Suppressing Pyroptosis Augments Post-Transplant Survival of Stem Cells and Cardiac Function Following Ischemic Injury.

The acute demise of stem cells following transplantation significantly compromises the efficacy of stem cell-based cell therapeutics for infarcted hearts. As the stem cells transplanted into the damaged heart are readily exposed to the hostile environment, it can be assumed that the acute death of the transplanted stem cells is also inflicted by the same environmental cues that caused massive death of the host cardiac cells. Pyroptosis, a highly inflammatory form of programmed cell death, has been added to the list of important cell death mechanisms in the damaged heart. However, unlike the well-established cell death mechanisms such as necrosis or apoptosis, the exact role and significance of pyroptosis in the acute death of transplanted stem cells have not been explored in depth. In the present study, we found that M1 macrophages mediate the pyroptosis in the ischemia/reperfusion (I/R) injured hearts and identified miRNA-762 as an important regulator of interleukin 1ß production and subsequent pyroptosis. Delivery of exogenous miRNA-762 prior to transplantation significantly increased the post-transplant survival of stem cells and also significantly ameliorated cardiac fibrosis and heart functions following I/R injury. Our data strongly suggest that suppressing pyroptosis can be an effective adjuvant strategy to enhance the efficacy of stem cell-based therapeutics for diseased hearts.

Single-Cell RNA Sequencing of Human Pluripotent Stem Cell-Derived Macrophages for Quality Control of The Cell Therapy Product.

Macrophages exhibit high plasticity to achieve their roles in maintaining tissue homeostasis, innate immunity, tissue repair and regeneration. Therefore, macrophages are being evaluated for cell-based therapeutics against inflammatory disorders and cancer. To overcome the limitation related to expansion of primary macrophages and cell numbers, human pluripotent stem cell (hPSC)-derived macrophages are considered as an alternative source of primary macrophages for clinical application. However, the quality of hPSC-derived macrophages with respect to the biological homogeneity remains still unclear. We previously reported a technique to produce hPSC-derived macrophages referred to as iMACs, which is amenable for scale-up. In this study, we have evaluated the biological homogeneity of the iMACs using a transcriptome dataset of 6,230 iMACs obtained by single-cell RNA sequencing. The dataset provides a valuable genomic profile for understanding the molecular characteristics of hPSC-derived macrophage cells and provide a measurement of transcriptomic homogeneity. Our study highlights the usefulness of single cell RNA-seq data in quality control of the cell-based therapy products.

Modelling Toxoplasma gondii infection in human cerebral organoids.

Pluripotent stem cell-derived cerebral organoids have the potential to recapitulate the pathophysiology of in vivo human brain tissue, constituting a valuable resource for modelling brain disorders, including infectious diseases. Toxoplasma gondii, an intracellular protozoan parasite, infects most warm-blooded animals, including humans, causing toxoplasmosis. In immunodeficient patients and pregnant women, infection often results in severe central nervous system disease and fetal miscarriage. However, understanding the molecular pathophysiology of the disease has been challenging due to limited in vitro model systems. Here, we developed a new in vitro model system of T. gondii infection using human brain organoids. We observed that tachyzoites can infect human cerebral organoids and are transformed to bradyzoites and replicate in parasitophorous vacuoles to form cysts, indicating that the T. gondii asexual life cycle is efficiently simulated in the brain organoids. Transcriptomic analysis of T. gondii-infected organoids revealed the activation of the type I interferon immune response against infection. In addition, in brain organoids, T. gondii exhibited a changed transcriptome related to protozoan invasion and replication. This study shows cerebral organoids as physiologically relevant in vitro model systems useful for advancing the understanding of T. gondii infections and host interactions.

Differentiation of adipose-derived stem cells into functional chondrocytes by a small molecule that induces Sox9.

Osteoarthritis (OA) is a common joint disease that results from the disintegration of joint cartilage and the underlying bone. Because cartilage and chondrocytes lack the ability to self-regenerate, efforts have been made to utilize stem cells to treat OA. Although various methods have been used to differentiate stem cells into functional chondrocytes, the currently available methods cannot induce stem cells to undergo differentiation into chondrocyte-like cells without inducing characteristics of hypertrophic chondrocytes, which finally lead to cartilage disintegration and calcification. Therefore, an optimized method to differentiate stem cells into chondrocytes that do not display undesired phenotypes is needed. This study focused on differentiating adipose-derived stem cells (ASCs) into functional chondrocytes using a small molecule that regulated the expression of Sox9 as a key factor in cartilage development and then explored its ability to treat OA. We selected ellipticine (ELPC), which induces chondrocyte differentiation of ASCs, using a GFP-Sox9 promoter vector screening system. An in vivo study was performed to confirm the recovery rate of cartilage regeneration with ASC differentiation into chondrocytes by ELPC in a collagenase-induced animal model of OA. Taken together, these data indicate that ellipticine induces ASCs to differentiate into mature chondrocytes without hypertrophic chondrocytes in vitro and in vivo, thus overcoming a problem encountered in previous studies. These results indicate that ELPC is a novel chondrocyte differentiation-inducing drug that shows potential as a cell therapy for OA.

