Cytoplasmic Intermediate Filaments in Cell Biology.

Intermediate filaments (IFs) are one of the three major elements of the cytoskeleton. Their stability, intrinsic mechanical properties, and cell type-specific expression patterns distinguish them from actin and microtubules. By providing mechanical support, IFs protect cells from external forces and participate in cell adhesion and tissue integrity. IFs form an extensive and elaborate network that connects the cell cortex to intracellular organelles. They act as a molecular scaffold that controls intracellular organization. However, IFs have been revealed as much more than just rigid structures. Their dynamics is regulated by multiple signaling cascades and appears to contribute to signaling events in response to cell stress and to dynamic cellular functions such as mitosis, apoptosis, and migration.

Perivascular cells support folliculogenesis in the developing ovary.

Supporting cells of the ovary, termed granulosa cells, are essential for ovarian differentiation and oogenesis by providing a nurturing environment for oocyte maintenance and maturation. Granulosa cells are specified in the fetal and perinatal ovary, and sufficient numbers of granulosa cells are critical for the establishment of follicles and the oocyte reserve. Identifying the cellular source from which granulosa cells and their progenitors are derived is an integral part of efforts to understand basic ovarian biology and the etiology of female infertility. In particular, the contribution of mesenchymal cells, especially perivascular cells, to ovarian development is poorly understood but is likely to be a source of new information regarding ovarian function. Here we have identified a cell population in the fetal ovary, which is a Nestin-expressing perivascular cell type. Using lineage tracing and ex vivo organ culture methods, we determined that perivascular cells are multipotent progenitors that contribute to granulosa, thecal, and pericyte cell lineages in the ovary. Maintenance of these progenitors is dependent on ovarian vasculature, likely reliant on endothelial-mesenchymal Notch signaling interactions. Depletion of Nestin+ progenitors resulted in a disruption of granulosa cell specification and in an increased number of germ cell cysts that fail to break down, leading to polyovular ovarian follicles. These findings highlight a cell population in the ovary and uncover a key role for vasculature in ovarian differentiation, which may lead to insights into the origins of female gonad dysgenesis and infertility.

Temporal and regional expression changes and co-staining patterns of metabolic and stemness-related markers during glioblastoma progression.

Glioblastomas (GBMs) are characterized by high heterogeneity, involving diverse cell types, including those with stem-like features contributing to GBM's malignancy. Moreover, metabolic alterations promote growth and therapeutic resistance of GBM. Depending on the metabolic state, antimetabolic treatments could be an effective strategy. Against this background, we investigated temporal and regional expression changes and co-staining patterns of selected metabolic markers [pyruvate kinase muscle isozyme 1/2 (PKM1/2), glucose transporter 1 (GLUT1), monocarboxylate transporter 1/4 (MCT1/4)] in a rodent model and patient-derived samples of GBM. To understand the cellular sources of marker expression, we also examined the connection of metabolic markers to markers related to stemness [Nestin, Krüppel-like factor 4 (KLF4)] in a regional and temporal context. Rat tumour biopsies revealed a temporally increasing expression of GLUT1, higher expression of MCT1/4, Nestin and KLF4, and lower expression of PKM1 compared to the contralateral hemisphere. Patient-derived tumours showed a higher expression of PKM2 and Nestin in the tumour centre vs. edge. Whereas rare co-staining of GLUT1/Nestin was found in tumour biopsies, PKM1/2 and MCT1/4 showed a more distinct co-staining with Nestin in rats and humans. KLF4 was mainly co-stained with GLUT1, MCT1 and PKM1/2 in rat and human tumours. All metabolic markers yielded individual co-staining patterns among themselves. Co-staining mainly occurred later in tumour progression and was more pronounced in tumour centres. Also, positive correlations were found amongst markers that showed co-staining. Our results highlight a link between metabolic alterations and stemness in GBM progression, with complex distinctions depending on studied markers, time points and regions.

Cdc7 kinase is required for postnatal brain development.

