Probes for INS

The mammalian epidermis undergoes constant renewal, replenished by a pool of stem cells and terminal differentiation of their progeny. This is accompanied by changes in gene expression and morphology that are orchestrated, in part, by epigenetic modifiers. Here, we define the role of the histone acetyltransferase KAT2A in epidermal homeostasis and provide a comparative analysis that reveals key functional divergence with its paralog KAT2B. In contrast to the reported function of KAT2B in epidermal differentiation, KAT2A supports the undifferentiated state in keratinocytes. RNA-seq analysis of KAT2A- and KAT2B- depleted keratinocytes revealed dysregulated epidermal differentiation. Depletion of KAT2A led to premature expression of epidermal differentiation genes in the absence of inductive signals, whereas loss of KAT2B delayed differentiation. KAT2A acetyltransferase activity was indispensable in regulating epidermal differentiation gene expression. The metazoan-specific N terminus of KAT2A was also required to support its function in keratinocytes. We further showed that the interplay between KAT2A- and KAT2B-mediated regulation was important for normal cutaneous wound healing in vivo. Overall, these findings reveal a distinct mechanism in which keratinocytes use a pair of highly homologous histone acetyltransferases to support divergent functions in self-renewal and differentiation processes.

Adult hippocampal neurogenesis (AHN) is a well-studied phenomenon that involves the derivation of new neurons from neural progenitor cells in the dentate gyrus region of the hippocampus, an area responsible for cognitive functions such as learning and memory storage. Moreover, the hippocampus is known to be implicated in neurological conditions such as Alzheimer's disease. Although AHN has been extensively observed in animal models for twenty years, its existence and persistence in humans have been widely debated in academia, heavily based on post-mortem immunohistochemical markers. Using the search engines PubMed and Google Scholar for “Adult Human Neurogenesis,” 143 articles that were most relevant to the history of AHN discovery, detection in rodents, immunohistochemical studies on post-mortem human sections, and therapeutic development targeting AHN were reviewed. This review article highlights the current understanding of AHN in rodents and humans, its implications in neurodegenerative diseases and therapeutics, and the inconsistencies and methodological variabilities encountered in studying AHN in humans. Furthermore, the correlation between AHN and diseases such as mood disorders and Alzheimer's disease is still not well established, with conflicting findings reported. Standardization of transcriptomic methodologies and increased availability of post-mortem human brain samples are crucial in advancing AHN research. This review article attempts to discover the fascinating and controversial world of adult human neurogenesis and its potential implications in treating neurological disorders. Apart from the discussion on AHN existence, tackling devastating diseases with this supplemental knowledge can lead to therapeutic advancements which greatly rely on understanding not only the presence of AHN but the mechanisms mediating its availability.

HIV-1 infection within the central nervous system (CNS) includes evolution of the virus, damaging inflammatory cascades, and the involvement of multiple cell types; however, our understanding of how Env tropism and inflammation can influence CNS infectivity is incomplete. In this study, we utilize macrophage-tropic and T cell-tropic HIV-1 Env proteins to establish accurate infection profiles for multiple CNS cells under basal and interferon alpha (IFN-α) or lipopolysaccharide (LPS)-induced inflammatory states. We found that macrophage-tropic viruses confer entry advantages in primary myeloid cells, including monocyte-derived macrophage, microglia, and induced pluripotent stem cell (iPSC)-derived microglia. However, neither macrophage-tropic or T cell-tropic HIV-1 Env proteins could mediate infection of astrocytes or neurons, and infection was not potentiated by induction of an inflammatory state in these cells. Additionally, we found that IFN-α and LPS restricted replication in myeloid cells, and IFN-α treatment prior to infection with vesicular stomatitis virus G protein (VSV G) Envs resulted in a conserved antiviral response across all CNS cell types. Further, using RNA sequencing (RNA-seq), we found that only myeloid cells express HIV-1 entry receptor/coreceptor transcripts at a significant level and that these transcripts in select cell types responded only modestly to inflammatory signals. We profiled the transcriptional response of multiple CNS cells to inflammation and found 57 IFN-induced genes that were differentially expressed across all cell types. Taken together, these data focus attention on the cells in the CNS that are truly permissive to HIV-1, further highlight the role of HIV-1 Env evolution in mediating infection in the CNS, and point to limitations in using model cell types versus primary cells to explore features of virus-host interaction. IMPORTANCE The major feature of HIV-1 pathogenesis is the induction of an immunodeficient state in the face of an enhanced state of inflammation. However, for many of those infected, there can be an impact on the central nervous system (CNS) resulting in a wide range of neurocognitive defects. Here, we use a highly sensitive and quantitative assay for viral infectivity to explore primary and model cell types of the brain for their susceptibility to infection using viral entry proteins derived from the CNS. In addition, we examine the ability of an inflammatory state to alter infectivity of these cells. We find that myeloid cells are the only cell types in the CNS that can be infected and that induction of an inflammatory state negatively impacts viral infection across all cell types.

