p53-dependent Nestin regulation links tumor suppression to cellular plasticity in liver cancer.

The p53 tumor suppressor coordinates a series of antiproliferative responses that restrict the expansion of malignant cells, and as a consequence, p53 is lost or mutated in the majority of human cancers. Here, we show that p53 restricts expression of the stem and progenitor-cell-associated protein nestin in an Sp1/3 transcription-factor-dependent manner and that Nestin is required for tumor initiation in vivo. Moreover, loss of p53 facilitates dedifferentiation of mature hepatocytes into nestin-positive progenitor-like cells, which are poised to differentiate into hepatocellular carcinomas (HCCs) or cholangiocarcinomas (CCs) in response to lineage-specific mutations that target Wnt and Notch signaling, respectively. Many human HCCs and CCs show elevated nestin expression, which correlates with p53 loss of function and is associated with decreased patient survival. Therefore, transcriptional repression of Nestin by p53 restricts cellular plasticity and tumorigenesis in liver cancer.

Clinical trial links oncolytic immunoactivation to survival in glioblastoma.

Immunotherapy failures can result from the highly suppressive tumour microenvironment that characterizes aggressive forms of cancer such as recurrent glioblastoma (rGBM)1,2. Here we report the results of a first-in-human phase I trial in 41 patients with rGBM who were injected with CAN-3110-an oncolytic herpes virus (oHSV)3. In contrast to other clinical oHSVs, CAN-3110 retains the viral neurovirulence ICP34.5 gene transcribed by a nestin promoter; nestin is overexpressed in GBM and other invasive tumours, but not in the adult brain or healthy differentiated tissue4. These modifications confer CAN-3110 with preferential tumour replication. No dose-limiting toxicities were encountered. Positive HSV1 serology was significantly associated with both improved survival and clearance of CAN-3110 from injected tumours. Survival after treatment, particularly in individuals seropositive for HSV1, was significantly associated with (1) changes in tumour/PBMC T cell counts and clonal diversity, (2) peripheral expansion/contraction of specific T cell clonotypes; and (3) tumour transcriptomic signatures of immune activation. These results provide human validation that intralesional oHSV treatment enhances anticancer immune responses even in immunosuppressive tumour microenvironments, particularly in individuals with cognate serology to the injected virus. This provides a biological rationale for use of this oncolytic modality in cancers that are otherwise unresponsive to immunotherapy (ClinicalTrials.gov: NCT03152318 ).

In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics.

Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage tracing in vivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multilineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal but ultimately leads to terminal astrocytic differentiation and RGL depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into in vivo properties of adult neural stem cells.

Nestin+ Mesenchymal Stromal Cells Fibrotic Transition Mediated by CD169+ Macrophages in Bone Marrow Chronic Graft-versus-Host Disease.

Chronic graft-versus-host disease (cGVHD) involves multiple organs, but little is known about bone marrow (BM) alterations caused by cGVHD. In mice and humans, we found that cGVHD is associated with BM fibrosis resulting in T cell infiltration, IgG deposition, and hematopoietic dysfunction. Macrophages and Nestin+ mesenchymal stromal cells (MSCs) participated in the process of BM fibrosis during BM cGVHD development. BM macrophage numbers were significantly increased in mice and humans with BM fibrosis associated with cGVHD. Amplified macrophages produced TGF-ß1, which recruited Nestin+ MSCs forming clusters, and Nestin+ MSCs later differentiated into fibroblasts, a process mediated by increased TGF-ß/Smad signaling. TLR4/MyD88-mediated activation of endoplasmic reticulum (ER) stress in macrophages is associated with fibrosis by increasing Nestin+ MSC migration and differentiation into fibroblasts. Depletion of macrophages by clodronate-containing liposomes and inhibition of ER stress by 4-phenylbutyric acid reversed BM fibrosis by inhibiting fibroblast differentiation. These studies provide insights into the pathogenesis of BM fibrosis during cGVHD development.

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.

TET1 Regulates Nestin Expression and Human Airway Smooth Muscle Proliferation.