The evolutionally conserved Cdc7 kinase plays crucial roles in initiation of DNA replication as well as in other chromosomal events. To examine the roles of Cdc7 in brain development, we have generated mice carrying Cdc7 knockout in neural stem cells by using Nestin-Cre. The Cdc7Fl/Fl NestinCre mice were born, but exhibited severe growth retardation and impaired postnatal brain development. These mice exhibited motor dysfunction within 9 days after birth and did not survive for more than 19 days. The cerebral cortical layer formation was impaired, although the cortical cell numbers were not altered in the mutant. In the cerebellum undergoing hypoplasia, granule cells (CGC) decreased in number in Cdc7Fl/F l NestinCre mice compared to the control at E15-18, suggesting that Cdc7 is required for DNA replication and cell proliferation of CGC at mid embryonic stage (before embryonic day 15). On the other hand, the Purkinje cell numbers were not altered but its layer formation was impaired in the mutant. These results indicate differential roles of Cdc7 in DNA replication/cell proliferation in brain. Furthermore, the defects of layer formation suggest a possibility that Cdc7 may play an additional role in cell migration during neural development.

Acetylated oligopeptide and N-acetylcysteine protect against iron overload-induced dentate gyrus hippocampal degeneration through upregulation of Nestin and Nrf2/HO-1 and downregulation of MMP-9/TIMP-1 and GFAP.

Iron accumulation in the brain causes oxidative stress, blood-brain barrier (BBB) breakdown, and neurodegeneration. We examined the preventive effects of acetylated oligopeptides (AOP) from whey protein on iron-induced hippocampal damage compared to N-acetyl cysteine (NAC). This 5-week study used 40 male albino rats. At the start, all rats received 150 mg/kg/day of oral NAC for a week. The 40 animals were then randomly divided into four groups: Group I (control) received a normal diet; Group II (iron overload) received 60 mg/kg/day intraperitoneal iron dextran 5 days a week for 4 weeks; Group III (NAC group) received 150 mg/kg/day NAC and iron dextran; and Group IV (AOP group) received 150 mg/kg/day AOP and iron dextran. Enzyme-linked immunosorbent assay, spectrophotometry, and qRT-PCR were used to measure MMP-9, tissue inhibitor metalloproteinase-1 (TIMP-1), MDA, reduced glutathione (GSH) levels, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Histopathological and immunohistochemical detection of nestin, claudin, caspase, and GFAP was also done. MMP-9, TIMP-1, MDA, caspase, and GFAP rose in the iron overload group, while GSH, Nrf2, HO-1, nestin, and claudin decreased. The NAC and AOP administrations improved iron overload-induced biochemical and histological alterations. We found that AOP and NAC can protect the brain hippocampus from iron overload, improve BBB disruption, and provide neuroprotection with mostly no significant difference from healthy controls.

Constitutive Neurogenesis and Neuronal Plasticity in the Adult Cerebellum and Brainstem of Rainbow Trout, Oncorhynchus mykiss.

The central nervous system of Pacific salmon retains signs of embryonic structure throughout life and a large number of neuroepithelial neural stem cells (NSCs) in the proliferative areas of the brain, in particular. However, the adult nervous system and neurogenesis studies on rainbow trout, Oncorhynchus mykiss, are limited. Here, we studied the localization of glutamine synthetase (GS), vimentin (Vim), and nestin (Nes), as well as the neurons formed in the postembryonic period, labeled with doublecortin (DC), under conditions of homeostatic growth in adult cerebellum and brainstem of Oncorhynchus mykiss using immunohistochemical methods and Western Immunoblotting. We observed that the distribution of vimentin (Vim), nestin (Nes), and glutamine synthetase (GS), which are found in the aNSPCs of both embryonic types (neuroepithelial cells) and in the adult type (radial glia) in the cerebellum and the brainstem of trout, has certain features. Populations of the adult neural stem/progenitor cells (aNSPCs) expressing GS, Vim, and Nes have different morphologies, localizations, and patterns of cluster formation in the trout cerebellum and brainstem, which indicates the morphological and, obviously, functional heterogeneity of these cells. Immunolabeling of PCNA revealed areas in the cerebellum and brainstem of rainbow trout containing proliferating cells which coincide with areas expressing Vim, Nes, and GS. Double immunolabeling revealed the PCNA/GS PCNA/Vim coexpression patterns in the neuroepithelial-type cells in the PVZ of the brainstem. PCNA/GS coexpression in the RG was detected in the submarginal zone of the brainstem. The results of immunohistochemical study of the DC distribution in the cerebellum and brainstem of trout have showed a high level of expression of this marker in various cell populations. This may indicate: (i) high production of the adult-born neurons in the cerebellum and brainstem of adult trout, (ii) high plasticity of neurons in the cerebellum and brainstem of trout. We assume that the source of new cells in the trout brain, along with PVZ and SMZ, containing proliferating cells, may be local neurogenic niches containing the PCNA-positive and silent (PCNA-negative), but expressing NSC markers, cells. The identification of cells expressing DC, Vim, and Nes in the IX-X cranial nerve nuclei of trout was carried out.

