Patient-derived Pluripotent Stem Cells Research Articles

Customized antisense oligonucleotide-based therapy for neurofilament-associated Charcot-Marie-Tooth disease.

DNA-based therapeutics have emerged as a revolutionary approach for addressing the treatment gap in rare inherited conditions by targeting the fundamental genetic causes of disease. Charcot-Marie-Tooth (CMT) disease, a group of inherited neuropathies, represents one of the most prevalent Mendelian disease groups in neurology and is characterized by diverse genetic etiology. Axonal forms of CMT, known as CMT2, are caused by dominant mutations in over 30 different genes which lead to degeneration of lower motor neuron axons. Recent advances in antisense oligonucleotide (ASO) therapeutics have shown promise in targeting neurodegenerative disorders. Here we elucidate pathomechanistic changes contributing to variant specific molecular phenotypes in CMT2E, caused by a single nucleotide substitution (p.N98S) in the neurofilament light chain gene (NEFL). We used a patient-derived pluripotent stem cell (iPSC)-induced motor neuron model, which recapitulates several cellular and biomarker phenotypes associated with CMT2E. Using an ASO treatment strategy targeting a heterozygous gain-of-function variant, we aimed to resolve molecular phenotypic changes observed in the CMT2E p.N98S subtype. To determine ASO therapeutic potential, we employed our treatment strategy in iPSC-derived motor neurons and used established as well as novel biomarkers of peripheral nervous system axonal degeneration. Our findings have demonstrated a significant decrease in clinically relevant biomarkers of axonal degeneration, presenting the first clinically viable genetic therapeutic for CMT2E. Similar strategies could be used to develop precision medicine approaches for otherwise untreatable gain of function inherited disorders.

Organoid Culture Applications: Mini Systematic Review

Recent scientific developments in the stem cell field have made it possible to create complex organoids, or structures that resemble complete organs, in vitro. In the majority of these methods, stem cells produced from stem cells or tissue progenitors are allowed to self-organize into three-dimensional (3D) structures using culture systems in three dimensions. The purpose of this study is to ascertain the use of organoid culture in the area of health. This kind of study employs the literature review methodology. The Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) criteria are referenced throughout the stages of the literature review performed in this study. The four processes that make up this level are identification, screening, eligibility, and acceptance. These discoveries showing that human stem cells and patient-derived pluripotent stem cells can be used to generate organoids open up a wide range of opportunities for modeling and disease development, toxicological research and medication discovery, and the field of regenerative medicine. Here, we discuss some of the most significant recent advancements in 3D human organoid production as well as the field's historical advancements. Finally, we discuss present restrictions and provide illustrations of how organoid technology may be used in the healthcare industry.

CRISPR-mediated correction of skeletal muscle Ca2+ handling in a novel DMD patient-derived pluripotent stem cell model

Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca2+ handling is a pathological feature of DMD, mechanisms underlying defective Ca2+ homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca2+ handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca2+ transients and mathematical modeling of Ca2+ dynamics reveal significantly reduced cytosolic Ca2+ clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca2+ flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wildtype PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca2+ homeostasis in DMD and suggests that Ca2+ signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.

Autologous treatment for ALS with implication for broad neuroprotection

BackgroundAmyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of motor neurons (MNs), leading to paralysis, respiratory failure and death within 2–5 years of diagnosis. The exact mechanisms of sporadic ALS, which comprises 90% of all cases, remain unknown. In familial ALS, mutations in superoxide dismutase (SOD1) cause 10% of cases.MethodsALS patient-derived human-induced pluripotent stem cells (ALS hiPSCs, harboring the SOD1AV4 mutation), were differentiated to MNs (ALS-MNs). The neuroprotective effects of conditioned medium (CM) of hESCs (H9), wt hiPSCs (WTC-11) and the ALS iPSCs, on MN apoptosis and viability, formation and maintenance of neurites, mitochondrial activity and expression of inflammatory genes, were examined. For in vivo studies, 200 μl of CM from the ALS iPSCs (CS07 and CS053) was injected subcutaneously into the ALS model mice (transgenic for the human SOD1G93A mutation). Animal agility and strength, muscle innervation and mass, neurological score, onset of paralysis and lifespan of the ALS mice were assayed. After observing significant disease-modifying effects, the CM was characterized biochemically by fractionation, comparative proteomics, and epigenetic screens for the dependence on pluripotency. CM of fibroblasts that were differentiated from the wt hiPSCs lacked any neuroprotective activity and was used as a negative control throughout the studies.ResultsThe secretome of PSCs including the ALS patient iPSCs was neuroprotective in the H2O2 model. In the model with pathogenic SOD1 mutation, ALS iPSC-CM attenuated all examined hallmarks of ALS pathology, rescued human ALS-MNs from denervation and death, restored mitochondrial health, and reduced the expression of inflammatory genes. The ALS iPSC-CM also improved neuro-muscular health and function, and delayed paralysis and morbidity in ALS mice. Compared side by side, cyclosporine (CsA), a mitochondrial membrane blocker that prevents the leakage of mitochondrial DNA, failed to avert the death of ALS-MNs, although CsA and ALS iPSC-CM equally stabilized MN mitochondria and attenuated inflammatory genes. Biochemical characterization, comparative proteomics, and epigenetic screen all suggested that it was the interactome of several key proteins from different fractions of PSC-CM that delivered the multifaceted neuroprotection.ConclusionsThis work introduces and mechanistically characterizes a new biologic for treating ALS and other complex neurodegenerative diseases.

DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières syndrome astrocytes.

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.

Genetic correction of concurrent \u03b1- and \u03b2-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology

BackgroundThalassemia is a genetic blood disorder characterized by decreased hemoglobin production. Severe anemia can damage organs and severe threat to life safety. Allogeneic transplantation of bone marrow-derived hematopoietic stem cell (HSCs) at present represents a promising therapeutic approach for thalassemia. However, immune rejection and lack of HLA-matched donors limited its clinical application. In recent years, human-induced pluripotent stem cells (hiPSCs) technology offers prospects for autologous cell-based therapy since it could avoid the immunological problems mentioned above.MethodsIn the present study, we established a new hiPSCs line derived from amniotic cells of a fetus with a homozygous β41-42 (TCTT) deletion mutation in the HBB gene and a heterozygous Westmead mutation (C > G) in the HBA2 gene. We designed a CRISPR-Cas9 to target these casual mutations and corrected them. Gene-corrected off-target analysis was performed by whole-exome capture sequencing. The corrected hiPSCs were analyzed by teratoma formation and erythroblasts differentiation assays.ResultsThese mutations were corrected with linearized donor DNA through CRISPR/Cas9-mediated homology-directed repair. Corrections of hiPSCs were validated by sequences. The corrected hiPSCs retain normal pluripotency. Moreover, they could be differentiated into hematopoietic progenitors, which proves that they maintain the multilineage differentiation potential.ConclusionsWe designed sgRNAs and demonstrated that these sgRNAs facilitating the CRISPR-Cas9 genomic editing system could be applied to correct concurrent α- and β-thalassemia in patient-derived hiPSCs. In the future, these corrected hiPSCs can be applied for autologous transplantation in patients with concurrent α- and β-thalassemia.

3152 – IDENTIFYING SELECTIVE REGULATORS OF HEMATOPOIETIC STEM CELLS IN DEVELOPMENT

Hematopoietic stem cells (HSCs) can sustain the entire adult blood system due to their multipotency and self-renewal properties. In contrast, embryonic HSC-independent progenitor cells (HPCs) have limited regeneration and restricted differentiation. Understanding what distinguishes HSCs from HPCs could reveal the key to multipotency and self-renewal. To dissect early differences between these cells during embryonic development, we established a temporally-regulated lineage tracing assay in zebrafish that allowed us to distinguish HSCs from HPCs based on distinct differentiation kinetics. We determined that T-cell and granulocyte production from HSCs was significantly delayed compared to HPCs. Using this system, we performed a focused reverse genetic screen for factors that modulate the timing of HSC-derived T-cell production. As a control, we demonstrated that loss of Runx1, a master transcription factor for definitive hematopoiesis, diminished both HSC and HPC-derived T-cells. We then targeted 30 novel genes using CRISPR/Cas9-mutagenesis and screened for acceleration or further delay in HSC differentiation to T-cells, identifying one positive and two negative HSC regulators. Mutagenesis of pa2g4b (proliferation-associated factor 4b) led to increased HSC-derived T-cells, but no change in HPC differentiation, suggesting it is an HSC-selective negative regulator. Beyond T-cells and granulocytes, we delineated the full differentiation repertoire of embryonic HSCs and HPCs using scRNA-seq of lineage-traced cells and found HPCs could contribute to all major blood lineages in larval zebrafish suggesting they might possess a broader differentiation potential. Identifying factors that regulate HSC's ability to differentiate and self-renew in development can aid the efforts for generating HSCs in vitro from alternative sources such as patient-derived pluripotent stem cells. Hematopoietic stem cells (HSCs) can sustain the entire adult blood system due to their multipotency and self-renewal properties. In contrast, embryonic HSC-independent progenitor cells (HPCs) have limited regeneration and restricted differentiation. Understanding what distinguishes HSCs from HPCs could reveal the key to multipotency and self-renewal. To dissect early differences between these cells during embryonic development, we established a temporally-regulated lineage tracing assay in zebrafish that allowed us to distinguish HSCs from HPCs based on distinct differentiation kinetics. We determined that T-cell and granulocyte production from HSCs was significantly delayed compared to HPCs. Using this system, we performed a focused reverse genetic screen for factors that modulate the timing of HSC-derived T-cell production. As a control, we demonstrated that loss of Runx1, a master transcription factor for definitive hematopoiesis, diminished both HSC and HPC-derived T-cells. We then targeted 30 novel genes using CRISPR/Cas9-mutagenesis and screened for acceleration or further delay in HSC differentiation to T-cells, identifying one positive and two negative HSC regulators. Mutagenesis of pa2g4b (proliferation-associated factor 4b) led to increased HSC-derived T-cells, but no change in HPC differentiation, suggesting it is an HSC-selective negative regulator. Beyond T-cells and granulocytes, we delineated the full differentiation repertoire of embryonic HSCs and HPCs using scRNA-seq of lineage-traced cells and found HPCs could contribute to all major blood lineages in larval zebrafish suggesting they might possess a broader differentiation potential. Identifying factors that regulate HSC's ability to differentiate and self-renew in development can aid the efforts for generating HSCs in vitro from alternative sources such as patient-derived pluripotent stem cells.

