Regeneration and reprogramming compared

<p>Abstract</p> <p>Background</p> <p>Dedifferentiation occurs naturally in mature cell types during epimorphic regeneration in fish and some amphibians. Dedifferentiation also occurs in the induction of pluripotent stem cells when a set of transcription factors (<it>Oct4, Sox2, Klf4 </it>and <it>c-Myc</it>) is over expressed in mature cell types.</p> <p>Results</p> <p>We hypothesised that there are parallels between dedifferentiation or reprogramming of somatic cells to induced pluripotent stem cells and the natural process of dedifferentiation during epimorphic regeneration. We analysed expression levels of the most commonly used pluripotency associated factors in regenerating and non-regenerating tissue and compared them with levels in a pluripotent reference cell. We found that some of the pluripotency associated factors (<it>oct4/pou5f1, sox2, c-myc, klf4, tert, sall4, zic3, dppa2/4 </it>and <it>fut1</it>, a homologue of <it>ssea1</it>) were expressed before and during regeneration and that at least two of these factors (<it>oct4, sox2</it>) were also required for normal fin regeneration in the zebrafish. However these factors were not upregulated during regeneration as would be expected if blastema cells acquired pluripotency.</p> <p>Conclusions</p> <p>By comparing cells from the regeneration blastema with embryonic pluripotent reference cells we found that induced pluripotent stem and blastema cells do not share pluripotency. However, during blastema formation some of the key reprogramming factors are both expressed and are also required for regeneration to take place. We therefore propose a link between partially reprogrammed induced pluripotent stem cells and the half way state of blastema cells and suggest that a common mechanism might be regulating these two processes.</p

Cord blood-derived neuronal cells by ectopic expression of SOX2 and c-MYC

The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific sets of transcrip- tion factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133+ cells lose their hematopoietic signature and are converted into CB-induced neu- ronal-like cells (CB-iNCs) by the ectopic expression of the transcrip- tion factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression analysis, immu- nophenotyping, and electrophysiological analysis show that CB- iNCs acquire a distinct neuronal phenotype characterized by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of future cell-replacement therapies

Increased dosage of tumor suppressors limits the tumorigenicity of iPS cells without affecting their pluripotency

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a promising therapeutic tool for many diseases, including aged tissues and organs at high risk of failure. However, the intrinsic self-renewal and pluripotency of ES and iPS cells make them tumorigenic, and hence, the risk of tumor development hinders their clinical application. Here, we present a novel approach to limit their tumorigenicity and increase their safety through increased copy number of tumor suppressors. iPS containing an extra copy of the p53 or Ink4a/ARF locus show normal pluripotency, as determined by in vitro and in vivo differentiation assays. Yet, while retaining full pluripotency, they also possess an improved engagement of the p53 pathway during teratocarcinoma formation, which leads to a reduced tumorigenic potential in various in vitro and in vivo assays. Furthermore, they show an improved response to anticancer drugs, which could aid in their elimination in case tumors arise with no adverse effects on cell function or aging. Our system provides a model for studying tumor suppressor pathways during reprogramming, differentiation, and cell therapy applications. This offers an improved understanding of the pathways involved in tumor growth from engrafted pluripotent stem cells, which could facilitate the use of ES and iPS cells in regenerative medicine

The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases.

Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes

A MT-TL1 variant identified by whole exome sequencing in an individual with intellectual disability, epilepsy, and spastic tetraparesis.

Funder: The Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779257The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC_012920.1:m.3291T > C; NC_012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line

Comprehensive reanalysis for CNVs in ES data from unsolved rare disease cases results in new diagnoses

We report the diagnostic results of a comprehensive copy number variant (CNV) reanalysis of 9,171 exome sequencing (ES) datasets from 5,757 families, including 6,143 individuals affected by a rare disease (RD). The data analysed was extremely heterogeneous, having been generated using 28 different exome enrichment kits, and sequenced on multiple short-read sequencing platforms, by 42 different research groups across Europe partnering in the Solve-RD project. Each of these research groups had previously undertaken their own analysis of the ES data but had failed to identify disease-causing variants.We applied three CNV calling algorithms to maximise sensitivity: ClinCNV, Conifer, and ExomeDepth. Rare CNVs overlapping genes of interest in custom lists provided by one of four partner European Reference Networks (ERN) were identified and taken forward for interpretation by clinical experts in RD. To facilitate interpretation, Integrative Genomics Viewer (IGV) screenshots incorporating a variety of custom-made tracks were generated for all prioritised CNVs.These analyses have resulted in a molecular diagnosis being provided for 51 families in this sample, with ClinCNV performing the best of the three algorithms in identifying disease-causing CNVs. We also identified pathogenic CNVs that are partially explanatory of the proband’s phenotype in a further 34 individuals. This work illustrates the value of reanalysing EScold casesfor CNVs even where analyses had been undertaken previously. Crucially, identification of these previously undetected CNVs has resulted in the conclusion of the diagnostic odyssey for these RD families, some of which had endured decades.3. Good health and well-bein

Analysis of Human and Mouse Reprogramming of Somatic Cells to Induced Pluripotent Stem Cells. What Is in the Plate?

