hESC- and hiPSC-derived Schwann cells are molecularly comparable and functionally equivalent.

Establishing robust models of human myelinating Schwann cells is critical for studying peripheral nerve injury and disease. Stem cell differentiation has emerged as a key human cell model and disease motivating development of Schwann cell differentiation protocols. Human embryonic stem cells (hESCs) are considered the ideal pluripotent cell but ethical concerns regarding their use have propelled the popularity of human induced pluripotent stem cells (hiPSCs). Given that the equivalence of hESCs and hiPSCs remains controversial, we sought to compare the molecular and functional equivalence of hESC- and hiPSC-derived Schwann cells generated with our previously reported protocol. We identified only modest transcriptome differences by RNA sequencing and insignificant proteome differences by antibody array. Additionally, both cell types comparably improved nerve regeneration and function in a chronic denervation and regeneration animal model. Our findings demonstrate that Schwann cells derived from hESCs and hiPSCs with our protocol are molecularly comparable and functionally equivalent.

Stem Cell-Based Approaches in Parkinson's Disease Research.

Parkinson's disease (PD) is a neurodegenerative condition characterized by the loss of midbrain dopaminergic neurons, leading to motor symptoms. While current treatments provide limited relief, they don't alter disease progression. Stem cell technology, involving patient-specific stem cell-derived neurons, offers a promising avenue for research and personalized regenerative therapies. This article reviews the potential of stem cell-based research in PD, summarizing ongoing efforts, their limitations, and introducing innovative research models. The integration of stem cell technology and advanced models promises to enhance our understanding and treatment strategies for PD.

Precision Medicine in Parkinson's Disease Using Induced Pluripotent Stem Cells.

Parkinson's disease (PD) is one of the most devastating neurological diseases; however, there is no effective cure yet. The availability of human induced pluripotent stem cells (iPSCs) provides unprecedented opportunities to understand the pathogenic mechanism and identification of new therapy for PD. Here a new model system of PD, including 2D human iPSC-derived midbrain dopaminergic (mDA) neurons, 3D iPSC-derived midbrain organoids (MOs) with cellular complexity, and more advanced microphysiological systems (MPS) with 3D organoids, is introduced. It is believed that successful integrations and applications of iPSC, organoid, and MPS technologies can bring new insight on PD's pathogenesis that will lead to more effective treatments for this debilitating disease.

Optogenetic Approach in Trigeminal Neuralgia and Potential Concerns: Preclinical Insights.

The integration of optogenetics in the trigeminal pain circuitry broadens and reinforces existing pain investigations. Similar to research on spinal neuropathic pain, the exploration of the underlying determinants of orofacial pain is expanding. Optogenetics facilitates more direct, specific, and subtle investigations of the neuronal circuits involved in orofacial pain. One of the most significant concerns of both dentistry and medicine is trigeminal neuralgia (TN) management due to its substantial impact on a patient's quality of life. Our objective is to gather insights from preclinical studies conducted in TN employing an optogenetic paradigm, thereby extending the prospects for in-depth neurobiological research. This review highlights optogenetic research in trigeminal pain circuitry involving TN. We outline the central and peripheral regions associated with pain-that have been investigated using optogenetics in the trigeminal pain circuitry. The study further reports its scope and limitations as well as its potential for future applications from bench to bedside.

Optogenetic induction of alpha-synuclein aggregation in human dopaminergic neurons to model Parkinson's disease pathology.

Alpha-synuclein (α-syn) aggregation is a principal factor in Parkinson's disease (PD) onset. Here, we present a protocol for optogenetic induction of α-syn aggregation in human midbrain dopaminergic (mDA) neurons, facilitating a detailed PD pathology study. We describe steps for nucleofection of the opto-α-syn construct, single colony selection and validation, alongside mDA neuron differentiation and rapid induction of toxic α-syn aggregates via blue light. This establishes a potent human induced pluripotent-stem-cell-based platform for PD drug testing and validation. For complete details on the use and execution of this protocol, please refer to Kim et al. (2023).1.

Advanced human iPSC-based preclinical model for Parkinson's disease with optogenetic alpha-synuclein aggregation.

