Search

The possibility of reprogramming human somatic cells to pluripotency has opened unprecedented opportunities for creating genuinely human experimental models of disease. Inborn errors of metabolism (IEMs) constitute a greatly heterogeneous class of diseases that appear, in principle, especially suited to be modeled by iPSC-based technology. Indeed, dozens of IEMs have already been modeled to some extent using patient-specific iPSCs. Here, we review the advantages and disadvantages of iPSC-based disease modeling in the context of IEMs, as well as particular challenges associated to this approach, together with solutions researchers have proposed to tackle them. We have structured this review around six lessons that we have learnt from those previous modeling efforts, and that we believe should be carefully considered by researchers wishing to embark in future iPSC-based models of IEMs. Supplementary Information The online version contains supplementary material available at 10.1007/s11154-021-09671-z.

Accumulation of misfolded α-synuclein (α-syn) is a hallmark of Parkinson's disease (PD) thought to play important roles in the pathophysiology of the disease. Dendritic systems, able to modulate the folding of proteins, have emerged as promising new therapeutic strategies for PD treatment. Dendrimers have been shown to be effective at inhibiting α-syn aggregation in cell-free systems and in cell lines. Here, we set out to investigate the effects of dendrimers on endogenous α-syn accumulation in disease-relevant cell types from PD patients. For this purpose, we chose cationic carbosilane dendrimers of bow-tie topology based on their performance at inhibiting α-syn aggregation in vitro. Dopamine neurons were differentiated from induced pluripotent stem cell (iPSC) lines generated from PD patients carrying the LRRK2G2019S mutation, which reportedly display abnormal accumulation of α-syn, and from healthy individuals as controls. Treatment of PD dopamine neurons with non-cytotoxic concentrations of dendrimers was effective at preventing abnormal accumulation and aggregation of α-syn. Our results in a genuinely human experimental model of PD highlight the therapeutic potential of dendritic systems and open the way to developing safe and efficient therapies for delaying or even halting PD progression.

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour. This protocol describes enhanced number and brightness (eN&B), an approach that uses fluorescence fluctuation spectroscopy data to directly measure the oligomerization state and dynamics of fluorescently tagged proteins in living cells.

Summary We generated patient-specific disease-free induced pluripotent stem cells (iPSCs) from peripheral blood CD34+ cells and differentiated them into functional endothelial cells (ECs) secreting factor VIII (FVIII) for gene and cell therapy approaches to cure hemophilia A (HA), an X-linked bleeding disorder caused by F8 mutations. iPSCs were transduced with a lentiviral vector carrying FVIII transgene driven by an endothelial-specific promoter (VEC) and differentiated into bona fide ECs using an optimized protocol. FVIII-expressing ECs were intraportally transplanted in monocrotaline-conditioned non-obese diabetic (NOD) severe combined immune-deficient (scid)-IL2rγ null HA mice generating a chimeric liver with functional human ECs. Transplanted cells engrafted and proliferated in the liver along sinusoids, in the long term showed stable therapeutic FVIII activity (6%). These results demonstrate that the hemophilic phenotype can be rescued by transplantation of ECs derived from HA FVIII-corrected iPSCs, confirming the feasibility of cell-reprogramming strategy in patient-derived cells as an approach for HA gene and cell therapy., Graphical Abstract, Highlights • Endothelial cells were differentiated from iPSCs obtained from hemophilic donors • LV transduced-hemophilic endothelial cells secreted FVIII in vitro and in vivo, Follenzi and colleagues show that endothelial cells can be obtained starting from blood CD34+ cells of both healthy and hemophilic donors passing through iPSC generation. During the differentiation process the cells can be genetically modified by LV to express FVIII; corrected endothelial cells secrete FVIII in vitro and ameliorate the hemophilic phenotype in a mouse model of the disease.

Photoreceptor loss is the principal cause of blindness in retinal degenerative diseases (RDDs). Whereas some therapies exist for early stages of RDDs, no effective treatment is currently available for later stages, and once photoreceptors are lost, the only option to rescue vision is cell transplantation. With the use of the Royal College of Surgeons (RCS) rat model of retinal degeneration, we sought to determine whether combined transplantation of human-induced pluripotent stem cell (hiPSC)-derived retinal precursor cells (RPCs) and retinal pigment epithelial (RPE) cells was superior to RPE or RPC transplantation alone in preserving retinal from degeneration. hiPSC-derived RPCs and RPE cells expressing (GFP) were transplanted into the subretinal space of rats. In vivo monitoring showed that grafted cells survived 12 weeks in the subretinal space, and rats treated with RPE + RPC therapy exhibited better conservation of the outer nuclear layer (ONL) and visual response than RPE-treated or RPC-treated rats. Transplanted RPE cells integrated in the host RPE layer, whereas RPC mostly remained in the subretinal space, although a limited number of cells integrated in the ONL. In conclusion, the combined transplantation of hiPSC-derived RPE and RPCs is a potentially superior therapeutic approach to protect retina from degeneration in RDDs., Graphical Abstract, In this article, Anna Duarri and colleagues aim to develop a novel cell-based therapeutic approach to preserve retinal structure and function in retinal degenerative diseases caused by dysfunctional retinal pigment epithelium (RPE). Giving to the eye support of healthy RPE together with healthy retinal cells protects the retina from degeneration

