Wnt signaling, focal adhesion, and PI3K-Akt-mTOR signaling. Nanog expression is presumed to play a vital role in deriving ES-like cells from SSCs in vitro. Finding its pivotal role in this path illuminates future research and clinical applications.


Nanog Homeobox Protein , Nanog Homeobox Protein/metabolism , Nanog Homeobox Protein/genetics , Animals , Male , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/cytology , Cell Differentiation , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Spermatogonia/cytology , Spermatogonia/metabolism , Computer Simulation , Gene Regulatory Networks , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Gene Expression Profiling , Computational Biology/methods , Humans
6.
Stem Cell Reports ; 19(5): 710-728, 2024 May 14.
Article En | MEDLINE | ID: mdl-38701780

Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.


Cellular Reprogramming , Histones , Induced Pluripotent Stem Cells , Kruppel-Like Factor 4 , Cellular Reprogramming/genetics , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Histones/metabolism , Cell Differentiation/genetics , Kruppel-Like Transcription Factors/metabolism , Kruppel-Like Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , SOXB1 Transcription Factors/genetics , Chromatin/metabolism , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Transcription Factors/metabolism , Transcription Factors/genetics , Transcriptome
7.
Nat Commun ; 15(1): 3931, 2024 May 10.
Article En | MEDLINE | ID: mdl-38729993

MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.


Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Pluripotent Stem Cells , Proto-Oncogene Proteins c-myc , Animals , Mice , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Transcription, Genetic , Embryo, Mammalian/metabolism , Embryonic Stem Cells/metabolism , Female , Male
8.
Cell Stem Cell ; 31(5): 583-585, 2024 May 02.
Article En | MEDLINE | ID: mdl-38701751

How nuclear RNA homeostasis impacts cellular functions remains elusive. In this issue of Cell Stem Cell, Han et al.1 utilized a controllable protein degradation system targeting EXOSC2 to perturb RNA homeostasis in mouse pluripotent embryonic stem cells, revealing its vital role in orchestrating crucial nuclear events for cellular fitness.


Homeostasis , RNA, Nuclear , Animals , Mice , RNA, Nuclear/metabolism , RNA, Nuclear/genetics , Exosome Multienzyme Ribonuclease Complex/metabolism , Exosome Multienzyme Ribonuclease Complex/genetics , Cell Nucleus/metabolism , Mouse Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/cytology , Humans , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA/metabolism , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology
9.
Dev Cell ; 59(9): 1093-1095, 2024 May 06.
Article En | MEDLINE | ID: mdl-38714156

In this issue of Developmental Cell, Fowler et al. applied genetic lineage-tracing mouse models to support the notion that artery endothelial cells are the predominant source of hematopoietic stem cells. They leveraged this and developed a method capable of efficiently differentiating human pluripotent stem cells into HLF+HOXA+ hematopoietic progenitors.


Cell Differentiation , Hematopoiesis , Hematopoietic Stem Cells , Pluripotent Stem Cells , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Animals , Humans , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Mice , Cell Lineage , Endothelial Cells/cytology , Endothelial Cells/metabolism
10.
Cell Mol Life Sci ; 81(1): 210, 2024 May 08.
Article En | MEDLINE | ID: mdl-38717553

The cytoophidium is an evolutionarily conserved subcellular structure formed by filamentous polymers of metabolic enzymes. In vertebrates, inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step in guanosine triphosphate (GTP) biosynthesis, is one of the best-known cytoophidium-forming enzymes. Formation of the cytoophidium has been proposed to alleviate the inhibition of IMPDH, thereby facilitating GTP production to support the rapid proliferation of certain cell types such as lymphocytes, cancer cells and pluripotent stem cells (PSCs). However, past studies lacked appropriate models to elucidate the significance of IMPDH cytoophidium under normal physiological conditions. In this study, we demonstrate that the presence of IMPDH cytoophidium in mouse PSCs correlates with their metabolic status rather than pluripotency. By introducing IMPDH2 Y12C point mutation through genome editing, we established mouse embryonic stem cell (ESC) lines incapable of forming IMPDH polymers and the cytoophidium. Our data indicate an important role of IMPDH cytoophidium in sustaining a positive feedback loop that couples nucleotide biosynthesis with upstream metabolic pathways. Additionally, we find that IMPDH2 Y12C mutation leads to decreased cell proliferation and increased DNA damage in teratomas, as well as impaired embryo development following blastocoel injection. Further analysis shows that IMPDH cytoophidium assembly in mouse embryonic development begins after implantation and gradually increases throughout fetal development. These findings provide insights into the regulation of IMPDH polymerisation in embryogenesis and its significance in coordinating cell metabolism and development.


