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.

Approaches to characterize the transcriptional trajectory of human myogenesis.

Human pluripotent stem cells (hPSCs) have attracted considerable interest in understanding the cellular fate determination processes and modeling a number of intractable diseases. In vitro generation of skeletal muscle tissues using hPSCs provides an essential model to identify the molecular functions and gene regulatory networks controlling the differentiation of skeletal muscle progenitor cells. Such a genetic roadmap is not only beneficial to understanding human myogenesis but also to decipher the molecular pathology of many skeletal muscle diseases. The combination of established human in vitro myogenesis protocols and newly developed molecular profiling techniques offers extensive insight into the molecular signatures for the development of normal and disease human skeletal muscle tissues. In this review, we provide a comprehensive overview of the current progress of in vitro skeletal muscle generation from hPSCs and relevant examples of the transcriptional landscape and disease-related transcriptional aberrations involving signaling pathways during the development of skeletal muscle cells.

GBA1 deficiency negatively affects physiological α-synuclein tetramers and related multimers.

Accumulating evidence suggests that α-synuclein (α-syn) occurs physiologically as a helically folded tetramer that resists aggregation. However, the mechanisms underlying the regulation of formation of α-syn tetramers are still mostly unknown. Cellular membrane lipids are thought to play an important role in the regulation of α-syn tetramer formation. Since glucocerebrosidase 1 (GBA1) deficiency contributes to the aggregation of α-syn and leads to changes in neuronal glycosphingolipids (GSLs) including gangliosides, we hypothesized that GBA1 deficiency may affect the formation of α-syn tetramers. Here, we show that accumulation of GSLs due to GBA1 deficiency decreases α-syn tetramers and related multimers and increases α-syn monomers in CRISPR-GBA1 knockout (KO) SH-SY5Y cells. Moreover, α-syn tetramers and related multimers are decreased in N370S GBA1 Parkinson's disease (PD) induced pluripotent stem cell (iPSC)-derived human dopaminergic (hDA) neurons and murine neurons carrying the heterozygous L444P GBA1 mutation. Treatment with miglustat to reduce GSL accumulation and overexpression of GBA1 to augment GBA1 activity reverse the destabilization of α-syn tetramers and protect against α-syn preformed fibril-induced toxicity in hDA neurons. Taken together, these studies provide mechanistic insights into how GBA1 regulates the transition from monomeric α-syn to α-syn tetramers and multimers and suggest unique therapeutic opportunities for PD and dementia with Lewy bodies.

Establishment of 3D Neuro-Organoids Derived from Pig Embryonic Stem-Like Cells.

Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies.

Neural induction of porcine-induced pluripotent stem cells and further differentiation using glioblastoma-cultured medium.

Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine-induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma- and Mad-related protein (SMAD) inhibitors, a TGF-ß inhibitor and BMP4 inhibitor. The dSMADi-derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid-treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma-cultured medium including injury-related cytokines treated porcine iPSC-NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.

Characterization of canine adipose tissue-derived mesenchymal stem cells immortalized by SV40-T retrovirus for therapeutic use.

Canine mesenchymal stem cells (cMSCs) are gaining popularity in the veterinary field as a regenerative therapy. But, their limited culture lifespan makes it an obstacle for preclinical investigation and therapeutic use. In this study, primary canine adipose tissue-derived MSCs (PCAT-MSCs) were isolated from adipose tissue and were transfected with the SV40-T retrovirus resulting in a life-extended immortalized canine adipose tissue-derived MSCs (ICAT-MSCs). A comparison was made through the characterization of both PCAT-MSCs and ICAT-MSCs. Both showed a fibroblastic morphology; ICAT-MSCs showed a higher potential of colony formation compared with PCAT-MSCs and a reduced population doubling time; stem cell markers SOX2 and NANOG were expressed in both cell lines; karyotyping analysis showed no abnormalities in both PCAT-MSCs and ICAT-MSCs; both cell lines were CD90+ , CD44 + , and CD45 - ; both generated chondrogenic pellet; in osteogenic differentiation both showed upregulation of Osterix, a master transcriptome of osteogenesis, but in PCAT-MSCs, an upregulation of SOX2 was also observed. In conclusion, ICAT-MSCs showed similar characteristics with PCAT-MSCs, thus established as an easy to access platform for studies on better understanding about cMSCs nature.

PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front.

Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.

Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation.