Asthma is characterized by aberrant airway smooth muscle (ASM) proliferation, which increases the thickness of the ASM layer within the airway wall and exacerbates airway obstruction during asthma attacks. The mechanisms that drive ASM proliferation in asthma are not entirely elucidated. Ten-eleven translocation methylcytosine dioxygenase (TET) is an enzyme that participates in the regulation of DNA methylation by catalyzing the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). The generation of 5-hmC disinhibits the gene silencing effect of 5-mC. In this study, TET1 activity and protein were enhanced in asthmatic human ASM cell cultures. Moreover, the concentration of 5-hmC was higher in asthmatic ASM cells than in nonasthmatic ASM cells. Knockdown (KD) of TET1, but not TET2, reduced the concentration of 5-hmC in asthmatic cells. Because the cytoskeletal protein nestin controls cell proliferation by modulating mTOR, we evaluated the effects of TET1 KD on this pathway. TET1 KD reduced nestin expression in ASM cells. In addition, TET1 inhibition alleviated the platelet-derived growth factor-induced phosphorylation of p70S6K, 4E-BP, S6, and Akt. TET1 inhibition also attenuated the proliferation of ASM cells. Taken together, these results suggest that TET1 drives ASM proliferation via the nestin-mTOR axis.

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.

Cardiac remodeling associated with chronic kidney disease is enhanced in a rat model of metabolic syndrome: Preparation of mesenchymal transition.

Cardiac alteration due to chronic kidney disease is described by tissue fibrosis. This remodeling involves myofibroblasts of various origins, including epithelial or endothelial to mesenchymal transitions. In addition, obesity and insulin resistance together or separately seem to exacerbate cardiovascular risk in chronic kidney disease (CKD). The main objective of this study was to assess if pre-existing metabolic disease exacerbates CKD-induced cardiac alterations. In addition, we hypothesised that endothelial to mesenchymal transition participates in this enhancement of cardiac fibrosis. Rats fed cafeteria type diet for 6 months underwent a subtotal nephrectomy at 4 months. Cardiac fibrosis was evaluated by histology and qRT-PCR. Collagens and macrophages were quantified by immunohistochemistry. Endothelial to mesenchymal transitions were assessed by qRT-PCR (CD31, VE-cadherin, α-SMA, nestin) and also by CD31 immunofluorescence staining. Rats fed with cafeteria type regimen were obese, hypertensive and insulin resistant. Cardiac fibrosis was predominant in CKD rats and was highly majored by cafeteria regimen. Collagen-1 and nestin expressions were higher in CKD rats, independently of regimen. Interestingly, in rats with CKD and cafeteria diet we found an increase of CD31 and α-SMA co-staining with suggest an implication of endothelial to mesenchymal transition during heart fibrosis. We showed that rats already obese and insulin resistant had an enhanced cardiac alteration to a subsequent renal injury. Cardiac fibrosis process could be supported by a involvement of the endothelial to mesenchymal transition phenomenon.

Homer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation.

OBJECTIVE: Proinflammatory necroptosis is the main pathological mechanism of ischemic stroke. Homer scaffolding protein 1 (Homer1) is a postsynaptic scaffolding protein that exerts anti-inflammatory effects in most central nervous system diseases. However, the relationship between Homer1 and proinflammatory necroptosis in ischemic stroke remains unclear. AIM: This study aimed to investigate the role of Homer1 in ischemia-induced necroptosis. METHODS: C57BL/6 mice were used to establish a model of permanent middle cerebral artery occlusion model (pMCAO). Homer1 knockdown mice were generated using adeno-associated virus (AAV) infection to explore the role of Homer1 and its impact on necroptosis in pMCAO. Finally, Homer1 protein was stereotaxically injected into the ischemic cortex of Homer1flox/flox/Nestin-Cre +/- mice, and the efficacy of Homer1 was investigated using behavioral assays and molecular biological assays to explore potential mechanisms. RESULTS: Homer1 expression peaked at 8 h in the ischemic penumbral cortex after pMCAO and colocalized with neurons. Homer1 knockdown promoted neuronal death by enhancing necroptotic signaling pathways and aggravating ischemic brain damage in mice. Furthermore, the knockdown of Homer1 enhanced the expression of proinflammatory cytokines. Moreover, injection of Homer1 protein reduced necroptosis-induced brain injury inhibited the expression of proinflammatory factors, and ameliorated the outcomes in the Homer1flox/flox/Nestin-Cre+/- mice after pMCAO. CONCLUSIONS: Homer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation. These data suggested that Homer1 is a novel regulator of neuronal death and neuroinflammation.

