hPSCreg - the human pluripotent stem cell registry

The human pluripotent stem cell registry (hPSCreg), accessible at http://hpscreg.eu, is a public registry and data portal for human embryonic and induced pluripotent stem cell lines (hESC and hiPSC). Since their first isolation the number of hESC lines has steadily increased to over 3000 and new iPSC lines are generated in a rapidly growing number of laboratories as a result of their potentially broad applicability in biomedicine and drug testing. Many of these lines are deposited in stem cell banks, which are globally established to store tens of thousands of lines from healthy and diseased donors. The Registry provides comprehensive and standardized biological and legal information as well as tools to search and compare information from multiple hPSC sources and hence addresses a translational research need. To facilitate unambiguous identification over different resources, hPSCreg automatically creates a unique standardized name for each cell line registered. In addition to biological information, hPSCreg stores extensive data about ethical standards regarding cell sourcing and conditions for application and privacy protection. hPSCreg is the first global registry that holds both, manually validated scientific and ethical information on hPSC lines, and provides access by means of a user-friendly, mobile-ready web application

Nanopatterns of surface-bound ephrinB1 produce multivalent ligand-receptor interactions that tune EphB2 receptor clustering

Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate with molecular sensitivity the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems

Pluripotent stem cells as research models : the examples of trinucleotide repeat instability and X-chromosome inactivatio

