RESUMO
CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.
Assuntos
Sistemas CRISPR-Cas/genética , Células-Tronco Embrionárias Humanas , RNA Guia de Cinetoplastídeos/genética , Deleção de Sequência/genética , Endonucleases/química , Endonucleases/genética , Escherichia coli/genética , Edição de Genes/métodos , Genoma Humano/genética , Genômica , Humanos , Ribonucleoproteínas/genéticaRESUMO
Vascularization plays a critical role in organ maturation and cell-type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.
RESUMO
BACKGROUND: NPHS2 variants are the most common cause of steroid-resistant nephrotic syndrome in children >1 month old. Missense NPHS2 variants were reported to cause mistrafficking of the encoded protein, PODOCIN, but this conclusion was on the basis of overexpression in some nonpodocyte cell lines. METHODS: We generated a series of human induced pluripotent stem cell (iPSC) lines bearing pathogenic missense variants of NPHS2 , encoding the protein changes p.G92C, p.P118L, p.R138Q, p.R168H, and p.R291W, and control lines. iPSC lines were also generated from a patient with steroid-resistant nephrotic syndrome (p.R168H homozygote) and a healthy heterozygous parent. All lines were differentiated into kidney organoids. Immunofluorescence assessed PODOCIN expression and subcellular localization. Podocytes were transcriptionally profiled and PODOCIN-NEPHRIN interaction interrogated. RESULTS: All variant lines revealed reduced levels of PODOCIN protein in the absence of reduced transcription. Although wild-type PODOCIN localized to the membrane, distinct variant proteins displayed unique patterns of subcellular protein trafficking, some unreported. P118L and R138Q were preferentially retained in the endoplasmic reticulum (ER); R168H and R291W accumulated in the Golgi. Podocyte profiling demonstrated minimal disease-associated transcriptional change. All variants displayed podocyte-specific apoptosis, which was not linked to ER stress. NEPHRIN-PODOCIN colocalization elucidated the variant-specific effect on NEPHRIN association and hence NEPHRIN trafficking. CONCLUSIONS: Specific variants of endogenous NPHS2 result in distinct subcellular PODOCIN localization within organoid podocytes. Understanding the effect of each variant on protein levels and localization and the effect on NEPHRIN provides additional insight into the pathobiology of NPHS2 variants. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_01_05_JASN2022060707.mp3.
Assuntos
Células-Tronco Pluripotentes Induzidas , Síndrome Nefrótica , Criança , Humanos , Lactente , Síndrome Nefrótica/genética , Síndrome Nefrótica/metabolismo , Rim/metabolismo , MutaçãoRESUMO
Human kidney tissue can now be generated via the directed differentiation of human pluripotent stem cells. This advance is anticipated to facilitate the modeling of human kidney diseases, provide platforms for nephrotoxicity screening, enable cellular therapy, and potentially generate tissue for renal replacement. All such applications will rely upon the accuracy and reliability of the model and the capacity for stem cell-derived kidney tissue to recapitulate both normal and diseased states. In this review, we discuss the models available, how well they recapitulate the human kidney, and how far we are from application of these cells for use in cellular therapies.
Assuntos
Rim/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/fisiologia , Humanos , Nefropatias/fisiopatologiaRESUMO
Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.
Assuntos
Organoides/citologia , Células-Tronco Pluripotentes/citologia , Retina/citologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Forma Celular , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Humanos , Interneurônios/citologia , Interneurônios/metabolismo , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/ultraestrutura , Reprodutibilidade dos Testes , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Sinapses/metabolismo , Tomografia de Coerência ÓpticaRESUMO
The utility of human pluripotent stem cell-derived kidney organoids relies implicitly on the robustness and transferability of the protocol. Here we analyze the sources of transcriptional variation in a specific kidney organoid protocol. Although individual organoids within a differentiation batch showed strong transcriptional correlation, we noted significant variation between experimental batches, particularly in genes associated with temporal maturation. Single-cell profiling revealed shifts in nephron patterning and proportions of component cells. Distinct induced pluripotent stem cell clones showed congruent transcriptional programs, with interexperimental and interclonal variation also strongly associated with nephron patterning. Epithelial cells isolated from organoids aligned with total organoids at the same day of differentiation, again implicating relative maturation as a confounder. This understanding of experimental variation facilitated an optimized analysis of organoid-based disease modeling, thereby increasing the utility of kidney organoids for personalized medicine and functional genomics.