Human induced pluripotent stem cells (hiPSCs) offer advantages for disease modeling and drug discovery. However, recreating innate cellular pathologies, particularly in late-onset neurodegenerative diseases with accumulated protein aggregates including Parkinson's disease (PD), has been challenging. To overcome this barrier, we developed an optogenetics-assisted α-synuclein (α-syn) aggregation induction system (OASIS) that rapidly induces α-syn aggregates and toxicity in PD hiPSC-midbrain dopaminergic neurons and midbrain organoids. Our OASIS-based primary compound screening with SH-SY5Y cells identified 5 candidates that were secondarily validated with OASIS PD hiPSC-midbrain dopaminergic neurons and midbrain organoids, leading us to finally select BAG956. Furthermore, BAG956 significantly reverses characteristic PD phenotypes in α-syn preformed fibril models in vitro and in vivo by promoting autophagic clearance of pathological α-syn aggregates. Following the FDA Modernization Act 2.0's emphasis on alternative non-animal testing methods, our OASIS can serve as an animal-free preclinical test model (newly termed "nonclinical test") for the synucleinopathy drug development.

Interleukin-7 enhances in vitro development and blastocyst quality in porcine parthenogenetic embryos.

Interleukin-7 (IL-7), a vital factor that affects cell development, proliferation, and survival, plays an important role in oocyte maturation. However, its role in embryonic development remains unknown. Therefore, in this study, we aimed to investigate the effects of IL-7 supplementation on in vitro culture (IVC) of porcine embryos after parthenogenetic activation (PA) based on characteristics such as cleavage, blastocyst formation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in cleaved embryos, total cell number, apoptosis rate, and cell lineage specification in blastocysts. Immunofluorescence revealed that IL-7 and its receptor, IL-7Rα (IL-7R) localized in the cytoplasm of porcine parthenote embryos. By supplementing the IVC medium (PZM5) with various concentrations of IL-7, an optimal concentration that enhanced embryonic development, promoted intracellular GSH, and decreased ROS levels in the cleavage stage during porcine embryo IVC was determined. Investigation of mRNA expression patterns via qRT-PCR suggested that IL-7 possibly regulated maternal mRNA clearance and zygotic genome activation. Furthermore, IL-7 supplementation reduced blastocyst apoptosis, enhanced the expression of the inner cell mass marker SOX2, and phosphorylated STAT5 levels in the blastocysts. Moreover, it altered the transcription patterns of genes that regulate apoptosis, IL-7 signaling, and development. Thus, we demonstrated the localization of IL-7 and IL-7R in porcine preimplantation embryos in vitro for the first time. Furthermore, we suggest that IL-7 supplementation can be employed to enhance embryonic development and blastocyst quality based on the activation of the transcripts of genes that are involved in developmental competence and IL-7 signaling during in vitro porcine embryo development following PA.

Engineering Skeletal Muscle Grafts with PAX7::GFP-Sorted Human Pluripotent Stem Cell-Derived Myogenic Progenitors on Fibrin Microfiber Bundles for Tissue Regeneration.

Tissue engineering strategies that combine human pluripotent stem cell-derived myogenic progenitors (hPDMs) with advanced biomaterials provide promising tools for engineering 3D skeletal muscle grafts to model tissue development in vitro and promote muscle regeneration in vivo. We recently demonstrated (i) the potential for obtaining large numbers of hPDMs using a combination of two small molecules without the overexpression of transgenes and (ii) the application of electrospun fibrin microfiber bundles for functional skeletal muscle restoration following volumetric muscle loss. In this study, we aimed to demonstrate that the biophysical cues provided by the fibrin microfiber bundles induce hPDMs to form engineered human skeletal muscle grafts containing multinucleated myotubes that express desmin and myosin heavy chains and that these grafts could promote regeneration following skeletal muscle injuries. We tested a genetic PAX7 reporter line (PAX7::GFP) to sort for more homogenous populations of hPDMs. RNA sequencing and gene set enrichment analyses confirmed that PAX7::GFP-sorted hPDMs exhibited high expression of myogenic genes. We tested engineered human skeletal muscle grafts derived from PAX7::GFP-sorted hPDMs within in vivo skeletal muscle defects by assessing myogenesis, engraftment and immunogenicity using immunohistochemical staining. The PAX7::GFP-sorted groups had moderately high vascular infiltration and more implanted cell association with embryonic myosin heavy chain (eMHC) regions, suggesting they induced pro-regenerative microenvironments. These findings demonstrated the promise for the use of PAX7::GFP-sorted hPDMs on fibrin microfiber bundles and provided some insights for improving the cell-biomaterial system to stimulate more robust in vivo skeletal muscle regeneration.

The role of KiSS1 gene on the growth and migration of prostate cancer and the underlying molecular mechanisms.