Cell Proliferation , IMP Dehydrogenase , Animals , IMP Dehydrogenase/metabolism , IMP Dehydrogenase/genetics , Mice , Fetal Development/genetics , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Female , Guanosine Triphosphate/metabolism , DNA Damage , Mice, Inbred C57BL
11.
Stem Cell Res Ther ; 15(1): 130, 2024 May 03.
Article En | MEDLINE | ID: mdl-38702837

BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.


Cell Differentiation , Hyaluronic Acid , Mesenchymal Stem Cells , Pluripotent Stem Cells , Humans , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Hyaluronic Acid/pharmacology , Hyaluronic Acid/metabolism , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Culture Media, Serum-Free/pharmacology , Cell Lineage , Cells, Cultured , Cell Culture Techniques/methods , Coculture Techniques
12.
Dev Cell ; 59(10): 1252-1268.e13, 2024 May 20.
Article En | MEDLINE | ID: mdl-38579720

The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.


Cell Differentiation , Gastrulation , Germ Layers , Animals , Mice , Germ Layers/cytology , Germ Layers/metabolism , T-Box Domain Proteins/metabolism , T-Box Domain Proteins/genetics , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Primitive Streak/cytology , Primitive Streak/metabolism , Fetal Proteins/metabolism , Fetal Proteins/genetics , Wnt Signaling Pathway , Cell Proliferation , Gene Expression Regulation, Developmental , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism
13.
In Vitro Cell Dev Biol Anim ; 60(5): 535-543, 2024 May.
Article En | MEDLINE | ID: mdl-38656570

Gastrulation is the first major differentiation process in animal embryos. However, the dynamics of human gastrulation remain mostly unknown owing to the ethical limitations. We studied the dynamics of the mesoderm and endoderm cell differentiation from human pluripotent stem cells for insight into the cellular dynamics of human gastrulation. Human pluripotent stem cells have properties similar to those of the epiblast, which gives rise to the three germ layers. The mesoderm and endoderm were induced with more than 75% purity from human induced pluripotent stem cells. Single-cell dynamics of pluripotent stem cell-derived mesoderm and endoderm cells were traced using time-lapse imaging. Both mesoderm and endoderm cells migrate randomly, accompanied by short-term directional persistence. No substantial differences were detected between mesoderm and endoderm migration. Computer simulations created using the measured parameters revealed that random movement and external force, such as the spread out of cells from the primitive streak area, mimicked the homogeneous discoidal germ layer formation. These results were consistent with the development of amniotes, which suggests the effectiveness of human pluripotent stem cells as a good model for studying human embryogenesis.


Cell Differentiation , Cell Movement , Endoderm , Mesoderm , Pluripotent Stem Cells , Humans , Endoderm/cytology , Mesoderm/cytology , Pluripotent Stem Cells/cytology , Computer Simulation
14.
Stem Cell Reports ; 19(5): 744-757, 2024 May 14.
Article En | MEDLINE | ID: mdl-38579711

Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.


Cell Lineage , Gene Knock-In Techniques , Pluripotent Stem Cells , Humans , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Cell Lineage/genetics , Gene Knock-In Techniques/methods , Genes, Reporter , CRISPR-Cas Systems , RNA, Guide, CRISPR-Cas Systems/genetics , Cell Line , Gene Editing/methods , RNA, Messenger/genetics , RNA, Messenger/metabolism
15.
Cell Stem Cell ; 31(5): 657-675.e8, 2024 May 02.
Article En | MEDLINE | ID: mdl-38642558

Alveolar epithelial type I cells (AT1s) line the gas exchange barrier of the distal lung and have been historically challenging to isolate or maintain in cell culture. Here, we engineer a human in vitro AT1 model system via directed differentiation of induced pluripotent stem cells (iPSCs). We use primary adult AT1 global transcriptomes to suggest benchmarks and pathways, such as Hippo-LATS-YAP/TAZ signaling, enriched in these cells. Next, we generate iPSC-derived alveolar epithelial type II cells (AT2s) and find that nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular, morphologic, and functional phenotype reminiscent of human AT1 cells, including the capacity to form a flat epithelial barrier producing characteristic extracellular matrix molecules and secreted ligands. Our results provide an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s.


Alveolar Epithelial Cells , Cell Differentiation , Induced Pluripotent Stem Cells , Humans , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Signal Transduction , Cells, Cultured , Transcriptome/genetics , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism
16.
Cell ; 187(9): 2143-2157.e15, 2024 Apr 25.
Article En | MEDLINE | ID: mdl-38670072

A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair.


