Prospects for clinical use of reprogrammed cells for autologous treatment of macular degeneration

Since the discovery of induced pluripotent stem cells (iPSC) in 2006, the symptoms of many human diseases have been reversed in animal models with iPSC therapy, setting the stage for future clinical development. From the animal data it is clear that iPSC are rapidly becoming the lead cell type for cell replacement therapy and for the newly developing field of iPSC-derived body organ transplantation. The first human pathology that might be treated in the near future with iPSC is age-related macular degeneration (AMD), which has recently passed the criteria set down by regulators for phase I clinical trials with allogeneic human embryonic stem cell-derived cell transplantation in humans. Given that iPSC are currently in clinical trial in Japan (RIKEN) to treat AMD, the establishment of a set of international criteria to make clinical-grade iPSC and their differentiated progeny is the next step in order to prepare for future autologous cell therapy clinical trials. Armed with clinical-grade iPSC, we can then specifically test for their threat of cancer, for proper and efficient differentiation to the correct cell type to treat human disease and then to determine their immunogenicity. Such a rigorous approach sets a far more relevant paradigm for their intended future use than non-clinical-grade iPSC. This review focuses on the latest developments regarding the first possible use of iPSC-derived retinal pigment epithelial cells in treating human disease, covers data gathered on animal models to date and methods to make clinical-grade iPSC, suggests techniques to ensure quality control and discusses possible clinical immune responses

Wnt antagonist secreted frizzled-related protein 4 upregulates adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells

With more than 1.4 billion overweight or obese adults worldwide, obesity and progression of the metabolic syndrome are major health and economic challenges. To address mechanisms of obesity, adipose tissue-derived mesenchymal stem cells (ADSCs) are being studied to detail the molecular mechanisms involved in adipogenic differentiation. Activation of the Wnt signalling pathway has inhibited adipogenesis from precursor cells. In our study, we examined this anti-adipogenic effect in further detail stimulating Wnt with lithium chloride (LiCl) and 6-bromo indirubin 3'oxime (BIO). We also examined the effect of Wnt inhibition using secreted frizzled-related protein 4 (sFRP4), which we have previously shown to be pro-apoptotic, anti-angiogenic, and anti-tumorigenic. Wnt stimulation in LiCl and BIOtreated ADSCs resulted in a significant reduction (2.7-fold and 12-fold respectively) in lipid accumulation as measured by Oil red O staining while Wnt inhibition with sFRP4 induced a 1.5-fold increase in lipid accumulation. Furthermore, there was significant 1.2-fold increase in peroxisome proliferator-activated receptor gamma (PPAR ?) and CCAAT/enhancer binding protein alpha (C/EBPa), and 1.3-fold increase in acetyl CoA carboxylase protein levels. In contrast, the expression of adipogenic proteins (PPAR?, C/EBPa, and acetyl CoA carboxylase) were decreased significantly with LiCl (by 1.6, 2.6, and 1.9-fold respectively) and BIO (by 7, 17, and 5.6-fold respectively) treatments. These investigations demonstrate interplay between Wnt antagonism and Wnt activation during adipogenesis and indicate pathways for therapeutic intervention to control this process

A novel microsurgical rodent model for the transplantation of engineered cardiac muscle flap

Background The survival of engineered cardiac muscle ‘grafts’ to the epicardium is limited by vascularization post‐transplantation in rat models. In this article, we describe the methodology of a novel rat model that allows for the transplantation of an engineered cardiac muscle flap (ECMF) onto the epicardium. Materials and Methods A total of 40 rats were used. Twenty‐four neonatal rats were used to harvest cardiomyocytes. At week 1, ECMF were generated by seeding cardiomyocytes into the arteriovenous loop (AVL) tissue engineering chamber implanted into the right groin of adult rats (n = 8). At week 6, the ECMF were harvested based on a pedicle along the femoral‐iliac‐abdominal vessel and anastomosed to the neck vessels of the recipient syngeneic adult rats (n = 8). The flaps were delivered into the thoracic cavity and onto the epicardium. The transplanted flaps were harvested at week 10. Survival of the flaps was assessed by the patency of anastomoses and viability of the cardiomyocytes through histological analysis (hematoxylin and eosin [H&E], desmin, and von Willebrand factor [vWF] immunostaining). Results Six out of 8 rats survived the transplantation procedure. These remaining 6 recipient rats survived until harvest time point at 4 weeks post‐transplantation. The mean area of the flap was 46.7mm2. Six out of 6 flaps harvested at week 10 showed viable cardiomyocytes using desmin immunostaining and vascular channels were seen at the interface between flap and epicardium. Conclusion This is a technically feasible model that will be useful for future assessment of different cardiac stem cell implants and their functional significance in rat heart models

Yearly rates of myocardial infraction and infraction index from 1953 to 2016.

