Impact of hydrogel stiffness on the induced neural stem cells modulation.

BACKGROUND: The induced neural stem cells (iNSCs) held great promises for cell replacement therapy, but iNSCs modulation need improvement. Matrix stiffness could control stem cell fates and might be effective to iNSCs modulations. Here the stiffness of hydrogel matrix on the adhesion, proliferation and differentiation of iNSCs were studied. METHODS: Hyaluronic acid (HA) hydrogels with gradient stiffness were prepared. The structure and stiffness of hydrogels were detected by scanning electron microscopy (SEM) and rheological test. iNSCs were generated from human blood mononuclear cells and cultured in the hydrogels. The cell adhesion, proliferation and differentiation on gradient stiffness hydrogels were examined by CCK-8 test and immunofluorescence staining. RESULTS: All hydrogels showed typical soft tissue, with the elastic modulus increasing with concentration (0.6-1.8%), ranging from 17 to 250 Pa. The iNSCs maintained growth and differentiation on all gels, but showed different behaviors to different stiffness. On the softer hydrogels, cells grew slowly at first but continuously and fast for long term, tending to differentiate into neurons; while on the harder hydrogels, cells adhered and grew faster at the early stage, tending to differentiate into glia cells after long term culture. CONCLUSIONS: The results suggested that hydrogels stiffness could regulate the key cellular processes of iNSCs. It was important for iNSCs modulation and application in the future.

Dysfunction of Orbitofrontal GABAergic Interneurons Leads to Impaired Reversal Learning in a Mouse Model of Obsessive-Compulsive Disorder.

Cognitive inflexibility is a cardinal symptom of obsessive-compulsive disorder (OCD) and often manifests as impaired reversal learning. Abnormal recruitment of the orbitofrontal cortex (OFC)-striatal circuit is implicated in reversal learning deficits in patients with OCD. However, the precise circuitry mechanism underlying normal and impaired reversal learning remains elusive. Using fiber photometry and optogenetics, we demonstrated cell-type-specific activity dynamics in the OFC-striatal circuit underlying normal reversal learning and cell-type-specific dysfunctions that causally lead to impaired reversal learning in an OCD mouse model (Sapap3 knockout mice). After contingency reversal, OFC GABAergic interneurons increase the activity in response to previously rewarded but currently non-reward cues to inhibit the elevated activity of OFC excitatory neurons encoding inappropriate cue-reward association. Striatal direct-pathway medium spiny neurons (D1-MSNs) gradually re-establish their response preference for rewarded versus non-reward cues. These activity dynamics together mediated normal reversal learning. In Sapap3 knockout OCD mouse model, the increase in activity of OFC GABAergic interneurons in response to previously rewarded but currently non-reward cues after contingency reversal was reduced, which resulted in insufficient inhibition on OFC excitatory neurons, which in turn led to a more severe inversion of the response preference of D1-MSNs for rewarded versus non-reward cues, ultimately resulting in slower reversal learning. These dysfunctions were causally involved in reversal learning impairments. Our findings identified OFC GABAergic interneurons as the key therapeutic target to treat cognitive inflexibility in OCD and may be generally applicable to cognitive inflexibility in other neuropsychiatric disorders.

GAP-43 is involved in the orientation of cell division by interacting with GΑI during neurogenesis.

Purpose: Recent studies have shown that growth-associated protein-43 (GAP-43) may influence the mitotic-spindle orientation of Madin-Darby Canine Kidney (MDCK) cells through interacting with G proteins in vitro. However, whether GAP-43 interacts with the G proteins under the influence of mitotic spindle positioning related to the orientation of cell division during neurogenesis remains unclear. In order to explore the molecular mechanism in vivo, the GAP-43 transgenic mice were produced and the angles of cell division in the ventricular zone (VZ) during neurogenesis (embryonic period between 13.5 and 17.5 days) were measured in both transgenic mice and wild type mice by spindle angle analysis.Materials and methods: The interaction of GAP-43 and Gαi was detected by co-immunoprecipitation (co-IP), whereas the localization of GAP-43 was determined by immunofluorescence.Results: The results obtained using co-IP and immunofluorescence showed that GAP-43 is localized on the cell membrane and interacts with Gαi. This interaction dramatically induced a significant increase in the proportion of horizontally and intermediately dividing cells during the embryonic period of 13.5 days in the transgenic mouse brain, as observed by spindle angle analysis.Conclusions: It can be concluded that GAP-43 is involved in the orientation of cell division by interacting with Gαi, and that this may be an important mechanism for neurogenesis in the mammalian brain.