The sodium/glucose cotransporters as potential therapeutic targets for CF lung diseases revealed by human lung organoid swelling assay

Cystic fibrosis (CF) is a lethal autosomal-recessive inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the present work, we derived human proximal lung organoids (HLOs) from patient-derived pluripotent stem cells (PSCs) carrying disease-causing CFTR mutations. We evaluated the forskolin (Fsk)-stimulated swellings of these HLOs in the presence of CFTR modulators (VX-770 and/or VX-809) and demonstrated that HLOs respond to CFTR modulators in a mutation-dependent manner. Using this assay, we examined the effects of the sodium-dependent glucose cotransporter 1/2 (SGLT1/2) inhibitor drugs phlorizin and sotagliflozin on the basis of our findings that SGLT1 expression is upregulated in CF HLOs and airway epithelial cells compared with their wild-type counterparts. Unexpectedly, both drugs promoted dF/dF HLO swelling. These results reveal SGLTs, especially SGLT1, as potential therapeutic targets for treating CF lung diseases and demonstrate the use of PSC-derived HLOs as a preclinical tool in CF drug development.

The promise of precision medicine in autism

Autism spectrum disorder (ASD) is a clinically and etiologically diverse developmental condition characterized by diminished social interactions, impaired communication, and repetitive and/or restrictive behaviors. Recent advances in ASD genetics pave the way for implementation of precision medicine in clinical management of autism.

Induced pluripotent stem cell-derived motor neurons of CMT type 2 patients reveal progressive mitochondrial dysfunction.

Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The types of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates its diagnosis and has inhibited the development of therapies. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrated neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC lines into peripheral sensory neurons only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveal shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2.

Inherited DNA Repair Defects Disrupt the Structure and Function of Human Skin.

Squamous cell carcinoma (SCC) is a global public health burden originating in epidermal stem and progenitor cells (ESPCs) of the skin and mucosa. To understand how genetic risk factors contribute to SCC, studies of ESPC biology are imperative. Children with Fanconi anemia (FA) are a paradigm for extreme SCC susceptibility caused by germline loss-of-function mutations in FA DNA repair pathway genes. To discover epidermal vulnerabilities, patient-derived pluripotent stem cells (PSCs) conditional for the FA pathway were differentiated into ESPCs and PSC-derived epidermal organotypic rafts (PSC-EORs). FA PSC-EORs harbored diminished cell-cell junctions and increased proliferation in the basal cell compartment. Furthermore, desmosome and hemidesmosome defects were identified in the skin of FA patients, and these translated into accelerated blistering following mechanically induced stress. Together, we demonstrate that a critical DNA repair pathway maintains the structure and function of human skin and provide 3D epidermal models wherein SCC prevention can now be explored.