Generation of a TLR7 heterozygous knockout line (WAe009-A-18) from human embryonic stem cells using CRISPR/Cas9.

Toll-like receptor 7 (TLR7) is a member of the toll-like receptor (TLR) family that is essential in the innate immune system. In this study, we established a heterozygous TLR7 knockout H9 cell line using CRISPR/Cas9. TLR7 knockout H9 cells maintained their pluripotency and exhibited the ability to differentiate into the three germ layers without any karyotype abnormalities.

Drug Discovery Platform Targeting M. tuberculosis with Human Embryonic Stem Cell-Derived Macrophages.

A major limitation in anti-tuberculosis drug screening is the lack of reliable and scalable models for homogeneous human primary macrophage cells of non-cancer origin. Here we report a modified protocol for generating homogeneous populations of macrophage-like cells from human embryonic stem cells. The induced macrophages, referred to as iMACs, presented similar transcriptomic profiles and characteristic immunological features of classical macrophages and were permissive to viral and bacterial infection, in particular Mycobacterium tuberculosis (Mtb). More importantly, iMAC production was amenable to scale up. To evaluate iMAC efficiency in high-throughput anti-tuberculosis drug screening, we performed a phenotypic screening against intracellular Mtb, involving a library of 3,716 compounds that included FDA-approved drugs and other bioactive compounds. Our primary screen identified 120 hits, which were validated in a secondary screen by dose-intracellular and -extracellular Mtb assays. Our confirmatory studies identified a novel anti-Mtb compound, 10-DEBC, also showing activity against drug-resistant strains.

Generation of a KSCBi005-A-5(TLR8KO-A10) homozygous knockout human induced pluripotent stem cell line using CRISPR/Cas9.

The Toll like Receptor (TLR) family plays an essential role in pathogen recognition and innate immunity activation. TLR8, an endosomal receptor, can recognize single-stranded RNA viruses, such as influenza virus, Sendai virus, Coxsackie B virus, HIV, and HCV. TLR8 binding to the viral RNA recruits MyD88 and leads to activation of the transcription factor NF-kB and antiviral response. We generated biallelic mutants of the TLR8 gene using a CRISPR-Cas9 genome editing method in human induced pluripotent stem cells (hiPSCs). The TLR8 homozygous-knockout hiPSCs retained normal morphology, gene expression, and in vivo differentiation potential.

Generation of a TLR7 homozygous knockout human induced pluripotent stem cell line using CRISPR/Cas9.

Toll Like Receptor (TLR) family plays an important role in the activation of innate immunity against pathogens. TLR7 mediates the recognition of single-stranded RNA viruses, such as human immunodeficiency virus, hepatitis C virus, and influenza virus in endosomes. Here, we generated a TLR7 homozygous knockout human induced pluripotent cell (hiPSC) line, hiPSC-TLR7KO-A59, using the CRISPR/Cas9 genome editing method. The hiPSC-TLR7KO-A59 line maintains normal morphology, pluripotency, and differentiation capacity into three germ layers.

TAK733 attenuates adrenergic receptor-mediated cardiomyocyte hypertrophy via inhibiting ErkThr188 phosphorylation.

BACKGROUND: Cardiac hypertrophy is an important risk factor for heart failure. The MEK-ERK axis has been reported as a major regulator in controlling cardiac hypertrophy. TAK733 is a potent and selective MEK inhibitor that suppresses cell growth in a broad range of cell lines. OBJECTIVE: Therefore, we aimed to investigate the anti-hypertrophic effect of TAK733 in cardiomyocytes. METHODS: Cardiomyocyte hypertrophy was induced with norepinephrine (NE) or phenylepinephrine (PE) using H9c2 cells. To confirm the cardiomyocyte hypertrophy, cell size and protein synthesis were measured and hypertrophy-related gene expression was estimated by reverse transcription polymerase chain reaction. To identify the signaling pathway involved, immunoblot analysis were performed. RESULTS: We observed that NE activated MEK-ERK signaling and increased ANP and BNP expression, resulting in cardiomyocyte hypertrophy. TAK733 significantly reduced cardiomyocyte hypertrophy by regulating NE-induced ERK1/2 and ERKThr188 activation, hypertrophy marker expression, and cardiomyocyte hypertrophy through depression of MEK activity. In addition, we examined that PE-induced cardiomyocyte hypertrophy was also attenuated by TAK733. CONCLUSIONS: Here, we report that TAK733 suppressed NE- or PE-induced cardiomyocyte hypertrophy by repressing a crucial component of cardiac hypertrophy-related pathways. These results suggest that TAK733 may be a useful therapeutics for cardiac hypertrophy and warrants further in vivo studies.