Pituitary tumours without distinct lineage differentiation express stem cell marker SOX2.

CONTEXT: The recent WHO 2022 Classification of pituitary tumours identified a novel group of 'plurihormonal tumours without distinct lineage differentiation (WDLD)'. By definition, these express multiple combinations of lineage commitment transcription factors, in a monomorphous population of cells. OBJECTIVES: To determine the expression of stem cell markers (SOX2, Nestin, CD133) within tumours WDLD, immature PIT-1 lineage and acidophil stem cell tumours, compared with committed cell lineage tumours. METHODS: Retrospective evaluation of surgically resected pituitary tumours from St Vincent's Hospital, Sydney. Patients were selected to cover a range of tumour types, based on transcription factor and hormone immunohistochemistry. Clinical data was collected from patient files. Radiology reports were reviewed for size and invasion. Samples were analysed by immunohistochemistry and RT-qPCR for SF-1, PIT-1, T-PIT, SOX2, Nestin and CD133. Stem cell markers were compared between tumours WDLD and those with classically "mature" types. RESULTS: On immunohistochemistry, SOX2 was positive in a higher proportion of tumours WDLD compared with those meeting WHO lineage criteria, 7/10 v 10/42 (70 v 23.4%, p = 0.005). CD133 was positive in 2/10 tumours WDLD but 0/41 meeting lineage criteria, P = 0.003. On RT-qPCR, there was no significant difference in relative expression of stem cell markers (SOX2, CD133, Nestin) between tumours with and WDLD. CONCLUSIONS: Our study is the first to biologically characterise pituitary tumours WDLD. We demonstrate that these tumours exhibit a higher expression of the stem cell marker SOX2 compared with other lineage-differentiated tumours, suggesting possible involvement of stem cells in their 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.

Immunohistochemical analyses of neural stem cell lineage markers in normal feline brains and glial tumors.

Neural stem cell (NSC) lineage cells have not been fully identified in feline brains, and the NSC-like nature of feline glial tumors has not been determined. In this study, 6 normal cat brains (3 newborn and 3 older cats) and 13 feline glial tumors were analyzed using immunohistochemical NSC lineage markers. The feline glial tumors were subjected to immunohistochemical scoring followed by hierarchical cluster analysis. In newborn brains, glial acidic fibrillary protein (GFAP)/nestin/sex-determining region Y-box transcription factor 2 (SOX2)-immunopositive NSCs, SOX2-immunopositive intermediate progenitor cells, oligodendrocyte transcription factor 2 (OLIG2)/platelet-derived growth factor receptor-α (PDGFR-α)-immunopositive oligodendrocyte precursor cells (OPCs), OLIG2/GFAP-immunopositive immature astrocytes, and neuronal nuclear (NeuN)/ß-3 tubulin-immunopositive mature neuronal cells were observed. The apical membrane of NSCs was also immunopositive for Na+/H+ exchanger regulatory factor 1 (NHERF1). In mature brains, the NSC lineage cells were similar to those of the newborn brains. A total of 13 glial tumors consisted of 2 oligodendrogliomas, 4 astrocytomas, 3 subependymomas, and 4 ependymomas. Astrocytomas, subependymomas, and ependymomas were immunopositive for GFAP, nestin, and SOX2. Subependymomas and ependymomas showed dot-like or apical membrane immunolabeling for NHERF1, respectively. Astrocytomas were immunopositive for OLIG2. Oligodendrogliomas and subependymomas were immunopositive for OLIG2 and PDGFR-α. Feline glial tumors also showed variable immunolabeling for ß-3 tubulin, NeuN, and synaptophysin. Based on these results, feline astrocytomas, subependymomas, and ependymomas appear to have an NSC-like immunophenotype. In addition, astrocytomas, subependymomas, and ependymomas have the characteristics of glial, oligodendrocyte precursor, and ependymal cells, respectively. Feline oligodendrogliomas likely have an OPC-like immunophenotype. In addition, feline glial tumors may have multipotential stemness for differentiation into neuronal cells. These preliminary results should be validated by gene expression analyses in future studies with larger case numbers.