Metastatic prostate cancers have a high mortality rate. KiSS1 was originally identified as a metastasis suppressor gene in metastatic melanoma and breast cancer, but its role in prostate cancer has been contradictory. This study was therefore undertaken to investigate the effects of KiSS1 overexpression on the growth and migration of human metastatic prostate cancer cells. We first tested the effect of KiSS1 overexpression on the growth and migration of DU145 human metastatic prostate cancer cells in vitro. DU145 cells were infected with the culture medium of 293T cells, which produce lentivirus particles containing KiSS1. A 2.5-fold increase in proliferation of KiSS1-overexpressing cancer cells was observed, and these cells formed tumor spheroids about 3 times larger than the vector control group. qPCR and immunoblotting revealed the association between increased cell growth and regulation of the PI3K/Akt and cell cycle genes, and also that increases in ß-catenin and CD133 contribute to tumor aggregation. KiSS1 overexpression resulted in upregulation of the ß-arrestin1/2 and Raf-MEK-ERK-NF-κB pathways via KiSS1R. Moreover, the migration and invasion of KiSS1-overexpressing cells were determined to be faster than the control group, along with 1.6-fold increased metastatic colonization of the KiSS1-overexpressing cancer cells. These were associated to the regulation of EMT gene expressions, such as E-cadherin and N-cadherin, and the upregulation of MMP9. In a xenograft mouse model inoculated with DU145 cells infected GFP or KiSS1 via a lentiviral vector, KiSS1 statistically significantly increased the tumor growth, with upregulation of PCNA and Ki-67 in the tumor tissues. In addition, KiSS1 increased the angiogenic capacity by upregulating VEGF-A and CD31, both in vitro and in vivo. Taken together, our results indicate that KiSS1 not only induces prostate cancer proliferation, but also promotes metastasis by increasing the migration, invasion, and angiogenesis of malignant cells.

Copper deficiency affects the developmental competence of porcine oocytes matured in vitro.

The trace element Cu is required for the activity of various enzymes essential for physiological processes. In this study, we elucidated the copper transport system in porcine follicular cells and investigated the effect of Cu chelation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). Cu chelation was induced by adding tetraethylenepentamine (TEPA) to the maturation media (TCM199-PVA). First, we identified the localization and relative levels of the copper transporter CTR1 in follicular cells. The level of CTR1 protein was the highest in mature cumulus cells; moreover, CTR1 was mainly localized in the cytoplasmic vesicular compartment in oocytes, whereas it was evenly distributed in the cytoplasm in cumulus cells. A total of 42 h after IVM, the TEPA-treated group showed reduced maturation rates compared to those of the control (p < 0.05). This negative effect of TEPA disappeared when it was added to the media with Cu (Cu + TEPA group). The TEPA treatment during IVM significantly increased the mRNA levels of the Has2 gene, which is related to cumulus expansion (p < 0.05). Both Cu supplementation and chelation significantly increased the reactive oxygen species (ROS) levels in porcine oocytes (p < 0.05). When we analyzed the transcript levels of folliculogenesis-related genes in Cu chelation conditions, only the expression of MAPK3 in cumulus cells significantly increased compared to that of the control. We also evaluated the subsequent embryonic development of PA embryos. TEPA-treated oocytes showed significantly decreased blastocyst formation rates compared to those of the control. The TEPA-induced toxic effect was alleviated when Cu was added with TEPA. Our findings suggest that the Cu transport system plays an important role in the porcine follicular development process and that the Cu deficiency negatively affects porcine oocyte maturation, as well as their subsequent developmental competence.

Physiological and Functional Roles of Neurotrophin-4 During In Vitro Maturation of Porcine Cumulus-Oocyte Complexes.

Neurotrophin-4 (NT-4), a granulosa cell-derived factor and a member of the neurotrophin family, is known to promote follicular development and oocyte maturation in mammals. However, the physiological and functional roles of NT-4 in porcine ovarian development are not yet known. The aim of this study was to investigate the physiological role of NT-4-related signaling in the in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COCs). The NT-4 protein and its receptors were detected in matured porcine COCs via immunofluorescence analysis. NT-4 was shown to promote the maturation of COCs by upregulating NFKB1 transcription via the neurotrophin/p75NTR signaling pathway. Notably, the mRNA expression levels of the oocyte-secreted factors GDF9 and BMP15, sperm-oocyte interaction regulator CD9, and DNA methylase DNMT3A were significantly upregulated in NT-4-treated than in untreated porcine oocytes. Concurrently, there were no significant differences in the levels of total and phosphorylated epidermal growth factor receptor and p38 mitogen-activated protein kinase between NT-4-treated and untreated cumulus cells (CCs); however, the level of phosphorylated ERK1/2 was significantly higher in NT-4-treated CCs. Both total and phosphorylated ERK1/2 levels were significantly higher in NT-4-treated than in untreated oocytes. In addition, NT-4 improved subsequent embryonic development after in vitro fertilization and somatic cell nuclear transfer. Therefore, the physiological and functional roles of NT-4 in porcine ovarian development include the promotion of oocyte maturation, CC expansion, and ERK1/2 phosphorylation in porcine COCs during IVM.

