Project description:Induced Pluripotent Stem Cells (iPSC) derived from healthy individuals are important controls for disease modeling studies. To create a resource of genetically annotated iPSCs, we reprogrammed footprint-free lines from four volunteers in the Personal Genome Project Canada (PGPC). Multilineage directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users further demonstrated line versatility by generating kidney organoids, T-lymphocytes and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole genome sequencing (WGS) based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harboured at least one pre-existing or acquired variant with cardiac, neurological or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cell types found in six tissues for disease modeling, and clinical annotation highlighted variant-preferred lines for use as unaffected controls in specific disease settings.
Project description:Induced Pluripotent Stem Cells (iPSC) derived from healthy individuals are important controls for disease modeling studies. To create a resource of genetically annotated iPSC, we reprogrammed footprint-free lines from four volunteers in the Personal Genome Project Canada (PGPC). Multilineage directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users further demonstrated line versatility by generating kidney organoids, T-cells and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited a hypertrophic phenotype as expected. WGS annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harboured at least one pre-existing or acquired variant with cardiac, neurological or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cell types found in six tissues for disease modelling, and clinical annotation highlighted variant-preferred lines for use as unaffected controls in specific disease settings.
Project description:Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids, T lymphocytes, and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac, neurological, or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling, and variant-preferred healthy control lines were identified for specific disease settings.
Project description:Tissue loss due to oral diseases requires the healing and regeneration of tissues of multiple lineages. While stem cells are native to oral tissues, a current major limitation to regeneration is the ability to direct their lineage-specific differentiation. This work utilizes polymeric scaffold systems with spatiotemporally controlled morphogen cues to develop precise morphogen fields to direct mesenchymal stem cell differentiation. First, a simple three-layer scaffold design was developed that presented two spatially segregated, lineage-specific cues (Dentinogenic TGF-?1 and Osteogenic BMP4). However, this system resulted in diffuse morphogen fields, as assessed by the in vitro imaging of cell-signaling pathways triggered by the morphogens. Mathematical modeling was then exploited, in combination with incorporation of specific inhibitors (neutralizing antibodies or a small molecule kinase inhibitor) into each morphogen in an opposing spatial pattern as the respective morphogen, to design a five-layer scaffold that was predicted to yield distinct, spatially segregated zones of morphogen signaling. To validate this system, undifferentiated MSCs were uniformly seeded in these scaffold systems, and distinct mineralized tissue differentiation were noted within these morphogen zones. Finally, to demonstrate temporal control over morphogen signaling, latent TGF-?1 was incorporated into one region of a concentric scaffold design, and laser treatment was used to activate the morphogen on-demand and to induce dentin differentiation solely within that specific spatial zone. This study demonstrates a significant advance in scaffold design to generate precise morphogen fields that can be used to develop in situ models to explore tissue differentiation and may ultimately be useful in engineering multi-lineage tissues in clinical dentistry.
Project description:MicroRNAs (miRNAs) are known to be crucial players in governing the differentiation of human induced pluripotent stem cells (hiPSCs). Despite their utter importance, identifying key lineage specifiers among the myriads of expressed miRNAs remains challenging. We believe that the current practice in mining miRNA specifiers via delineating dynamic fold-changes only is inadequate. Our study, therefore, provides evidence to pronounce "lineage specificity" as another important attribute to qualify for these lineage specifiers. Adopted hiPSCs were differentiated into representative lineages (hepatic, nephric and neuronal) over all three germ layers whilst the depicted miRNA expression changes compiled into an integrated atlas. We demonstrated inter-lineage analysis shall aid in the identification of key miRNAs with lineage-specificity, while these shortlisted candidates were collectively known as "lineage-specific miRNAs". Subsequently, we followed through the fold-changes along differentiation via computational analysis to identify miR-192 and miR-372-3p, respectively, as representative candidate key miRNAs for the hepatic and nephric lineages. Indeed, functional characterization validated that miR-192 and miR-372-3p regulate lineage differentiation via modulation of the expressions of lineage-specific genes. In summary, our presented miRNA atlas is a resourceful ore for the mining of key miRNAs responsible for lineage specification.