Blastocyst-like Structures in the Peripheral Retina of Young Adult Beagles.

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

Nestin identifies a subpopulation of rat ventricular fibroblasts and participates in cell migration.

Fibroblast progenitor cells migrate to the endocardial region during cardiogenesis, and the migration of ventricular fibroblasts to the ischemically damaged region of the infarcted adult heart is a seminal event of reparative fibrosis. The intermediate filament protein nestin is implicated in cell migration and expression identified in a subpopulation of scar-derived myofibroblasts. The present study tested the hypothesis that fibroblast progenitor cells express nestin, and the intermediate filament protein drives the migratory phenotype of ventricular fibroblasts. Transcription factor 21 (Tcf21)- and Wilms tumor 1 (WT1)-fibroblast progenitor cells identified in the epicardial/endocardial regions of the E12.5- to E13.5-day embryonic mouse heart predominantly expressed nestin. Nuclear Tcf21/WT1 staining was identified in neonatal rat ventricular fibroblasts (NNVFbs), and a subpopulation coexpressed nestin. Nuclear Tcf21/WT1 expression persisted in adult rat ventricular fibroblasts, whereas nestin protein levels were downregulated. Nestin-expressing NNVFbs exhibited a unique phenotype as the subpopulation was refractory to cell cycle reentry in response to selective stimuli. Nestin(-)- and nestin(+)-scar-derived rat myofibroblasts plated in Matrigel unmasked a migratory phenotype characterized by the de novo formation of lumen-like structures. The elongated membrane projections emanating from scar myofibroblasts delineating the boundary of lumen-like structures expressed nestin. Lentiviral short-hairpin RNA (shRNA)-mediated nestin depletion inhibited the in vitro migratory response of NNVFbs as the wound radius was significantly larger compared with NNVFbs infected with the empty lentivirus. Thus, nestin represents a marker of embryonic Tcf21/WT1(+)-fibroblast progenitor cells. The neonatal rat heart contains a distinct subpopulation of nestin-immunoreactive Tcf21/WT1(+) fibroblasts refractory to cell cycle reentry, and the intermediate filament protein may preferentially facilitate ventricular fibroblast migration during physiological/pathological remodeling.NEW & NOTEWORTHY Tcf21/WT1(+)-fibroblast progenitor cells of the embryonic mouse heart predominantly express the intermediate filament protein nestin. A subpopulation of Tcf21/WT1(+)-neonatal rat ventricular fibroblasts express nestin and are refractory to selective stimuli influencing cell cycle reentry. Scar-derived myofibroblasts plated in Matrigel elicit the formation of lumen-like structures characterized by the appearance of nestin(+)-membrane projections. Lentiviral shRNA-mediated nestin depletion in a subpopulation of neonatal rat ventricular fibroblasts suppressed the migratory response following the in vitro scratch assay.

Distinct protein kinase C isoforms drive the cell cycle re-entry of two separate populations of neonatal rat ventricular cardiomyocytes.

