Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products.

Background Aims: Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products.

Methods: Our standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform.

CRB1 is required for recycling by RAB11A+ vesicles in human retinal organoids.

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals.

AAV-mediated gene augmentation therapy of CRB1 patient-derived retinal organoids restores the histological and transcriptional retinal phenotype.

Retinitis pigmentosa and Leber congenital amaurosis are inherited retinal dystrophies that can be caused by mutations in the Crumbs homolog 1 (CRB1) gene. CRB1 is required for organizing apical-basal polarity and adhesion between photoreceptors and Müller glial cells. CRB1 patient-derived induced pluripotent stem cells were differentiated into CRB1 retinal organoids that showed diminished expression of variant CRB1 protein observed by immunohistochemical analysis.

Introduction of a Geminin mScarlet Reporter into H2B-mTurq2 hiPSCs for Live-cell Imaging of Proliferation and Cell Cycling.

We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ubiquitously transcribed mScarletI-Geminin (GMMN) (1-110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle.

Generation of AAVS1 and CLYBL STRAIGHT-IN v2 acceptor human iPSC lines for integrating DNA payloads.

STRAIGHT-IN is a platform to precisely integrate DNA payloads into the genome of cells, including hiPSCs. Here, we generated two hiPSC acceptor lines each with one copy of an upgraded landing pad (LP). This improved design allows more efficient (∼100 %) and rapid (∼2-3 weeks) generation of genetically modified hiPSC lines containing the desired payloads.

Generation of LUMCi041-A-2: Equipping a PAX3 reporter iPSC line with doxycycline inducible H2B-mTurquoise2 for live cell imaging.

An induced pluripotent stem cell (iPSC) line, in which a H2B-fluorescent protein fusion is temporally expressed, is a valuable tool to track cells and study cell divisions and apoptosis. To this end we introduced a 3rd generation "all-in-one" doxycycline-inducible H2B-mTurquoise2 vector into the AAVS1 locus of PAX3-Venus iPSCs via CRISPR/Cas9. H2B-mTurquoise2 expression is absent but readily induced by doxycycline allowing quantification of cell divisions and imaging of living cells.

Generation of human induced pluripotent stem cell line EURACi006-A and its isogenic gene-corrected line EURACi006-A-1 from an arrhythmogenic cardiomyopathy patient carrying the c.1643delG PKP2 mutation.

Arrhythmogenic Cardiomyopathy (ACM) is a rare genetic cardiac disease predominantly associated with mutations in genes of the desmosomes and characterized by arrhythmia and fibro-fatty replacement of the myocardium. We generated human induced pluripotent stem cells (hiPSCs) from one patient affected by ACM carrying the heterozygous c.1643delG (p.

Generation of three human induced pluripotent stem cell lines, LUMCi024-A, LUMCi025-A, and LUMCi026-A, from two patients with combined oxidative phosphorylation deficiency 8 and a related control.

Combined Oxidative Phosphorylation Deficiency 8 (COXPD8) is an autosomal recessive disorder causing lethal childhood-onset hypertrophic cardiomyopathy. Homozygous or compound heterozygous mutations in the nuclear-encoded mitochondrial alanyl-tRNA synthetase 2 (AARS2) gene underly the pathology. We generated induced pluripotent stem cells (hiPSCs) from two patients carrying the heterozygous compound c.

Reprogramming Urine-Derived Cells using Commercially Available Self-Replicative RNA and a Single Electroporation.

We describe a protocol for efficient generation of human-induced pluripotent stem cells (hiPSCs) from urine-derived cells (UDCs) obtained from adult donors using self-replicative RNA containing the reprogramming factors OCT3/4, SOX2, KLF4, GLIS1, and c-MYC (ReproRNA-OKSGM). After electroporation, transfection efficiency is quantified by measuring OCT3/4-expressing UDCs using flow cytometry and should be ≥0.1%.

Generation of human induced pluripotent stem cell line LUMCi027-A and its isogenic gene-corrected line from a patient affected by arrhythmogenic cardiomyopathy and carrying the c.2013delC PKP2 mutation.

Arrhythmogenic Cardiomyopathy (ACM) is a rare inherited heart muscle disease characterised by progressive fibro-fatty replacement of the ventricular myocardium leading to life-threatening arrhythmias. We generated human induced pluripotent stem cells (hiPSCs) from a patient affected by ACM and carrying the heterozygous c.2013delC (p.

Generation of two human induced pluripotent stem cell lines, LUMCi020-A and LUMCi021-A, from two patients with Catecholaminergic Polymorphic Ventricular Tachycardia carrying heterozygous mutations in the RYR2 gene.

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a malignant channelopathy associated with exercise- and stress-induced cardiac sudden death. The autosomal dominant form of CPVT is due to mutations in the ryanodine receptor 2 (RYR2) gene. We generated induced pluripotent stem cells (hiPSCs) from skin fibroblasts of two patients carrying the c.