Exosomes with Engineered Brain Derived Neurotrophic Factor on Their Surfaces Can Proliferate Menstrual Blood Derived Mesenchymal Stem Cells: Targeted Delivery for a Protein Drug.

Despite the efficacy of brain derived neurotrophic factor (BDNF) in neuro-regenerative medicine, it can't pass the blood-brain barrier. Recently, exosomes have been harnessed for targeted delivery of therapeutics into brain. Given these facts, an engineered exosome capable of BDNF expression on the surface would be an amenable tool for drug delivery. The BDNF gene was cloned into a plex-lamp lentiviral vector and virus particles were packaged using the Torano method. HEK293T cells were transduced by the purified viruses to produce and purify recombinant exosomes displaying the fusion protein on their surfaces. Western blot, Zeta sizer, TEM, and ELISA methods were used for exosome characterization. The effect of engineered exosomes on menstrual blood-derived mesenchymal stem cells (Mens-MSCs) proliferation was evaluated by cell counting assay, MTT assay, and qPCR on the bcl2 and nestin genes. Approximately, 1.8 × 108 TdU/ml of the viral particles was purified from the transfected cells and transduced into the HEK293T. Western blot and ELISA methods confirmed the surface display of the LAMP-BDNF fusion. TEM graphs and Zeta sizer results confirmed the morphology and the size of purified exosomes. Treatment of Mens-MSCs with the targeted exosomes augmented the expression level of bcl2 and nestin genes, increased the cell proliferation, and elevated the cell number. Chimeric BDNF on the exosome surface could retain its biological activity and elevate the expression of bcl2 and nestin genes. Moreover, these exosomes are capable of elevating the Mens-MSCs proliferation.

A Systematic Review of Delta-9-Tetrahydrocannabinol (∆9-THC) in Astrocytic Markers.

BACKGROUND: Astrocytic reactivity in substance use disorders (SUDs) has been extensively studied, yet the molecular effect of delta-9-tetrahydrocannabinol (∆9-THC, the main psychoactive compound in cannabis) on glial cells, especially astrocytes, remains poorly understood. Exploring ∆9-THC's impact on astrocytic markers can provide insight into its effects on brain functions such as homeostasis, synaptic transmission, and response to neuronal injury. This systematic review synthesizes findings from studies investigating ∆9-THC's impact on astrocytic markers. METHODS: A systematic review was conducted using EMBASE, Medline, and PsychoInfo via the OvidSP platform. Studies reporting astrocytic markers following ∆9-THC exposure in animals and humans were included. Data were extracted from twelve eligible full-text articles, and the risk of bias was assessed using the Systematic Review Center for Laboratory Animal Experimentation. RESULTS: This research identified several astrocytic markers, including glial fibrillary acidic protein (GFAP), nestin, and glutamate-aspartate transporter (GLAST). Both GFAP and nestin expressions increased in adulthood following adolescence and adult ∆9-THC exposure. An increase in GLAST expression was also noted during early development after ∆9-THC exposure. CONCLUSIONS: This review indicates varying levels of astrocytic reactivity to ∆9-THC across different developmental stages, including adolescence and adulthood. ∆9-THC appears to impact maturation, particularly during early developmental stages, and exhibits sex-dependent effects.

The effects of primary cilia-mediated mechanical stimulation on nestin+-BMSCs during bone-tendon healing.