The present study tested the hypothesis that protein kinase C-α (PKC-α) recruitment in the presence of the p38α/ß MAPK inhibitor SB203580 facilitated the appearance and cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes (NNVMs) and induced a transcript profile delineating a proliferative phenotype. Phorbol 12,13-dibutyrate (PDBu) treatment did not induce de novo nestin expression or increase the cell cycle re-entry of 1-day-old NNVMs but significantly increased runt-related transcription factor 1 (Runx1) and p16 cell cycle inhibitor (CDKN2a) mRNA levels and downregulated epithelial cell transforming 2 (ECT2) mRNA expression. SB203580 administration to PDBu-treated NNVMs induced de novo nestin expression, preferentially increased the density (normalized to 500 NNVMs) of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu, 1.4 ± 3 vs. PDBu/SB203580, 128 ± 34; n = 5 independent litters), significantly inhibited CDKN2a and Runx1 mRNA upregulation and reversed ECT2 mRNA downregulation. PDBu treatment of NNVMs reduced PKC-α, protein kinase-δ (PKC-δ) and protein kinase-ε (PKC-ε) protein levels and GF109203X (conventional PKC isoform inhibitor) selectively attenuated PKC-α protein downregulation. GF109203X administration to PDBu/SB203580-treated NNVMs significantly reduced the density of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 40 ± 46; n = 5). Moreover, GF109203X/PDBu/SB203580 treatment unmasked the predominant appearance of a separate NNVM population that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 192 ± 42; n = 5) delineated by the absence of de novo nestin expression. Sotrastaurin (conventional/novel PKC isoform inhibitor) administration to PDBu/SB203580-treated NNVMs significantly attenuated the density of nestin(+)-NNVMs (PDBu/SB203580/sotrastaurin, 8 ± 10; n = 4) and nestin(-)-NNVMs (PDBu/SB203580/sotrastaurin, 64 ± 30; n = 4) that incorporated 5-bromo-2'-deoxyuridine. These data reveal that the neonatal rat heart contains at least two separate populations of NNVMs that re-enter the cell cycle and the preferential appearance of nestin(+)- or nestin(-)-NNVMs is driven by distinct PKC isoforms in the presence of SB203580.NEW & NOTEWORTHY The appearance of nestin(+)-neonatal rat ventricular cardiomyocytes that re-entered the cell cycle following phorbol ester stimulation in the presence of p38α/ß MAPK inhibitor SB203580 was associated with the inhibition of Runx1 and CDKN2a mRNA upregulation. PKC-α selectively induced the cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes. Pharmacological inhibition of PKC-α with concomitant p38α/ß MAPK suppression unmasked the cell cycle re-entry of a second population of neonatal rat ventricular cardiomyocytes in the absence of nestin expression.

Perturbations in neural stem cell function during a neurotropic viral infection in juvenile mice.

Viral infections of the central nervous system (CNS) often cause worse neurological outcomes in younger hosts. Throughout childhood, the brain undergoes extensive development and refinement to produce functional neural networks. Network function is maintained partly with the help of neural stem cells (NSCs) that replace neuronal and glia subtypes in the two neurogenic niches of the brain (the hippocampus and subventricular zone). Accumulating evidence suggests that viruses disrupt NSC function in adulthood and infancy, but the in vivo impact of childhood infections on acute and long-term NSC function is unknown. Using a juvenile mouse model of measles virus (MeV) infection, where only mature neurons in the brain are infected, we defined the effects of the antiviral immune response on NSCs from juvenile to adult stages of life. We found that (a) virus persists in the brains of survivors despite an anti-viral immune response; (b) NSC numbers decrease dramatically during early infection, but ultimately stabilize in adult survivors; (c) infection is associated with mild apoptosis throughout the juvenile brain, but NSC proliferation is unchanged; (d) the loss of NSC numbers is dependent upon the stage of NSC differentiation; and (e) immature neurons increase early during infection, concurrent with depletion of NSC pools. Collectively, we show that NSCs are exquisitely sensitive to the inflammatory microenvironment created during neuron-restricted MeV infection in juveniles, responding with an early loss of NSCs but increased neurogenesis. These studies provide insight into potential cellular mechanisms associated with long-term neurological deficits in survivors of childhood CNS infections.

Nestin expression in osteocytes following myeloablation and during bone marrow metastasis.

Nestin is an intermediate filament protein, which was originally detected in neuroepithelial stem cells. Besides its use as a phenotypic marker of mesenchymal stem cells in the hematopoeitic stem cell niche, the functional interpretation of nestin+ cells remains elusive. We investigated the cellular expression of nestin in bone marrow trephine biopsies of MPN patients, following myeloablation at a stage of hypocellularity during early regeneration. Here, nestin is highly expressed in mature osteocytes, arteriolar endothelial and perivascular cells and small capillaries within the bone marrow space, but not in sinusoid lining cells. This is in stark contrast to nestin expression pattern in myeloproliferative neoplasms that show hypercellularity due to oncogenic driver mutations. Here, nestin is expressed exclusively in endothelial cells of arterioles, but not in osteocytes or small capillaries. Thus, the pattern of nestin expression following myeloablation inversely correlates with cellularity in the bone marrow. This nestin expression pattern is mimicking early postnatal transcriptional programming during bone marrow development. We show that nestin expression in osteocytes occurs across different species following transplant and also in bone marrow metastasis.