Neuropathic pain is amongst the most common non-communicable disorders and the poor effectiveness of current treatment is an unmet need. Although pain is a universal experience, there are significant inter-individual phenotypic differences. Developing models that can accurately recapitulate the clinical pain features is crucial to better understand underlying pathophysiological mechanisms and find innovative treatments. Current data from heterologous expression systems that investigate properties of specific molecules involved in pain signaling, and from animal models, show limited success with their translation into the development of novel treatments for pain. This is in part because they do not recapitulate the native environment in which a particular molecule functions, and due to species-specific differences in the properties of several key molecules that are involved in pain signaling. The limited availability of post-mortem tissue, in particular dorsal root ganglia (DRG), has hampered research using human cells in pre-clinical studies. Human induced-pluripotent stem cells (iPSCs) have emerged as an exciting alternative platform to study patient-specific diseases. Sensory neurons that are derived from iPSCs (iPSC-SNs) have provided new avenues towards elucidating peripheral pathophysiological mechanisms, the potential for development of personalized treatments, and as a cell-based system for high-throughput screening for discovering novel analgesics. Nevertheless, reprogramming and differentiation protocols to obtain nociceptors have mostly yielded immature homogenous cell populations that do not recapitulate the heterogeneity of native sensory neurons. To close the gap between native human tissue and iPSCs, alternative strategies have been developed. We will review here recent developments in differentiating iPSC-SNs and their use in pre-clinical translational studies. Direct conversion of stem cells into the cells of interest has provided a more cost- and time-saving method to improve reproducibility and diversity of sensory cell types. Furthermore, multi-cellular strategies that mimic in vivo microenvironments for cell maturation, by improving cell contact and communication (co-cultures), reproducing the organ complexity and architecture (three-dimensional organoid), and providing iPSCs with the full spatiotemporal context and nutrients needed for acquiring a mature phenotype (xenotransplantation), have led to functional sensory neuron-like systems. Finally, this review touches on novel prospective strategies, including fluorescent-tracking to select the differentiated neurons of relevance, and dynamic clamp, an electrophysiological method that allows direct manipulation of ionic conductances that are missing in iPSC-SNs.

The milestone achievement of reprogramming a human somatic cell into a pluripotent stem cell by Yamanaka and Takahashi in 2007 has changed the stem cell research landscape tremendously. Their discovery opened the unprecedented opportunity to work with human-induced pluripotent stem cells and the differentiated progeny thereof, without major ethical restrictions. Additionally, the new method offers the possibility to generate pluripotent stem cells from patients with various genetic diseases which is of great importance (a) to understand the basic mechanisms of a specific disease in a human cellular context and (b) to help find suitable therapies for the persons concerned. In individual cases, this can even help to develop personalized treatment options. Chronic pain is a disease that affects roughly one in five people worldwide, but its onset is rarely based upon genetic alterations. Nevertheless, the work with sensory-like neurons derived from human pluripotent stem cells has become a more widely used tool also in the field of pain research, as during the past years several differentiation procedures have been published that describe the generation of different types of sensory-like neurons and their useful contribution to studying mechanisms of sensitization. Especially also to complement and verify cellular and molecular mechanisms identified in rodent model systems, the model of choice for decades. Although a sole cellular system is not able to mimic a disease as complex as pain, it is a valid tool to understand basic mechanisms of sensitization in specific subsets of human neurons that might be at the onset of the disease. In addition, the creativity of basic researchers and the more and more advanced available technologies will most likely find ways to implement the derived human cells in more complex networks. In this chapter, I want to introduce a selection of published differentiation strategies that result in the generation of human sensory-like neurons. Additionally, I will point out some studies whose results helped to further understand pain-related mechanisms and which were conducted using the aforementioned differentiation procedures.