Mechanisms of neuronal infection by varicella-zoster virus (VZV) have been challenging to study due to the relatively strict human tropism of the virus and the paucity of tractable experimental models. Cellular mitogen-activated protein kinases (MAPKs) have been shown to play a role in VZV infection of nonneuronal cells, with distinct consequences for infectivity in different cell types. Here, we utilize several human neuronal culture systems to investigate the role of one such MAPK, the c-Jun N-terminal kinase (JNK), in VZV lytic infection and reactivation. We find that the JNK pathway is specifically activated following infection of human embryonic stem cell-derived neurons and that this activation of JNK is essential for efficient viral protein expression and replication. Inhibition of the JNK pathway blocked viral replication in a manner distinct from that of acyclovir, and an acyclovir-resistant VZV isolate was as sensitive to the effects of JNK inhibition as an acyclovir-sensitive VZV isolate in neurons. Moreover, in a microfluidic-based human neuronal model of viral latency and reactivation, we found that inhibition of the JNK pathway resulted in a marked reduction in reactivation of VZV. Finally, we utilized a novel technique to efficiently generate cells expressing markers of human sensory neurons from neural crest cells and established a critical role for the JNK pathway in infection of these cells. In summary, the JNK pathway plays an important role in lytic infection and reactivation of VZV in physiologically relevant cell types and may provide an alternative target for antiviral therapy.IMPORTANCE Varicella-zoster virus (VZV) has infected over 90% of people worldwide. While primary infection leads to the typically self-limiting condition of chickenpox, the virus can remain dormant in the nervous system and may reactivate later in life, leading to shingles or inflammatory diseases of the nervous system and eye with potentially severe consequences. Here, we take advantage of newer stem cell-based technologies to study the mechanisms by which VZV infects human neurons. We find that the c-Jun N-terminal kinase (JNK) pathway is activated by VZV infection and that blockade of this pathway limits lytic replication (as occurs during primary infection). In addition, JNK inhibition limits viral reactivation, exhibiting parallels with herpes simplex virus reactivation. The identification of the role of the JNK pathway in VZV infection of neurons reveals potential avenues for the development of alternate antiviral drugs.

Isolation and characterization of GFAP-positive porcine neural stem/progenitor cells derived from a GFAP-CreERT2 transgenic piglet.

BACKGROUND: The porcine brain is gyrencephalic with similar gray and white matter composition and size more comparable to the human rather than the rodent brain; however, there is lack of information about neural progenitor cells derived from this model. RESULTS: Here, we isolated GFAP-positive porcine neural stem cells (NSCs) from the brain explant of a transgenic piglet, with expression of CreERT2 under the control of the GFAP promoter (pGFAP-CreERT2). The isolated pGFAP-CreERT2 NSCs showed self-renewal and expression of representative NSC markers such as Nestin and Sox2. Pharmacological inhibition studies revealed that Notch1 signaling is necessary to maintain NSC identity, whereas serum treatment induced cell differentiation into reactive astrocytes and neurons. CONCLUSIONS: Collectively, these results indicate that GFAP promoter-driven porcine CreERT2 NSCs would be a useful tool to study neurogenesis of the porcine adult central nervous system and furthers our understanding of its potential clinical application in the future. ᅟ.

Cellular stage specific functional analysis of REX1: In human embryonic stem cells.

As transcription and translation are dynamic and can vary among the cell types and conditions, proteomics may reveal the tissue-specific functions of a protein, more relevant to its genuine functions on cellular mechanisms. The new proteome analysis by Son et al. [Proteomics 2015, 15, 2220-2229] identified the functions of the pluripotency marker protein, REX1 in hESCs, and unraveling its regulatory network orchestrating pluripotency. Compared to the previous transcriptome analysis that showed mechanisms irrelevant to pluripotency, Son et al. employed a proteome analysis determined convincing and meaningful mechanisms of REX. In addition to demonstrating the biological importance of REX1, this research by Son et al. is also a compelling example of the conceptual significance of connecting proteomics with stem cell biology.

Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.

The isolation of human induced pluripotent stem cells (iPSCs) offers a new strategy for modelling human disease. Recent studies have reported the derivation and differentiation of disease-specific human iPSCs. However, a key challenge in the field is the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease in iPSCs. Familial dysautonomia (FD) is a rare but fatal peripheral neuropathy, caused by a point mutation in the IKBKAP gene involved in transcriptional elongation. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood owing to the lack of an appropriate model system. Here we report the derivation of patient-specific FD-iPSCs and the directed differentiation into cells of all three germ layers including peripheral neurons. Gene expression analysis in purified FD-iPSC-derived lineages demonstrates tissue-specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript, suggesting a mechanism for disease specificity. FD pathogenesis is further characterized by transcriptome analysis and cell-based assays revealing marked defects in neurogenic differentiation and migration behaviour. Furthermore, we use FD-iPSCs for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration. Our study illustrates the promise of iPSC technology for gaining new insights into human disease pathogenesis and treatment.

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.

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.

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.

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.

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.

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.

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.

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.