RESUMEN
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.
Asunto(s)
Riñón/metabolismo , Organoides/metabolismo , Diferenciación Celular/genética , Células Clonales , Células Epiteliales/citología , Perfilación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/citología , Enfermedades Renales/genética , Enfermedades Renales/patología , Modelos Biológicos , Organoides/citología , Reproducibilidad de los Resultados , Análisis de la Célula Individual , Transcripción GenéticaRESUMEN
Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable ß-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. Moreover, we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigel-coated surface. Thus, we provided a totally defined condition from hESC culture to insulin-producing cell differentiation, and the derived cells could be a therapeutic resource for diabetic patients in the future.
Asunto(s)
Acrilatos/química , Células Madre Embrionarias/fisiología , Insulina/biosíntesis , Fragmentos de Péptidos/química , Materiales Biocompatibles/química , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Colágeno/química , Medios de Cultivo/química , Combinación de Medicamentos , Humanos , Laminina/química , Imitación Molecular , Proteoglicanos/química , Propiedades de Superficie , Vitronectina/químicaRESUMEN
Bardet-Biedl syndrome (BBS) is a pleiotropic, genetically heterogeneous disorder characterized by obesity, retinopathy, polydactyly, cognitive impairment, renal and cardiac anomalies, as well as hypertension and diabetes. Multiple genes are known to independently cause BBS. These genes do not appear to code for the same functional category of proteins; yet, mutation of each results in a similar phenotype. Gene knockdown of different BBS genes in zebrafish shows strikingly overlapping phenotypes including defective melanosome transport and disruption of the ciliated Kupffer's vesicle. Here, we demonstrate that individual knockdown of bbs1 and bbs3 results in the same prototypical phenotypes as reported previously for other BBS genes. We utilize the zebrafish system to comprehensively determine whether simultaneous pair-wise knockdown of BBS genes reveals genetic interactions between BBS genes. Using this approach, we demonstrate eight genetic interactions between a subset of BBS genes. The synergistic relationships between distinct combinations are not due to functional redundancy but indicate specific interactions within a multi-subunit BBS complex. In addition, we utilize the zebrafish model system to investigate limb development. Human polydactyly is a cardinal feature of BBS not reproduced in BBS-mouse models. We evaluated zebrafish fin bud patterning and observed altered Sonic hedgehog (shh) expression and subsequent changes to fin skeletal elements. The SHH fin bud phenotype was also used to confirm specific genetic interactions between BBS genes. This study reveals an in vivo requirement for BBS function in limb bud patterning. Our results provide important new insights into the mechanism and biological significance of BBS.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/fisiopatología , Tipificación del Cuerpo , Modelos Animales de Enfermedad , Extremidades/embriología , Proteínas de Pez Cebra/genética , Animales , Cartílago/patología , Embrión no Mamífero/metabolismo , Embrión no Mamífero/fisiología , Extremidades/fisiopatología , Regulación de la Expresión Génica , Silenciador del Gen , Proteínas Hedgehog/metabolismo , Humanos , Fenotipo , Polidactilia/genética , Polidactilia/fisiopatología , Especificidad de la Especie , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
The identification of mutations in genes that cause human diseases has largely been accomplished through the use of positional cloning, which relies on linkage mapping. In studies of rare diseases, the resolution of linkage mapping is limited by the number of available meioses and informative marker density. One recent advance is the development of high-density SNP microarrays for genotyping. The SNP arrays overcome low marker informativity by using a large number of markers to achieve greater coverage at finer resolution. We used SNP microarray genotyping for homozygosity mapping in a small consanguineous Israeli Bedouin family with autosomal recessive Bardet-Biedl syndrome (BBS; obesity, pigmentary retinopathy, polydactyly, hypogonadism, renal and cardiac abnormalities, and cognitive impairment) in which previous linkage studies using short tandem repeat polymorphisms failed to identify a disease locus. SNP genotyping revealed a homozygous candidate region. Mutation analysis in the region of homozygosity identified a conserved homozygous missense mutation in the TRIM32 gene, a gene coding for an E3 ubiquitin ligase. Functional analysis of this gene in zebrafish and expression correlation analyses among other BBS genes in an expression quantitative trait loci data set demonstrate that TRIM32 is a BBS gene. This study shows the value of high-density SNP genotyping for homozygosity mapping and the use of expression correlation data for evaluation of candidate genes and identifies the proteasome degradation pathway as a pathway involved in BBS.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Mapeo Cromosómico , Análisis Mutacional de ADN , Genoma Humano , Homocigoto , Humanos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Bardet-Biedl syndrome (BBS) is characterized by obesity, retinopathy, polydactyly, cognitive impairment, renal and cardiac anomalies as well as hypertension and diabetes. The nine known BBS genes do not appear to belong to the same functional category; yet mutation of these genes results in a nearly identical pleiotropic phenotype. Although the precise functions of the BBS proteins have yet to be determined, current data support a role in cilia function and intraflagellar transport. To gain insight into the biological processes controlled by BBS genes, we embarked on studies of six BBS orthologues from zebrafish. Knockdown of zebrafish bbs2, bbs4, bbs5, bbs6, bbs7 or bbs8 results in disruption of Kupffer's vesicle (KV), a ciliated organ thought to play a role in left-right patterning. KV defects are due to a progressive loss of cilia within the vesicle and result in subsequent alterations to organ laterality. We also note a specific defect altering retrograde melanosome transport. These studies are the first to comprehensively compare the diverse group of BBS genes in parallel and demonstrate a common role in intracellular trafficking, indicating that BBS proteins are involved in general organelle trafficking.
Asunto(s)
Síndrome de Bardet-Biedl/genética , Cilios/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo , Cafeína/farmacología , Clonación Molecular , Embrión no Mamífero , Epinefrina/farmacología , Ojo/embriología , Ojo/ultraestructura , Flagelos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Melanosomas/efectos de los fármacos , Melanosomas/metabolismo , Microscopía Confocal , Oligonucleótidos Antisentido/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Bardet-Biedl syndrome (BBS, OMIM 209900) is a genetic disorder with the primary features of obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation and hypogenitalism. Individuals with BBS are also at increased risk for diabetes mellitus, hypertension and congenital heart disease. What was once thought to be a homogeneous autosomal recessive disorder is now known to map to at least six loci: 11q13 (BBS1), 16q21 (BBS2), 3p13 p12 (BBS3), 15q22.3 q23 (BBS4), 2q31 (BBS5) and 20p12 (BBS6). There has been considerable interest in identifying the genes that underlie BBS, because some components of the phenotype are common. Cases of BBS mapping ro BBS6 are caused by mutations in MKKS; mutations in this gene also cause McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly and congenital heart defects). In addition, we recently used positional cloning to identify the genes underlying BBS2 (ref. 16) and BBS4 (ref. 17). The BBS6 protein has similarity to a Thermoplasma acidophilum chaperonin, whereas BBS2 and BBS4 have no significant similarity to chaperonins. It has recently been suggested that three mutated alleles (two at one locus, and a third at a second locus) may be required for manifestation of BBS (triallelic inheritance). Here we report the identification of the gene BBS1 and show that a missense mutation of this gene is a frequent cause of BBS. In addition, we provide data showing that this common mutation is not involved in triallelic inheritance.