The rapid advancement of genome editing technologies has opened up new possibilities in the field of medicine. Nuclease-based techniques such as the CRISPR/Cas9 system are now used to target ...genetically linked disorders that were previously hard-to-treat. The CRISPR/Cas9 gene editing approach wields several advantages over its contemporary editing systems, notably in the ease of component design, implementation and the option of multiplex genome editing. While results from the early phase clinical trials have been encouraging, the small patient population recruited into these trials hinders a conclusive assessment on the safety aspects of the CRISPR/Cas9 therapy. Potential safety concerns include the lack of fidelity in the CRISPR/Cas9 system which may lead to unintended DNA modifications at non-targeted gene loci. This review focuses modifications to the CRISPR/Cas9 components that can mitigate off-target effects in in vitro and preclinical models and its translatability to gene therapy in patient populations.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The rising interest in human induced pluripotent stem cell (hiPSC)-derived organoid culture has stemmed from the manipulation of various combinations of directed multi-lineage differentiation and ...morphogenetic processes that mimic organogenesis. Organoids are three-dimensional (3D) structures that are comprised of multiple cell types, self-organized to recapitulate embryonic and tissue development in vitro. This model has been shown to be superior to conventional two-dimensional (2D) cell culture methods in mirroring functionality, architecture, and geometric features of tissues seen in vivo. This review serves to highlight recent advances in the 3D organoid technology for use in modeling complex hereditary diseases, cancer, host-microbe interactions, and possible use in translational and personalized medicine where organoid cultures were used to uncover diagnostic biomarkers for early disease detection via high throughput pharmaceutical screening. In addition, this review also aims to discuss the advantages and shortcomings of utilizing organoids in disease modeling. In summary, studying human diseases using hiPSC-derived organoids may better illustrate the processes involved due to similarities in the architecture and microenvironment present in an organoid, which also allows drug responses to be properly recapitulated in vitro.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The skin is made up of a plethora of cells arranged in multiple layers with complex and intricate vascular networks, creating a dynamic microenvironment of cells-to-matrix interactions. With limited ...donor sites, engineered skin substitute has been in high demand for many therapeutic purposes. Over the years, remarkable progress has occurred in the skin tissue-engineering field to develop skin grafts highly similar to native tissue. However, the major hurdle to successful engraftment is the incorporation of functional vasculature to provide essential nutrients and oxygen supply to the embedded cells. Limitations of traditional tissue engineering have driven the rapid development of vascularized skin tissue production, leading to new technologies such as 3D bioprinting, nano-fabrication and micro-patterning using hydrogel based-scaffold. In particular, the key hope to bioprinting would be the generation of interconnected functional vessels, coupled with the addition of specific cell types to mimic the biological and architectural complexity of the native skin environment. Additionally, stem cells have been gaining interest due to their highly regenerative potential and participation in wound healing. This review briefly summarizes the current cell therapies used in skin regeneration with a focus on the importance of vascularization and recent progress in 3D fabrication approaches to generate vascularized network in the skin tissue graft.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Motor neurons (MNs) are highly energetic cells and recent studies suggest that altered energy metabolism precede MN loss in amyotrophic lateral sclerosis (ALS), an age-onset neurodegenerative ...disease. However, clear mechanistic insights linking altered metabolism and MN death are still missing. In this study, induced pluripotent stem cells from healthy controls, familial ALS, and sporadic ALS patients were differentiated toward spinal MNs, cortical neurons, and cardiomyocytes. Metabolic flux analyses reveal an MN-specific deficiency in mitochondrial respiration in ALS. Intriguingly, all forms of familial and sporadic ALS MNs tested in our study exhibited similar defective metabolic profiles, which were attributed to hyper-acetylation of mitochondrial proteins. In the mitochondria, Sirtuin-3 (SIRT3) functions as a mitochondrial deacetylase to maintain mitochondrial function and integrity. We found that activating SIRT3 using nicotinamide or a small molecule activator reversed the defective metabolic profiles in all our ALS MNs, as well as correct a constellation of ALS-associated phenotypes.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The versatility of pluripotent stem cells, attributable to their unlimited self-renewal capacity and plasticity, has sparked a considerable interest for potential application in regenerative ...medicine. Over the past decade, the concept of replenishing the lost cardiomyocytes, the crux of the matter in ischemic heart disease, with pluripotent stem cell-derived cardiomyocytes (PSC-CM) has been validated with promising pre-clinical results. Nevertheless, clinical translation was hemmed in by limitations such as immature cardiac properties, long-term engraftment, graft-associated arrhythmias, immunogenicity, and risk of tumorigenicity. The continuous progress of stem cell-based cardiac therapy, incorporated with tissue engineering strategies and delivery of cardio-protective exosomes, provides an optimistic outlook on the development of curative treatment for heart failure. This review provides an overview and current status of stem cell-based therapy for heart regeneration, with particular focus on the use of PSC-CM. In addition, we also highlight the associated challenges in clinical application and discuss the potential strategies in developing successful cardiac-regenerative therapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Spinal Muscular Atrophy (SMA) is caused by genetic mutations in the SMN1 gene, resulting in drastically reduced levels of Survival of Motor Neuron (SMN) protein. Although SMN is ubiquitously ...expressed, spinal motor neurons are one of the most affected cell types. Previous studies have identified pathways uniquely activated in SMA motor neurons, including a hyperactivated ER stress pathway, neuronal hyperexcitability, and defective spliceosomes. To investigate why motor neurons are more affected than other neural types, we developed a spinal organoid model of SMA. We demonstrate overt motor neuron degeneration in SMA spinal organoids, and this degeneration can be prevented using a small molecule inhibitor of CDK4/6, indicating that spinal organoids are an ideal platform for therapeutic discovery.