INTRODUCTION: Mechanical stimulation has been proven to promote bone-tendon interface (BTI) healing, but the mechanism remains unclear. OBJECTIVE: To investigate the effects of mechanical stimulation on the biological behavior of nestin+-bone mesenchymal stem cells (BMSCs) during the BTI healing, and to reveal the mechanisms of mechanical stimulation affecting BTI healing by primary cilia on the nestin+-BMSCs. METHODS: Transgenic tracing mice (nestin creERT2:: IFT88fl/fl/ROSA26 YFP) with primary cilia on nestin+-BMSCs conditioned knocked out were constructed, and the littermates (nestin creERT2:: ROSA26 YFP) with normal cilia on nestin+-BMSCs were the control. After establishing mouse supraspinatus insertion injury models, samples were collected at week-2 (n = 5 per group), 4 and 8 (n = 15 per group, respectively). In vivo, the repair efficiency was evaluated by histology, imaging, biomechanics, and the migration of nestin+-BMSCs, detected by immunofluorescence staining. In vitro, nestin+ BMSCs were sorted and stimulated by tensile force to study the mechanisms of primary cilium-mediated mechanosensitive basis. RESULTS: Mechanical stimulation (MS) accelerated the recruitment of nestin+-BMSCs and promoted osteogenic and chondrogenic capacity. Histological, imaging and biomechanical results showed that the BTI healing quality of the IFT88+/+, MS group was better than that of the other groups. After the conditionally knockout IFT88 in nestin+-BMSCs, the repair ability of the BTI was obviously deteriorated, even though mechanical stimulation did not increase significantly (IFT88-/-, MS group). In vitro results showed the tensile loading enhanced the proliferation, migration and osteogenic or chondrogenic gene expression of nestin+-BMSCs with normal cilia. On the other hand, osteogenesis and chondrogenic expression were significantly decreased after inhibiting actin- Hippo/YAP pathway components. CONCLUSION: The primary cilia mediated mechanical stimulation regulated osteogenic and chondrogenic differentiation potential of nestin+-BMSCs through the actin- Hippo/YAP pathway, and then promoted the BTI healing process.

p75ECD-Fc reverses neonatal hypoxic-ischemic encephalopathy-induced neurological deficits and inhibits apoptosis associated with Nestin.

A recent study has introduced a recombinant fusion protein, consisting of the extracellular domain (ECD) of p75 and the Fc fragment of human immunoglobulin IgG1 (p75ECD-Fc), as a multifaceted agent within the nervous system. This research aimed to assess the effects of p75ECD-Fc on neuronal growth and the restoration of neurological functions in rats afflicted with neonatal hypoxic-ischemic encephalopathy (NHIE). In vitro analyses revealed that 1 µM p75ECD-Fc treatment markedly increased cell viability and facilitated neurite outgrowth in neurons exposed to oxygen-glucose deprivation (OGD). Subsequent in vivo studies determined that a dose of 78.6 µg/3 µl of p75ECD-Fc significantly mitigated brain damage and both acute and long-term neurological impairments, outperforming the therapeutic efficacy of hypothermia, as evidenced through behavioral assessments. Additionally, in vivo immunostaining showed that p75ECD-Fc administration enhanced neuronal survival and regeneration, and reduced astrocytosis and microglia activation in the cortex and hippocampus of NHIE rats. A noteworthy shift from A1 to A2 astrocyte phenotypes and from M1 to M2 microglia phenotypes was observed after p75ECD-Fc treatment. Furthermore, a co-expression of the p75 neurotrophin receptor (p75NTR) and Nestin was identified, with an overexpression of Nestin alleviating the neurological dysfunction induced by NHIE. Mechanistically, the neuroprotective effects of p75ECD-Fc, particularly its inhibition of neuronal apoptosis post-OGD, may be attributed to Nestin. Taken together, these results highlight the neuroprotective and anti-inflammatory effects of p75ECD-Fc treatment through the modulation of glial cell phenotypes and the Nestin-mediated inhibition of neuronal apoptosis, positioning it as a viable therapeutic approach for NHIE.

Regulation of a novel circATP8B4/miR-31-5p/nestin ceRNA crosstalk in proliferation, motility, invasion and radiosensitivity of human glioma cells.

Deregulation of circular RNAs (circRNAs) is frequent in human glioma. Although circRNA ATPase phospholipid transporting 8B4 (circATP8B4) is highly expressed in glioma, its precise action in glioma development is still not fully understood. The relationship of microRNA (miR)-31-5p and circATP8B4 or nestin (NES) was predicted by bioinformatic analysis and confirmed by RNA pull-down and Dual-luciferase reporter assays. CircATP8B4, miR-31-5p and NES were quantified by qRT-PCR or western blot. Cell functional behaviors were assessed by EdU, wound-healing and transwell invasion assays. Xenograft model experiments were performed to define circATP8B4's activity in vivo. CircATP8B4, a true circular transcript, was upregulated in human glioma. CircATP8B4 downregulation weakened glioma cell growth, motility, and invasion and facilitated radiosensitivity. Mechanistically, circATP8B4 and NES 3'UTR harbored a shared miR-31-5p pairing site, and circATP8B4 involved the post-transcriptional NES regulation by functioning as a competing endogenous RNA (ceRNA). Furthermore, the miR-31-5p/NES axis participated in circATP8B4's activity in glioma cell proliferation, motility, invasion and radiosensitivity. Additionally, circATP8B4 loss diminished tumor growth and enhanced the anticancer effect of radiotherapy in vivo. We have uncovered an uncharacterized ceRNA cascade, circATP8B4/miR-31-5p/NES axis, underlying glioma development and radiosensitivity. Targeting the ceRNA crosstalk may have potential to improve the outcome of glioma patients.