Assuntos
Rim/metabolismo , Organoides/metabolismo , Diferenciação Celular/genética , Células Clonais , Células Epiteliais/citologia , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/citologia , Nefropatias/genética , Nefropatias/patologia , Modelos Biológicos , Organoides/citologia , Reprodutibilidade dos Testes , Análise de Célula Única , Transcrição GênicaRESUMO
Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.
Assuntos
Bioimpressão , Túbulos Renais Proximais/metabolismo , Organoides/metabolismo , Células-Tronco Pluripotentes/metabolismo , Humanos , Túbulos Renais Proximais/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologiaRESUMO
Despite the increasing diagnostic rate of genomic sequencing, the genetic basis of more than 50% of heritable kidney disease remains unresolved. Kidney organoids differentiated from induced pluripotent stem cells (iPSCs) of individuals affected by inherited renal disease represent a potential, but unvalidated, platform for the functional validation of novel gene variants and investigation of underlying pathogenetic mechanisms. In this study, trio whole-exome sequencing of a prospectively identified nephronophthisis (NPHP) proband and her parents identified compound-heterozygous variants in IFT140, a gene previously associated with NPHP-related ciliopathies. IFT140 plays a key role in retrograde intraflagellar transport, but the precise downstream cellular mechanisms responsible for disease presentation remain unknown. A one-step reprogramming and gene-editing protocol was used to derive both uncorrected proband iPSCs and isogenic gene-corrected iPSCs, which were differentiated to kidney organoids. Proband organoid tubules demonstrated shortened, club-shaped primary cilia, whereas gene correction rescued this phenotype. Differential expression analysis of epithelial cells isolated from organoids suggested downregulation of genes associated with apicobasal polarity, cell-cell junctions, and dynein motor assembly in proband epithelial cells. Matrigel cyst cultures confirmed a polarization defect in proband versus gene-corrected renal epithelium. As such, this study represents a "proof of concept" for using proband-derived iPSCs to model renal disease and illustrates dysfunctional cellular pathways beyond the primary cilium in the setting of IFT140 mutations, which are established for other NPHP genotypes.
Assuntos
Cílios/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/patologia , Organoides/patologia , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Reprogramação Celular/genética , Ataxia Cerebelar/genética , Células Epiteliais/metabolismo , Feminino , Fibroblastos/patologia , Flagelos/metabolismo , Edição de Genes , Perfilação da Expressão Gênica , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Rim/diagnóstico por imagem , Fenótipo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Retinose Pigmentar/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Sequenciamento do ExomaRESUMO
Nephron formation continues throughout kidney morphogenesis in both mice and humans. Lineage tracing studies in mice identified a self-renewing Six2-expressing nephron progenitor population able to give rise to the full complement of nephrons throughout kidney morphogenesis. To investigate the origin of nephrons within human pluripotent stem cell-derived kidney organoids, we performed a similar fate-mapping analysis of the SIX2-expressing lineage in induced pluripotent stem cell (iPSC)-derived kidney organoids to explore the feasibility of investigating lineage relationships in differentiating iPSCs in vitro Using CRISPR/Cas9 gene-edited lineage reporter lines, we show that SIX2-expressing cells give rise to nephron epithelial cell types but not to presumptive ureteric epithelium. The use of an inducible (CreERT2) line revealed a declining capacity for SIX2+ cells to contribute to nephron formation over time, but retention of nephron-forming capacity if provided an exogenous WNT signal. Hence, while human iPSC-derived kidney tissue appears to maintain lineage relationships previously identified in developing mouse kidney, unlike the developing kidney in vivo, kidney organoids lack a nephron progenitor niche capable of both self-renewal and ongoing nephrogenesis.