PDGF receptor signal mediates the contribution of Nestin-positive cell lineage to subcutaneous fat development.

The beiging of white adipose tissue (WAT) is expected to improve systemic metabolic conditions; however, the regulation and developmental origin of this process remain insufficiently understood. In the present study, the implication of platelet-derived growth factor receptor alpha (PDGFRα) was examined in the beiging of inguinal WAT (ingWAT) of neonatal mice. Using in vivo Nestin expressing cell (Nestin+) lineage tracing and deletion mouse models, we found that, in the mice with Pdgfra gene inactivation in Nestin+ lineage (N-PRα-KO mice), the growth of inguinal WAT (ingWAT) was suppressed during neonatal periods as compared with control wild-type mice. In the ingWAT of N-PRα-KO mice, the beige adipocytes appeared earlier that were accompanied by the increased expressions of both adipogenic and beiging markers compared to control wild-type mice. In the perivascular adipocyte progenitor cell (APC) niche of ingWAT, many PDGFRα+ cells of Nestin+ lineage were recruited in Pdgfra-preserving control mice, but were largely decreased in N-PRα-KO mice. This PDGFRα+ cell depletion was replenished by PDGFRα+ cells of non-Nestin+ lineage, unexpectedly resulting in an increase of total PDGFRα+ cell number in APC niche of N-PRα-KO mice over that of control mice. These represented a potent homeostatic control of PDGFRα+ cells between Nestin+ and non-Nestin+ lineages that was accompanied by the active adipogenesis and beiging as well as small WAT depot. This highly plastic nature of PDGFRα+ cells in APC niche may contribute to the WAT remodeling for the therapeutic purpose against metabolic diseases.

Muscle hypertrophy and neuroplasticity in the small bowel in short bowel syndrome.

Short bowel syndrome (SBS) is a severe, life-threatening condition and one of the leading causes of intestinal failure in children. Here we were interested in changes in muscle layers and especially in the myenteric plexus of the enteric nervous system (ENS) of the small bowel in the context of intestinal adaptation. Twelve rats underwent a massive resection of the small intestine to induce SBS. Sham laparotomy without small bowel transection was performed in 10 rats. Two weeks after surgery, the remaining jejunum and ileum were harvested and studied. Samples of human small bowel were obtained from patients who underwent resection of small bowel segments due to a medical indication. Morphological changes in the muscle layers and the expression of nestin, a marker for neuronal plasticity, were studied. Following SBS, muscle tissue increases significantly in both parts of the small bowel, i.e., jejunum and ileum. The leading pathophysiological mechanism of these changes is hypertrophy. Additionally, we observed an increased nestin expression in the myenteric plexus in the remaining bowel with SBS. Our human data also showed that in patients with SBS, the proportion of stem cells in the myenteric plexus had risen by more than twofold. Our findings suggest that the ENS is tightly connected to changes in intestinal muscle layers and is critically involved in the process of intestinal adaptation to SBS.

Nestin expression in intact and hypertrophic myocardium of spontaneously hypertensive rats during aging.