Simultaneous Suppression of Multiple Programmed Cell Death Pathways by miRNA-105 in Cardiac Ischemic Injury.

Recent studies have shown that several upstream signaling elements of apoptosis and necroptosis are closely associated with acute injury in the heart. In our study, we observed that miR-105 was notably dysregulated in rat hearts with myocardial infarction (MI). Thus, the purpose of this study was to test the hypothesis that miR-105 participates in the regulation of RIP3/p-MLKL- and BNIP3-dependent necroptosis/apoptosis in H9c2 cells and MI rat hearts. Our results show that the RIP3/p-MLKL necroptotic pathway and BNIP3-dependent apoptosis signaling are enhanced in H9c2 cells under hypoxic conditions, whereas, compared with these pathways in the controls, those in miR-105-treated H9c2 cells are suppressed. Mechanistically, we identified miR-105 as the miRNA directly suppressing the expression of RIP3 and BNIP3, two important mediators involved in cell necroptosis and apoptosis. Furthermore, MI rat hearts injected with miR-105 had decreased infarct sizes, indicating that miR-105 is among three miRNAs that function simultaneously to suppress necroptotic/apoptotic cell death pathways and to inhibit MI-induced cardiomyocyte cell death at multiple levels. Taken together, miR-105 may constitute a new therapeutic strategy for cardioprotection in ischemic heart disease.

Multipoint targeting of TGF-ß/Wnt transactivation circuit with microRNA 384-5p for cardiac fibrosis.

Cardiac fibrosis is a common precursor to ventricular dysfunction and eventual heart failure, and cardiac fibrosis begins with cardiac fibroblast activation. Here we have demonstrated that the TGF-ß signaling pathway and Wnt signaling pathway formed a transactivation circuit during cardiac fibroblast activation and that miR-384-5p is a key regulator of the transactivation circuit. The results of in vitro study indicated that TGF-ß activated an auto-positive feedback loop by increasing Wnt production in cardiac fibroblasts, and Wnt neutralizing antibodies disrupted the feedback loop. Also, we demonstrated that miR-384-5p simultaneously targeted the key receptors of the TGF-ß/Wnt transactivation circuit and significantly attenuated both TGF-ß-induced cardiac fibroblast activation and ischemia-reperfusion-induced cardiac fibrosis. In addition, small molecule that prevented pro-fibrogenic stimulus-induced downregulation of endogenous miR-384-5p significantly suppressed cardiac fibroblast activation and cardiac fibrosis. In conclusion, modulating a key endogenous miRNA targeting multiple components of the TGF-ß/Wnt transactivation circuit can be an effective means to control cardiac fibrosis and has great therapeutic potential.

Priming stem cells with protein kinase C activator enhances early stem cell-chondrocyte interaction by increasing adhesion molecules.

BACKGROUND: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)-induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. RESULTS: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). CONCLUSION: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.

Corrigendum: Small molecule-mediated up-regulation of microRNA targeting a key cell death modulator BNIP3 improves cardiac function following ischemic injury.

This corrects the article DOI: 10.1038/srep23472.

TAK-733 inhibits inflammatory neointimal formation by suppressing proliferation, migration, and inflammation in vitro and in vivo.