The PPAR-γ inhibitor T0070907 normalizes impaired electro-mechanical properties of human-induced pluripotent stem cell-derived cardiomyocytes in DSC2 arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) primarily involves right ventricle impairment and lethal arrhythmias. Hereditary forms of ACM are mostly caused by mutations in genes coding for desmosomal proteins. Histopathological hallmark includes fibrofatty replacement of myocardial tissue, which may involve transdifferentiation of cardiomyocytes into adipocyte cells through the PPAR-γ pathway. Comprehensive investigation of cardiomyocytes electro-mechanical properties during this process has never been undertaken. We aimed to assess the implication of the PPAR-γ pathway inhibition using T0070907 inhibitor. We used cardiomyocytes (hiPSC-CM) differentiated from an ACM patient-derived pluripotent stem cells (hiPSC) harbouring a mutation (R132 C) in the DSC2 gene. The control and DSC2 hiPSC were reprogrammed and differentiated into hiPSC-CM to examine gene expression, electro-mechanical properties (patch-clamp and video analysis) and calcium handling (fluorescence imaging) profiles. The DSC2 hiPSC-CM were cultured for 40 days in presence or absence of 1 μM T0070907. The RT-qPCR of both DSC2 patient's heart samples and hiPSC-CM showed an increase of PPAR-γ expression when compared to control cells. When compared to control hiPSC-CM, the DSC2 hiPSC-CM exhibited a shorter action potential duration, due to an increase of K+ current density. Culture of DSC2 hiPSC-CM with the PPAR-γ inhibitor normalized both the action potential duration and K+ current density. Differences were also found in both the calcium transient and mechanical properties between control, DSC2 and DSC2 with PPAR-γ hiPSC-CM. PPAR-γ pathway inhibition by T0070907 normalized the disrupted electro-mechanical properties in DSC2 hiPSC-CM. This result opens interesting perspectives for further studies of the adipogenesis pathway to prevent appearance of fibro-fatty adipose tissue in the ACM patient's heart.

GATA6 Cooperates with EOMES/SMAD2/3 to Deploy the Gene Regulatory Network Governing Human Definitive Endoderm and Pancreas Formation.

SummaryHeterozygous de novo mutations in GATA6 are the most frequent cause of pancreatic agenesis in humans. In mice, however, a similar phenotype requires the biallelic loss of Gata6 and its paralog Gata4. To elaborate the human-specific requirements for GATA6, we chose to model GATA6 loss in vitro by combining both gene-edited and patient-derived pluripotent stem cells (hPSCs) and directed differentiation toward β-like cells. We find that GATA6 heterozygous hPSCs show a modest reduction in definitive endoderm (DE) formation, while GATA6-null hPSCs fail to enter the DE lineage. Consistent with these results, genome-wide studies show that GATA6 binds and cooperates with EOMES/SMAD2/3 to regulate the expression of cardinal endoderm genes. The early deficit in DE is accompanied by a significant reduction in PDX1+ pancreatic progenitors and C-PEPTIDE+ β-like cells. Taken together, our data position GATA6 as a gatekeeper to early human, but not murine, pancreatic ontogeny.

Fosl1 overexpression directly activates trophoblast-specific gene expression programs in embryonic stem cells

During early development in placental mammals, proper trophoblast lineage development is essential for implantation and placentation. Defects in this lineage can cause early pregnancy failures and other pregnancy disorders. However, transcription factors controlling trophoblast development remain poorly understood. Here, we utilize Fosl1, previously implicated in trophoblast giant cell development as a member of the AP-1 complex, to trans-differentiate embryonic stem (ES) cells to trophoblast lineage-like cells. We first show that the ectopic expression of Fosl1 is sufficient to induce trophoblast-specific gene expression programs in ES cells. Surprisingly, we find that this transcriptional reprogramming occurs independently of changes in levels of ES cell core factors during the cell fate change. This suggests that Fosl1 acts in a novel way to orchestrate the ES to trophoblast cell fate conversion compared to previously known reprogramming factors. Mapping of Fosl1 targets reveals that Fosl1 directly activates TE lineage-specific genes as a pioneer factor. Our work suggests Fosl1 may be used to reprogram ES cells into differentiated cell types in trophoblast lineage, which not only enhances our knowledge of global trophoblast gene regulation but also may provide a future therapeutic tool for generating induced trophoblast cells from patient-derived pluripotent stem cells.

Prospects for Modeling Abnormal Neuronal Function in Schizophrenia Using Human Induced Pluripotent Stem Cells.

