Generation and characterization of GATA6-specific EGFP expressing human induced pluripotent stem cell line, KSCBi017-A-1, using CRISPR/Cas9.

GATA6 is expressed during early embryogenesis and localizes to endoderm- and mesoderm-derived tissues during later embryogenesis. Here, we established a human induced pluripotent stem cell (hiPSC) line expressing EGFP under GATA6 gene. EGFP coding sequence was introduced into the C-terminus of GATA6 in KSCBi017-A hiPSCs through homologous recombination using CRISPR/Cas9 system. The successfully edited line, KSCBi017-A-1, was selected and confirmed by sequencing. The line had a normal karyotype and exhibited potential to differentiate into three germ layers while it expressed EGFP upon endoderm induction. KSCBi017-A-1 cells can be used to monitor the expression of GATA6 during differentiation. This cell line is available from Korea National Stem Cell Bank.

A Study on the Protective Effect of sRAGE-MSCs in a Rodent Reperfusion Model of Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the major leading causes of death in humans globally. Recently, increased levels of recruited macrophages and AGE-albumin were observed in the hearts of humans and animals with acute myocardial infarction. Thus, the purposes of this study were to investigate whether the elevated levels of AGE-albumin from activated macrophage cells are implicated in ischemia-induced cardiomyocyte death and to develop therapeutic strategies for AMI based on its underlying molecular mechanisms with respect to AGEs. The present study demonstrated that activated macrophages and AGE-albumin were observed in heart tissues obtained from humans and rats with AMI incidences. In the cellular model of AMI, it was found that increased expression of AGE-albumin was shown to be co-localized with macrophages, and the presence of AGE-albumin led to increased expression of RAGE through the mitogen-activated protein kinase pathway. After revealing cardiomyocyte apoptosis induced by toxicity of the AGE-RAGE system, sRAGE-secreting MSCs were generated using the CRISPR/Cas9 platform to investigate the therapeutic effects of sRAGE-MSCs in an AMI rat model. Gene-edited sRAGE-MSCs showed greater therapeutic effects against AMI pathogenesis in rat models compared to mock MSCs, and promising results of the functional improvement of stem cells could result in significant improvements in the clinical management of cardiovascular diseases.

BMP4 signaling plays critical roles in self-renewal of R2i mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state under R2i culture conditions that inhibit the TGF-ß and ERK signaling pathways. BMP4 is another member of the TGF-ß family that plays a crucial role in maintaining the pluripotency state of mESCs. It has been reported that inhibition of BMP4 caused the death of R2i-grown cells. In this study, we used the loss-of-function approach to investigate the role of BMP4 signaling in mESC self-renewal. Inhibition of this pathway with Noggin and dorsomorphin, two bone morphogenetic protein (BMP) antagonists, elicited a quick death of the R2i-grown cells. We showed that the canonical pathway of BMP4 (BMP/SMAD) was dispensable for self-renewal and maintaining pluripotency of these cells. Transcriptome analysis of the BMPi-treated cells revealed that the p53 signaling and two adhesion (AD) and apoptotic mitochondrial change (MT) pathways could be involved in the cell death of the BMPi-treated cells. According to our results, inhibition of BMP4 signaling caused a decrease in cell adhesion and ECM detachment, which triggered anoikis in the R2i-grown cells. Altogether, these findings demonstrate that endogenous BMP signaling is required for the survival of mESCs under the R2i condition.

Targeting Scavenger Receptors in Inflammatory Disorders and Oxidative Stress.

Oxidative stress and inflammation cannot be considered as diseases themselves; however, they are major risk factors for the development and progression of the pathogenesis underlying many illnesses, such as cancer, neurological disorders (including Alzheimer's disease and Parkinson's disease), autoimmune and metabolic disorders, etc. According to the results obtained from extensive studies, oxidative stress-induced biomolecules, such as advanced oxidation protein products, advanced glycation end products, and advanced lipoxidation end products, are critical for an accelerated level of inflammation and oxidative stress-induced cellular damage, as reflected in their strong affinity to a wide range of scavenger receptors. Based on the limitations of antioxidative and anti-inflammatory molecules in practical applications, targeting such interactions between harmful molecules and their cellular receptors/signaling with advances in gene engineering technology, such as CRISPR or TALEN, may prove to be a safe and effective alternative. In this review, we summarize the findings of recent studies focused on the deletion of scavenger receptors under oxidative stress as a development in the therapeutic approaches against the diseases linked to inflammation and the contribution of advanced glycation end products (AGEs), advanced lipid peroxidation products (ALEs), and advanced oxidation protein products (AOPPs).