Cardiac tissue engineering is very much in a current focus of regenerative medicine research as it represents a promising strategy for cardiac disease modelling, cardiotoxicity testing and cardiovascular repair. Advances in this field over the last two decades have enabled the generation of human engineered cardiac tissue constructs with progressively increased functional capabilities. However, reproducing tissue-like properties is still a pending issue, as constructs generated to date remain immature relative to native adult heart. Moreover, there is a high degree of heterogeneity in the methodologies used to assess the functionality and cardiac maturation state of engineered cardiac tissue constructs, which further complicates the comparison of constructs generated in different ways. Here, we present an overview of the general approaches developed to generate functional cardiac tissues, discussing the different cell sources, biomaterials, and types of engineering strategies utilized to date. Moreover, we discuss the main functional assays used to evaluate the cardiac maturation state of the constructs, both at the cellular and the tissue levels. We trust that researchers interested in developing engineered cardiac tissue constructs will find the information reviewed here useful. Furthermore, we believe that providing a unified framework for comparison will further the development of human engineered cardiac tissue constructs displaying the specific properties best suited for each particular application.

Background iPSC (induced pluripotent stem cells) banks of iPSC lines with homozygous HLA (human leukocyte antigen) haplotypes (haplobanks) are proposed as an affordable and off-the-shelf approach to allogeneic transplantation of iPSC derived cell therapies. Cord blood banks offer an extensive source of HLA-typed cells suitable for reprogramming to iPSC. Several initiatives worldwide have been undertaken to create national and international iPSC haplobanks that match a significant part of a population. Methods To create an iPSC haplobank that serves the Spanish population (IPS-PANIA), we have searched the Spanish Bone Marrow Donor Registry (REDMO) to identify the most frequently estimated haplotypes. From the top ten donors identified, we estimated the population coverage using the criteria of zero mismatches in HLA-A, HLA-B, and HLA-DRB1 with different stringencies: high resolution, low resolution, and beneficial mismatch. Results We have calculated that ten cord blood units from homozygous donors stored at the Spanish cord blood banks can provide HLA-A, HLA-B, and HLA-DRB1 matching for 28.23% of the population. Conclusion We confirm the feasibility of using banked cord blood units to create an iPSC haplobank that will cover a significant percentage of the Spanish and international population for future advanced therapy replacement strategies.

Aims: Hypertrophic cardiomyopathy (HCM) is often accompanied by increased trabeculated myocardium (TM)&mdash, which clinical relevance is unknown. We aim to measure the left ventricular (LV) mass and proportion of trabeculation in an HCM population and to analyze its clinical implication. Methods and Results: We evaluated 211 patients with HCM (mean age 47.8 &plusmn, 16.3 years, 73.0% males) with cardiac magnetic resonance (CMR) studies. LV trabecular and compacted mass were measured using dedicated software for automatic delineation of borders. Mean compacted myocardium (CM) was 160.0 &plusmn, 62.0 g and trabecular myocardium (TM) 55.5 &plusmn, 18.7 g. The percentage of trabeculated myocardium (TM%) was 26.7% &plusmn, 6.4%. Females had significantly increased TM% compared to males (29.7 &plusmn, 7.2 vs. 25.6 &plusmn, 5.8, p &lt, 0.0001). Patients with LVEF &lt, 50% had significantly higher values of TM% (30.2% &plusmn, 6.0% vs. 26.6% &plusmn, 6.4%, p = 0.02). Multivariable analysis showed that female gender and neutral pattern of hypertrophy were directly associated with TM%, while dynamic obstruction, maximal wall thickness and LVEF% were inversely associated with TM%. There was no association between TM% with arterial hypertension, physical activity, or symptoms. Atrial fibrillation and severity of hypertrophy were the only variables associated with cardiovascular death. Multivariable analysis failed to demonstrate any correlation between TM% and arrhythmias. Conclusions: Approximately 25% of myocardium appears non-compacted and can automatically be measured in HCM series. Proportion of non-compacted myocardium is increased in female, non-obstructives, and in those with lower contractility. The amount of trabeculation might help to identify HCM patients prone to systolic heart failure.

A deeper understanding of early disease mechanisms occurring in Parkinson’s disease (PD) is needed to reveal restorative targets. Here we report that human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) obtained from healthy individuals or patients harboring LRRK2 PD-causing mutation can create highly complex networks with evident signs of functional maturation over time. Compared to control neuronal networks, LRRK2 PD patients’ networks displayed an elevated bursting behavior, in the absence of neurodegeneration. By combining functional calcium imaging, biophysical modeling, and DAn-lineage tracing, we found a decrease in DAn neurite density that triggered overall functional alterations in PD neuronal networks. Our data implicate early dysfunction as a prime focus that may contribute to the initiation of downstream degenerative pathways preceding DAn loss in PD, highlighting a potential window of opportunity for pre-symptomatic assessment of chronic degenerative diseases., Research from the authors’ laboratories is supported by the European Research Council-ERC (2012-StG-311736-PD-HUMMODEL), the MESOBRAIN project from the European Union’s Horizon 2020 research and innovation (grant agreement No. 713140), the Spanish Ministry of Economy and Competitiveness-MINECO (RTI2018-095377-B-100, FIS2016-78507-C2-2-P, PID2019-108842GB-C21) the Spanish Ministry of Economy and Competitiveness-MINECO/AEI/FEDER, UE (BFU2016-80870-P) and the Spanish Ministry of Economy and Competitiveness- AEI/10.13039/501100011033 (PID2019-108792GB-I00), Instituto de Salud Carlos III-ISCIII/FEDER (Red de Terapia Celular - TerCel RD16/0011/0024 and RD16/0011/0025), AGAUR (2017-SGR-899 and 2017-SGR-1061), and CERCA Program/Generalitat de Catalunya. M.P.E. was partially supported by a Beatriu de Pinós fellowship from the Agency for Management of University and Research Grants (AGAUR) of the Government of Catalonia (2017 BP 00133). L.B. is the recipient of a pre-doctoral fellowship FPI (BES-2017-080579) from the Spanish Ministry of Economy and Competitiveness (MINECO). G.C. was partially supported by pre-doctoral fellowship from Spanish Economy and Competitiveness-MINECO (BES-2014-069603).