Heterotopic ossification (HO) is one of the most intractable conditions following injury to the musculoskeletal system. In recent years, much attention has been paid to the role of lncRNA in musculoskeletal disorders, but its role in HO was still unclear. Therefore, this study attempted to determine the role of lncRNA MEG3 in the formation of post-traumatic HO and further explore the underlying mechanisms.On the basis of high-throughput sequencing and qPCR validation, elevated expression of the lncRNA MEG3 was shown during traumatic HO formation. Accordingly, in vitro experiments demonstrated that lncRNA MEG3 promoted aberrant osteogenic differentiation of tendon-derived stem cells (TDSCs). Mechanical exploration through RNA pulldown, luciferase reporter gene assay and RNA immunoprecipitation assay identified the direct binding relationship between miR-129-5p and MEG3, or miR-129-5p and TCF4. Further rescue experiments confirmed the miR-129-5p/TCF4/β-catenin axis to be downstream molecular cascade responsible for the osteogenic-motivating effects of MEG3 on the TDSCs. Finally, experiments in a mouse burn/tenotomy model corroborated the promoting effects of MEG3 on the formation of HO through the miR-129-5p/TCF4/β-catenin axis.Our study demonstrated that the lncRNA MEG3 promoted osteogenic differentiation of TDSCs and thus the formation of heterotopic ossification, which could be a potential therapeutic target.

A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer. Here, we report that Discs large 1 (Dlg1), although dispensable for polarity and cellular turnover during intestinal homeostasis, is required for ISC survival in the context of increased Wnt signaling. RNA sequencing (RNA-seq) and genetic mouse models demonstrated that DLG1 regulates the cellular response to increased canonical Wnt ligands. This occurs via the transcriptional regulation of Arhgap31, a GTPase-activating protein that deactivates CDC42, an effector of the non-canonical Wnt pathway. These findings reveal a DLG1-ARHGAP31-CDC42 axis that is essential for the ISC response to increased niche Wnt signaling.

Fibro/adipogenic progenitors (FAPs) are skeletal muscle stromal cells that support regeneration of injured myofibers and their maintenance in healthy muscles. FAPs are related to mesenchymal stem cells (MeSCs) found in other adult tissues, but there is poor understanding of the extent of similarity between these cells. Using single cell RNA sequencing (scRNA-seq) datasets from multiple mouse tissues we have performed comparative transcriptomic analysis between these cells. This identified remarkable transcriptional similarity between FAPs and MeSCs and confirmed the suitability of PDGFRα as a reliable reporter for FAPs. It also identified extracellular proteolysis as a new function of FAPs. Using PDGFRα as a cell surface marker, we isolated FAPs from healthy and dysferlinopathic mouse muscles and performed scRNA-seq analysis. This identified decreased FAP-mediated Wnt signaling as a potential driver of FAP dysfunction in dysferlinopathic muscles. Analysis of FAPs in dysferlin- and dystrophindeficient muscles identified distinct FAP-specific gene expression changes between these diseases that show existence of reciprocal relationship between muscle pathology and FAP gene expression.