Brain-Derived Neurotrophic Factor Secreting Human Mesenchymal Stem Cells Improve Outcomes in Rett Syndrome Mouse Models.

Rett syndrome (RTT) is a severe X-linked dominant neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene; MeCP2 regulates the expression of brain-derived neurotrophic factor (BDNF) and increasing BDNF levels ameliorates RTT symptoms. However, the clinical application of BDNF is limited, because of its short half-life and low penetrance across the blood-brain barrier. In this study, we generated BDNF-secreting mesenchymal stem cells (MSCs) from the human umbilical cord cells, using CRISPR-Cas9. We studied the effects of BDNF-MSCs in MECP2 knockout and MECP2-deficient mice. BDNF-MSCs upregulated the expression of BDNF, pAKT, and pERK1/2 and downregulated that of pp38, both in vitro and in vivo. In our in vivo experiments, BDNF-MSCs increased the body and brain weights in mice. BDNF-MSCs increased the neuronal cell numbers in the hippocampus, cortex, and striatum; in addition, they increased the number of synapses. BDNF-MSCs upregulated BDNF and the activity of BDNF downstream effectors, such as pAKT and pERK 1/2; this upregulation was persistent. In conclusion, BDNF-MSCs generated using CRISPR-Cas9 could be a therapeutic strategy for treating RTT.

Recent advances in stem cells and gene editing: Drug discovery and therapeutics.

The recently introduced genome editing technology has had a remarkable impact on genetic medicine. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas nucleases are the three major platforms used for priming of stem cells or correction of mutated genes. Among these nucleases, CRISPR/Cas is the most easily applicable. Various CRISPR/Cas variants such as base editors, prime editors, mad7 nucleases, RESCUE, REPAIR, digenome sequencing, and SHERLOCK are being developed and considered as a promising tool for gene therapy and drug discovery. These advances in the CRISPR/Cas platform have enabled the correction of gene mutations from DNA to RNA level and validation of the safety of genome editing performance at a very precise level by allowing the detection of one base-pair mismatch. These promising alternatives of the CRISPR/Cas system can benefit millions of patients with intractable diseases. Although the therapeutic effects of stem cells have been confirmed in a wide range of disease models, their safety still remains an issue. Hence, scientists are concentrating on generating functionally improved stem cells by using programmable nucleases such as CRISPR. Therefore, in this chapter, we have summarized the applicable options of the CRISPR/Cas platforms by weighing their advantages and limitations in drug discovery and gene therapy.

CUPRAC-Reactive Advanced Glycation End Products as Prognostic Markers of Human Acute Myocardial Infarction.

Cardiovascular disorders, especially acute coronary syndromes, are among the leading causes of mortality worldwide, and advanced glycation end products (AGEs) are associated with cardiovascular disease and serve as biomarkers for diagnosis and prediction. In this study, we investigated the utility of AGEs as prognostic biomarkers for acute myocardial infarction (AMI). We measured AGEs in serum samples of AMI patients (N = 27) using the cupric ion reducing antioxidant capacity (CUPRAC) method on days 0, 2, 14, 30, and 90 after AMI, and the correlation of serum AGE concentration and post-AMI duration was determined using Spearman's correlation analysis. Compared to total serum protein, the level of CUPRAC reactive AGEs was increased from 0.9 to 2.1 times between 0-90 days after AMI incident. Furthermore, the glycation pattern and Spearman's correlation analysis revealed four dominant patterns of AGE concentration changes in AMI patients: stable AGE levels (straight line with no peak), continuous increase, single peak pattern, and multimodal pattern (two or more peaks). In conclusion, CUPRAC-reactive AGEs can be developed as a potential prognostic biomarker for AMI through long-term clinical studies.

Recent advances in genome editing of stem cells for drug discovery and therapeutic application.