As a potent and selective allosteric inhibitor of MEK, TAK-733 has been shown to exert anti-cancer effects for a wide range of cancers both in vitro and in vivo. However, its effects on inhibiting growth have never been investigated in the cardiovascular system, where regulation of abnormal vascular smooth muscle cell growth in neointimal hyperplasia is an important area of focus. Angiotensin II was used to mimic inflammatory neointimal hyperplasia in an in vitro environment, and balloon catheter-induced injury with an infusion of angiotensin II was used to generate an in vivo rat restenosis model under inflammatory conditions. TAK-733 exerted anti-proliferative and anti-migratory effects on human vascular smooth muscle cells. These multiple effects of TAK-733 were evaluated using various assays, such as cell cycle analysis and wound healing. Interestingly, TAK-733 did not induce apoptosis in smooth muscle cells but only reduced the proliferation rate; additionally, it did not affect EC viability. TAK-733 also exhibited anti-inflammatory activity, as observed by attenuated monocyte adhesion to smooth muscle cells via inhibition of ICAM1 and VCAM1 overexpression. The in vivo study demonstrated that neointimal hyperplasia after balloon injury and angiotensin II stimulation was suppressed by TAK-733, and downregulation of the inflammatory signal and enhanced re-endothelialization were observed. TAK-733 may have therapeutic potential for treating neointimal hyperplasia by attenuating smooth muscle cell proliferation, migration, and inflammation. Thus, TAK-733 could be a promising drug candidate for treating patients with restenosis.

Extract of Oxytropis pseudoglandulosa inhibits vascular smooth muscle cell proliferation and migration via suppression of ERK1/2 and Akt signaling pathways1.

Excessive vascular smooth muscle cell (VSMC) proliferation and migration accelerate the development of occlusive vascular disease. Therefore, finding a means to control the aberrant proliferation and migration of VSMCs has own clinical significance. In the present study, we examined the feasibility of using extract from medicinal plant Oxytropis pseudoglandulosa (OG) to control pathologic proliferation and migration of VSMCs, which never have been tested. Our data indicate that the extract of OG significantly suppressed proliferation and migration of VSMCs without cytotoxic effect, suggesting the OG extract may be an alternative agent to effectively control the aberrant VSMC proliferation and migration without any serious adverse effect. These data suggest that the extract of OG may be a potent therapeutic agent for the treatment of occlusive vascular disease and warrant further studies to identify the major acting ingredient and to validate in vivo efficacy.

microRNA-133a attenuates cardiomyocyte hypertrophy by targeting PKCδ and Gq.

During the past decade, microRNAs have continuously been suggested as a promising therapeutic tool due to their beneficial effects, such as their multi-targets and multi-functions in pathologic conditions. As a pathologic phenotype is generally regulated by multiple signaling pathways, in this study we identified a microRNA regulating multiple target genes within cardiac hypertrophic signaling pathways. microRNA-133a is known to play a crucial role in cardiac hypertrophy. However, the role of microRNA-133a, which may regulate several signaling pathways in norepinephrine-induced cardiac hypertrophy via multi-targeting, has not been investigated. In the current study, we showed that microRNA-133a can protect cardiomyocyte hypertrophy against norepinephrine stimulation in neonatal rat ventricular cardiomyocytes via new targets, PKCδ and Gq, all of which are related to downstream signaling pathways of the α1-adrenergic receptor. Taken together, these results suggest the advantages of the therapeutic use of microRNAs as an effective potential drug regulating multiple signaling pathways under pathologic conditions.

Priming stem cells with protein kinase C activator enhances early stem cell-chondrocyte interaction by increasing adhesion molecules

BACKGROUND: Osteoarthritis (OA) can be defined as degradation of articular cartilage of the joint, and is the most common degenerative disease. To regenerate the damaged cartilage, different experimental approaches including stem cell therapy have been tried. One of the major limitations of stem cell therapy is the poor post-transplantation survival of the stem cells. Anoikis, where insufficient matrix support and adhesion to extracellular matrix causes apoptotic cell death, is one of the main causes of the low post-transplantation survival rate of stem cells. Therefore, enhancing the initial interaction of the transplanted stem cells with chondrocytes could improve the therapeutic efficacy of stem cell therapy for OA. Previously, protein kinase C activator phorbol 12-myristate 13-acetate (PMA)- induced increase of mesenchymal stem cell adhesion via activation of focal adhesion kinase (FAK) has been reported. In the present study, we examine the effect PMA on the adipose-derived stem cells (ADSCs) adhesion and spreading to culture substrates, and further on the initial interaction between ADSC and chondrocytes. RESULTS: PMA treatment increased the initial adhesion of ADSC to culture substrate and cellular spreading with increased expression of adhesion molecules, such as FAK, vinculin, talin, and paxillin, at both RNA and protein level. Priming of ADSC with PMA increased the number of ADSCs attached to confluent layer of cultured chondrocytes compared to that of untreated ADSCs at early time point (4 h after seeding). CONCLUSION: Taken together, the results of this study suggest that priming ADSCs with PMA can increase the initial interaction with chondrocytes, and this proof of concept can be used to develop a non-invasive therapeutic approach for treating OA. It may also accelerate the regeneration process so that it can relieve the accompanied pain faster in OA patients. Further in vivo studies examining the therapeutic effect of PMA pretreatment of ADSCs for articular cartilage damage are required.

