GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells

Pompe disease is a metabolic myopathy caused by deficiency of the acid α-glucosidase (GAA) enzyme and results in progressive wasting of skeletal muscle cells. The c.-32-13T>G (IVS1) GAA variant promotes exon 2 skipping during pre-mRNA splicing and is the most common variant for the childhood/adult disease form. We previously identified antisense oligonucleotides (AONs) that promoted GAA exon 2 inclusion in patient-derived fibroblasts. It was unknown how these AONs would affect GAA splicing in skeletal muscle cells. To test this, we expanded induced pluripotent stem cell (iPSC)-derived myogenic progenitors and differentiated these to multinucleated myotubes. AONs restored splicing in myotubes to a similar extent as in fibroblasts, suggesting that they act by modulating the action of shared splicing regulators. AONs targeted the putative polypyrimidine tract of a cryptic splice acceptor site that was part of a pseudo exon in GAA intron 1. Blocking of the cryptic splice donor of the pseudo exon with AONs likewise promoted GAA exon 2 inclusion. The simultaneous blocking of the cryptic acceptor and cryptic donor sites restored the majority of canonical splicing and alleviated GAA enzyme deficiency. These results highlight the relevance of cryptic splicing in human disease and its potential as therapeutic target for splicing modulation using AONs

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors

peer reviewedIn vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single-cell comparison to human fetal and adult myogenic progenitor /satellite cells reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH−/− iPS cell lines, we aggregated FAH−/−-iPS cells with tetraploid embryos and obtained entirely FAH−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH−/− mice. Then, we transduced FAH cDNA into the FAH−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models

Creation of a ubiquitous vector for expression oh hSOD1G93Ain pig

Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease that may occur in two forms: sporadic and familial, the latter linked to a mutation in the Cu/Zn superoxide dismutase (SOD1) gene. Rodents expressing the human mutated form of SOD1 (hSOD1G93A) fail to faithfully reproduce the biology of the disease. Moreover, results of drug tests in mice have never been translated into humans. Aim of this work is to create a novel model for ALS, closer related to man, by overexpressing the hSOD1G93A cDNA in pig. We have previously developed an ubiquitous EGFP expression vector, driven by the pCAGGS hybrid promoter (CMV-IE enhancer ? chicken beta actin promoter) that maintains high level of expression through the next generation of pigs (Brunetti et al. 2008). We created a Destination Vector (pMGMARneoP-OrfA) inserting the Conversion cassette (OrfA) of Multisite Gateway system (Invitrogen) into the ubiquitous expression vector. This vector had the pCAGGS promoter inserted between two insulators (MAR of chicken lysozyme gene) to prevent different silencing effects (positional or copy number effects). On the same structure there is a \ufb02oxed PGK-neo cassette to select the transfected clones but keeping the possibility to remove the selection cassette using the Cre recombinase. We initially inserted the hSOD1G93A cDNA into the Entry clone of Gateway system obtaining the pENTRL1L2-hSODG93A. The SalI-BamHI fragment of this construct was removed and the resulting pENTRL1L2-hSODG93AdelSB was veri\ufb01ed by sequencing before using it in a LR exchange reaction with the Destination Vector pMG MARneoP-OrfA mediated by the LR Clonase. The obtained exchange reaction was used to transform chemically competent E.coli cells (One Shot Mach1-Invitrogen) and the resulting pMGMARneoP-CXhSODG93A vector was puri\ufb01ed and analyzed by different restriction enzymes and con\ufb01rmed by sequencing. Speci\ufb01c PCR reaction and DIGlabelled probe have been developed and validated. The linearized expression vector, with the veri\ufb01ed correct sequence is currently being transfected to immortalized PK15 pig cells. We have also screened by ICC an antibody (human anti-SOD1, Millipore) and found that it does not cross react with wild type pig cells and therefore it will be used to detect and quantify the hSOD1G93A expression in combination with RT-PCR, Northern Blot, Southern Blot, Western Blot. Data will be presented on the expression levels of hSOD1G93A in PK15 cells and in \ufb01broblasts that will be subsequently destined for SCNT to create the swine model

Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors

Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1-11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring and that retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.close53353

A central role for TFIID in the pluripotent transcription circuitry

Item does not contain fulltextEmbryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states