<p>(A) Number of papers retrieved for each year from a Scopus “all fields” search for “myocardial infraction”. (Accessed January 4, 2017). (B) The infraction index over the same time frame, calculated as a percentage of errors from the same search for “myocardial infarction”. (Accessed January 4, 2017).</p

Using RNA-seq to identify suitable housekeeping genes for hypoxia studies in human adipose-derived stem cells

Abstract Background Hypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of some commonly used housekeeping genes, such as GAPDH and PGK1, can confound the relative gene expression analysis. Thus, there is ongoing debate as to which housekeeping gene is appropriate for studies investigating hypoxia-induced cell responses. Specifically, there is still contradicting information for which housekeeping genes are stable in hypoxia cultures of mesenchymal stem cells. In this study, candidate housekeeping genes curated from the literature were matched to RNAseq data of normoxic and hypoxic human adipose-derived stem cell cultures to determine if gene expression was modulated by hypoxia or not. Expression levels of selected candidates were used to calculate coefficient of variation. Then, accounting for the mean coefficient of variation, and normalised log twofold change, genes were ranked and shortlisted, before validating with qRT-PCR. Housekeeping gene suitability were then determined using GeNorm, NormFinder, BestKeeper, comparative $$\Delta Ct$$ Δ C t , RefFinder, and the Livak method. Results Gene expression levels of 78 candidate genes identified in the literature were analysed in the RNAseq dataset generated from hADSC cultured under Nx and Hx conditions. From the dataset, 15 candidates with coefficient of variation ≤ 0.15 were identified, where differential expression analysis results further shortlisted 8 genes with least variation in expression levels. The top 4 housekeeping gene candidates, ALAS1, RRP1, GUSB, and POLR2B, were chosen for qRT-PCR validation. Additionally, 18S, a ribosomal RNA commonly used as housekeeping gene but not detected in the RNAseq method, was added to the list of housekeeping gene candidates to validate. From qRT-PCR results, 18S and RRP1 were determined to be stably expressed in cells cultured under hypoxic conditions. Conclusions We have demonstrated that 18S and RRP1 are suitable housekeeping genes for use in hypoxia studies with human adipose-derived stem cell and should be used in combination. Additionally, these data shown that the commonly used GAPDH and PGK1 are not suitable housekeeping genes for investigations into the effect of hypoxia in human adipose-derived stem cell

The top ten journals ranked by infraction index, based on numbers of papers returning at least one “myocardial infraction” error in a Scopus search (1953 to 2017).

<p>The top ten journals ranked by infraction index, based on numbers of papers returning at least one “myocardial infraction” error in a Scopus search (1953 to 2017).</p

Mechanical strain stimulates a mitogenic response in coronary vascular smooth muscle cells via release of basic fibroblast growth factor

Mechanical strain has been shown to induce mitogenesis in a rat neonatal vascular smooth muscle (VSM) cell line in a response mediated predominantly by transcription, expression, and release of platelet-derived growth factor (PDGF). We examined the effect of cyclic mechanical strain and growth factor production on mitogenic response in ovine coronary artery smooth muscle cells. Vascular smooth muscle cells were cultured from explants of left anterior descending (LAD) coronary arteries from young sheep. Cells for experiments were grown on wells with silicone-elastomer bottoms, and subjected to strain (60 cycles/min) using a vacuum actuated strain device. Tritiated thymidine incorporation was used as a measure of DNA synthesis. Cell membrane damage was assessed with differentially permeable nuclear staining dyes. We observed an increase in tritiated thymidine incorporation in response to strain with a temporal response identical to that observed in response to exogenous growth factors (PDGF-BB and basic fibroblast growth factor [bFGF]). Supernatant medium obtained from stretched cells induced a twofold increase in DNA synthesis in unstretched cells. The mitogenic response was abolished by monoclonal antibodies to bFGF, but not by antibodies to PDGF-AB. Studies of fluorescent dye exclusion indicated the stretching protocol caused no cell membrane damage. Thus, mechanical strain is an important stimulus for growth factor release in coronary VSM cells. The mitogenic response is mediated by release of bFGF