MicroRNA (miRNA) misregulation in peripheral blood has been linked to Parkinson disease (PD) but its role in the disease progression remains elusive. We performed an explorative genome-wide study of miRNA expression levels in dopaminergic neurons (DAn) from PD patients generated by somatic cell reprogramming and induced pluripotent stem cells differentiation. We quantified expression levels of 377 miRNAs in DAn from 3 sporadic PD patients (sPD), 3 leucine-rich repeat kinase 2-associated PD patients (L2PD) (total 6 PD), and 4 healthy controls. We identified differential expression of 10 miRNA of which 5 were upregulated in PD (miR-9-5p, miR-135a-5p, miR-135b-5p, miR-449a, and miR-449b-5p) and 5 downregulated (miR-141-3p, miR-199a-5p, miR-299-5p, miR-518e-3p, and miR-519a-3p). Changes were similar in sPD and L2PD. Integrative analysis revealed significant correlations between miRNA/mRNA expression. Moreover, upregulation of miR-9-5p and miR-135b-5p was associated with downregulation of transcription factors related to the DNA hypermethylation of enhancer elements in PD DAn (FOXA1 and NR3C1). In summary, miRNA changes are associated with monogenic L2PD and sPD and co-occur with epigenetic changes in DAn from PD patients.

Over the past 10 years’ significant research developments have taken place on human pluripotent stem cells and human embryonic stem cells to exploit the future potential in gene therapy and other focused treatments. There remains concerns around ethics of research and the fate of the human embryo used in such studies. European Board and College of Obstetrics and Gynaecology urge upon all scientists and the research bodies to adhere to the highest ethical principles of confidentiality and their actions should meet the criteria as set out by the international society for stem cell research.

Multiple sclerosis (MS) is considered a chronic autoimmune disease of the central nervous system that leads to gliosis, demyelination, axonal damage and neuronal death. The MS disease aetiology is unknown, though a polymorphism of the TNFRSF1A gene, rs1800693, is known to confer an increased risk for MS. Using retroviral delivery of reprogramming transgenes, we generated six MS patient-specific iPSC lines with two distinct genotypes, CC or TT, of the polymorphism rs1800693. iPSC lines had normal karyotype, expressed pluripotency genes and differentiated into the three germ layers. These lines offer a good tool to study MS pathomechanisms and for drug testing.

Mesenchymal stromal cells (MSCs) have been shown to have useful properties for cell therapy and have been proposed for treatment of neurodegenerative diseases, including Parkinson's disease. However, the mechanisms involved in recovering dopaminergic neurons are not clear. The present study aims to evaluate the pathways and molecules involved in the neuroprotective effect of MSCs. We analyzed the viability of dopaminergic cells from different sources in response to conditioned medium derived from bone marrow MSC (MSC-CM). MSC-CM increased the viability of dopaminergic cells of rat and human origins, having both neuroprotective and neurorescue activities against effects of dopaminergic neurotoxin 6-hydroxydopamine. We found that lipid removal, inhibition of the prostaglandin E2 receptor 2 (EP2), and its signaling pathway were able to block the effects of MSC-CM on a pure population of dopaminergic neurons. Moreover, in primary mesencephalic cultures and hiPSC-derived neurons, inhibition of EP2 signaling caused a reduction in the number of dopaminergic neurons obtained in culture. Taken together, our results demonstrate for the first time the involvement of prostaglandin signaling from MSC in dopaminergic neuron survival through EP2 receptors, and suggest new approaches for treatment of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease that lacks therapies to prevent progressive neurodegeneration. Impaired energy metabolism and reduced ATP levels are common features of PD. Previous studies revealed that terazosin (TZ) enhances the activity of phosphoglycerate kinase 1 (PGK1), thereby stimulating glycolysis and increasing cellular ATP levels. Therefore, we asked whether enhancement of PGK1 activity would change the course of PD. In toxin-induced and genetic PD models in mice, rats, flies, and induced pluripotent stem cells, TZ increased brain ATP levels and slowed or prevented neuron loss. The drug increased dopamine levels and partially restored motor function. Because TZ is prescribed clinically, we also interrogated 2 distinct human databases. We found slower disease progression, decreased PD-related complications, and a reduced frequency of PD diagnoses in individuals taking TZ and related drugs. These findings suggest that enhancing PGK1 activity and increasing glycolysis may slow neurodegeneration in PD.