Assuntos
Mapeamento Cromossômico , Perfilação da Expressão Gênica , Genes Reporter , Néfrons/embriologia , Néfrons/metabolismo , Organogênese/genética , Biomarcadores , Sistemas CRISPR-Cas , Técnicas de Cultura de Células , Diferenciação Celular , Proteínas de Homeodomínio/genética , Humanos , Proteínas do Tecido Nervoso/genética , Organoides , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Célula ÚnicaRESUMO
BACKGROUND: The generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development in vivo, its application in human induced pluripotent stem cells (iPSCs) is now feasible. METHODS: We used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics. RESULTS: Each iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments. CONCLUSIONS: We generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.
Assuntos
Células-Tronco Pluripotentes Induzidas , Rim/citologia , Organoides , Animais , Feminino , Camundongos , OrganogêneseRESUMO
All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost close to the time of birth. The generation of new nephron progenitors and their maintenance in culture are central to the success of kidney regenerative strategies. Using a lentiviral screening approach, we previously generated a human induced nephron progenitor-like state in vitro using a pool of six transcription factors. Here, we sought to develop a more efficient approach for direct reprogramming of human cells that could be applied in vivo. PiggyBac transposons are a non-viral integrating gene delivery system that is suitable for in vivo use and allows for simultaneous delivery of multiple genes. Using an inducible piggyBac transposon system, we optimized a protocol for the direct reprogramming of HK2 cells to induced nephron progenitor-like cells with expression of only 3 transcription factors (SNAI2, EYA1, and SIX1). Culture in conditions supportive of the nephron progenitor state further increased the expression of nephron progenitor genes. The refined protocol was then applied to primary human renal epithelial cells, which integrated into developing nephron structures in vitro and in vivo. Such inducible reprogramming to nephron progenitor-like cells could facilitate direct cellular reprogramming for kidney regeneration.
Assuntos
Reprogramação Celular/genética , Elementos de DNA Transponíveis/genética , Engenharia Genética/métodos , Néfrons/fisiologia , Regeneração/genética , Células Cultivadas , Técnicas de Transferência de Genes , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Cultura Primária de Células , Proteínas Tirosina Fosfatases/genética , Fatores de Transcrição da Família Snail/genéticaRESUMO
Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Epitélio Pigmentado da Retina/patologia , Atrofia , Proteínas do Olho/metabolismo , Humanos , Fatores de Crescimento Neural/metabolismo , Fagocitose , Pigmentação , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Serpinas/metabolismoRESUMO
AIMS: We identified a novel homozygous truncating mutation in the gene encoding alpha kinase 3 (ALPK3) in a family presenting with paediatric cardiomyopathy. A recent study identified biallelic truncating mutations of ALPK3 in three unrelated families; therefore, there is strong genetic evidence that ALPK3 mutation causes cardiomyopathy. This study aimed to clarify the mutation mechanism and investigate the molecular and cellular pathogenesis underlying ALPK3-mediated cardiomyopathy. METHODS AND RESULTS: We performed detailed clinical and genetic analyses of a consanguineous family, identifying a new ALPK3 mutation (c.3792G>A, p.W1264X) which undergoes nonsense-mediated decay in ex vivo and in vivo tissues. Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. Multi-electrode array analysis and calcium imaging demonstrated an extended field potential duration and abnormal calcium handling in mutant contractile cultures. CONCLUSIONS: This study validates the genetic evidence, suggesting that mutations in ALPK3 can cause familial cardiomyopathy and demonstrates loss of function as the underlying genetic mechanism. We show that ALPK3-deficient cardiomyocytes derived from pluripotent stem cell models recapitulate the ultrastructural and electrophysiological defects observed in vivo. Analysis of differentiated contractile cultures identified abnormal calcium handling as a potential feature of cardiomyocytes lacking ALPK3, providing functional insights into the molecular mechanisms underlying ALPK3-mediated cardiomyopathy.