Nestin is a unique intermediate filament expressed for a short period in the developing heart. It was also documented in several cell types of the adult myocardium under pathological conditions such as myocardial infarction or fibrosis. However, circumstances of nestin re-occurrence in the diseased or aging heart have not been elucidated yet. In this work we immunohistochemically detected nestin to determine its expression and distribution pattern in the left ventricular myocardium of normotensive Wistar Kyoto (WKY) rats and in the hypertrophic ones of spontaneously hypertensive (SHR) rats, both at the age of 1 and 1.5 year. No nestin+ cells were identified in the intact myocardium of 1-year-old WKY rats, whereas in the aged 1.5-year-old WKY rats nestin+ endothelial cells in some blood vessels were discovered. In the hypertrophic myocardium of all SHR rats, nestin was rarely detected in desmin+ vimentin- cardiomyocytes and in some vimentin+ interstitial cells often accumulated in clusters, varying in intensity of desmin immunoreactivity. Moreover, nestin was infrequently expressed in the endothelial cells of some myocardial blood vessels in 1-year-old SHR rats, but not in 1.5-year-old ones. Quantitative image analysis of nestin expression in the myocardium confirmed significant increase in 1.5-year-old WKY rats and in SHR rats of both ages compared to the intact 1-year-old WKY rats. This study firstly documents nestin re-expression indicating cytoskeletal remodelling in different cell types of the aging intact and chronically pressure over-loaded hypertrophied myocardium. Our findings confirm nestin involvement in complex changes during myocardial hypertrophy and progressive aging.

Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial-mesenchymal transition in lung endothelial cells.

BACKGROUND: Nestin, an intermediate filament protein, participates in various pathophysiological processes, including wound healing, angiogenesis, endothelial-mesenchymal transition (EndoMT), and fibrosis. However, the pathophysiological roles of lung nestin-expressing cells remain unclear due to conflicting reports. The objective of this study is to elucidate the characteristics and functions of lung nestin-expressing cells. METHODS: We conducted a series of in vitro and in vivo experiments using endothelial cell line MS1 and nestin-GFP mice. This animal model allows for nestin-expressing cell detection without the use of anti-nestin antibodies. RESULTS: Lung nestin-expressing cells occurred in approximately 0.2% of CD45- cells and was co-expressed with epithelial, endothelial, and mesenchymal cell-surface markers. Importantly, virtually all nestin-expressing cells co-expressed CD31. When compared to lung nestin-nonexpressing endothelial cells, nestin-expressing endothelial cells showed robust angiogenesis with frequent co-expression of PDGFRß and VEGFR2. During TGFß-mediated EndoMT, the elevation of Nes mRNA expression preceded that of Col1a1 mRNA, and nestin gene silencing using nestin siRNA resulted in further upregulation of Col1a1 mRNA expression. Furthermore, Notch3 expression was regulated by nestin in vitro and in vivo; nestin siRNA resulted in reduced Notch3 expression accompanied with enhanced EndoMT. Contrary to previous reports, neither Nes mRNA expression nor nestin-expressing cells were increased during pulmonary fibrosis. CONCLUSIONS: Our study showed that (1) lung nestin-expressing cells are an endothelial lineage but are distinct from nestin-nonexpressing endothelial cells; (2) nestin regulates Notch3 and they act collaboratively to regulate angiogenesis, collagen production, and EndoMT; and (3) nestin plays novel roles in lung angiogenesis and fibrosis. Video Abstract.

Immune Privileges as a Result of Mutual Regulation of Immune and Stem Systems.

Immune privileges of cancer stem cells is a well-known and widely studied problem, as presence of such cells in tumors is associated with refractoriness, recurrence, and metastasis. Accumulating evidence also suggests presence of immune privileges in non-pathological stem cells in addition to their other defense mechanisms against damaging factors. This similarity between pathological and normal stem cells raises the question of why stem cells have such a potentially dangerous property. Regulation of vital processes of autoimmunity control and regeneration realized through interactions between immune cells, stem cells, and their microenvironment are reviewed in this work as causes of formation of the stem cell immune privilege. Deep mutual integration between regulations of stem and immune cells is noted. Considering diversity and complexity of mutual regulation of stem cells, their microenvironment, and immune system, I suggest the term "stem system".

PDGFRα signaling drives adipose tissue fibrosis by targeting progenitor cell plasticity.