Summary In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates., Graphical Abstract, Highlights • GATA2 promotes hemogenic emergence during human iPSC differentiation • GATA2 enhances hematopoietic differentiation of human iPSCs • GATA2 acts as a direct repressor of cardiac fates during mesoderm specification, Giorgetti A. and colleagues demonstrate that GATA2 induction in early mesodermal cells leads to the robust generation of hemogenic endothelial and hematopoietic cells from human iPSCs. They show the ability of GATA2 to instruct mesodermal precursors toward a hematopoietic cell fate and concurrently inhibit cardiac fates. This study expands our understanding of the regulatory networks that control early human hematopoiesis.

Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson’s disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus. After differentiation of the TH-mOrange reporter iPSC line, we confirmed that mOrange expression faithfully mimicked endogenous TH expression in iPSC-derived DAn. We also employed calcium imaging techniques to determine the intrinsic functional differences between dopaminergic and non-dopaminergic ventral midbrain neurons. Crucially, the brightness of mOrange allowed direct visualization of TH-expressing cells in heterogeneous cultures, and enabled us to isolate live mOrange-positive cells through fluorescence-activated cell sorting, for further differentiation. This technique, coupled to refined imaging and data processing tools, could advance the investigation of PD pathogenesis and might offer a platform to test potential new therapeutics for PD and other neurodegenerative diseases., Research from the authors’ laboratories is supported by the European Research Council-ERC (2012-StG-311736-PD-HUMMODEL), the European Union’s Horizon 2020 Program (MESOBRAIN project; grant agreement No 713140), the Spanish Ministry of Economy and Competitiveness-MINECO (SAF2015-69706-R; BFU2017-83066-P; and BFU2016-80870-P), Instituto de Salud Carlos III-ISCIII/FEDER (Red de Terapia Celular - TerCel RD16/0011/0003 and RD16/0011/0024; PIE12/00061), AGAUR (2017-SGR-899 and 2017-SGR-1061), and CERCA Program/Generalitat de Catalunya. C.C. is supported by the PD-HUMMODEL European Research Council (ERC)- Ideas PhD fellowship. G.C. is partially supported by pre-doctoral fellowship from Spanish Economy and Competitiveness-MINECO (BES-2014-069603).

Zebrafish can regenerate their hearts. The role of the extracellular matrix in this process is largely unknown. We have analyzed the proteome in control hearts and at different times of regeneration. Decellularization of samples allowed for enrichment of extracellular matrix proteins, increasing their detection. The results reported dynamic changes in specific proteins associated with specific stages of the regenerative process. Biomechanical analysis by atomic force microscopy revealed concomitant changes in matrix stiffness during this process., Graphical Abstract Highlights We have developed a decellularization protocol for ECM protein enrichment. We have characterized the proteome of adult zebrafish heart ECM. We describe dynamic changes in heart ECM proteome during regeneration. We describe changes in heart ECM stiffness during regeneration., Adult zebrafish, in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored. Here, we set out to characterize the ECM protein composition in adult zebrafish hearts, and whether it changed during the regenerative response. For this purpose, we first established a decellularization protocol of adult zebrafish ventricles that significantly enriched the yield of ECM proteins. We then performed proteomic analyses of decellularized control hearts and at different times of regeneration. Our results show a dynamic change in ECM protein composition, most evident at the earliest (7 days postamputation) time point analyzed. Regeneration associated with sharp increases in specific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopy that the changes in ECM composition translated to decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration.

Summary In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Here, we report on the design and implementation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSCs), which we term “CardioSlice.” PSC-derived cardiomyocytes, together with human fibroblasts, are seeded into large 3D porous scaffolds and cultured using a parallelized perfusion bioreactor with custom-made culture chambers. Continuous electrical stimulation for 2 weeks promotes cardiomyocyte alignment and synchronization, and the emergence of cardiac tissue-like properties. These include electrocardiogram-like signals that can be readily measured on the surface of CardioSlice constructs, and a response to proarrhythmic drugs that is predictive of their effect in human patients., Highlights • Human iPSC-derived cardiomyocytes are cultured in ∼1-mm-thick constructs • Continuous electrical stimulation greatly enhances maturation at the tissue level • Electrostimulated constructs (CardioSlice) generate ECG-like bioelectrical signals • CardioSlice response to proarrhythmic drugs is predictive of effect in patients, In this article, Raya and colleagues design and implement a cardiac tissue-engineering platform for the production of macrotissues from human pluripotent stem cells. These human cardiac macrotissues can be electrically stimulated, and their electric activity recorded from the surface of the constructs. After 2 weeks of electrical stimulation, distinct cardiac tissue-like properties appear such as electrocardiogram-like signals and patient-like response to cardiotoxic drugs.

Summary Neurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(−/−) cells, descendants from the tumor originating cell. These NF1(−/−)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(−/−) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available., Graphical Abstract, Highlights • We generated iPSCs from neurofibromatosis type 1 plexiform neurofibroma (PNF) cells • PNF-derived iPSCs were differentiated into neural crest and Schwann cells (SCs) • iPSC-differentiated NF1(−/−) SCs exhibit a high proliferation rate and form spheres • Sphere-forming SCs express the same markers as their primary PNF counterparts, In this article, Eduard Serra and colleagues describe the generation of iPSCs directly from plexiform neurofibromas (PNFs), benign Schwann cell (SC) tumors associated with neurofibromatosis type 1. iPSCs bearing the double inactivation of the NF1 gene were differentiated into SCs that exhibited a high proliferation rate, a poor myelination ability, and a tendency to form spheres, resembling PNF-derived SCs.