Chemical Coaxing of Mesenchymal Stromal Cells by Drug Repositioning for Nestin Induction.

Mesenchymal stromal cells (MSCs) display heterogeneity in origin and functional role in tissue homeostasis. Subsets of MSCs derived from the neural crest express nestin and serve as niches in bone marrow, but the possibility of coaxing MSCs into nestin-expresing cells for enhanced supportive activity is unclear. In this study, as an approach to the chemical coaxing of MSC functions, we screened libraries of clinically approved chemicals to identify compounds capable of inducing nestin expression in MSCs. Out of 2000 clinical compounds, we chose vorinostat as a candidate to coax the MSCs into neural crest-like fates. When treated with vorinostat, MSCs exhibited a significant increase in the expression of genes involved in the pluripotency and epithelial-mesenchymal transition (EMT), as well as nestin and CD146, the markers for pericytes. In addition, these nestin-induced MSCs exhibited enhanced differentiation towards neuronal cells with the upregulation of neurogenic markers, including SRY-box transcription factor 2 (Sox2), SRY-box transcription factor 10 (Sox10) and microtubule associated protein 2 (Map2) in addition to nestin. Moreover, the coaxed MSCs exhibited enhanced supporting activity for hematopoietic progenitors without supporting leukemia cells. These results demonstrate the feasibility of the drug repositioning of MSCs to induce neural crest-like properties through the chemical coaxing of cell fates.

CD26 Is Differentially Expressed throughout the Life Cycle of Infantile Hemangiomas and Characterizes the Proliferative Phase.

Infantile hemangiomas (IHs) are benign vascular neoplasms of childhood (prevalence 5-10%) due to the abnormal proliferation of endothelial cells. IHs are characterized by a peculiar natural life cycle enclosing three phases: proliferative (≤12 months), involuting (≥13 months), and involuted (up to 4-7 years). The mechanisms underlying this neoplastic disease still remain uncovered. Twenty-seven IH tissue specimens (15 proliferative and 12 involuting) were subjected to hematoxylin and eosin staining and a panel of diagnostic markers by immunohistochemistry. WT1, nestin, CD133, and CD26 were also analyzed. Moreover, CD31pos/CD26pos proliferative hemangioma-derived endothelial cells (Hem-ECs) were freshly isolated, exposed to vildagliptin (a DPP-IV/CD26 inhibitor), and tested for cell survival and proliferation by MTT assay, FACS analysis, and Western blot assay. All IHs displayed positive CD31, GLUT1, WT1, and nestin immunostaining but were negative for D2-40. Increased endothelial cell proliferation in IH samples was documented by ki67 labeling. All endothelia of proliferative IHs were positive for CD26 (100%), while only 10 expressed CD133 (66.6%). Surprisingly, seven involuting IH samples (58.3%) exhibited coexisting proliferative and involuting aspects in the same hemangiomatous lesion. Importantly, proliferative areas were characterized by CD26 immunolabeling, at variance from involuting sites that were always CD26 negative. Finally, in vitro DPP-IV pharmacological inhibition by vildagliptin significantly reduced Hem-ECs proliferation through the modulation of ki67 and induced cell cycle arrest associated with the upregulation of p21 protein expression. Taken together, our findings suggest that CD26 might represent a reliable biomarker to detect proliferative sites and unveil non-regressive IHs after a 12-month life cycle.

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.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Predictivism and avoidance of ad hoc-ness: An empirical study.

Predictivism is the thesis that evidence successfully predicted by a scientific theory counts more (or ought to count more) in the confirmation of that theory than already known evidence would. One rationale that has been proposed for predictivism is that predictive success guards against ad hoc hypotheses. Despite the intuitive attraction of predictivism, there is historical evidence that speaks against it. As valuable as the historical evidence may be, however, it is largely indirect evidence for the epistemic attitudes of individual - albeit prominent - scientists. This paper presents the results of an empirical study of scientists' attitudes toward predictivism and ad hoc-ness (n = 492), which will put the debate on a more robust empirical footing. The paper also draws attention to a tension between the ad hoc-ness avoidance rationale of predictivism and the ways philosophers have spelled out the notion of ad hoc-ness.

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.