Human pluripotent stem cell-derived myogenic progenitors undergo maturation to quiescent satellite cells upon engraftment.

Human pluripotent stem cell (hPSC)-derived myogenic progenitor cell (MPC) transplantation is a promising therapeutic approach for a variety of degenerative muscle disorders. Here, using an MPC-specific fluorescent reporter system (PAX7::GFP), we demonstrate that hPSC-derived MPCs can contribute to the regeneration of myofibers in mice following local injury and in mice deficient of dystrophin (mdx). We also demonstrate that a subset of PAX7::GFP MPCs engraft within the basal lamina of regenerated myofibers, adopt a quiescent state, and contribute to regeneration upon reinjury and in mdx mouse models. This subset of PAX7::GFP MPCs undergo a maturation process and remodel their molecular characteristics to resemble those of late-stage fetal MPCs/adult satellite cells following in vivo engraftment. These in-vivo-matured PAX7::GFP MPCs retain a cell-autonomous ability to regenerate and can repopulate in the niche of secondary recipient mice, providing a proof of principle for future hPSC-based cell therapy for muscle disorders.

Direct Conversion to Achieve Glial Cell Fates: Oligodendrocytes and Schwann Cells.

Glia have been known for its pivotal roles in physiological and pathological conditions in the nervous system. To study glial biology, multiple approaches have been applied to utilize glial cells for research, including stem cell-based technologies. Human glial cells differentiated from pluripotent stem cells are now available, allowing us to study the structural and functional roles of glia in the nervous system, although the efficiency is still low. Direct conversion is an advanced strategy governing fate conversion of diverse cell types directly into the desired lineage. This novel strategy stands as a promising approach for preliminary research and regenerative medicine. Direct conversion employs genetic and environmental cues to change cell fate to that with the required functional cell properties while retaining maturity-related molecular features. As an alternative method, it is now possible to obtain a variety of mature cell populations that could not be obtained using conventional differentiation methods. This review summarizes current achievements in obtaining glia, particularly oligodendrocytes and Schwann cells.

Heart neurons use clock genes to control myocyte proliferation.

Neurons can regulate the development, pathogenesis, and regeneration of target organs. However, the role of neurons during heart development and regeneration remains unclear. We genetically inhibited sympathetic innervation in vivo, which resulted in heart enlargement with an increase in cardiomyocyte number. Transcriptomic and protein analysis showed down-regulation of the two clock gene homologs Period1/Period2 (Per1/Per2) accompanied by up-regulation of cell cycle genes. Per1/Per2 deletion increased heart size and cardiomyocyte proliferation, recapitulating sympathetic neuron­deficient hearts. Conversely, increasing sympathetic activity by norepinephrine treatment induced Per1/Per2 and suppressed cardiomyocyte proliferation. We further found that the two clock genes negatively regulate myocyte mitosis entry through the Wee1 kinase pathway. Our findings demonstrate a previously unknown link between cardiac neurons and clock genes in regulation of cardiomyocyte proliferation and heart size and provide mechanistic insights for developing neuromodulation strategies for cardiac regen5eration.

Multi-omic analysis of selectively vulnerable motor neuron subtypes implicates altered lipid metabolism in ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating disorder in which motor neurons degenerate, the causes of which remain unclear. In particular, the basis for selective vulnerability of spinal motor neurons (sMNs) and resistance of ocular motor neurons to degeneration in ALS has yet to be elucidated. Here, we applied comparative multi-omics analysis of human induced pluripotent stem cell-derived sMNs and ocular motor neurons to identify shared metabolic perturbations in inherited and sporadic ALS sMNs, revealing dysregulation in lipid metabolism and its related genes. Targeted metabolomics studies confirmed such findings in sMNs of 17 ALS (SOD1, C9ORF72, TDP43 (TARDBP) and sporadic) human induced pluripotent stem cell lines, identifying elevated levels of arachidonic acid. Pharmacological reduction of arachidonic acid levels was sufficient to reverse ALS-related phenotypes in both human sMNs and in vivo in Drosophila and SOD1G93A mouse models. Collectively, these findings pinpoint a catalytic step of lipid metabolism as a potential therapeutic target for ALS.