Background: Parkinson’s disease (PD) is characterized by the loss of midbrain dopaminergic neurons (DAn). Previously, we described the presence of DNA hyper- and hypo-methylation alterations in induced pluripotent stem cells (iPSC)-derived DAn from PD patients using the Illumina 450K array which prominently covers gene regulatory regions. Methods: To expand and contextualize previous findings, we performed the first whole-genome DNA bisulfite sequencing (WGBS) using iPSC-derived DAn from representative PD subjects: one sporadic PD (sPD) patient, one monogenic LRRK2-associated PD patient (L2PD), and one control. Results: At the whole-genome level, we detected global DNA hyper-methylation in the PD which was similarly spread across the genome in both sPD and L2PD and mostly affected intergenic regions. Conclusion: This study implements previous epigenetic knowledge in PD at a whole genome level providing the first comprehensive and unbiased CpG DNA methylation data using iPSC-derived DAn from PD patients. Our results indicate that DAn from monogenic or sporadic PD exhibit global DNA hyper-methylation changes. Findings from this exploratory study are to be validated in further studies analyzing other PD cell models and patient tissues. This work was supported by the Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III (ISCIII) to M.E. (grant # PI14/00426), the Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED) to the Movement Disorders Unit of the Neurology Service from the Hospital Clínic de Barcelona to E.T., M.-J.M., R.F.-S., and M.E. (grant # PRI-16-2017). R.F.S. was supported by a Jóvenes Investigadores grant (#SAF2015-73508-JIN) through the Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad (Plan Estatal de I+D+I 2013–2016) of the Spanish Ministry of Economy and Competitiveness (MINECO), and the Agencia Estatal de Investigación (AEI), which is cofunded by FEDER (AEI/FEDER/UE). Other funding included the grants BFU2013-49157-P, BFU2016-80870-P, and the European Research Council (ERC) 2012-StG (311736- PD-HUMMODEL) to A.C; ISCIII/FEDER (RD16/0011/0024) and Generalitat de Catalunya (iPS4BioMed SGR 2017–2019) to A.R.; and ISCIII/FEDER (PIE14/00061) to A.R.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are a promising cell source for cardiac repair after myocardial infarction (MI) because they offer several advantages such as potential to remuscularize infarcted tissue, integration in the host myocardium, and paracrine therapeutic effects. However, cell delivery issues have limited their potential application in clinical practice, showing poor survival and engraftment after transplantation. In this work, we hypothesized that the combination of hiPSC-CMs with microparticles (MPs) could enhance long-term cell survival and retention in the heart and consequently improve cardiac repair. CMs were obtained by differentiation of hiPSCs by small-molecule manipulation of the Wnt pathway and adhered to biomimetic poly(lactic-co-glycolic acid) MPs covered with collagen and poly(d-lysine). The potential of the system to support cell survival was analyzed in vitro, demonstrating a 1.70-fold and 1.99-fold increase in cell survival after 1 and 4 days, respectively. The efficacy of the system was tested in a mouse MI model. Interestingly, 2 months after administration, transplanted hiPSC-CMs could be detected in the peri-infarct area. These cells not only maintained the cardiac phenotype but also showed in vivo maturation and signs of electrical coupling. Importantly, cardiac function was significantly improved, which could be attributed to a paracrine effect of cells. These findings suggest that MPs represent an excellent platform for cell delivery in the field of cardiac repair, which could also be translated into an enhancement of the potential of cell-based therapies in other medical applications.

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43 days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

SummaryCurrent sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41+, display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.

Summary Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development., Graphical Abstract, Highlights • Gata1, Tal1, Lmo2, and c-Myc reprogram fibroblasts to erythroid progenitors (iEPs) • iEP gene expression is more similar to that of primitive than definitive erythroblasts • Klf1 or Myb overexpression induces adult hemoglobin expression in iEPs, Capellera-Garcia et al. show that Gata1, Tal1, Lmo2, and c-Myc directly convert murine and human fibroblasts into erythroid progenitor/precursor cells. This finding defines a conserved transcriptional program instructing erythroid cell fate in mammals.