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.

The Role of Serum Nestin in Diagnosis and Prognosis of Breast Cancer: A Prospective Observational Study.

BACKGROUND/AIM: The molecular classification of breast cancer has enabled targeted therapy for specific molecular subtypes. Nestin, which has been studied for its role in oncogenesis, could contribute to this direction. This study aimed to investigate the differences between serum nestin levels and molecular profiling, as well as histopathological tumor types, in women who underwent surgery for breast cancer. PATIENTS AND METHODS: Women who underwent surgery for breast cancer at the Breast Unit of the 1st Propaedeutic Department of Surgery, Hippocration General Hospital, National and Kapodistrian University of Athens were prospectively included. Patients' demographic data were recorded and serum nestin levels were measured. Molecular biomarker analysis was performed, as well as histopathologic assessment. RESULTS: Seventy patients were included in the analysis. Among patients with breast cancer, 93% were estrogen receptor (ER) positive, 91% were progesterone receptor (PR) positive, and 43% were human epidermal growth factor receptor 2 (HER2) positive. Ki67 was expressed in 16% of patients and p53 was expressed in 32% of patients. Invasive ductal carcinoma was diagnosed in 80% of patients, with 44% of tumors classified as T1 and 46% as T2. Additionally, 43% were G1 and 56% were N0, while 34% were N1. No statistically significant difference was observed between serum nestin levels and ER, PR, HER2, Ki67, and p53 expression. Furthermore, no difference was observed between serum nestin levels and breast cancer histological type, size, N-stage, and grading. CONCLUSION: The diagnostic and prognostic role of circulating nestin for breast cancer was not confirmed and no correlation with immunohistochemistry results was observed. Thus, the necessity of larger prospective studies is enhanced.

Significance of Nestin and CD133 as cancer stem cell markers in diffuse glioma and association with p53 expression and IDH status.

BACKGROUND: Recent evidence suggests that the tumor stem cells that are responsible for the pathogenesis of gliomas have similar properties to those of neural stem cells. We have studied two of the most consistently expressed stem cell markers in gliomas, i.e., CD133 and Nestin, and compared them with respect to p53 expression and IDH status. OBJECTIVES: To assess the level of expression of Nestin and CD133, and identify a correlation among various grades of diffuse glioma with IDH status and expression of p53. MATERIALS AND METHODS: A cross-sectional retrospective study with 102 subjects for the expression of cancer stem cell markers; CD133 and Nestin and the correlation of their expression with that of p53 and IDH1 status in adult diffuse glioma. The study was conducted in the Departments of Pathology and Neurosurgery. The expression was assessed by immunohistochemistry on formalin-fixed paraffin-embedded sections. The scoring of expression of CD133 and Nestin was adapted from Zhang et al. The scoring for p53 was adopted from Aruna et al. Results: The diffuse gliomas were graded based on WHO into grade II (30.3%), grade III (28.4%), and grade IV (41.3%). Among WHO grade IV, 59.4% were primary, and 40.4% were secondary glioblastomas. 73% of the diffuse gliomas were IDH mutant, and p53 showed an overall expression of 76.4%. The expression of CD133 and Nestin were compared with the increasing grades of diffuse gliomas, which, when plotted on ROC curves, had AUCs of 0.6806 and 0.6119, respectively. Their expression showed a positive correlation with the IDH status of the tumor. CONCLUSIONS: Cancer stem cell markers CD133 and Nestin are expressed in diffuse glioma and have a higher expression with increasing WHO grade of malignancy. These cancer stem cell markers have shown significant association with the IDH-1 mutant status of diffuse gliomas. Hence, it can be inferred that diffuse gliomas with a higher expression of CD133 and Nestin have a poorer prognosis. Further, these cancer stem cell markers may be used as therapeutic targets in the future.

The Effect of Bone Marrow Mesenchymal Stem Cells on Nestin and Sox-2 Gene Expression and Spatial Learning (Percent Alternation Y-Maze Test) against AlCl3-Induced Alzheimer's-like Pathology in a Rat Model.