Genome engineering technologies right from viral vector-mediated to protein-based editing- which include zinc finger nucleases, TALENs, and CRISPR/Cas systems-have been improved significantly. These technologies have facilitated drug discovery and have resulted in the development of potential curative therapies for many intractable diseases. They can efficiently correct genetic errors; however, these technologies have limitations, such as off-target effects and possible safety issues, which need to be considered when employing these techniques in humans. Significant efforts have been made to overcome these limitations and to accelerate the clinical implementation of these technologies. In this review, we focus on the recent technological advancements in genome engineering and their applications in stem cells to enable efficient discovery of drugs and treatment of intractable diseases.

CRISPR/Cas9 Edited sRAGE-MSCs Protect Neuronal Death in Parkinson’s Disease Model.

BACKGROUND AND OBJECTIVES: Parkinson’s disease (PD) is a fatal and progressive degenerative disease of the nervous system. Until recently, its promising treatment and underlying mechanisms for neuronal death are poorly understood. This study was investigated to identify the molecular mechanism of neuronal death in the substantia nigra and corpus striatum of PD. METHODS: The soluble RAGE (sRAGE) secreting Umbilical Cord Blood-derived Mesenchymal Stem Cell (UCB-MSC) was generated by gene editing method using clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9). These cells were transplanted into Corpus Striatum of rotenone-induced PD animal models then behavioral test, morphological analysis, and immunohistochemical experiments were performed to determine the neuronal cell death and recovery of movement. RESULTS: The neuronal cell death in Corpus Striatum and Substantia Nigra was dramatically reduced and the movement was improved after sRAGE secreting UCB-MSC treatment in PD mice by inhibition of RAGE in neuronal cells. CONCLUSIONS: We suggest that sRAGE secreting UCB-MSC based therapeutic approach could be a potential treatment strategy for neurodegenerative disease including PD.

MicroRNA-340 inhibits the proliferation and promotes the apoptosis of colon cancer cells by modulating REV3L.

DNA Directed Polymerase Zeta Catalytic Subunit (REV3L) has recently emerged as an important oncogene. Although the expressions of REV3L are similar in normal and cancer cells, several mutations in REV3L have been shown to play important roles in cancer. These mutations cause proteins misfolding and mislocalization, which in turn alters their interactions and biological functions. miRNAs play important regulatory roles during the progression and metastasis of several human cancers. This study was undertaken to determine how changes in the location and interactions of REV3L regulate colon cancer progression. REV3L protein mislocalization confirmed from the immunostaining results and the known interactions of REV3L was found to be broken as seen from the PLA assay results. The mislocalized REV3L might interact with new proteins partners in the cytoplasm which in turn may play role in regulating colon cancer progression. hsa-miR-340 (miR-340), a microRNA down-regulated in colon cancer, was used to bind to and downregulate REV3L, and found to control the proliferation and induce the apoptosis of colon cancer cells (HCT-116 and DLD-1) via the MAPK pathway. Furthermore, this down-regulation of REV3L also diminished colon cancer cell migration, and down-regulated MMP-2 and MMP-9. Combined treatment of colon cancer cells with miR-340 and 5-FU enhanced the inhibitory effects of 5-FU. In addition, in vivo experiments conducted on nude mice revealed tumor sizes were smaller in a HCT-116-miR-340 injected group than in a HCT-116-pCMV injected group. Our findings suggest mutations in REV3L causes protein mislocalization to the cytoplasm, breaking its interaction and is believed to form new protein interactions in cytoplasm contributing to colon cancer progression. Accordingly, microRNA-340 appears to be a good candidate for colon cancer therapy.

Advanced glycation end-product (AGE)-albumin from activated macrophage is critical in human mesenchymal stem cells survival and post-ischemic reperfusion injury.