BACKGROUND: With the recent development of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing technology, the possibility to genetically manipulate the human germline (gametes and embryos) has become a distinct technical possibility. Although many technical challenges still need to be overcome in order to achieve adequate efficiency and precision of the technology in human embryos, the path leading to genome editing has never been simpler, more affordable, and widespread. OBJECTIVE AND RATIONALE: In this narrative review we seek to understand the possible impact of CRISR/Cas9 technology on human reproduction from the technical and ethical point of view, and suggest a course of action for the scientific community. SEARCH METHODS: This non-systematic review was carried out using Medline articles in English, as well as technical documents from the Human Fertilisation and Embryology Authority and reports in the media. The technical possibilities of the CRISPR/Cas9 technology with regard to human reproduction are analysed based on results obtained in model systems such as large animals and laboratory rodents. Further, the possibility of CRISPR/Cas9 use in the context of human reproduction, to modify embryos, germline cells, and pluripotent stem cells is reviewed based on the authors' expert opinion. Finally, the possible uses and consequences of CRISPR/cas9 gene editing in reproduction are analysed from the ethical point of view. OUTCOMES: We identify critical technical and ethical issues that should deter from employing CRISPR/Cas9 based technologies in human reproduction until they are clarified. WIDER IMPLICATIONS: Overcoming the numerous technical limitations currently associated with CRISPR/Cas9 mediated editing of the human germline will depend on intensive research that needs to be transparent and widely disseminated. Rather than a call to a generalized moratorium, or banning, of this type of research, efforts should be placed on establishing an open, international, collaborative and regulated research framework. Equally important, a societal discussion on the risks, benefits, and preferred applications of the new technology, including all relevant stakeholders, is urgently needed and should be promoted, and ultimately guide research priorities in this area. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Using a lentivirus-mediated labeling method, we investigated whether the adult hippocampus retains long-lasting, self-renewing neural stem cells (NSCs). We first showed that a single injection of a lentiviral vector expressing a green fluorescent protein (LV PGK-GFP) into the subgranular zone (SGZ) of the adult hippocampus enabled an efficient, robust, and long-term marking of self-renewing NSCs and their progeny. Interestingly, a subset of labeled cells showed the ability to proliferate multiple times and give rise to Sox2+ cells, clearly suggesting the ability of NSCs to self-renew for an extensive period of time (up to 6 months). In addition, using GFP+ cells isolated from the SGZ of mice that received a LV PGK-GFP injection 3 months earlier, we demonstrated that some GFP+ cells displayed the essential properties of NSCs, such as self-renewal and multipotency. Furthermore, we investigated the plasticity of NSCs in a perforant path transection, which has been shown to induce astrocyte formation in the molecular layer of the hippocampus. Our lentivirus (LV)-mediated labeling study revealed that hippocampal NSCs are not responsible for the burst of astrocyte formation, suggesting that signals released from the injured perforant path did not influence NSC fate determination. Therefore, our studies showed that a gene delivery system using LVs is a unique method to be used for understanding the complex nature of NSCs and may have translational impact in gene therapy by efficiently targeting NSCs.

Posttranscriptional modifications of histones constitute an epigenetic mechanism that is closely linked to both gene silencing and activation events. Trimethylation of Histone3 at lysine 27 (H3K27me3) is a repressive mark that associates with developmental gene regulation during differentiation programs. In the developing pancreas, expression of the transcription factor Neurogenin3 in multipotent progenitors initiates endocrine differentiation that culminates in the generation of all pancreatic islet cell lineages, including insulin-producing beta cells. Previously, we showed that Neurogenin3 promoted the removal of H3K27me3 marks at target gene promoters in vitro, suggesting a functional connection between this factor and regulators of this chromatin mark. In the present study, we aimed to specifically evaluate whether targeting the activity of these histone modifiers can be used to modulate pancreatic endocrine differentiation. Our data show that chemical inhibition of the H3K27me3 demethylases Jmjd3/Utx blunts Neurogenin3-dependent gene activation in vitro. Conversely, inhibition of the H3K27me3 methyltransferase Ezh2 enhances both the transactivation ability of Neurogenin3 in cultured cells and the formation of insulin-producing cells during directed differentiation from pluripotent cells. These results can help improve current protocols aimed at generating insulin-producing cells for beta cell replacement therapy in diabetes.

Parkinson’s disease is associated with intracellular α-synuclein accumulation and ventral midbrain dopaminergic neuronal death in the Substantia Nigra of brain patients. The Rho GTPase pathway, mainly linking surface receptors to the organization of the actin and microtubule cytoskeletons, has been suggested to participate to Parkinson’s disease pathogenesis. Nevertheless, its exact contribution remains obscure. To unveil the participation of the Rho GTPase family to the molecular pathogenesis of Parkinson’s disease, we first used C elegans to demonstrate the role of the small GTPase RAC1 (ced-10 in the worm) in maintaining dopaminergic function and survival in the presence of alpha-synuclein. In addition, ced-10 mutant worms determined an increase of alpha-synuclein inclusions in comparison to control worms as well as an increase in autophagic vesicles. We then used a human neuroblastoma cells (M17) stably over-expressing alpha-synuclein and found that RAC1 function decreased the amount of amyloidogenic alpha-synuclein. Further, by using dopaminergic neurons derived from patients of familial LRRK2-Parkinson’s disease we report that human RAC1 activity is essential in the regulation of dopaminergic cell death, alpha-synuclein accumulation, participates in neurite arborization and modulates autophagy. Thus, we determined for the first time that RAC1/ced-10 participates in Parkinson’s disease associated pathogenesis and established RAC1/ced-10 as a new candidate for further investigation of Parkinson’s disease associated mechanisms, mainly focused on dopaminergic function and survival against α-synuclein-induced toxicity. Electronic supplementary material The online version of this article (10.1007/s12035-018-0881-7) contains supplementary material, which is available to authorized users.

Decreasing ECM stiffness rescues the regeneration ability of the neonatal mouse heart., Neonatal mice have been shown to regenerate their hearts during a transient window of time of approximately 1 week after birth. However, experimental evidence for this phenomenon is not undisputed, because several laboratories have been unable to detect neonatal heart regeneration. We first confirmed that 1-day-old neonatal mice are indeed able to mount a robust regenerative response after heart amputation. We then found that this regenerative ability sharply declines within 48 hours, with hearts of 2-day-old mice responding to amputation with fibrosis, rather than regeneration. By comparing the global transcriptomes of 1- and 2-day-old mouse hearts, we found that most differentially expressed transcripts encode extracellular matrix components and structural constituents of the cytoskeleton. These results suggest that the stiffness of the local microenvironment, rather than cardiac cell-autonomous mechanisms, crucially determines the ability or inability of the heart to regenerate. Testing this hypothesis by pharmacologically decreasing the stiffness of the extracellular matrix in 3-day-old mice, we found that decreased matrix stiffness rescued the ability of mice to regenerate heart tissue after apical resection. Together, our results identify an unexpectedly restricted time window of regenerative competence in the mouse neonatal heart and open new avenues for promoting cardiac regeneration by local modification of the extracellular matrix stiffness.