Background: Alzheimer's disease (AD) is a neurodegenerative condition characterized by gradual cognitive impairment, including loss of synapses and nerve cells involved in learning, memory, and habit formation processes. Bone Marrow Mesenchymal Stem Cells (BM-MSCs) are multipotent cells. Because of their self-renewable, differentiation, and immunomodulatory capabilities, they are commonly used to treat many disorders. Hence, the current study intends to examine the effect of BM-MSCs transplantation on Aluminum chloride (AlCl3)-induced cognitive problems, an experimental model resembling AD's hallmarks in rats. Methods: The study was conducted in 2022 at The Biomedical Laboratory Faculty of Medicine, Andalas University, Indonesia. Adult male Wistar rats (three groups: negative control; no intervention+treatment with PBS; positive control: AlCl3+treatment with aqua dest; AlCl3+BM-MSCs: AlCl3+treatment with BM-MSCs, n=5 each) were treated daily with AlCl3 orally for five days. Stem cells were intraperitoneally injected into rats at a dose of 1x106 cells/rat. The same quantity of phosphate-buffered saline was given to the control group. One month after stem cell injection, the rat brain tissue was removed and placed in the film bottles that had been created. The expression of neural progenitor cell markers, including nestin and sex-determining Y-box 2 (SOX-2), was analyzed using real-time polymerase chain reaction (RT-PCR). Rats' cognitive and functional memory were examined using Y-maze. Data were analyzed using SPSS software (version 26.0) with a one-way analysis of variance (ANOVA) test. Results: The gene expression of nestin (29.74±0.42), SOX-2 (31.44±0.67), and percent alternation of Y-maze (67.04±2.28) increased in the AlCl3+BM-MSCs group compared to that in the positive control group. RT-PCR analysis indicated that nestin (P<0.001) and SOX-2 (P<0.001) were significantly enhanced in the AlCl3+BM-MSCs group compared to the positive control group. This group also indicated an increased percent alternation of Y-maze (P<0.001) in the AlCl3+BM-MSCs group compared to the positive control group. Conclusion: Due to its potential effects on cell therapy, BM-MSCs were found effective in a rat model of AD on the impairment of the rats' behavior and increased expression of neural progenitor cell markers.

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.

Combined hepatocellular cholangiocarcinoma: A clinicopathological update.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer associated with an appalling prognosis. The diagnosis and management of this entity have been challenging to physicians, radiologists, surgeons, pathologists, and oncologists alike. The diagnostic and prognostic value of biomarkers such as the immunohistochemical expression of nestin, a progenitor cell marker, have been explored recently. With a better understanding of biology and the clinical course of cHCC-CCA, newer treatment modalities like immune checkpoint inhibitors are being tried to improve the survival of patients with this rare disease. In this review, we give an account of the recent developments in the pathology, diagnostic approach, and management of cHCC-CCA.

Exposure to Low-Frequency Radiation Changes the Expression of Nestin, VEGF, BCRP and Apoptosis Markers During Glioma Treatment Strategy: An In Vitro Study.

BACKGROUND: Exposure to physical contamination during chemotherapy, including non-ionizing electromagnetic fields, raises concerns about the widespread sources of exposure to this type of radiation. Glioblastoma multiforme (GBM) is an aggressive central nervous system tumor that is hard to treat due to resistance to drugs such as temozolomide (TMZ). OBJECTIVE: Electromagnetic fields (EMF) and haloperidol (HLP) may have anticancer effects. In this study, we investigated the effects of TMZ, HLP, and EMF on GBM cell lines and analyzed the association between non-ionizing radiation and the risk of change in drug performance. METHODS: Cell viability and reactive oxygen species (ROS) generation were measured by MTT and NBT assay, respectively. Then, the expression levels of breast cancer-resistant protein (BCRP), Bax, Bcl2, Nestin, vascular endothelial growth factor (VEGF) genes, and P53, Bax, and Bcl2 Proteins were evaluated by real-time PCR and western blot. RESULTS: Co-treatment of GBM cells by HLP and TMZ enhanced apoptosis in T-98G and A172 cells by increasing the expression of P53 and Bax and decreasing Bcl-2. Interestingly, exposure of GBM cells to EMF decreased apoptosis in the TMZ+HLP group. CONCLUSION: In conclusion, EMF reduced the synergistic effect of TMZ and HLP. This hypothesis that patients who are treated for brain tumors and suffer from depression should not be exposed to EMF is proposed in the present study. There appears to be an urgent need to reconsider exposure limits for low-frequency magnetic fields, based on experimental and epidemiological research, the relationship between exposure to non-ionizing radiation and adverse human health effects.