Post-ischemic reperfusion injury (PIRI) triggers an intense inflammatory response which is essential for repair but is also implicated in pathogenesis of post-ischemic remodeling in several organs in human. Stem cell therapy has recently emerged as a promising method for treatment of PIRI in human. However, satisfactory results have not been reported due to severe loss of injected stem cells in PIRI including critical limb ischemia (CLI). For investigating the advanced glycation end-product-albumin (AGE-albumin) from activated macrophages is critical in both muscle cell and stem cell death, we evaluated the recovery of PIRI-CLI by injection of human bone marrow derived mesenchymal stem cells (hBD-MSCs) with or without soluble receptor for AGEs (sRAGE). Our results showed that activated M1 macrophages synthesize and secrete AGE-albumin, which induced the skeletal muscle cell death and injected hBD-MSCs in PIRI-CLI through RAGE increase. Combined injection of sRAGE and hBD-MSCs resulted in enhanced survival of hBD-MSCs and angiogenesis in PIRI-CLI mice. Taken together, AGE-albumin from activated macrophages is critical for both skeletal muscle cell and hBD-MSCs death in PIRI-CLI. Therefore, the inhibition of AGE-albumin from activated macrophages could be a successful therapeutic strategy for treatment of PIRI including CLI with or without stem cell therapy.

Microglial AGE-albumin is critical for neuronal death in Parkinson's disease: a possible implication for theranostics.

Advanced glycation end products (AGEs) are known to play an important role in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD), by inducing protein aggregation and cross-link, formation of Lewy body, and neuronal death. In this study, we observed that AGE-albumin, the most abundant AGE product in the human PD brain, is synthesized in activated microglial cells and accumulates in the extracellular space. AGE-albumin synthesis in human-activated microglial cells is distinctly inhibited by ascorbic acid and cytochalasin treatment. Accumulated AGE-albumin upregulates the receptor to AGE, leading to apoptosis of human primary dopamine (DA) neurons. In animal experiments, we observed reduced DA neuronal cell death by treatment with soluble receptor to AGE. Our study provides evidence that activated microglial cells are one of the main contributors in AGE-albumin accumulation, deleterious to DA neurons in human and animal PD brains. Finally, activated microglial AGE-albumin could be used as a diagnostic and therapeutic biomarker with high sensitivity for neurodegenerative disorders, including PD.

Amyloid-beta-activated human microglial cells through ER-resident proteins.

Microglial activation in the central nervous system is a key event in the neuroinflammation that accompanies neurodegenerative diseases such as Alzheimer's disease (AD). Among cytokines involved in microglial activation, amyloid ß (Aß) peptide is known to be a key molecule in the induction of diverse inflammatory products, which may lead to chronic inflammation in AD. However, proteomic studies of microglia in AD are limited due to lack of proper cell or animal model systems. In this study, we performed a proteomic analysis of Aß-stimulated human microglial cells using SILAC (stable isotope labeling with amino acids in cell culture) combined with LC-MS/MS. Results showed that 60 proteins increased or decreased their abundance by 1.5 fold or greater. Among these, ER-resident proteins such as SERPINH1, PDIA6, PDIA3, and PPIB were revealed to be key molecular biomarkers of human microglial activation by validation of the proteomic results by immunostaining, PCR, ELISA, and Western blot. Taken together, our data suggest that ER proteins play an essential role in human microglial activation by Aß and may be important molecular therapeutic targets for treatment of AD.

Microglial AGE-albumin is critical in promoting alcohol-induced neurodegeneration in rats and humans.

Alcohol is a neurotoxic agent, since long-term heavy ingestion of alcohol can cause various neural diseases including fetal alcohol syndrome, cerebellar degeneracy and alcoholic dementia. However, the molecular mechanisms of alcohol-induced neurotoxicity are still poorly understood despite numerous studies. Thus, we hypothesized that activated microglial cells with elevated AGE-albumin levels play an important role in promoting alcohol-induced neurodegeneration. Our results revealed that microglial activation and neuronal damage were found in the hippocampus and entorhinal cortex following alcohol treatment in a rat model. Increased AGE-albumin synthesis and secretion were also observed in activated microglial cells after alcohol exposure. The expressed levels of receptor for AGE (RAGE)-positive neurons and RAGE-dependent neuronal death were markedly elevated by AGE-albumin through the mitogen activated protein kinase pathway. Treatment with soluble RAGE or AGE inhibitors significantly diminished neuronal damage in the animal model. Furthermore, the levels of activated microglial cells, AGE-albumin and neuronal loss were significantly elevated in human brains from alcoholic indivisuals compared to normal controls. Taken together, our data suggest that increased AGE-albumin from activated microglial cells induces neuronal death, and that efficient regulation of its synthesis and secretion is a therapeutic target for preventing alcohol-induced neurodegeneration.