Gerstmann-Sträussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia, spastic paraparesis, extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene, patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation, as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis, increased phospho-Tau, altered microtubule-associated transport and cell death. However, they failed to generate proteinase K-resistant prion. In this study we set out to test, for the first time, whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e, tauopathy) identified in the GSS patient., SCOPUS: ar.j, info:eu-repo/semantics/published

Determining causal relationships between distinct chromatin features and gene expression, and ultimately cell behavior, remains a major challenge. Recent developments in targetable epigenome-editing tools enable us to assign direct transcriptional and functional consequences to locus-specific chromatin modifications. This Protocol Review discusses the unprecedented opportunity that CRISPR/Cas9 technology offers for investigating and manipulating the epigenome to facilitate further understanding of stem cell biology and engineering of stem cells for therapeutic applications. We also provide technical considerations for standardization and further improvement of the CRISPR/Cas9-based tools to engineer the epigenome.

The combination of biomatrices and induced pluripotent stem cell (iPSC) derivatives to aid repair and myocardial scar formation may soon become a reality for cardiac regenerative medicine. However, the tumor risk associated with residual undifferentiated cells remains an important safety concern of iPSC-based therapies. This concern is not satisfactorily addressed in xenotransplantation, which requires immune suppression of the transplanted animal. In this study, we assessed the safety of transplanting undifferentiated iPSCs in an allogeneic setting. Given that swine are commonly used as large animal models in cardiac medicine, we used porcine iPSCs (p-iPSCs) in conjunction with bioengineered constructs that support recovery after acute myocardial infarction. Histopathology analyses found no evidence of p-iPSCs or p-iPSC-derived cells within the host myocardium or biomatrices after 30 and 90 days of follow-up. Consistent with the disappearance of the implanted cells, we could not observe functional benefit of these treatments in terms of left ventricular ejection fraction, cardiac output, ventricular volumes, or necrosis. We therefore conclude that residual undifferentiated iPSCs should pose no safety concern when used on immune-competent recipients in an allogeneic setting, at least in the context of cardiac regenerative medicine.

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. SummaryBackground The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level.

Patient-specific iPSC are being intensively exploited as experimental disease models. Even for late-onset diseases of complex genetic influence, such as Parkinson's disease (PD), the use of iPSC-based models is beginning to provide important insights into the genetic bases of PD heritability. Here, we present an update on recently reported genetic risk factors associated with PD. We discuss how iPSC technology, combined with targeted edition of the coding or noncoding genome, can be used to address clinical observations such as incomplete penetrance, and variability in phenoconversion or age-at-onset in familial PD. Finally, we also discuss the relevance of advanced iPSC/CRISPR/Cas9 disease models to ascertain causality in genotype-to-phenotype correlation studies of sporadic PD.

Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization–condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients.

A skin biopsywas obtained froma 25-year-old female patientwith autosomal recessive Alport syndrome (ARAS) with the homozygous COL4A3 mutation c. 345delG, p.(P166Lfs* 37). Dermal fibroblasts were derived and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53shRNA. The generated induced Pluripotent Stem Cell (iPSC) clone AS FiPS1 Ep6F-2 was free of genomically integrated reprogramming genes, had the specific homozygous mutation, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. This iPSC line offers a useful resource to study Alport syndrome pathomechanisms and drug testing. (c) 2017 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license.

Skin biopsies were obtained from two male patients with X-linked Alport syndrome (XLAS) with hemizygous COL4A5 mutations in exon 41 or exon 46. Dermal fibroblastswere extracted and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53 shRNA. The generated induced Pluripotent Stem Cell (iPSC) lines AS-FiPS2-Ep6F-28 and AS-FiPS3-Ep6F-9 were free of genomically integrated reprogramming genes, had the specific mutations, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. These iPSC lines offer a useful resource to study Alport syndrome pathomechanisms and drug testing. (C) 2017 The Authors. Published by Elsevier B.V.

Induced pluripotent stem cells (iPSCs) represent an invaluable tool in a chromosomal instability syndrome such as Fanconi anemia (FA), as they can allow to study of the molecular defects underlying this disease. Many other applications, such as its use as a platform to test different methods or compounds, could also be of interest. But the greatest impact of iPSCs may be in bone marrow failure diseases, as iPSCs could represent an unlimited source of autologous cells to apply in advanced treatments such as gene therapy. At the same time, genome editing constitutes the next generation of technology to further develop a safer, personalized, targeted gene therapy. Despite the promising advantages that these two technologies would present in a disease such as FA, the specific characteristics of the disease make both of these processes especially challenging. Efficient and safer FA-hiPSC (human induced pluripotent stem cell) generation methods, robust and reliable differentiation protocols for iPSCs, as well as really efficient delivery methods to perform targeted gene correction should be developed.

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD.

