Eye on a Dish Models to Evaluate Splicing Modulation.

Inherited retinal dystrophies, such as Leber congenital amaurosis, Stargardt disease, and retinitis pigmentosa, are characterized by photoreceptor dysfunction and death and currently have few treatment options. Recent technological advances in induced pluripotent stem cell (iPSC) technology and differentiation methods mean that human photoreceptors can now be studied in vitro. For example, retinal organoids provide a platform to study the development of the human retina and mechanisms of diseases in the dish, as well as being a potential source for cell transplantation. Here, we describe differentiation protocols for 3D cultures that produce retinal organoids containing photoreceptors with rudimentary outer segments. These protocols can be used as a model to understand retinal disease mechanisms and test potential therapies, including antisense oligonucleotides (AONs) to alter gene expression or RNA processing. This "retina in a dish" model is well suited for use with AONs, as the organoids recapitulate patient mutations in the correct genomic and cellular context, to test potential efficacy and examine off-target effects on the translational path to the clinic.

Proteomic analysis identifies key differences in the cardiac interactomes of dystrophin and micro-dystrophin.

ΔR4-R23/ΔCT micro-dystrophin (µDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, µDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of µDys, we compared by mass spectrometry the composition of purified dystrophin and µDys protein complexes in the mouse heart. We report that compared to dystrophin, µDys has altered associations with α1- and ß2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavin-1 and cavin-4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx5cv mice, a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx5cv mouse heart. Expression of µDys in mdx5cv mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients.

Publisher Correction: Human mid-trimester amniotic fluid (stem) cells lack expression of the pluripotency marker OCT4A.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human mid-trimester amniotic fluid (stem) cells lack expression of the pluripotency marker OCT4A.

Expression of OCT4A is one of the hallmarks of pluripotency, defined as a stem cell's ability to differentiate into all the lineages of the three germ layers. Despite being defined as non-tumorigenic cells with high translational potential, human mid-trimester amniotic fluid stem cells (hAFSCs) are often described as sharing features with embryonic stem cells, including the expression of OCT4A, which could hinder their clinical potential. To clarify the OCT4A status of hAFSCs, we first undertook a systematic review of the literature. We then performed extensive gene and protein expression analyses to discover that neither frozen, nor fresh hAFSCs cultivated in multipotent stem cell culture conditions expressed OCT4A, and that the OCT4A positive results from the literature are likely to be attributed to the expression of pseudogenes or other OCT4 variants. To address this issue, we provide a robust protocol for the assessment of OCT4A in other stem cells.

Regulation of the dystrophin-associated glycoprotein complex composition by the metabolic properties of muscle fibres.

The dystrophin-glycoprotein complex (DGC) links the muscle cytoskeleton to the extracellular matrix and is responsible for force transduction and protects the muscle fibres from contraction induced damage. Mutations in components of the DGC are responsible for muscular dystrophies and congenital myopathies. Expression of DGC components have been shown to be altered in many myopathies. In contrast we have very little evidence of whether adaptive changes in muscle impact on DGC expression. In this study we investigated connection between muscle fibre phenotype and the DGC. Our study reveals that the levels of DGC proteins at the sarcolemma differ in highly glycolytic muscle compared to wild-type and that these changes can be normalised by the super-imposition of an oxidative metabolic programme. Importantly we show that the metabolic properties of the muscle do not impact on the total amount of DGC components at the protein level. Our work shows that the metabolic property of a muscle fibre is a key factor in regulating the expression of DGC proteins at the sarcolemma.

Embryonic Stem Cell-Derived Mesenchymal Stem Cells (MSCs) Have a Superior Neuroprotective Capacity Over Fetal MSCs in the Hypoxic-Ischemic Mouse Brain.