Project description:Human naïve pluripotent stem cells (PSCs) share features with the pre-implantation epiblast. They therefore provide an unmatched opportunity for characterising the developmental programme of pluripotency in Homo sapiens Here, we confirm that naïve PSCs do not respond directly to germ layer induction, but must first acquire competence. Capacitation for multi-lineage differentiation occurs without exogenous growth factor stimulation and is facilitated by inhibition of Wnt signalling. Whole-transcriptome profiling during this formative transition highlights dynamic changes in gene expression, which affect many cellular properties including metabolism and epithelial features. Notably, naïve pluripotency factors are exchanged for postimplantation factors, but competent cells remain devoid of lineage-specific transcription. The gradual pace of transition for human naïve PSCs is consistent with the timespan of primate development from blastocyst to gastrulation. Transcriptome trajectory during in vitro capacitation of human naïve cells tracks the progression of the epiblast during embryogenesis in Macaca fascicularis, but shows greater divergence from mouse development. Thus, the formative transition of naïve PSCs in a simple culture system may recapitulate essential and specific features of pluripotency dynamics during an inaccessible period of human embryogenesis.
Project description:Genetic patterns of inter-population variation are a result of different demographic and adaptive histories, which gradually shape the frequency distribution of the variants. However, the study of clinically relevant mutations has a Eurocentric bias. The Romani, the largest transnational minority ethnic group in Europe, originated in South Asia and received extensive gene flow from West Eurasia. Most medical genetic studies have only explored founder mutations related to Mendelian disorders in this population. Here we analyze exome sequences and genome-wide array data of 89 healthy Spanish Roma individuals to study complex traits and disease. We apply a different framework and focus on variants with both increased and decreased allele frequencies, taking into account their local ancestry. We report several OMIM traits enriched for genes with deleterious variants showing increased frequencies in Roma or in non-Roma (e.g., obesity is enriched in Roma, with an associated variant linked to South Asian ancestry; while non-insulin dependent diabetes is enriched in non-Roma Europeans). In addition, previously reported pathogenic variants also show differences among populations, where some variants segregating at low frequency in non-Roma are virtually absent in the Roma. Lastly, we describe frequency changes in drug-response variation, where many of the variants increased in Roma are clinically associated with metabolic and cardiovascular-related drugs. These results suggest that clinically relevant variation in Roma cannot only be characterized in terms of founder mutations. Instead, we observe frequency differences compared to non-Roma: some variants are absent, while other have drifted to higher frequencies. As a result of the admixture events, these clinically damaging variants can be traced back to both European and South Asian-related ancestries. This can be attributed to a different prevalence of some genetic disorders or to the fact that genetic susceptibility variants are mostly studied in populations of European descent, and can differ in individuals with different ancestries.
Project description:BACKGROUND:The goal of this study is to identify and characterize treatment resistant tumor initiating cells (TRTICs) using orthotopic xenografts. METHODS:TRTICs were enriched from GBM cell lines using mouse xenografts treated with fractionated doses of radiation and temozolomide. TRTICs were characterized by neurosphere clonogenicity and self-renewal, serial xenotransplantation, differentiation potential, and mRNA & miRNA transcriptomic profiling. We use an unbiased approach to identify antigens encoding TRTIC and glioma stem cells (GSC) populations. Co-culture experiments of TRTIC and differentiated cells were conducted to evaluate the reliance of TRTIC differentiation on the secretome of differentiated cells. FINDINGS:TRTICs acquire stem-like gene expression signatures and increased side population staining resulting from the activation of multi-drug resistance genes. Genetic and functional characterization of TRTICs shows a striking resemblance with GSCs. TRTICs can differentiate towards specific progeny in the neural stem cell lineage. TRTIC-derived tumors display all the histological hallmarks of glioblastoma (GBM) and exhibit a miRNA-transcript and mRNA-transcriptomic profile associated with aggressiveness. We report that CD24+/CD44+ antigens are expressed in TRTICs and patient-derived GSCs. Double positive CD24+/CD44+ exhibit treatment resistance and enhanced tumorigenicity. Interestingly, co-culture experiments with TRTICs and differentiated cells indicated that the regulation of TRTIC differentiation could rely on the secretome in the tumor niche. INTERPRETATION:Radiation and temozolomide treatment enriches a population of cells that have increased iPSC gene expression. As few as 500 cells produced aggressive intracranial tumors resembling patient GBM. CD24+/CD44+ antigens are increased in TRTICs and patient-derived GSCs. The enrichment for TRTICs may result in part from the secretome of differentiated cells. FUND: NIH/NCI 1RC2CA148190, 1R01CA108633, 1R01CA188228, and The Ohio State University Comprehensive Cancer Center.