TRIP12 ubiquitination of glucocerebrosidase contributes to neurodegeneration in Parkinson's disease.

Impairment in glucocerebrosidase (GCase) is strongly associated with the development of Parkinson's disease (PD), yet the regulators responsible for its impairment remain elusive. In this paper, we identify the E3 ligase Thyroid Hormone Receptor Interacting Protein 12 (TRIP12) as a key regulator of GCase. TRIP12 interacts with and ubiquitinates GCase at lysine 293 to control its degradation via ubiquitin proteasomal degradation. Ubiquitinated GCase by TRIP12 leads to its functional impairment through premature degradation and subsequent accumulation of α-synuclein. TRIP12 overexpression causes mitochondrial dysfunction, which is ameliorated by GCase overexpression. Further, conditional TRIP12 knockout in vitro and knockdown in vivo promotes the expression of GCase, which blocks α-synuclein preformed fibrils (α-syn PFFs)-provoked dopaminergic neurodegeneration. Moreover, TRIP12 accumulates in human PD brain and α-synuclein-based mouse models. The identification of TRIP12 as a regulator of GCase provides a new perspective on the molecular mechanisms underlying dysfunctional GCase-driven neurodegeneration in PD.

Nectin-1 Is an Entry Mediator for Varicella-Zoster Virus Infection of Human Neurons.

Varicella-zoster virus (VZV) maintains lifelong latency in neurons following initial infection and can subsequently be reactivated to result in herpes zoster or severe neurological manifestations such as encephalitis. Mechanisms of VZV neuropathogenesis have been challenging to study due to the strict human tropism of the virus. Although neuronal entry mediators of other herpesviruses, including herpes simplex virus, have been identified, little is known regarding how VZV enters neurons. Here, we utilize a human stem cell-based neuronal model to characterize cellular factors that mediate entry. Through transcriptional profiling of infected cells, we identify the cell adhesion molecule nectin-1 as a candidate mediator of VZV entry. Nectin-1 is highly expressed in the cell bodies and axons of neurons. Either knockdown of endogenous nectin-1 or incubation with soluble forms of nectin-1 produced in mammalian cells results in a marked decrease in infectivity of neurons. Notably, while addition of soluble nectin-1 during viral infection inhibits infectivity, addition after infection has no effect on infectivity. Ectopic expression of human nectin-1 in a cell line resistant to productive VZV infection confers susceptibility to infection. In summary, we have identified nectin-1 as a neuronal entry mediator of VZV. IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox, gains access to neurons during primary infection where it resides lifelong, and can later be reactivated. Reactivation is associated with shingles and postherpetic neuralgia, as well as with severe neurologic complications, including vasculitis and encephalitis. Although the varicella vaccine substantially decreases morbidity and mortality associated with primary infection, the vaccine cannot prevent the development of neuronal latency, and vaccinated populations are still at risk for reactivation. Furthermore, immunocompromised individuals are at higher risk for VZV reactivation and associated complications. Little is known regarding how VZV enters neurons. Here, we identify nectin-1 as an entry mediator of VZV in human neurons. Identification of nectin-1 as a neuronal VZV entry mediator could lead to improved treatments and preventative measures to reduce VZV related morbidity and mortality.

Inhibition of the Combinatorial Signaling of Transforming Growth Factor-Beta and NOTCH Promotes Myotube Formation of Human Pluripotent Stem Cell-Derived Skeletal Muscle Progenitor Cells.

Understanding the signaling pathways that regulate the final differentiation of human myoblasts is essential for successful cell transplantation and drug screening for the treatment of muscular dystrophy. In an effort to improve myotube formation from hiPSC-derived myoblasts, we validated a collection of 13 small molecules in a newly established in vitro screening platform for the assessment of myotube formation. The analysis of myotube formation as measured by the fusion index showed that the combinational inhibition of the TGFß signaling with NOTCH signaling enhances the ability of multi-nucleated myotube production. Combinational treatment of inhibitors for TGFß and NOTCH signaling pathways improved myotube formation in a dose-dependent manner. This effect was achieved by inhibiting the combinatorial mechanism of signaling. The combination treatment of small molecules effective in inducing multinucleated myotubes was validated in healthy human primary myoblasts. In addition, it was also applied to DMD patient iPSC-derived myoblasts to enhance the generation of multinucleated myotubes.