SummaryLarge-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines.
Despite a significant improvement in the availability of therapeutic options to treat lung diseases, pulmonary disease still remains a major cause of morbidity and mortality around the world. Currently there are limited opportunities to study human lung disease either in vivo and in vitro. Using induced pluripotent stem cells (iPSC) we have generated a reproducible differentiation protocol to make mature post‐mitotic multiciliated cells in a functional airway epithelium. iPSC were generated from human skin biopsies and differentiated via FOXA2+SOX17+ definitive endoderm (>90% efficiency) to FOXA2+NKx2.1+ anterior foregut endoderm, FOXA2+NKx2.1+SOX2+ (~50% efficiency) pulmonary endoderm and then matured in an air liquid interface. Robust multiciliogenesis occurred when Notch signaling was inhibited and was confirmed by; i) the assembly of multiple pericentrin stained centrioles at the apical surface, ii) expression of transcription factor FOXJ1 and iii) presence of multiple acetylated tubulin labeled cilia projections in individual cells. The presence of NKx2.1+CC10+ Clara cells, MUC5A/C+ goblet cells and FOXA2+p63+ basal cells was also confirmed showing we are generating a complete polarized epithelial cell layer comprised of all relevant cell types. Functional cAMP activated and CFTRinh‐172 sensitive CFTR currents were recorded in isolated epithelial cells by whole cell patch clamp technique. Furthermore, we have corrected the deltaF508 mutation in the CFTR gene (>80% of all cases of CF) using a combination of CRISPR‐Cas9 endonuclease‐mediated genome editing and piggyBac transposase technologies, in the CF patient‐derived iPSC. The generation of mature multiciliated cells in a human iPSC differentiated respiratory epithelium and the ability to correct disease causing mutations provides a significant advancement toward modeling a number of human respiratory diseases in vitro. Grant Funding Source: Supported in part by CIRM and the Berger Foundation
SUMMARY Lung disease is a major cause of death in the USA, with current therapeutic approaches only serving to manage symptoms. The most common chronic and life-threatening genetic disease of the lung is Cystic fibrosis (CF) caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). We have generated induced pluripotent stem cells (iPSC) from CF patients carrying a homozygous deletion of F508 in the CFTR gene, which results in defective processing of CFTR to the cell membrane. This mutation was precisely corrected using CRISPR to target corrective sequences to the endogenous CFTR genomic locus, in combination with a completely excisable selection system which significantly improved the efficiency of this correction. The corrected iPSC were subsequently differentiated to mature airway epithelial cells where recovery of normal CFTR expression and function was demonstrated. This isogenic iPSC-based model system for CF could be adapted for the development of new therapeutic approaches.
Exposure of skin tissue to UV radiation has been shown to cause DNA photodamage. If this damaged DNA is allowed to replicate, carcinogenesis may occur. DNA damage is prevented from being passed on to daughter cells by upregulation of the protein p21. p21 halts the cells cycle allowing the cell to undergo apoptosis, or repair its DNA before replication. Previous work suggested that milk phospholipids may possess protective properties against UV damage. In this study, we observed cell morphology, cell apoptosis, and p21 expression in tissue engineered epidermis through the use of Hematoxylin and Eosin staining, confocal microscopy, and western blot respectively. Tissues were divided into four treatment groups including: a control group with no UV and no milk phospholipid treatment, a group exposed to UV alone, a group incubated with milk phospholipids alone, and a group treated with milk phospholipids and UV. All groups were incubated for twenty-four hours after treatment. Tissues were then fixed, processed, and embedded in paraffin. Performing western blots resulted in visible p21 bands for the UV group only, implying that in every other group, p21 expression was lesser. Numbers of apoptotic cells were determined by observing the tissues treated with Hoechst dye under a confocal microscope, and counting the number of apoptotic and total cells to obtain a percentage of apoptotic cells. We found a decrease in apoptotic cells in tissues treated with milk phospholipids and UV compared to tissues exposed to UV alone. Collectively, these results suggest that milk phospholipids protect cell DNA from damage incurred from UV light.
Immune cell subtype population frequencies can have a large effect on the efficacy of T cell therapies. Current methods, like flow cytometry, have specific sample requirements, high sample input, are low throughput, and are difficult to standardize, all of which are detrimental to characterization of cell therapy products during their development and manufacturing. The assays described herein accurately identify and quantify immune cell types in a heterogeneous mixture of cells using isolated genomic DNA (gDNA). DNA methylation patterns are revealed through bisulfite conversion, a process in which unmethylated cytosines are converted to uracils. Unmethylated DNA regions are detected through qPCR amplification using primers targeting converted areas. One unique locus per assay is measured and serves as an accurate identifier for a specific cell type. The assays are robust and identify CD8+, regulatory, and Th17 T cells in a high throughput manner. These optimized assays can potentially be used for in-process and product release testing for cell therapy process.
Despite a significant improvement in the availability of therapeutic options to treat lung diseases, pulmonary disease still remains a major cause of morbidity and mortality around the world. Currently there are limited opportunities to study human lung disease either in vivo and in vitro. Using induced pluripotent stem cells (iPSC) we have generated a reproducible differentiation protocol to make mature post‐mitotic multiciliated cells in a functional airway epithelium. iPSC were generated from human skin biopsies and differentiated via FOXA2+SOX17+ definitive endoderm (>90% efficiency) to FOXA2+NKx2.1+ anterior foregut endoderm, FOXA2+NKx2.1+SOX2+ (~50% efficiency) pulmonary endoderm and then matured in an air liquid interface. Robust multiciliogenesis occurred when Notch signaling was inhibited and was confirmed by; i) the assembly of multiple pericentrin stained centrioles at the apical surface, ii) expression of transcription factor FOXJ1 and iii) presence of multiple acetylated tubulin labeled cilia projections in individual cells. The presence of NKx2.1+CC10+ Clara cells, MUC5A/C+ goblet cells and FOXA2+p63+ basal cells was also confirmed showing we are generating a complete polarized epithelial cell layer comprised of all relevant cell types. Functional cAMP activated and CFTRinh‐172 sensitive CFTR currents were recorded in isolated epithelial cells by whole cell patch clamp technique. Furthermore, we have corrected the deltaF508 mutation in the CFTR gene (>80% of all cases of CF) using a combination of CRISPR‐Cas9 endonuclease‐mediated genome editing and piggyBac transposase technologies, in the CF patient‐derived iPSC. The generation of mature multiciliated cells in a human iPSC differentiated respiratory epithelium and the ability to correct disease causing mutations provides a significant advancement toward modeling a number of human respiratory diseases in vitro. Grant Funding Source: Supported in part by CIRM and the Berger Foundation
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