Human mesenchymal stem cells (MSCs) have huge potential for regenerative medicine. In particular, the use of pluripotent stem cell-derived mesenchymal stem cells (PSC-MSCs) overcomes the hurdle of replicative senescence associated with the in vitro expansion of primary cells and has increased therapeutic benefits in comparison to the use of various adult sources of MSCs in a wide range of animal disease models. On the other hand, fetal MSCs exhibit faster growth kinetics and possess longer telomeres and a wider differentiation potential than adult MSCs. Here, for the first time, we compare the therapeutic potential of PSC-MSCs (ES-MSCs from embryonic stem cells) to fetal MSCs (AF-MSCs from the amniotic fluid), demonstrating that ES-MSCs have a superior neuroprotective potential over AF-MSCs in the mouse brain following hypoxia-ischemia. Further, we demonstrate that nuclear factor (NF)-κB-stimulated interleukin (IL)-13 production contributes to an increased in vitro anti-inflammatory potential of ES-MSC-conditioned medium (CM) over AF-MSC-CM, thus suggesting a potential mechanism for this observation. Moreover, we show that induced pluripotent stem cell-derived MSCs (iMSCs) exhibit many similarities to ES-MSCs, including enhanced NF-κB signaling and IL-13 production in comparison to AF-MSCs. Future studies should assess whether iMSCs also exhibit similar neuroprotective potential to ES-MSCs, thus presenting a potential strategy to overcome the ethical issues associated with the use of embryonic stem cells and providing a potential source of cells for autologous use against neonatal hypoxic-ischemic encephalopathy in humans. Stem Cells Translational Medicine 2018;7:439-449.

Link between MHC Fiber Type and Restoration of Dystrophin Expression and Key Components of the DAPC by Tricyclo-DNA-Mediated Exon Skipping.

Exon skipping mediated by tricyclo-DNA (tc-DNA) antisense oligonucleotides has been shown to induce significant levels of dystrophin restoration in mdx, a mouse model of Duchenne muscular dystrophy. This translates into significant improvement in key disease indicators in muscle, cardio-respiratory function, heart, and the CNS. Here we examine the relationship between muscle fiber type, based on myosin heavy chain (MHC) profile, and the ability of tc-DNA to restore not only dystrophin but also other members of the dystrophin-associated glycoprotein complex (DAPC). We first profiled this relationship in untreated mdx muscle, and we found that all fiber types support reversion events to a dystrophin-positive state, in an unbiased manner. Importantly, we show that only a small fraction of revertant fibers expressed other members of the DAPC. Immunoblot analysis of protein levels, however, revealed robust expression of these components, which failed to correctly localize to the sarcolemma. We then show that tc-DNA treatment leads to nearly all fibers expressing not only dystrophin but also other key components of the DAPC. Of significance, our work shows that MHC fiber type does not bias the expression of any of these important proteins. This work also highlights that the improved muscle physiology following tc-DNA treatment reported previously results from the complete restoration of the dystrophin complex in all MHCII fibers with equal efficiencies.

TGFß-induced osteogenic potential of human amniotic fluid stem cells via CD73-generated adenosine production.

The human amniotic fluid stem cell (hAFSC) population consists of two morphologically distinct subtypes, spindle-shaped and round-shaped cells (SS-hAFSCs and RS-hAFSCs). Whilst SS-hAFSCs are routinely expanded in mesenchymal-type (MT) conditions, we previously showed that they acquire broader differentiation potential when cultured under embryonic-type (ET) conditions. However, the effects of culture conditions on RS-hAFSCs have not been determined. Here, we show that culturing RS-hAFSCs under ET conditions confers faster proliferation and enhances the efficiency of osteogenic differentiation of the cells. We show that this occurs via TGFß-induced activation of CD73 and the associated increase in the generation of extracellular adenosine. Our data demonstrate that culture conditions are decisive for the expansion of hAFSCs and that TGFß present in ET conditions causes the phenotype of RS-hAFSCs to revert to an earlier state of stemness. Cultivating RS-hAFSCs in ET conditions with TGFß may therefore increase their therapeutic potential for clinical applications.