Fibrosis is a common disease process in which profibrotic cells disturb organ function by secreting disorganized extracellular matrix (ECM). Adipose tissue fibrosis occurs during obesity and is associated with metabolic dysfunction, but how profibrotic cells originate is still being elucidated. Here, we use a developmental model to investigate perivascular cells in white adipose tissue (WAT) and their potential to cause organ fibrosis. We show that a Nestin-Cre transgene targets perivascular cells (adventitial cells and pericyte-like cells) in WAT, and Nestin-GFP specifically labels pericyte-like cells. Activation of PDGFRα signaling in perivascular cells causes them to transition into ECM-synthesizing profibrotic cells. Before this transition occurs, PDGFRα signaling up-regulates mTOR signaling and ribosome biogenesis pathways and perturbs the expression of a network of epigenetically imprinted genes that have been implicated in cell growth and tissue homeostasis. Isolated Nestin-GFP(+) cells differentiate into adipocytes ex vivo and form WAT when transplanted into recipient mice. However, PDGFRα signaling opposes adipogenesis and generates profibrotic cells instead, which leads to fibrotic WAT in transplant experiments. These results identify perivascular cells as fibro/adipogenic progenitors in WAT and show that PDGFRα targets progenitor cell plasticity as a profibrotic mechanism.

Endogenous Neural Stem Cell Activation after Low-Intensity Focused Ultrasound-Induced Blood-Brain Barrier Modulation.

Endogenous neural stem cells (eNSCs) in the adult brain, which have the potential to self-renew and differentiate into functional, tissue-appropriate cell types, have raised new expectations for neurological disease therapy. Low-intensity focused ultrasound (LIFUS)-induced blood-brain barrier modulation has been reported to promote neurogenesis. Although these studies have reported improved behavioral performance and enhanced expression of brain biomarkers after LIFUS, indicating increased neurogenesis, the precise mechanism remains unclear. In this study, we evaluated eNSC activation as a mechanism for neurogenesis after LIFUS-induced blood-brain barrier modulation. We evaluated the specific eNSC markers, Sox-2 and nestin, to confirm the activation of eNSCs. We also performed 3'-deoxy-3'[18F] fluoro-L-thymidine positron emission tomography ([18F] FLT-PET) to evaluate the activation of eNSCs. The expression of Sox-2 and nestin was significantly upregulated 1 week after LIFUS. After 1 week, the upregulated expression decreased sequentially; after 4 weeks, the upregulated expression returned to that of the control group. [18F] FLT-PET images also showed higher stem cell activity after 1 week. The results of this study indicated that LIFUS could activate eNSCs and induce adult neurogenesis. These results show that LIFUS may be useful as an effective treatment for patients with neurological damage or neurological disorders in clinical settings.

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.

BACKGROUND & AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs. METHODS: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling. RESULTS: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies. CONCLUSION: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. LAY SUMMARY: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.

Is nestin a diagnostic marker for combined hepatocellular-cholangiocarcinoma?

BACKGROUND AND AIMS: The type IV intermediate filament, nestin, may be a candidate diagnostic marker for combined hepatocellular-cholangiocarcinoma (cHCC-CCA). Therefore, the significance of nestin as a diagnostic marker for cHCC-CCA categorized by the World Health Organization (WHO) 2019 classification and its relationship with clinicopathological features were examined in the present study. METHODS AND RESULTS: Nestin expression was immunohistochemically assessed in the liver sections from 75 patients with cHCC-CCA, 22 with small duct-type intrahepatic cholangiocarcinoma (iCCA), 20 with large duct-type iCCA and 35 with hepatocellular carcinoma (HCC). Nestin expression and its relationship with clinicopathological features and genetic alterations were investigated in cHCC-CCA. Nestin expression was detected in significantly more patients with cHCC-CCA (66.7%) than in those with large duct-type iCCA (5%) (P < 0.01), HCC (2.9%) (P < 0.01) and small duct-type iCCA (40.9%) (P < 0.05). Nestin expression was partly associated with neural cell adhesion molecule (NCAM) and vimentin expression. Nestin expression was also observed in significantly more patients with small duct-type iCCA than in those with large duct-type iCCA and HCC (P < 0.01). Nestin-positive cHCC-CCA was characterized by a smaller tumour size, the more frequent presence of cholangiolocellular carcinoma (CLC) components, a higher rate of p53 overexpression and a higher rate of multiple genetic alterations (P < 0.05). Furthermore, p53 overexpression was associated with a higher histological grade and multiple genetic alterations (P < 0.05) in nestin-positive cHCC-CCA. CONCLUSION: Nestin may be a useful diagnostic marker for a specific subgroup of cHCC-CCA and small duct-type iCCA associated with CLC components, p53 mutations and multiple genetic alterations, which are related to stemness and multipotent differentiation.