The standard graft material for alveolar cleft repair (ACR) is autogenous iliac crest. However, a promising alternative potential graft adjunct - newborn human umbilical cord mesenchymal stem cells (h-UCMSC) - has yet to be explored in vivo. Their capacity for self-renewal, multipotent differentiation, and proliferation allows h-UCMSC to be harnessed for regenerative medicine. Our study seeks to evaluate the efficacy of using tissue-derived h-UCMSC and their osteogenic capabilities in a murine model to improve ACR.Foxn1 mice were separated into three groups with the following calvarial defects: (1) no-treatment (empty defect; n=6), (2) poly (D,L-lactide-co-glycolide) (PLGA) scaffold (n=6), and (3) h-UCMSC with PLGA (n=4). Bilateral 2-mm diameter parietal bone critical-sized defects were created using a dental drill. Micro-CT imaging occurred at 1, 2, 3, and 4 weeks postoperatively. The mice were euthanized 4 weeks postoperatively for RNAscope analysis, immunohistochemistry, and histology.No mice experienced complications during the follow-up period. Micro-CT and histology demonstrated that the no-treatment (1) and PLGA-only (2) defects remained patent without significant defect size differences across groups. In contrast, the h-UCMSC with PLGA group (3) had significantly greater bone fill on micro-CT and histology.We demonstrate a successful calvarial defect model for the investigation of h-UCMSC-mediated osteogenesis and bone repair. Furthermore, evidence reveals that PLGA alone has neither short-term effects on bone formation nor any unwanted side effects, making it an attractive scaffold. Further investigation using h-UCMSC with PLGA in larger animals is warranted to advance future translation to patients requiring ACR.Our results demonstrate a successful murine calvarial defect model for the investigation of h-UCMSC-mediated osteogenesis and bone repair and provide preliminary evidence for the safe and efficacious use of this graft adjunct in alveolar cleft repair.

Background: The standard graft material for alveolar cleft repair (ACR) is autogenous iliac crest. However, a promising alternative potential graft adjunct - newborn human umbilical cord mesenchymal stem cells (h-UCMSC) - has yet to be explored in vivo. Their capacity for selfrenewal, multipotent differentiation, and proliferation allows h-UCMSC to be harnessed for regenerative medicine. Our study seeks to evaluate the efficacy of using tissue-derived hUCMSC and their osteogenic capabilities in a murine model to improve ACR. Methods: Foxn1 mice were separated into three groups with the following calvarial defects: (1) no-treatment (empty defect; n=6), (2) poly (D,L-lactide-co-glycolide) (PLGA) scaffold (n=6), and (3) h-UCMSC with PLGA (n=4). Bilateral 2-mm diameter parietal bone critical-sized defects were created using a dental drill. Micro-CT imaging occurred at 1, 2, 3, and 4 weeks postoperatively. The mice were euthanized 4 weeks postoperatively for RNAscope analysis, immunohistochemistry, and histology. Results: No mice experienced complications during the follow-up period. Micro-CT and histology demonstrated that the no-treatment (1) and PLGA-only (2) defects remained patent without significant defect size differences across groups. In contrast, the h-UCMSC with PLGA group (3) had significantly greater bone fill on micro-CT and histology. Conclusions: We demonstrate a successful calvarial defect model for the investigation of hUCMSC-mediated osteogenesis and bone repair. Furthermore, evidence reveals that PLGA alone has neither short-term effects on bone formation nor any unwanted side effects, making it an 3 attractive scaffold. Further investigation using h-UCMSC with PLGA in larger animals is warranted to advance future translation to patients requiring ACR. Clinical Relevance Statement: Our results demonstrate a successful murine calvarial defect model for the investigation of h-UCMSC-mediated osteogenesis and bone repair and provide preliminary evidence for the safe and efficacious use of this graft adjunct in alveolar cleft repair.

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

IDH wild-type glioblastomas (GBM) are one of the most malignant and complex tumors for treatment. The urgent question of new therapeutic and diagnostic tools searching should be resolved based on cellular and molecular pathogenesis mechanisms, which remain insufficiently studied. In this study, we aimed to investigate GBM pathogenesis.Using the isolation of different GBM cell populations and the cell cultures, animal models, and molecular genetic methods, we tried to clarify the picture of GBM pathogenesis by constructing a projection from different glioma stem cells types to an integral neoplasm.We have shown a potential transformation pathway for both glioma stem cells and four definitive cell populations during gliomagenesis. Moreover, we have characterized each population, taking into account its place in the pathogenetic continuum, with a description of the most fundamental molecular and functional properties.Finally, we have formed a complex holistic concept of the pathogenetic evolution of GBM at the cell-population level by integrating our results with the data of the world literature.