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.

Expression profiling of stemness markers in testicular germline stem cells from neonatal and adult Swiss albino mice during their transdifferentiation in vitro.

BACKGROUND: Spermatogonial stem cells (SSCs) were considered to be stem cells with limited potencies due to their existence in adult organisms. However, the production of spermatogonial stem cell colonies with broader differentiation capabilities in primary germ cell cultures from mice of select genetic backgrounds (C57BL6/Tg14, ddY, FVB and 129/Ola) indicated that SSCs from these strains were pluripotent. METHODS: We established primary cultures of SSCs from neonatal and adult Swiss 3T3 Albino mice. Stemness of SSC colonies were evaluated by performing real-time PCR and immunofluorescence analysis for a panel of chosen stemness markers. Differentiation potentials of SSCs were examined by attempting the generation of embryoid bodies and evaluating the expression of ectodermal, mesodermal and endodermal markers using immunofluorescence and real-time PCR analysis. RESULTS: Spermatogonial stem cells from neonatal and mature mice testes colonised in vitro and formed compact spermatogonial stem cell colonies in culture. The presence of stem cell markers ALPL, ITGA6 and CD9 indicated stemness in these colonies. The differentiation potential of these SSC colonies was demonstrated by their transformation into embryoid bodies upon withdrawal of growth factors from the culture medium. SSC colonies and embryoid bodies formed were evaluated using immunofluorescence and real-time PCR analysis. Embryoid body like structures derived from both neonatal and adult mouse testis were quite similar in terms of the expression of germ layer markers. CONCLUSION: These results strongly suggest that SSC-derived EB-like structures could be used for further differentiation into cells of interest in cell-based therapeutics.

Neural and Neural Crest-Derived Stem Cell Distribution in the Adult Human Dental Pulp / Distribución de Células Madre Neurales y Derivadas de la Cresta Neural en la Pulpa Dental Humana Adulta

SUMMARY: Despite comprehensive studies and reports about the properties of dental pulp stem cells (DPSCs) in vitro, we still need to confirm whether these in vitro characteristics coincide with the nature of DPSCs in situ. The anatomical location of DPSCs populations in the dental pulp has yet to be investigated. Moreover, the mesenchymal DPSCs have been much more studied than the neural crest-derived DPSCs. In this study, well-recognized neural/neural crest stem cell markers NCAM1, Nestin, SNAIL/SLUG, SOX9, and S100 are being investigated by immunohistochemistry to localize the precise location of these populations of DPSCs within the human adult dental pulp.All previously mentioned markers were expressed in the dental pulp, and their intensity and location of expression were reported.

A pesar de estudios e informes exhaustivos sobre las propiedades de las células madre de la pulpa dental (DPSC) in vitro, todavía necesitamos confirmar si estas características in vitro coinciden con la naturaleza de las DPSC in situ. La ubicación anatómica de las poblaciones de DPSC en la pulpa dental aún no se ha investigado. Además, las DPSC mesenquimales han sido mucho más estudiadas que las DPSC derivadas de la cresta neural. En este estudio, se están investigando mediante inmunohisto química marcadores de células madre de la cresta neural/ neural NCAM1, Nestin, SNAIL/SLUG, SOX9 y S100 para localizar la ubicación precisa de estas poblaciones de DPSC dentro de la pulpa dental humana adulta. Todos los marcadores mencionados anteriormente se expresaron en la pulpa dental y se informó su intensidad y ubicación de expresión.

Expression of fibroblast growth factor receptor 2 (FGFR2) in combined hepatocellular-cholangiocarcinoma and intrahepatic cholangiocarcinoma: clinicopathological study.

Genetic alterations including fusions in fibroblast growth factor receptor 2 (FGFR2) are detected in 10-20% of intrahepatic cholangiocarcinoma (iCCA), and FGFR2 inhibitors are effective for the treatment of iCCA. We examined a prevalence of FGFR2 genetic alterations and their clinicopathological significance in combined hepatocellular-cholangiocarcinoma (cHCC-CCA). FGFR2 expression, which is a surrogate marker for FGFR2 genetic alterations, was immunohistochemically assessed in the liver sections from 75 patients with cHCC-CCA, 35 with small duct-type iCCA, 30 with large duct-type iCCA, and 35 with hepatocellular carcinoma (HCC). FGFR2 genetic alterations were detected by reverse transcription-PCR and direct sequence. An association of FGFR2 expression with clinicopathological features was investigated in cHCC-CCAs. FGFR2 expression was detected in significantly more patients with cHCC-CCA (21.3%) and small duct-type iCCA (25.7%), compared to those with large duct-type iCCA (3.3%) and HCC (0%) (p < 0.05). FGFR2-positive cHCC-CCAs were significantly smaller size (p < 0.05), with more predominant cholangiolocarcinoma component (p < 0.01) and less nestin expression (p < 0.05). Genetic alterations of ARID1A and BAP1 and multiple genes were significantly more frequent in FGFR2-positive cHCC-CCAs (p < 0.05). 5'/3' imbalance in FGFR2 genes indicating exon18-truncated FGFR2 was significantly more frequently detected in FGFR2-positive cHCC-CCAs and small duct iCCAs, compared to FGFR2-negative ones (p < 0.05). FGFR2::BICC fusion was detected in a case of cHCC-CCAs. FGFR2 genetic alterations may be prevalent in cHCC-CCAs as well as small duct-type iCCAs, which suggest cHCC-CCAs may also be a possible therapeutic target of FGFR2 inhibitors.

Ovarian Microcystic Stromal Tumor with Significant Nestin Expression: A Unique Case.

Ovary microcystic stromal tumor (MCST) is an extremely rare subtype of sex cord-stromal neoplasm, and only 57 cases have been reported. We herein report a unique case of ovarian MCST with positive nestin expression in a 39-year-old Chinese woman. The tumor showed microcystic stromal histological structures and characteristically expressed the CD10, WT-1, and Ki67 proteins. A molecular analysis identified a point mutation (c.110C > T) in exon 3 of the CTNNB1 gene. To our knowledge, no report has described a case of ovarian MCST with positive staining for nestin protein. Our study provides new insights into the tumor biology of ovarian MCST.

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.

Developmental Anomalies in Human Teeth: Odontoblastic Differentiation in Hamartomatous Calcifying Hyperplastic Dental Follicles Presenting with DSP, Nestin, and HES1.

Hyperplastic dental follicles (HDFs) represent odontogenic hamartomatous lesions originating from the pericoronal tissues and are often associated with impacted or embedded teeth. These lesions may occasionally feature unique calcifying bodies, known as calcifying whorled nodules (CWNs), characterized by stromal cells arranged in a whorled or spiral fashion. CWNs are typically observed in multiple calcifying hyperplastic dental follicles or regional odontodysplasia. In our study, we examined 40 cases of HDFs, including nine instances with characteristics of CWNs, referred to as calcifying hyperplastic dental follicles (CHDFs), which are infrequently accompanied by odontodysplasia. The median ages of the HDFs and CHDFs were 16 (ranging from 3 to 66) and 15 (ranging from 11 to 50) years, respectively. The lower third molars were the most frequently affected by HDSFs and CHDFs, followed by the upper canines. A histological examination was conducted on all 40 cases, with an immunohistochemical analysis performed on 21 of them. Among the cases with CWN, nine affected a single embedded tooth, with one exception. CWNs exhibited diverse calcifications featuring sparse or entirely deposited psammoma bodies, and some displayed dentinoid formation. Immunohistochemically, the stromal cells of HDFs were frequently positive for CD56 and nestin. By contrast, CWNs were negative for CD56 but positive for nestin as well as hairy and enhancer split 1 (HES1), with a few dentin sialoprotein (DSP)-positive calcified bodies. Our results revealed that hamartomatous CHDFs can impact multiple and single-embedded teeth. CWNs composed of nestin and HES1-positive ectomesenchymal cells demonstrated the potential to differentiate into odontoblasts and contribute to dentin matrix formation under the influence of HES1. This study is the first report documenting odontoblastic differentiation in HDFs. The rare occurrence of HDFs and CHDFs contributes to limited comprehension. To prevent misdiagnosis, a better understanding of these conditions is necessary.