Human Amniocytes Are Receptive to Chemically Induced Reprogramming to Pluripotency.

Restoring pluripotency using chemical compounds alone would be a major step forward in developing clinical-grade pluripotent stem cells, but this has not yet been reported in human cells. We previously demonstrated that VPA_AFS cells, human amniocytes cultivated with valproic acid (VPA) acquired functional pluripotency while remaining distinct from human embryonic stem cells (hESCs), questioning the relationship between the modulation of cell fate and molecular regulation of the pluripotency network. Here, we used single-cell analysis and functional assays to reveal that VPA treatment resulted in a homogeneous population of self-renewing non-transformed cells that fulfill the hallmarks of pluripotency, i.e., a short G1 phase, a dependence on glycolytic metabolism, expression of epigenetic modifications on histones 3 and 4, and reactivation of endogenous OCT4 and downstream targets at a lower level than that observed in hESCs. Mechanistic insights into the process of VPA-induced reprogramming revealed that it was dependent on OCT4 promoter activation, which was achieved independently of the PI3K (phosphatidylinositol 3-kinase)/AKT/mTOR (mammalian target of rapamycin) pathway or GSK3ß inhibition but was concomitant with the presence of acetylated histones H3K9 and H3K56, which promote pluripotency. Our data identify, for the first time, the pluripotent transcriptional and molecular signature and metabolic status of human chemically induced pluripotent stem cells.

Counteracting bone fragility with human amniotic mesenchymal stem cells.

The impaired maturation of bone-forming osteoblasts results in reduced bone formation and subsequent bone weakening, which leads to a number of conditions such as osteogenesis imperfecta (OI). Transplantation of human fetal mesenchymal stem cells has been proposed as skeletal anabolic therapy to enhance bone formation, but the mechanisms underlying the contribution of the donor cells to bone health are poorly understood and require further elucidation. Here, we show that intraperitoneal injection of human amniotic mesenchymal stem cells (AFSCs) into a mouse model of OI (oim mice) reduced fracture susceptibility, increased bone strength, improved bone quality and micro-architecture, normalised bone remodelling and reduced TNFα and TGFß sigalling. Donor cells engrafted into bones and differentiated into osteoblasts but importantly, also promoted endogenous osteogenesis and the maturation of resident osteoblasts. Together, these findings identify AFSC transplantation as a countermeasure to bone fragility. These data have wider implications for bone health and fracture reduction.

Human Chorionic Stem Cells: Podocyte Differentiation and Potential for the Treatment of Alport Syndrome.

Alport syndrome (AS) is a hereditary glomerulopathy caused by a mutation in type IV collagen genes, which disrupts glomerular basement membrane, leading to progressive glomerulosclerosis and end-stage renal failure. There is at present no cure for AS, and cell-based therapies offer promise to improve renal function. In this study, we found that human first trimester fetal chorionic stem cells (CSC) are able to migrate to glomeruli and differentiate down the podocyte lineage in vitro and in vivo. When transplanted into 7-week-old Alport 129Sv-Col4α3(tm1Dec)/J (-/-) mice, a single intraperitoneal injection of CSC significantly lowered blood urea and urine proteinuria levels over the ensuing 2 weeks. In addition, nearly two-thirds of transplanted -/- mice maintained their weight above the 80% welfare threshold, with both males and females weighing more than age-matched nontransplanted -/- mice. This was associated with less renal cortical fibrosis and interstitial inflammation compared to nontransplanted mice as shown by reduction in murine CD4, CD68, and CD45.2 cells. Transplanted CSC homed to glomeruli, where they expressed CR1, VEGFA, SYNAPTOPODIN, CD2AP, and PODOCIN at the RNA level and produced PODOCIN, CD2AP, and COLIVα3 proteins in nontransplanted -/- mice, indicating that CSC have adopted a podocyte phenotype. Together, these data indicate that CSC may be used to delay progression of renal pathology by a combination of anti-inflammatory effects and replacement of the defective resident podocytes.