After the hope and controversy brought by embryonic stem cells two decades ago for regenerative medicine, a new turn has been taken in pluripotent cells research when, in 2006, Yamanaka's group reported the reprogramming of fibroblasts to pluripotent cells with the transfection of only four transcription factors. Since then many researchers have managed to reprogram somatic cells from diverse origins into pluripotent cells, though the cellular and genetic consequences of reprogramming remain largely unknown. Furthermore, it is still unclear whether induced pluripotent stem cells (iPSCs) are truly functionally equivalent to embryonic stem cells (ESCs) and if they demonstrate the same differentiation potential as ESCs. There are a large number of reprogramming experiments published so far encompassing genome-wide transcriptional profiling of the cells of origin, the iPSCs and ESCs, which are used as standards of pluripotent cells and allow us to provide here an in-depth analysis of transcriptional profiles of human and mouse cells before and after reprogramming. When compared to ESCs, iPSCs, as expected, share a common pluripotency/self-renewal network. Perhaps more importantly, they also show differences in the expression of some genes. We concentrated our efforts on the study of bivalent domain-containing genes (in ESCs) which are not expressed in ESCs, as they are supposedly important for differentiation and should possess a poised status in pluripotent cells, i.e. be ready to but not yet be expressed. We studied each iPSC line separately to estimate the quality of the reprogramming and saw a correlation of the lowest number of such genes expressed in each respective iPSC line with the stringency of the pluripotency test achieved by the line. We propose that the study of expression of bivalent domain-containing genes, which are normally silenced in ESCs, gives a valuable indication of the quality of the iPSC line, and could be used to select the best iPSC lines out of a large number of lines generated in each reprogramming experiment

Systematic Collaborative Reanalysis of Genomic Data Improves Diagnostic Yield in Neurologic Rare Diseases

Altres ajuts: Generalitat de Catalunya, Departament de Salut; Generalitat de Catalunya, Departament d'Empresa i Coneixement i CERCA Program; Ministerio de Ciencia e Innovación; Instituto Nacional de Bioinformática; ELIXIR Implementation Studies (CNAG-CRG); Centro de Investigaciones Biomédicas en Red de Enfermedades Raras; Centro de Excelencia Severo Ochoa; European Regional Development Fund (FEDER).Many patients experiencing a rare disease remain undiagnosed even after genomic testing. Reanalysis of existing genomic data has shown to increase diagnostic yield, although there are few systematic and comprehensive reanalysis efforts that enable collaborative interpretation and future reinterpretation. The Undiagnosed Rare Disease Program of Catalonia project collated previously inconclusive good quality genomic data (panels, exomes, and genomes) and standardized phenotypic profiles from 323 families (543 individuals) with a neurologic rare disease. The data were reanalyzed systematically to identify relatedness, runs of homozygosity, consanguinity, single-nucleotide variants, insertions and deletions, and copy number variants. Data were shared and collaboratively interpreted within the consortium through a customized Genome-Phenome Analysis Platform, which also enables future data reinterpretation. Reanalysis of existing genomic data provided a diagnosis for 20.7% of the patients, including 1.8% diagnosed after the generation of additional genomic data to identify a second pathogenic heterozygous variant. Diagnostic rate was significantly higher for family-based exome/genome reanalysis compared with singleton panels. Most new diagnoses were attributable to recent gene-disease associations (50.8%), additional or improved bioinformatic analysis (19.7%), and standardized phenotyping data integrated within the Undiagnosed Rare Disease Program of Catalonia Genome-Phenome Analysis Platform functionalities (18%)

Exome reanalysis and proteomic profiling identified TRIP4 as a novel cause of cerebellar hypoplasia and spinal muscular atrophy (PCH1)

Abstract: TRIP4 is one of the subunits of the transcriptional coregulator ASC-1, a ribonucleoprotein complex that participates in transcriptional coactivation and RNA processing events. Recessive variants in the TRIP4 gene have been associated with spinal muscular atrophy with bone fractures as well as a severe form of congenital muscular dystrophy. Here we present the diagnostic journey of a patient with cerebellar hypoplasia and spinal muscular atrophy (PCH1) and congenital bone fractures. Initial exome sequencing analysis revealed no candidate variants. Reanalysis of the exome data by inclusion in the Solve-RD project resulted in the identification of a homozygous stop-gain variant in the TRIP4 gene, previously reported as disease-causing. This highlights the importance of analysis reiteration and improved and updated bioinformatic pipelines. Proteomic profile of the patient’s fibroblasts showed altered RNA-processing and impaired exosome activity supporting the pathogenicity of the detected variant. In addition, we identified a novel genetic form of PCH1, further strengthening the link of this characteristic phenotype with altered RNA metabolism

A MT-TL1 variant identified by whole exome sequencing in an individual with intellectual disability, epilepsy, and spastic tetraparesis

Funder: The Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779257Abstract: The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC_012920.1:m.3291T > C; NC_012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line