Excitatory dopaminergic neurons, inhibitory GABAergic neurons, microglia, and oligodendrocytes have all been implicated in schizophrenia (SZ) network pathology. Still, SZ has been a difficult disorder to study, not only because of the limitations of animal models in capturing the complexity of the human mind, but also because it is greatly polygenic, with high rates of variability across the population. The advent of patient-derived pluripotent stem cells and induced neural and glial cultures has brought hope for modeling the molecular dysfunction underlying SZ pathology in a patient-specific manner. Here I review the successes of the patient-specific induced cultures in generating different cell types for the study of SZ, with special emphasis on the utility of co-culture techniques, both two- and three-dimensional, for modeling network dysfunction in disease.

P.387 - Patient-derived pluripotent stem cells: an in vitro model for neuromuscular diseases and high-throughput drug screening

P.387 - Patient-derived pluripotent stem cells: an in vitro model for neuromuscular diseases and high-throughput drug screening

CRISPR knock-out of alpha-synuclein in patient-derived pluripotent stem cell model of Parkinson’s disease

CRISPR knock-out of alpha-synuclein in patient-derived pluripotent stem cell model of Parkinson’s disease

Retraction Note: Robust method for TALEN-edited correction of pF508del in patient-specific induced pluripotent stem cells

Cystic fibrosis is one of the most frequent inherited rare diseases, caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. Apart from symptomatic treatments, therapeutic protocols for curing the disease have not yet been established. The regeneration of genetically corrected, disease-free epithelia in cystic fibrosis patients is envisioned by designing a stem cell/genetic therapy in which patient-derived pluripotent stem cells are genetically corrected, from which target tissues are derived. In this framework, we present an efficient method for seamless correction of pF508del mutation in patient-specific induced pluripotent stem cells by gene edited homologous recombination. Gene edition has been performed by transcription activator-like effector nucleases and a homologous recombination donor vector which contains a PiggyBac transposon-based double selectable marker cassette. This new method has been designed to partially avoid xenobiotics from the culture system, improve cell culture efficiency and genome stability by using a robust culture system method, and optimize timings. Overall, once the pluripotent cells have been amplified for the first nucleofection, the procedure can be completed in 69 days, and can be easily adapted to edit and change any gene of interest.

Generation of human organs in pigs via interspecies blastocyst complementation

More than eighteen years have passed since the first derivation of human embryonic stem cells (ESCs), but their clinical use is still met with several challenges, such as ethical concerns regarding the need of human embryos, tissue rejection after transplantation and tumour formation. The generation of human induced pluripotent stem cells (iPSCs) enables the access to patient-derived pluripotent stem cells (PSCs) and opens the door for personalized medicine as tissues/organs can potentially be generated from the same genetic background as the patient recipients, thus avoiding immune rejections or complication of immunosuppression strategies. In this regard, successful replacement, or augmentation, of the function of damaged tissue by patient-derived differentiated stem cells provides a promising cell replacement therapy for many devastating human diseases. Although human iPSCs can proliferate unlimitedly in culture and harbour the potential to generate all cell types in the adult body, currently, the functionality of differentiated cells is limited. An alternative strategy to realize the full potential of human iPSC for regenerative medicine is the in vivo tissue generation in large animal species via interspecies blastocyst complementation. As this technology is still in its infancy and there remains more questions than answers, thus in this review, we mainly focus the discussion on the conceptual framework, the emerging technologies and recent advances involved with interspecies blastocyst complementation, and will refer the readers to other more in-depth reviews on dynamic pluripotent stem cell states, genome editing and interspecies chimeras. Likewise, other emerging alternatives to combat the growing shortage of human organs, such as xenotransplantation or tissue engineering, topics that has been extensively reviewed, will not be covered here.

Robust method for TALEN-edited correction of pF508del in patient-specific induced pluripotent stem cells.

Cystic fibrosis is one of the most frequent inherited rare diseases, caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. Apart from symptomatic treatments, therapeutic protocols for curing the disease have not yet been established. The regeneration of genetically corrected, disease-free epithelia in cystic fibrosis patients is envisioned by designing a stem cell/genetic therapy in which patient-derived pluripotent stem cells are genetically corrected, from which target tissues are derived. In this framework, we present an efficient method for seamless correction of pF508del mutation in patient-specific induced pluripotent stem cells by gene edited homologous recombination. Gene edition has been performed by transcription activator-like effector nucleases and a homologous recombination donor vector which contains a PiggyBac transposon-based double selectable marker cassette.This new method has been designed to partially avoid xenobiotics from the culture system, improve cell culture efficiency and genome stability by using a robust culture system method, and optimize timings. Overall, once the pluripotent cells have been amplified for the first nucleofection, the procedure can be completed in 69 days, and can be easily adapted to edit and change any gene of interest.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0275-6) contains supplementary material, which is available to authorized users.