Small molecule-mediated induction of miR-9 suppressed vascular smooth muscle cell proliferation and neointima formation after balloon injury.

Pathologic proliferation and migration of vascular smooth muscle cells (VSMCs) exacerbate cardiovascular disease. MicroRNAs (miRNAs), as endogenous inhibitors of protein synthesis, are expected to modulate pathologic proliferation of VSMCs. Here we report that both platelet-derived growth factor receptor (PDGFR) targeting miR-9 and a small molecule that increases miR-9 can inhibit the serum-induced proliferation of VSMCs. First, based on miRNA-target prediction databases and empirical data, we have selected miR-9 as a potent anti-proliferative miRNA in VSMCs. Further examination indicated that miR-9 directly targets PDGFR disrupting downstream signaling cascades, and this resulted in inhibition of VSMC proliferation and migration. Exogenous delivery of miR-9 inhibited VSMC proliferation in vitro, and a small molecule that increased miR-9 expression also inhibited neointima formation following balloon injury in vivo. We provide evidence of miRNA-mediated modulation of VSMC proliferation and further demonstrate that small molecule-mediated regulation of miRNA targeting a key regulator of VSMC proliferation is a viable therapeutic strategy for treating vascular disease involving pathologic VSMC proliferation such as restenosis.

Rapid Induction of Osteogenic Markers in Mesenchymal Stem Cells by Adipose-Derived Stromal Vascular Fraction Cells.

BACKGROUND/AIMS: Stromal vascular fraction (SVF) cells are a mixed cell population, and their regenerative capacity has been validated in various therapeutic models. The purpose of this study was to investigate the regenerative mechanisms utilized by implanted SVF cells. Using an in vitro co-culture system, we sought to determine whether SVF implantation into impaired tissue affects endogenous mesenchymal stem cell (MSC) differentiation; MSCs can differentiate into a variety of cell types, and they have a strong regenerative capacity despite their low numbers in impaired tissue. METHODS: Adipose-derived SVF cells obtained from four donors were co-cultured with bone marrow-derived MSCs, and the differential expression of osteogenic markers and osteogenic differentiation inducers over time was analyzed in mono-cultured MSCs and MSCs co-cultured with SVF cells. RESULTS: The co-cultivation of MSCs with SVF cells significantly and mutually induced the expression of osteogenic-specific markers via paracrine and/or autocrine regulation but did not induce adipocyte, chondrocyte or myoblast marker expression. More surprisingly, subsequent osteogenesis and/or comparable effects were rapidly induced within 48 h. CONCLUSION: To the best of our knowledge, this is the first study in which osteogenesis and/or comparable effects were rapidly induced in bone marrow-derived MSCs and adipose-derived SVF cells through co-cultivation. Our findings suggest that the positive effects of SVF implantation into impaired bone may be attributed to the rapid induction of MSC osteogenesis, and the transplantation of co-cultured and preconditioned SVF cells and/or MSCs may be more effective than the transplantation of untreated cells for the treatment of bone defects.

Adipose-derived stem cell-released osteoprotegerin protects cardiomyocytes from reactive oxygen species-induced cell death.

BACKGROUND: The paracrine effect is likely the major mechanism of the adipose-derived stem cell (ASC)-mediated cardioprotective effect. However, the exact composition and nature of ASC-released paracrine factors remain elusive. In the present study, we examined the effect of osteoprotegerin (OPG), a stem cell-released decoy receptor for death ligand, on the survival of cardiomyocytes exposed to oxidative stress. METHODS: The production of OPG from ASCs under oxidative stress was determined by ELISA and immunohistochemistry. The effects of OPG and the OPG-containing conditioned media of ASCs on the survival of cardiomyocytes were determined using a cell viability assay. RESULTS: Hydrogen peroxide (H2O2) significantly increased OPG production from ASCs in vitro, and OPG production from the ASCs transplanted into the ischemia-reperfusion-injured heart was also observed. OPG significantly attenuated cardiomyocyte death in vitro. OPG-containing conditioned media of ASCs also significantly protected cardiomyocytes. Delivery of siRNA specific to OPG significantly decreased the OPG production of ASCs, and also offset the protective effect of the conditioned media of ASCs. CONCLUSIONS: Our study strongly suggests that OPG is one of the prosurvival factors released from ASCs that may contribute to the ASC-mediated cardioprotection and calls for further studies to elucidate detailed underlying mechanisms.