9 pages, 2 figures, 2 tables, While vitrification has become the method of choice for preservation of human oocytes and embryos, cryopreservation of complex tissues and of large yolk-containing cells, remains largely unsuccessful. One critical step in such instances is appropriate permeation while avoiding potentially toxic concentrations of cryoprotectants. Permeation of water and small non-charged solutes, such as those used as cryoprotectants, occurs largely through membrane channel proteins termed aquaporins (AQPs). Substitution of a Thr by an Ala residue in the pore-forming motif of the zebrafish (Dario rerio) Aqp3b paralog resulted in a mutant (DrAqp3b-T85A) that when expressed in Xenopus or porcine oocytes increased their permeability to ethylene glycol at pH 7.5 and 8.5. The main objective of this study was to test whether ectopic expression of DrAqp3b-T85A also conferred higher resistance to cryoinjury. For this, DrAqp3b-T85A + eGFP (reporter) cRNA, or eGFP cRNA alone, was microinjected into in vivo fertilized 1-cell mouse zygotes. Following culture to the 2-cell stage, appropriate membrane expression of DrAqp3b-T85A was confirmed by immunofluorescence microscopy using a primary specific antibody directed against the C-terminus of DrAqp3b. Microinjected 2-cell embryos were then cryopreserved using a fast-freezing rate and low concentration (1.5 M) of ethylene glycol in order to highlight any benefits from DrAqp3b-T85A expression. Notably, post-thaw survival rates were higher (P, Dr Sylvia J. Bedford-Guaus was supported and this research was partially funded by the Subprograma Ramón y Cajal (RYC-2012–10053), Ministerio de Economía y Competitividad (MINECO), Spain. This work was also partially funded by the Instituto de Salud Carlos III, FEDER, Plan Estatal de Investigación Científica y Técnica y de Innovación (PEICTI) 2013–2016, PI14/02184 (to SJB-G) and the MINECO project AGL2013-41196-R (to JC)

The generation of patient-specific induced pluripotent stem cells (iPSCPSCPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSCPSCPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCPSCPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCPSCPSCs can be obtained in 4–5 months.

During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency., Reprogramming of somatic cells is a stepwise process where cells must overcome several barriers before reaching the pluripotent state. Here the authors show that mitochondrial fission in response to ERK1/2 signalling is an important early step during reprogramming to pluripotency.

Adult zebrafish have the remarkable ability to regenerate their heart upon injury, a process that involves limited dedifferentiation and proliferation of spared cardiomyocytes (CMs), and migration of their progeny. During regeneration, proliferating CMs are detected throughout the myocardium, including areas distant to the injury site, but whether all of them are able to contribute to the regenerated tissue remains unknown. Here, we developed a CM-specific, photoinducible genetic labelling system, and show that CMs labelled in embryonic hearts survive and contribute to all three (primordial, trabecular and cortical) layers of the adult zebrafish heart. Next, using this system to investigate the fate of CMs from different parts of the myocardium during regeneration, we show that only CMs immediately adjacent to the injury site contributed to the regenerated tissue. Finally, our results show an extensive predetermination of CM fate during adult heart regeneration, with cells from each myocardial layer giving rise to cells that retain their layer identity in the regenerated myocardium. Overall, our results indicate that adult heart regeneration in the zebrafish is a rather static process governed by short-range signals, in contrast to the highly dynamic plasticity of CM fates that takes place during embryonic heart regeneration.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. We found LRRK2 to be degraded in lysosomes by chaperone-mediated autophagy (CMA), whereas the most common pathogenic mutant form of LRRK2, G2019S, was poorly degraded by this pathway. In contrast to the behavior of typical CMA substrates, lysosomal binding of both wild-type and several pathogenic mutant LRRK2 proteins was enhanced in the presence of other CMA substrates, which interfered with the organization of the CMA translocation complex, resulting in defective CMA. Cells responded to such LRRK2-mediated CMA compromise by increasing levels of the CMA lysosomal receptor, as seen in neuronal cultures and brains of LRRK2 transgenic mice, induced pluripotent stem cell-derived dopaminergic neurons and brains of Parkinson's disease patients with LRRK2 mutations. This newly described LRRK2 self-perpetuating inhibitory effect on CMA could underlie toxicity in Parkinson's disease by compromising the degradation of α-synuclein, another Parkinson's disease-related protein degraded by this pathway.

International audience; Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo. Two-photon inducible activators provide spatial resolution for superficial cells, but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis. Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures, but whether it can be used for photoactivation remains to be tested. Here we show, both theoretically and experimentally, that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules. We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage. This technique allows for a noninvasive genetic manipulation in vivo with spatial, temporal and cell-type specificity, and may have wide applicability in experimental biology.

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson’s disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients’ genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type., AS-D was partially supported by a pre-doctoral fellowship from MEC. Additional support was provided by grants from MICINN (BFU2009- 13277, PLE2009-0144 and ACI2010-1117 to AR; RyC-2008- 02772 and BFU2010-21823 to AC; SAF2008-04360, to JA; SAF2009-07774 and PLE2009-0089, to JMC), FIS (PI10/00849 to MV; RD06/0010/0006 to JMC), NIH/NIA AG031782/ AG038072 (to AMC) and Fondazione Guido Berlucchi (to AC). The Cell Therapy Program was supported by the CMRB (Promt-0901 to JMC). The work was also part of a CIBERNED Cooperative Project (to JA, JL-B, MV, ET and AR).