RESUMO
Maximizing the potential of human liver organoids (LOs) for modeling human septic liver requires the integration of innate immune cells, particularly resident macrophage Kupffer cells. In this study, we present a strategy to generate LOs containing Kupffer cells (KuLOs) by recapitulating fetal liver hematopoiesis using human induced pluripotent stem cell (hiPSC)-derived erythro-myeloid progenitors (EMPs), the origin of tissue-resident macrophages, and hiPSC-derived LOs. Remarkably, LOs actively promote EMP hematopoiesis toward myeloid and erythroid lineages. Moreover, supplementing with macrophage colony-stimulating factor (M-CSF) proves crucial in sustaining the hematopoietic population during the establishment of KuLOs. Exposing KuLOs to sepsis-like endotoxins leads to significant organoid dysfunction that closely resembles the pathological characteristics of the human septic liver. Furthermore, we observe a notable functional recovery in KuLOs upon endotoxin elimination, which is accelerated by using Toll-like receptor-4-directed endotoxin antagonist. Our study represents a comprehensive framework for integrating hematopoietic cells into organoids, facilitating in-depth investigations into inflammation-mediated liver pathologies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Hepatopatias , Sepse , Humanos , Células de Kupffer , Fígado/patologia , Hepatopatias/patologia , Organoides , Sepse/patologia , Endotoxinas , Diferenciação CelularRESUMO
Human iPSC-derived liver organoids (LO) or hepatic spheroids (HS) have attracted widespread interest, and the numerous studies on them have recently provided various production protocols. However, the mechanism by which the 3D structures of LO and HS are formed from the 2D-cultured cells and the mechanism of the LO and HS maturation remain largely unknown. In this study, we demonstrate that PDGFRA is specifically induced in the cells that are suitable for HS formation and that PDGF receptors and signaling are required for HS formation and maturation. Additionally, in vivo, we show that the localization of PDGFRα is in complete agreement with mouse E9.5 hepatoblasts, which begin to form the 3D-structural liver bud from the single layer. Our results present that PDGFRA play important roles for 3D structure formation and maturation of hepatocytes in vitro and in vivo and provide a clue to elucidate the hepatocyte differentiation mechanism.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Técnicas de Cultura de Células/métodos , Fígado , Hepatócitos , Diferenciação Celular , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Esferoides CelularesRESUMO
Cellular membrane fluidity is a critical modulator of cell adhesion and migration, prompting us to define the systematic landscape of lineage-specific cellular fluidity throughout differentiation. Here, we have unveiled membrane fluidity landscapes in various lineages ranging from human pluripotency to differentiated progeny: (1) membrane rigidification precedes the exit from pluripotency, (2) membrane composition modulates activin signaling transmission, and (3) signatures are relatively germ layer specific presumably due to unique lipid compositions. By modulating variable lineage-specific fluidity, we developed a label-free "adhesion sorting (AdSort)" method with simple cultural manipulation, effectively eliminating pluripotent stem cells and purifying target population as a result of the over 1,150 of screened conditions combining compounds and matrices. These results underscore the important role of tunable membrane fluidity in influencing stem cell maintenance and differentiation that can be translated into lineage-specific cell purification strategy.
Assuntos
Linhagem da Célula , Fluidez de Membrana , Adesão Celular , Diferenciação Celular , Membrana Celular/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , CinéticaRESUMO
BACKGROUND AIMS: We have previously reported the generation of a current Good Manufacture Practice (cGMP)-compliant induced pluripotent stem cell (iPSC) line for clinical applications. Here we show that multiple cellular products currently being considered for therapy can be generated from a single master cell bank of this or any other clinically compliant iPSC line METHODS: Using a stock at passage 20 prepared from the cGMP-compliant working cell bank (WCB), we tested differentiation into therapeutically relevant cell types of the three germ layers using standardized but generic protocols. Cells that we generated include (i) neural stem cells, dopaminergic neurons and astrocytes; (ii) retinal cells (retinal pigment epithelium and photoreceptors); and (iii) hepatocyte, endothelial and mesenchymal cells. To confirm that these generic protocols can also be used for other iPSC lines, we tested the reproducibility of our methodology with a second clinically compliant line RESULTS: Our results confirmed that well-characterized iPSC lines have broad potency, and, despite allelic variability, the same protocols could be used with minimal modifications with multiple qualified lines. In addition, we introduced a constitutively expressed GFP cassette in Chr13 safe harbor site using a standardized previously described method and observed no significant difference in growth and differentiation between the engineered line and the control line indicating that engineered products can be made using a standardized methodology CONCLUSIONS: We believe that our demonstration that multiple products can be made from the same WCB and that the same protocols can be used with multiple lines offers a path to a cost-effective strategy for developing cellular products from iPSC lines.
Assuntos
Engenharia Celular/métodos , Engenharia Celular/normas , Linhagem da Célula , Fidelidade a Diretrizes , Células-Tronco Pluripotentes Induzidas/citologia , Astrócitos/citologia , Astrócitos/fisiologia , Diferenciação Celular , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/fisiologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Fidelidade a Diretrizes/normas , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Mesoderma/citologia , Mesoderma/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Guias de Prática Clínica como Assunto/normas , Padrões de Referência , Reprodutibilidade dos Testes , Retina/citologia , Bancos de Tecidos/normasRESUMO
Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Fígado/enzimologia , Organoides/citologia , Organoides/embriologia , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Fígado/citologiaRESUMO
The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals.
Assuntos
Animais Geneticamente Modificados , Galinhas/genética , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento , Transgenes , Animais , Linhagem Celular , Embrião de Galinha , Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Transfecção , Transposases/biossíntese , Transposases/genéticaRESUMO
We describe a systematic screening to search for molecules that act as an extracellular signal during somitogenesis in vertebrates. Somitogenesis, which gives rise to segmented structures of axial bones and muscles, is a consequence of cooperative morphogenetic movements caused by precisely regulated cell and tissue interactions. We employed a strategy that combined subtractive hybridization to enrich paraxial mesoderm/somite-specific cDNAs and the signal sequence trap method, which selects signal sequence-containing molecules. Ninety-two independent cDNAs found to possess a putative signal sequence or a transmembrane domain are presented with a data base accession number for each. These clones include cDNAs which were previously identified with a function characterized, cDNAs previously identified with an undetermined function, and also cDNAs with no similarity to known sequences. Among them, 16 clones exhibited peculiar patterns of expression in the presomitic mesoderm/somites revealed by whole-mount and section in situ hybridization techniques, with some clones also being expressed in the forming neural tube. This is the first report in which an elaborate strategy combining three different screening steps was employed to identify signaling molecules relevant to a particular morphogenetic process.