Assuntos
Miócitos Cardíacos , Cálcio , Cardiomiopatias , Células-Tronco Embrionárias Humanas , Humanos , Células-Tronco Pluripotentes Induzidas , Proteínas Musculares , Proteínas QuinasesRESUMO
Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model system.
Assuntos
Células-Tronco Embrionárias/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Células-Tronco Neurais/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Células-Tronco Neurais/citologia , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/embriologia , Fatores de Transcrição/metabolismoRESUMO
Genome engineering in human pluripotent stem cells (hPSCs) holds great promise for biomedical research and regenerative medicine. Recently, an RNA-guided, DNA-cleaving interference pathway from bacteria [the type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) pathway] has been adapted for use in eukaryotic cells, greatly facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus pyogenes and Streptococcus thermophilus), each with their own distinct targeting requirements and limitations, have been developed for genome editing thus far. Furthermore, limited information exists about homology-directed repair (HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis, we demonstrate efficient targeting of an endogenous gene in three hPSC lines using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) recognizes a 5'-NNNNGATT-3' protospacer adjacent motif (PAM) different from those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9 and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a single-guide RNA (sgRNA) to direct its activity. Because of its distinct protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases the sequence contexts amenable to RNA-directed genome editing.
Assuntos
Proteínas de Bactérias/metabolismo , Engenharia Genética/métodos , Genoma Humano/genética , Neisseria meningitidis/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Deleção de Genes , Marcação de Genes , Humanos , Mamíferos , Dados de Sequência Molecular , RNA/metabolismo , Edição de RNA/genética , Pequeno RNA não TraduzidoRESUMO
The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.
Assuntos
Células-Tronco Embrionárias/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Serina/metabolismo , Treonina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Células Cultivadas , Células HEK293 , Humanos , Imunoprecipitação , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/genética , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição SOXB1/metabolismo , Homologia de Sequência de Aminoácidos , Serina/química , Serina/genética , Treonina/química , Treonina/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.
Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Biópsia , Bovinos , Proliferação de Células , Sobrevivência Celular , Materiais Revestidos Biocompatíveis , Meios de Cultura Livres de Soro/química , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Expressão Gênica , Substâncias de Crescimento , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariotipagem , Soroalbumina Bovina , Pele/citologia , VitronectinaRESUMO
Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.
Assuntos
Atrofia Girata/enzimologia , Atrofia Girata/genética , Ornitina-Oxo-Ácido Transaminase/genética , Ornitina-Oxo-Ácido Transaminase/metabolismo , Células-Tronco Pluripotentes/enzimologia , Células Cultivadas , Marcação de Genes/métodos , Estudo de Associação Genômica Ampla , Instabilidade Genômica/genética , Atrofia Girata/patologia , Atrofia Girata/terapia , Humanos , Células-Tronco Pluripotentes/patologia , Recombinação GenéticaRESUMO
The transcription factor WT1 plays a critical role in several embryonic developmental processes such as gonadogenesis, nephrogenesis, and cardiac development. We generated a homozygous (MCRIi031-A-3) WT1 knockout induced pluripotent stem cell (iPSC) line from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. The cells exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of WT1 in critical developmental processes.
Assuntos
Homozigoto , Células-Tronco Pluripotentes Induzidas , Proteínas WT1 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas WT1/genética , Proteínas WT1/metabolismo , Linhagem Celular , Sistemas CRISPR-Cas , Diferenciação Celular , Técnicas de Inativação de Genes , Edição de GenesRESUMO
The transcription factor SOX9 plays a critical role in several embryonic developmental processes such as gonadogenesis, chrondrogenesis, and cardiac development. We generated heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of SOX9 in critical developmental processes.