Els models de malalties són una eina bàsica per la comprensió de les malalties humanes. Actualment, la majoria de la informació de la que disposem de malalties humanes es basa en models animals. Tot i això, els models animals difereixen molecular i fenotípicament dels humans, i no sempre reprodueixen fidelment la malaltia humana. En les últimes dècades, les cèl·lules mare humanes s'han establert com una opció molt interessant en el camp de la modelització cel·lular. En aquest treball hem volgut caracteritzar les cèl·lules mare embrionàries com a models per a l'estudi de la inestabilitat de la repetició de trinucleotids a la distròfia miotonica tipus 1 (DM1) i la malaltia de Huntington (HD). Així mateix, hem volgut estudiar la inactivació del cromosoma X amb la intenció de fer servir linees cel·lulars com a models per l'estudi del desenvolupament embrionàri humà. A la primera part d'aquest treball, hem observant una inestabilitat de repeticions de trinucleotids significativa al locus de la malaltia DM1 de les cèl·lules mare estudiades. La diferenciació d'aquestes cèl·lules va estabilitzar el número de repeticions. L'estabilització de les repeticions va ser concomitant amb la regulació a la baixa de l'expressió de gens involucrats en els mecanismes de reparació cel·lular. Posteriorment a la publicació del nostre article, altres grups varen reproduir els nostres resultats, però en aquest cas utilitzant cèl·lules mare induïdes. Els estudis recolzen la reproductibilitat dels nostres resultats, suggerint que poden ser extrapol·lats a altres linees de cèl·lules mare arreu del mon. Referent a la mutació de HD, varem trobar que era estable en totes les condicions estudiades, en cèl·lules indiferenciades, diferenciades a progenitors d'os, teratomes i progenitors neurals. Aquests resultats estan en concordancia amb els resultats obtinguts per altres grups que descriuen un baix nombre de repeticions al locus de HD. Per altra banda, varis grups han descrit la presencia de inestabilitat de les repeticions en cèl·lules diferenciades a la linea neural. La discrepància entre els nostres resultats i aquests últims podria ser deguda a la obtenció de cèl·lules neurals menys madures en el moment del nostre estudi. A la segona part d'aquesta tesis hem estudiat la inactivació del cromosoma X en 23 línies femenines de cèl·lules mare pluripotents. Vàrem observar una ràpida progressió de les cèl·lules de dependència de XIST en la inactivació del cromosoma X cap a un estat d'adaptació al cultiu que es caracteritza per un estadi de inactivació independent de l'expressió de XIST i amb una erosió de la metilació. També describim un patró d'inactivació esbiaixat en la majoria de les línies estudiades, contrari al patró aleatori observat en cèl·lules femenines adultes. A més a més, aquest patró és independent de XIST, de l'origen del cromosoma X i d'aberracions cromosòmiques. Aquests resultats suggereixen que el patró esbiaixat observant esta dirigit provablement per l'activació o repressió d'al·lels específics que es troben en el cromosoma X i que li confereixen a la cèl·lula un avantatge respecte a les altres cèl·lules. En conclusió, les cèl·lules mare pluripotents semblen ser un bon model in vitro per a l'estudi d'ambdues malalties, DM1 i HD, ja que presenten el mateix patró d'inestabilitat de la repetició del trinucleotid que s'observa in vivo. Cal remarcar també la depencia Overall, hPSC appear to be a good in vitro model for the study of both DM1 and HD TNR instability, as the repeat follows in vitro the same patterns as found in vivo, including its dependency of the 12R machinery, particularly in the case of DM1. However, our results on the study of the X chromosome inactivation (XCI) state suggest caution when using hPSC as early human developmental research models. The eroded state of XCI found in many of the hPSC lines, and the frequency of skewed XCI patterns suggests that these cells are not a good proxy to early embryonic cells, at least what XCI is concerned. Conversely, they may still provide an interesting model to study gene function and mechanisms implicatedDisease modelling is an essential tool for the understanding of human disease. Currently, much of the information we have on human diseases is based on animal models. However, animal models differ molecularly and phenotypically from humans, and are not always suitable to reproduce with fidelity human diseases. In the past decades, human pluripotent stem cells (hPSC) have emerged as an interesting option in the field of cellular modelling, this development recently having taken up much momentum. In this work, we aimed at characterizing hPSC as models for the study of Myotonic dystrophy type 1 (DM1) and Huntington's disease (HD) trinucleotide repeat (TNR) instability and to investigate the status of the X-chromosome inactivation with an eye on using these cells as models for early human development. In the first part of our work, we observed a significant TNR instability for the DM1 locus in hESC, and that differentiation resulted in a stabilization of the repeat. This stabilization was concommitant with a downregulation of the mismatch repair (MMR). Our results were later replicated in hiPSC by other researchers, showing their reproducibility and suggesting they may be extrapolated to other hPSC lines worldwide. Regarding the HD repeat, we found it was very stable in all conditions studied, both in undifferentiated hESC and cells differentiated into osteogenic progenitor-like cells, teratoma cells and neural progenitors. This is in line with other studies showing that hESC show very limited TNR in the HD locus. On the other hand, some groups have now reported some instability of this locus in cells differentiated into the neuronal lineage. The instability seen in neuronal lineage in later studies, not in our study, is probably explained by the use of hPSC derived neurons more similar to the cells showing in vivo instability than the ones we were able to generate at the time of the study. In the second part of the thesis we studied the X-chromosome inactivation in 23 female hPSC lines. We found that hPSC rapidly progress from a XIST-dependent XCI state to a culture-adapted, XIST-independent XCI state with loss of repressive histone modifications and erosion of methylation. We also report a remarkably high incidence of non-random XCI patterns, and that this skewing of the methylation patterns is independent from the transition to the XIST-independent XCI state, the origin of the X chromosome or chromosomal aberrations. These results suggest that XCI skewing is possibly driven by the activation or repression of a specific allele on the X chromosome, conferring a growth or survival advantage to the cells. Overall, hPSC appear to be a good in vitro model for the study of both DM1 and HD TNR instability, as the repeat follows in vitro the same patterns as found in vivo, including its dependency of the MMR machinery, particularly in the case of DM1. However, our results on the study of the X chromosome inactivation (XCI) state suggest caution when using hPSC as early human developmental research models. The eroded state of XCI found in many of the hPSC lines, and the frequency of skewed XCI patterns suggests that these cells are not a good proxy to early embryonic cells, at least what XCI is concerned. Conversely, they may still provide an interesting model to study gene function and mechanisms implicated

Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

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

MSH3 polymorphisms and protein levels affect CAG repeat instability in huntington's disease mice

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)~100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases

Eph-ephrin signaling modulated by polymerization and condensation of receptors

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

Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats