Neurogenic Differentiation Research Articles

Three-Dimensional Nanostructured Architectures Enable Efficient Neural Differentiation of Mesenchymal Stem Cells via Mechanotransduction.

Cell morphology and geometry affect cellular processes such as stem cell differentiation, suggesting that these parameters serve as fundamental regulators of biological processes within the cell. Hierarchical architectures featuring micro- and nanotopographical features therefore offer programmable systems for stem cell differentiation. However, a limited number of studies have explored the effects of hierarchical architectures due to the complexity of fabricating systems with rationally tunable micro- and nanostructuring. Here, we report three-dimensional (3D) nanostructured microarchitectures that efficiently regulate the fate of human mesenchymal stem cells (hMSCs). These nanostructured architectures strongly promote cell alignment andefficient neurogenic differentiation where over 85% of hMSCs express microtubule-associated protein 2 (MAP2), a mature neural marker, after 7 days of culture on the nanostructured surface. Remarkably, we found that the surface morphology of nanostructured surface is a key factor that promotes neurogenesis and that highly spiky structures promote more efficient neuronal differentiation. Immunostaining and gene expression profiling revealed significant upregulation of neuronal markers compared to unpatterned surfaces. These findings suggest that the 3D nanostructured microarchitectures can play a critical role in defining stem cell behavior.

Interleukin 6 promotes an in vitro mineral deposition by stem cells isolated from human exfoliated deciduous teeth.

Interleukin 6 (IL-6) plays various roles including stem cell regulation. The present study investigated the effect of IL-6 on cell proliferation, colony forming unit ability, stem cell marker expression and differentiation ability in stem cells isolated from human exfoliated deciduous teeth (SHEDs). We reported that the isolated cells from dental pulp tissues for deciduous teeth expressed CD44, CD90 and CD105 but not CD45. These cells were able to differentiate into osteoblasts, adipocytes and neuronal-like cells. IL-6 treatment resulted in the significant increase of NANOG, SOX2 and REX1 mRNA expression. However, IL-6 had no effect on cell proliferation and colony forming unit ability. IL-6 did not alter adipogenic and neurogenic differentiation potency. IL-6 supplementation in osteogenic medium led to a significant increase of mineralization. Furthermore, IL-6 upregulated ALP, ANKH and PIT1 mRNA levels. In conclusion, IL-6 participates in the regulation of pluripotent marker expression and is also involved in mineralization process of SHEDs. Hence, IL-6 could be employed as a supplementary substance in culture medium to maintain stemness and to induce osteogenic induction in SHEDs for future regenerative cell therapy.

Amyloid β Peptide Compromises Neural Stem Cell Fate by Irreversibly Disturbing Mitochondrial Oxidative State and Blocking Mitochondrial Biogenesis and Dynamics.

Alzheimer's disease (AD) is the most common neurodegenerative disease and is characterized by the accumulation of amyloid β peptide (Aβ). Although most AD mouse models present a decline in neurogenesis, they express mutated genes which regulate neurogenesis per se and are not present in most AD patients, thus masking the real impact of Aβ on adult neurogenesis. Mitochondrion, a well-known target of Aβ in neurons, is a main regulator of neural stem cell (NSC) fate. Here, we aimed to investigate the impact of Aβ on NSC mitochondria and cell fate decisions, namely whether and how Aβ affects neurogenesis. NSC fate and mitochondrial parameters, including biogenesis, dynamics, and oxidative stress, were evaluated. Our results showed that Aβ impaired NSC viability and proliferation and indirectly blocked neurogenic differentiation, by disrupting mitochondrial signaling of self-renewing NSCs. Importantly, Aβ decreased ATP levels, generated oxidative stress, and affected the radical scavenger system through SOD2 and SIRT3. Aβ also reduced mtDNA and mitochondrial biogenesis proteins, such as Tfam, PGC-1α, and NRF1, and inhibited activation of PGC-1α-positive regulator CREB. Moreover, Aβ triggered mitochondrial fragmentation in self-renewing NSCs and reduced mitochondrial fusion proteins, such as Mfn2 and ERRα. Notably, Aβ compromised NSC commitment and survival by irreversibly impairing mitochondria and thwarting any neurogenic rescue through mitochondrial biogenesis, dynamics, or radical scavenger system. Altogether, this study brings new perspective to rethink the molecular targets relevant for endogenous NSC-based strategies in AD.

Neurogenic Differentiation of Bone Marrow Mesenchymal-Like Stem Cell Induced by Delonix regia Flowers Extract

Stem cell technology has great potential in the effort to cure degenerative diseases. This study was done to determine optimum dose of flamboyant (Delonix regia) flower extract to induce proliferation and differentiation of mice (Mus musculus) bone marrow mesenchymal-like stem cell. Bone marrow cells were collected from mice by aspiration. Cells suspension (1 x 106) were poured into petri dishes containing 2 ml of modified Dulbecco's Modified Eagle's Media (mDMEM) and incubated overnight at 37 °C in a 5% CO2 incubator and microscopically observed. In quadriplicate, MSC were cultured in mDMEM containing D. regia flower extract of 0.0 (control), 0.4, 0.6, 0.8, and 1.0 mg/ml and incubated at 37 °C for 9 days. Population doubling time (PDT) and differentiated cell type were microscopically observed using HE staining on day 1 and 10. Data obtained were analyzed by ANOVA and Tukey test. The results showed that the addition of D regia flowers extracts 0.8 and 1.0 mg/ml significantly reduced PDT compared to that of 0.4, 0.6 and control. The extract, at 0.4 and 0.6 mg/ml, were able to induce MSC differentiation into fibroblast-like and nerve-like cells. In conclusion, D. regia flower extracts of 0.6, 0.8 and 1.0 mg/ml were able to stimulate MSC proliferation, but optimum dose for neurogenic differentiation was 0.6 mg/ml. This is the first to show potential of D. regia flower extract as neurogenic differentiatian inducer on mice MSC. These findings can be used as preliminary information for using the extract as cellular differentian inducer in basic and applicative reseach using stem cells.

Circulation perfusion culture of insulin-producing cells using rat decellularized pancreatic scaffolds

Objective To cultivate mouse bone marrow mesenchymal stem cells (mBMSCs) drived insulin-producing cells (IPCs) in rat decellularized pancreatic scaffolds and evaluate the growth and function of cells. Methods Decellularized pancreatic scaffolds were obtained by continuous perfusion of detergent by the splenic artery. HE staining, Masson staining, DNA content, glycosaminoglycan (GAG) content, extracellular matrix (ECM), biological compatibility were adopted to evaluate the result. mBMSCs were differentiated into IPCs via infected by pancreatic and duodenal homeobox-1 (PDX-1), neurogenic differentiation-1 (NeuroD1) and V-mar musculoaponeurotic fibrosarcoma oncogene homologue A (MafA). Immunofluorescence and real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) were detected of insulin expression of the IPCs. The IPCs were exposed to a glucose gradient and insulin release was measured by enzyme linked immunosorbent assay (ELISA) assay. The IPCs were cultivated in the decellularized pancreatic scaffolds in the circulation perfusion device. Hematoxylin-eosin staining (HE) and immunofluorescence were implemented to evaluate the growth and function of cells. The insulin gene expression was compared with traditional culture by FQ-PCR. Results After perfused of detergent, HE staining and Masson staining demonstrated no residual cells remained and most of the collagen fiber components were well retained. DNA content of decellularized scaffolds was (44.92±3.89) ng/mg (t=15.160, P=0.001)and GAG content was (30.27±2.70) ng/mg, (t=2.862, P=0.046). Immunohistochemical staining showed collagen I and fibronectin were retained. Scaffolds were desirable biocompatibility. Immunofluorescence and FQ-PCR showed gene and protein expression of insulin after infected by PDX-1, NeuroD1 and MafA. The IPCs were glucose responsiveness to different glucose concentration according to ELISA assay. After cultivated in the decellularized pancreatic scaffolds, HE and immunofluorescence demostrated that the IPCs grew well in the scaffolds. FQ-PCR showed that insulin gene expression was increased compared with the traditional two-dimensional culture (t=15.030, P=0.004). Conclusion The rat decellularized pancreatic scaffolds not only preserved the ECM and structure of vascular, but also good biocompatibility. The IPCs differentiated from mBMSCs had the gene expression of insulin expression. The decellularized pancreatic scaffolds not only supported the growth and function of IPCs, but provided a favourable platform compared with the two-dimensional culture. Key words: Pancreas; Decellularized scaffolds; Diabetes; Tissue engineering

In vitro osteoblastic differentiation of mesenchymal stem cells generates cell layers with distinct properties

BackgroundDifferentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties.MethodsThrough the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers.ResultsThe cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization.ConclusionsMSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.

Neurogenic Differentiation of Human Dental Pulp Stem Cells on Graphene-Polycaprolactone Hybrid Nanofibers.

Stem cells derived from dental tissues—dental stem cells—are favored due to their easy acquisition. Among them, dental pulp stem cells (DPSCs) extracted from the dental pulp have many advantages, such as high proliferation and a highly purified population. Although their ability for neurogenic differentiation has been highlighted and neurogenic differentiation using electrospun nanofibers (NFs) has been performed, graphene-incorporated NFs have never been applied for DPSC neurogenic differentiation. Here, reduced graphene oxide (RGO)-polycaprolactone (PCL) hybrid electrospun NFs were developed and applied for enhanced neurogenesis of DPSCs. First, RGO-PCL NFs were fabricated by electrospinning with incorporation of RGO and alignments, and their chemical and morphological characteristics were evaluated. Furthermore, in vitro NF properties, such as influence on the cellular alignments and cell viability of DPSCs, were also analyzed. The influences of NFs on DPSCs neurogenesis were also analyzed. The results confirmed that an appropriate concentration of RGO promoted better DPSC neurogenesis. Furthermore, the use of random NFs facilitated contiguous junctions of differentiated cells, whereas the use of aligned NFs facilitated an aligned junction of differentiated cells along the direction of NF alignments. Our findings showed that RGO-PCL NFs can be a useful tool for DPSC neurogenesis, which will help regeneration in neurodegenerative and neurodefective diseases.

Insulin-like growth factor binding protein 4 inhibits proliferation of bone marrow mesenchymal stem cells and enhances growth of neurospheres derived from the stem cells.

Insulin-like growth factor binding protein 4 (IGFBP-4) was reported to trigger cellular senescence and reduce cell growth of bone marrow mesenchymal stem cells (BMSCs), but its contribution to neurogenic differentiation of BMSCs remains unknown. In the present study, BMSCs were isolated from the femur and tibia of young rats to investigate effects of IGFBP-4 on BMSC proliferation and growth of neurospheres derived from BMSCs. Bone marrow mesenchymal stem cell proliferation was assessed using CCK-8 after treatment with IGFBP-4 or blockers of IGF-IR and β-catenin. Phosphorylation levels of Akt, Erk, and p38 in BMSCs were analysed by Western blotting. Bone marrow mesenchymal stem cells were induced into neural lineages in NeuroCult medium; the number and the size of BMSC-derived neurospheres were counted after treatment with IGFBP-4 or the blockers. It was shown that addition of IGFBP-4 inhibited BMSC proliferation and immunodepletion of IGFBP-4 increased the proliferation. The blockade of IGF-IR with AG1024 increased BMSC proliferation and reversed IGFBP-4-induced proliferation inhibition; however, blocking of β-catenin with FH535 did not. p-Erk was significantly decreased in IGFBP-4-treated BMSCs. IGFBP-4 promoted the growth of neurospheres derived from BMSCs, as manifested by the increases in the number and the size of the derived neurospheres. Both AG1024 and FH535 inhibited the formation of NeuroCult-induced neurospheres, but FH535 significantly inhibited the growth of neurospheres in NeuroCult medium with EGF, bFGF, and IGFBP-4. The data suggested that IGFBP-4 inhibits BMSC proliferation through IGF-IR pathway and promotes growth of BMSC-derived neurospheres via stabilizing β-catenin.

Toxic Effects of Trichloroethylene on Rat Neuroprogenitor Cells.

Trichloroethylene (TCE) is a common volatile organic solvent which is considered as an ubiquitous environmental pollutant. It is claimed to be a developmental neurotoxicant. Our group evaluated previously its impact on three-dimensional neurospheres in vitro. The current work aims to investigate the neurotoxic effects of a lower concentration of TCE on the same system. To perform the experiment, neural progenitor cells were obtained from the brains of nine newborn rats. Afterward, these cells were cultured in both growth and differentiation media to get the neurospheres. Cell cultures were divided into two groups: group 1 (control), group 2 (exposed to 0.25 μM TCE). Neurospheres were photographed at different durations and assessment of the morphological changes such as proliferation and differentiation of neurospheres was done. In addition, cell viability, apoptosis, and necrosis were analyzed using flow cytometry to clarify the mechanism of involved cytotoxicity. The results revealed that TCE-treated neurospheres showed significantly decreased proliferation on days 7 and 14. These cells failed to show the neurogenic differentiation seen in the neurospheres of the control group. Furthermore, a highly significant decrease in viability and a significant increase in the number of apoptotic cells were observed in the treated cells in comparison to the control group. The present work confirmed that TCE, at very low doses relevant to daily life exposure in humans, caused neurotoxic effects in 3D neurosphere model through the affection of neural proliferation and differentiation as well as disturbance of cell viability and apoptosis.

Inhibiting RHOA Signaling in Mice Increases Glucose Tolerance and Numbers of Enteroendocrine and Other Secretory Cells in the Intestine

Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation. We collected intestinal tissues from GLU-Venus, GPR41-RFP, and Neurog3-RFP mice, in which the endocrine lineage is fluorescently labeled, for invitro culture and histologic analysis. Small intestine organoids derived from these mice were cultured with Y-27632 and we measured percentages of L cells, expression of intestinal cell-specific markers, and secretion of GLP1 in medium. Mice were fed a normal chow or a high-fat diet and given Y-27632 or saline (control) and blood samples were collected for measurement of GLP1, insulin, and glucose. Incubation of intestinal organoids with Y-27632 increased numbers of L cells and secretion of GLP1. These increases were associated with upregulated expression of genes encoding intestinal hormones, neurogenin 3, neurogenic differentiation factor 1, forkhead box A1 and A2, and additional markers of secretory cells. Mice fed the normal chow diet and given Y-27632 had increased numbers of L cells in intestinal tissues, increased plasma levels of GLP1 and insulin, and lower blood levels of glucose compared withmice fed the normal chow diet and given saline. In mice with insulin resistance induced by the high-fat diet, administration of Y-27632 increased secretion of GLP1 and glucosetolerance compared with administration of saline. In mouse intestinal organoids, an inhibitor of RhoA signaling increased the differentiation of the secretory lineage and the development of enteroendocrine cells. Inhibitors of RhoA signaling or other strategies to increase numbers of Lcells might be developed for treatment of patients with type 2 diabetes or for increasing glucose tolerance.

Enhanced brain expression of genes related to cell proliferation and neural differentiation is associated with cortisol receptor expression in fishes

Stress enhances or inhibits neurogenesis in mammals and some fish species. The link between the two processes is still unclear. Most studies have been performed in very specific stressful or altered environments. Despite the known inter-individual divergence in coping abilities within populations, the relationship between the stress axis and neurogenesis has never been addressed in unstressed individuals. Here we correlate brain expression of the pcna (proliferating cell nuclear antigen) and neurod1 (neurogenic differentiation factor 1) genes, two markers of neurogenesis, with transcripts of cortisol receptors in three fish species living in very distinct environments. Within the three species, individuals with the highest expression of neurogenesis genes were also those that expressed the high levels of cortisol receptors. Based on these correlations and the hypothesis that mRNA levels are proxies of protein levels, we hypothesize that within unstressed animals, individuals sensitive to cortisol perceive a similar environment to be more stimulating, leading to increased neurogenesis. Although it is difficult to determine whether it is sensitivity to cortisol that affects neurogenesis capacities or the opposite, the proposed pathway is a potentially fruitful avenue that warrants further mechanistic experiments.

PR361: Neurogenic, angiogenic and osteogenic differentiation potential of healthy human periodontal ligament stem cells

PR361: Neurogenic, angiogenic and osteogenic differentiation potential of healthy human periodontal ligament stem cells

CELL THERAPY OF EPILEPSIA. CLINICAL AND IMMUNOLOGICAL ASPECTS

The aim is to develop and implement a method for the treatment of drug-resistant epilepsy using autologous mesenchymal stem cells and the neuroimaging, immunological and neurophysiological predictors of the brain function. Material and Methods . Twenty patients (12 males and 8 females) with symptomatic drug-resistant epilepsy participated in the study. The patient age varied from 23 to 46 years; and the duration of epilepsy was 7-29 years. Autologous mesenchymal stem cells of the bone marrow were characterized using cultural, morphological, immunological, molecular-genetic, clinical-functional, laboratory, pathopsychological, and neurophysiological methods. The standard parametric and nonparametric statistical tests were used to evaluate the results. Results . The study resulted in producing of cultured autologous mesenchymal stem cells of the bone marrow (AMSCBM) sufficient to conduct 20 courses of cell therapy. In total, 40 transplantation procedures using AMSCBM were performed (20 intravenous and 20 endolumbar injections). Cellularity index in the intravenous inoculate ranged from 39.5 to 110.0 million and that for the endolumbar injection – from 5.1 to 10.0 million with viability not less than 95%. The distribution of AMSCBM by key surface markers (CD105+, CD90+, CD45-, CD34-) matched the criteria of the International Association for cell therapy (ISCT). The cell injections were well tolerated and did not cause any severe adverse effects. To monitor the process of neurogenic differentiation, the expression of the surface markers was determined. In most samples with confirmed neural differentiation, a significant increase in the expression of neuron-specific enolase, nestin and MAR-2 was detected. In patients with symptomatic epilepsy, the most significant deviations from normal values were found for the numbers of cytotoxic and activated cells, natural killer (NK) cells, and T-cells with the NK activity. After a course of cell therapy, a significant decrease in CD4+CD8+, CD3+CD8+, CD3+CD95+, and CD8+CD25+ cells was noted. Also decreased were the numbers of NK cells and T-cells with the NK activity, however, their levels remained relatively high as compared with the control group. Following the treatment, we continued to monitor the patients for 3, 6, and 12 months after the cell administration as well as the patients from the group of comparison. Conclusion . For the first time in the Republic of Belarus, cell therapy in patients with epilepsy was conducted. An intravenous injection of AMSCBM and endolumbar administration of neuro-induced AMSCBM can serve an effective additional therapy of choice in patients with drug-resistant epilepsy.

Neurogenic differentiation of adipose derived stem cells on graphene-based mat

Adipose derived stem cells (ADSCs) have been proved as an abundant and accessible cell source with the ability to differentiate into neuron-like cells. However, the low differentiation efficiency puts forward an important challenge to practical applications in clinic. Considering of the good biocompatibility of graphene-based materials and the potential interaction between graphene and cells mentioned in previous studies, herein, we investigated the effect of graphene oxide (GO) and reduced graphene oxide (rGO) mats on neurogenic differentiation of the ADSCs. We demonstrated the excellent capabilities of graphene-based mats, especially GO to support the neural differentiation of ADSCs. By comparing the observation under an optical microscope and fluorescence microscope, the conversion rate of neuron-like cells reached about 90%. We consider that GO mat is better for promoting the differentiation of ADSCs into neuron-like cells, which compared to rGO based platforms. Meanwhile, we made an analysis of the mechanism by which graphene induced the differentiation of ADSCs to neuron-like cells. The data obtained here highlight the effect of GO mat on neurogenic differentiation of ADSCs and implicate the potential of graphene-based materials in application of neural tissue engineering for the limited self-repair capability of nerve cells.

OP-34 - Amyloid-β peptide irreversibly blocks mitochondrial biogenesis and dynamics of self-renewing neural stem cells compromising neurogenesis

In Alzheimer’s disease (AD), amyloid-β peptide (Aβ) seeding leads to neuronal death through mitochondria. AD brains and most AD mouse models present a decline in neurogenesis. Mitochondria have been highlighted as a main regulator of neurogenesis. We investigated the impact of Aβ on neurogenesis via mitochondria, in a NSC line. Aβ impairs NSC viability and proliferation and indirectly blocks neurogenic differentiation, by disrupting mitochondria. Aβ decreases ATP levels whereas generates oxidative stress and reduces SOD2 and SIRT3 expression. Aβ decreases mitochondrial biogenesis markers such as mtDNA, Tfam, PGC-1α and NRF1, and promotes mitochondrial fragmentation by reducing the expression of proteins associated to mitochondrial fusion, such as Mfn2 and ERRα. However, once Aβ impairs mitochondria, NSC cell fate cannot be rescued neither through overexpressing PGC-1α, Mfn2 or Sirt3. Briefly, Aβ directly blocks viability and proliferation of NSCs, contributing for a decline of NSC pool. Moreover, Aβ irreversibly modifies mitochondrial pathways associated to mitochondrial biogenesis, dynamics and oxidative state in self-renewing NSCs, compromising neurogenic differentiation. This study warns against the use of endogenous NSCs in NSC-based therapies for AD.

Aligned laminin core-polydioxanone/collagen shell fiber matrices effective for neuritogenesis

Neural tissue regeneration is a significant challenge, because severe nerve injury is quite difficult to regenerate spontaneously. Although, many studies have been devoted to promote nerve regeneration, there are still many technical challenges to achieve satisfactory results. In this study, we designed biomimetic matrices composed of aligned laminin core-polydioxanone/collagen shell (Lam-PDO/Col) fibers, which can provide both topographical and biochemical cues for promoting neuritogenesis. The aligned Lam-PDO/Col core-shell fiber matrices were fabricated by magnetic field-assisted electrospinning with the coaxial system, and their potential as biofunctional scaffolds for promoting neuritogenesis was explored. It was demonstrated that the aligned Lam-PDO/Col core-shell fibers were successfully fabricated, and the laminin in the core of fibers was steadily and continuously released from fibers. In addition, the cellular behaviors of hippocampal neuronal cells on the matrices were significantly enhanced. Moreover, the aligned Lam-PDO/Col fiber matrices effectively improved and guided neurite outgrowth as well as the neurogenic differentiation by providing both topographical and biochemical cues through aligned fiber structure and sustained release of laminin. Collectively, it is suggested that the aligned Lam-PDO/Col core-shell fiber matrices are one of the most promising approaches for promoting neuritogenesis and neural tissue regeneration.

The Effect of Recombinant Tyrosine Hydroxylase Expression on the Neurogenic Differentiation Potency of Mesenchymal Stem Cells

ObjectiveTyrosine hydroxylase (TH) is a rate-limiting enzyme in dopamine synthesis, making the enhancement of its activity a target for ensuring sufficient dopamine levels. Rat bone marrow mesenchymal stem cells (rBM-MSCs) are known to synthesize TH after differentiating into neuronal cells through chemical induction, but the effect of its ectopic expression on these cells has not yet been determined. This study investigated the effects of ectopic recombinant TH expression on the stemness characteristics of rBM-MSCs. MethodsAfter cloning, a cell line with stable TH expression was maintained, and the proliferation, the gene expression profile, and differentiation potential of rBM-MSCs were analyzed. Analysis of the cells showed an increment in the proliferation rate that could be reversed by the neutralization of TH. ResultsThe constitutive expression of TH in rBM-MSCs was successfully implemented, without significantly affecting their osteogenic and adipogenic differentiation potential. TH expression improved the expression of other neuronal markers, such as glial fibrillary acidic protein, β-tubulin, nestin, and c-Fos, confirming the neurogenic differentiation capacity of the stem cells. The expression of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) significantly increased after the chemical induction of neurogenic differentiation. ConclusionIn this study, the expression of recombinant TH improved the neuroprotective effect of MSCs by upregulating the expression of BDNF and CNTF. Although the neuronal markers were upregulated, the expression of recombinant TH alone in rBM-MSCs was not sufficient for MSCs to differentiate into neurogenic cell lines.

Nature-Based Tourism Elicits a Phenotypic Shift in the Coping Abilities of Fish.

Nature-based tourism is gaining extensive popularity, increasing the intensity and frequency of human-wildlife contacts. As a consequence, behavioral and physiological alterations were observed in most exposed animals. However, while the majority of these studies investigated the effects of punctual exposure to tourists, the consequences of constant exposition to humans in the wild remains overlooked. This is an important gap considering the exponential interest for recreational outdoor activities. To infer long-term effects of intensive tourism, we capitalized on Odontostilbe pequira, a short-lived sedentary Tetra fish who spends its life close to humans, on which it feeds on dead skin. Hence, those fish are constantly exposed to tourists throughout their lifecycle. Here we provide an integrated picture of the whole phenomenon by investigating, for the first time, the expression of genes involved in stress response and neurogenesis, as well as behavioral and hormonal responses of animals consistently exposed to tourists. Gene expression of the mineralocorticoid (and cortisol) receptor (mr) and the neurogenic differentiation factor (NeuroD) were significantly higher in fish sampled in the touristic zone compared to those sampled in the control zone. Additionally, after a simulated stress in artificial and controlled conditions, those fish previously exposed to visitors produced more cortisol and presented increased behavioral signs of stress compared to their non-exposed conspecifics. Overall, nature-based tourism appeared to shift selection pressures, favoring a sensitive phenotype that does not thrive under natural conditions. The ecological implications of this change in coping style remain, nevertheless, an open question.

Neural stem cell encapsulation and differentiation in strain promoted crosslinked polyethylene glycol-based hydrogels.

Encapsulated cell viability within crosslinked hydrogels is a critical factor to consider in regenerative medicine/cell delivery applications. Herein, a "click" hydrogel system is presented encompassing 4-dibenzocyclooctynol functionalized polyethylene glycol, a four arm polyethylene glycol tetraazide crosslinker, tethered native protein attachment ligands (laminin), and a tethered potent neurogenic differentiation factor (interferon-γ). With this approach, hydrogel formation occurs via strain-promoted, metal-free, azide-alkyne cycloaddition in an aqueous buffer. This system demonstrated safe encapsulation of neural stem cells in biological conditions without chemical initiators/ultraviolet light, achieving high cell viability. Cell viability in click gels was nearly double that of ultraviolet exposed gels after 1 d as well as 14 d of subsequent culture; demonstrating the sensitivity of neural stem cells to ultraviolet light damage, as well as the need to develop safer encapsulation strategies. Finally, protein immobilized click hydrogel neural stem cell in vitro differentiation over 2 weeks demonstrated that the click gels specified primarily neurons without the need for additional protein differentiation factor media supplementation.

Neurogenic differentiation by hippocampal neural stem and progenitor cells is biased by NFIX expression.

Our understanding of the transcriptional programme underpinning adult hippocampal neurogenesis is incomplete. In mice, under basal conditions, adult hippocampal neural stem cells (AH-NSCs) generate neurons and astrocytes, but not oligodendrocytes. The factors limiting oligodendrocyte production, however, remain unclear. Here, we reveal that the transcription factor NFIX plays a key role in this process. NFIX is expressed by AH-NSCs, and its expression is sharply upregulated in adult hippocampal neuroblasts. Conditional ablation of Nfix from AH-NSCs, coupled with lineage tracing, transcriptomic sequencing and behavioural studies collectively reveal that NFIX is cell-autonomously required for neuroblast maturation and survival. Moreover, a small number of AH-NSCs also develop into oligodendrocytes following Nfix deletion. Remarkably, when Nfix is deleted specifically from intermediate progenitor cells and neuroblasts using a Dcx-creERT2 driver, these cells also display elevated signatures of oligodendrocyte gene expression. Together, these results demonstrate the central role played by NFIX in neuroblasts within the adult hippocampal stem cell neurogenic niche in promoting the maturation and survival of these cells, while concomitantly repressing oligodendrocyte gene expression signatures.