Neurons , Animals , Mice , Rats , Neurons/metabolism , Neurons/cytology , Neurons/physiology , Blastocyst/metabolism , Blastocyst/cytology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Brain/cytology , Brain/physiology , Female , Hippocampus/cytology , Hippocampus/physiology , Species Specificity , Mice, Inbred C57BL , Male
17.
Cell Mol Life Sci ; 81(1): 197, 2024 Apr 25.
Article En | MEDLINE | ID: mdl-38664263

Congenital heart defects are associated with significant health challenges, demanding a deep understanding of the underlying biological mechanisms and, thus, better devices or platforms that can recapitulate human cardiac development. The discovery of human pluripotent stem cells has substantially reduced the dependence on animal models. Recent advances in stem cell biology, genetic editing, omics, microfluidics, and sensor technologies have further enabled remarkable progress in the development of in vitro platforms with increased fidelity and efficiency. In this review, we provide an overview of advancements in in vitro cardiac development platforms, with a particular focus on technological innovation. We categorize these platforms into four areas: two-dimensional solid substrate cultures, engineered substrate architectures that enhance cellular functions, cardiac organoids, and embryos/explants-on-chip models. We conclude by addressing current limitations and presenting future perspectives.


Drug Evaluation, Preclinical , Heart , Tissue Engineering , Humans , Animals , Drug Evaluation, Preclinical/methods , Tissue Engineering/methods , Organoids/metabolism , Organoids/cytology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Heart Defects, Congenital/genetics , Lab-On-A-Chip Devices
18.
Cell Rep ; 43(4): 114031, 2024 Apr 23.
Article En | MEDLINE | ID: mdl-38583153

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.


Cell Differentiation , Lateral Ventricles , Leukemia Inhibitory Factor , Organoids , Pluripotent Stem Cells , Humans , Organoids/metabolism , Organoids/cytology , Leukemia Inhibitory Factor/metabolism , Leukemia Inhibitory Factor/pharmacology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Lateral Ventricles/cytology , Lateral Ventricles/metabolism , STAT3 Transcription Factor/metabolism , Neuroglia/metabolism , Neuroglia/cytology , Signal Transduction
19.
Nat Cell Biol ; 26(5): 719-730, 2024 May.
Article En | MEDLINE | ID: mdl-38594587

During embryonic development, blood cells emerge from specialized endothelial cells, named haemogenic endothelial cells (HECs). As HECs are rare and only transiently found in early developing embryos, it remains difficult to distinguish them from endothelial cells. Here we performed transcriptomic analysis of 28- to 32-day human embryos and observed that the expression of Fc receptor CD32 (FCGR2B) is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and human pluripotent stem cell-derived endothelial cells revealed that robust multilineage haematopoietic potential is harboured within CD32+ endothelial cells and showed that 90% of CD32+ endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a haematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and human pluripotent stem cell cultures, thus allowing the efficient generation of haematopoietic cells in vitro.


Embryonic Development , Receptors, IgG , Humans , Embryonic Development/genetics , Receptors, IgG/metabolism , Receptors, IgG/genetics , Hemangioblasts/metabolism , Hemangioblasts/cytology , Cell Differentiation , Endothelial Cells/metabolism , Endothelial Cells/cytology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Cell Lineage , Cells, Cultured , Gene Expression Regulation, Developmental , Hematopoiesis , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/cytology , Transcriptome , Gene Expression Profiling , Embryo, Mammalian/metabolism , Embryo, Mammalian/cytology
20.
EMBO J ; 43(10): 1965-1989, 2024 May.
Article En | MEDLINE | ID: mdl-38605224

The transition of mouse embryonic stem cells (ESCs) between serum/LIF and 2i(MEK and GSK3 kinase inhibitor)/LIF culture conditions serves as a valuable model for exploring the mechanisms underlying ground and confused pluripotent states. Regulatory networks comprising core and ancillary pluripotency factors drive the gene expression programs defining stable naïve pluripotency. In our study, we systematically screened factors essential for ESC pluripotency, identifying TEAD2 as an ancillary factor maintaining ground-state pluripotency in 2i/LIF ESCs and facilitating the transition from serum/LIF to 2i/LIF ESCs. TEAD2 exhibits increased binding to chromatin in 2i/LIF ESCs, targeting active chromatin regions to regulate the expression of 2i-specific genes. In addition, TEAD2 facilitates the expression of 2i-specific genes by mediating enhancer-promoter interactions during the serum/LIF to 2i/LIF transition. Notably, deletion of Tead2 results in reduction of a specific set of enhancer-promoter interactions without significantly affecting binding of chromatin architecture proteins, CCCTC-binding factor (CTCF), and Yin Yang 1 (YY1). In summary, our findings highlight a novel prominent role of TEAD2 in orchestrating higher-order chromatin structures of 2i-specific genes to sustain ground-state pluripotency.


Chromatin , DNA-Binding Proteins , Mouse Embryonic Stem Cells , TEA Domain Transcription Factors , Transcription Factors , Animals , Mice , TEA Domain Transcription Factors/metabolism , Chromatin/metabolism , Chromatin/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Mouse Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Promoter Regions, Genetic , Enhancer Elements, Genetic
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