Mutations in mitochondrial DNA (mtDNA), typically maternally inherited, can result in severe neurological conditions. There is currently no cure for mitochondrial DNA diseases and treatments focus on ...management of the symptoms rather than correcting the defects downstream of the mtDNA mutation. Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is one such mitochondrial disease that affects many bodily systems, particularly the central nervous system and skeletal muscles. Given the motor deficits seen in MELAS patients, we investigate the contribution of motor neuron pathology to MELAS. Using a spinal cord organoid system derived from induced pluripotent stem cells of a MELAS patient, as well as its isogenically corrected control, we found that high levels of Notch signaling underlie neurogenesis delays and neurite outgrowth defects that are associated with MELAS neural cultures. Furthermore, we demonstrate that the gamma-secretase inhibitor DAPT can reverse these neurodevelopmental defects.
Identification of the factors critical to the tumor-initiating cell (TIC) state may open new avenues in cancer therapy. Here we show that the metabolic enzyme glycine decarboxylase (GLDC) is critical ...for TICs in non-small cell lung cancer (NSCLC). TICs from primary NSCLC tumors express high levels of the oncogenic stem cell factor LIN28B and GLDC, which are both required for TIC growth and tumorigenesis. Overexpression of GLDC and other glycine/serine enzymes, but not catalytically inactive GLDC, promotes cellular transformation and tumorigenesis. We found that GLDC induces dramatic changes in glycolysis and glycine/serine metabolism, leading to changes in pyrimidine metabolism to regulate cancer cell proliferation. In the clinic, aberrant activation of GLDC correlates with poorer survival in lung cancer patients, and aberrant GLDC expression is observed in multiple cancer types. This link between glycine metabolism and tumorigenesis may provide novel targets for advancing anticancer therapy.
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► Non-small cell lung cancer (NSCLC) tumor-initiating cells express high levels of glycine decarboxylase (GLDC) ► GLDC is an oncogene that promotes cellular transformation ► GLDC activity regulates pyrimidine metabolism in cancer cells ► GLDC expression predicts mortality in NSCLC patients
An enzyme involved in glycine metabolism acts as an oncogene to drive cancer stem cell proliferation and tumorigenesis in non-small cell lung cancer. Aberrant expression of this enzyme occurs in many cancers and correlates with mortality in lung cancer.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Development of the complex human heart is tightly regulated at multiple levels, maintaining multipotency and proliferative state in the embryonic cardiovascular progenitors and thereafter suppressing ...progenitor characteristics to allow for terminal differentiation and maturation. Small regulatory microRNAs (miRNAs) are at the level of post-transcriptional gene suppressors, which enhance the degradation or decay of their target protein-coding mRNAs. These miRNAs are known to play roles in a large number of biological events, cardiovascular development being no exception. A number of critical cardiac-specific miRNAs have been identified, of which structural developmental defects have been linked to dysregulation of miRNAs in the proliferating cardiac stem cells. These miRNAs present in the stem cell niche are lost when the cardiac progenitors terminally differentiate, resulting in the postnatal mitotic arrest of the heart. Therapeutic applications of these miRNAs extend to the realm of heart failure, whereby the death of heart cells in the ageing heart cannot be replaced due to the arrest of cell division. By utilizing miRNA therapy to control cell cycling, the regenerative potential of matured myocardium can be restored. This review will address the various cardiac progenitor-related miRNAs that control the development and proliferative potential of the heart.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. Because this gene is expressed ubiquitously, it remains poorly understood why motor neurons (MNs) are one of the most affected ...cell types. To address this question, we carried out RNA sequencing studies using fixed, antibody-labeled, and purified MNs produced from control and SMA patient-derived induced pluripotent stem cells (iPSCs). We found SMA-specific changes in MNs, including hyper-activation of the ER stress pathway. Functional studies demonstrated that inhibition of ER stress improves MN survival in vitro even in MNs expressing low SMN. In SMA mice, systemic delivery of an ER stress inhibitor that crosses the blood-brain barrier led to the preservation of spinal cord MNs. Therefore, our study implies that selective activation of ER stress underlies MN death in SMA. Moreover, the approach we have taken would be broadly applicable to the study of disease-prone human cells in heterogeneous cultures.
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•Fixed and antibody-labeled motor neurons (MNs) are purified efficiently for RNA-seq•Analysis of purified MNs, not mixed cultures, reveals elevated ER stress in SMA•Abnormal splicing of DNAJC10 in SMA MNs is responsible for increased ER stress•Pharmacological and genetic intervention of the ER stress pathway prevents SMA MN death
Loss of the protein SMN causes spinal muscular atrophy, but it is unclear why deficiency in the ubiquitously expressed protein primarily affects motor neurons. In this study, Ng et al. show that a hyperactive ER stress pathway in SMA motor neurons could explain this selective neuronal vulnerability.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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