Lateral orbitofrontal dysfunction in the Sapap3 knockout mouse model of obsessive­compulsive disorder

Background: Obsessive­compulsive disorder (OCD) is a common psychiatric disorder that affects about 2% of the population, but the underlying neuropathophysiology of OCD is not well understood. Although increasing lines of evidence implicate dysfunction of the orbitofrontal cortex (OFC) in OCD, a detailed understanding of the functional alterations in different neuronal types in the OFC is still elusive. Methods: We investigated detailed activity pattern changes in putative pyramidal neurons and interneurons, as well as local field potential oscillations, in the lateral OFC underlying OCD-relevant phenotypes. We applied in vivo multichannel recording in an awake OCD mouse model that carried a deletion of the Sapap3 gene, and in wildtype littermates. Results: Compared with wildtype mice, the lateral OFC of Sapap3 knockout mice exhibited network dysfunction, demonstrated by decreased power of local field potential oscillations. The activity of inhibitory and excitatory neurons in the lateral OFC showed distinct perturbations in Sapap3 knockout mice: putative interneurons exhibited increased activity; putative pyramidal neurons exhibited enhanced bursting activity; and both putative pyramidal neurons and interneurons exhibited enhanced discharge variability and altered synchronization. Limitations: To exclude motor activity confounders, this study examined functional alterations in lateral OFC neurons only when the mice were stationary. Conclusion: We provide, to our knowledge, the first direct in vivo electrophysiological evidence of detailed functional alterations in different neuronal types in the lateral OFC of an OCD mouse model. These findings may help in understanding the underlying neuropathophysiology and circuitry mechanisms for phenotypes relevant to OCD, and may help generate and refine hypotheses about potential biomarkers for further investigation.

MicroRNA-23a inhibits melanoma cell proliferation, migration, and invasion in mice through a negative feedback regulation of sdcbp and the MAPK/ERK signaling pathway.

Melanoma is the main cause of death associated with skin cancer. Surgical resection and adjuvant therapy are currently effective treatments, but the recurrence rate is very high. The understanding of microRNA (miR) dynamics after surgical resection of melanoma is essential to accurately explain the changes in the recurrence of melanoma. In this study, we hypothesized that microRNA-23a (miR-23a) affects the cell proliferation, migration, and invasion of melanoma with a mechanism related to SDCBP and the MAPK/ERK signaling pathway. To validate this, we performed a series of experiments in cells of melanoma modeled. Initially, positive expression of SDCBP and morphology of normal and melanoma tissues and cells were observed. Expression of miR-23a, SDCBP, and MAPK/ERK signaling pathway-related genes was identified in melanoma tissues. Melanoma cells transfected with mimic or inhibitor of miR-23a or si-SDCBP were detected to validate effect of miR-23a on SDCBP and the MAPK/ERK signaling pathway. MTT assay, scratch test, transwell assay, and flow cytometry were performed to evaluate cell viability, invasion, metastasis, and apoptosis in vitro, respectively. Tumorigenicity assay in nude mice was conducted to test the tumorigenesis of the transfected cells in vivo. High positive expression of SDCBP and abnormal morphology were observed in melanoma tissues and cells. Reduced expression of miR-23a and increased expression of SDCBP and MAPK/ERK signaling pathway-related genes were identified in the melanoma tissues of melanoma mice. Overexpressed miR-23a dampened SDCBP and the MAPK/ERK signaling pathway. The melanoma cells with overexpressed miR-23a presented ascended cell apoptosis and descended cell proliferation, migration, invasion as well as tumor size. Taken together, our study demonstrated that miR-23a could inhibit the development of melanoma in mice through a negative feedback regulation of SDCBP and the MAPK/ERK signaling pathway. © 2018 IUBMB Life, 71(5):587-600, 2019.

Culture of pyramidal neural precursors, neural stem cells, and fibroblasts on various biomaterials.

A combinatory approach using biomaterials together with cells may improve the efficacy of cell therapy for treatment of various diseases/indications. In the current study, we cultured pyramidal neural precursors (PNPs), neural stem cells (NSCs), and fibroblasts on different materials that included fibrin, collagen, hyaluronic acid (HA), sciatic nerves, and matrigel, to search for the most suitable biomaterial for culture of each cell type. Collagen was fabricated in both an aligned collagen-poly (lactic-co-glycolic acid) (PLGA) composite and an alveolate form; fibrin and hyaluronic acid were made in an aligned form only. Pyramidal neurons have strong projection ability and have potentials in neural circuit reconstruction. However, PNPs showed difficulty in attaching to and growing neurites on most of the materials tested, except for matrigel, in which neurite growth was observed in a three dimentional culture. NSCs and derivatives hold promise in treating neurological diseases. On aligned fibrin, NSCs could differentiate and grow neurites in a directional manner before fibrin was degraded in 2 days. On aligned collagen-PLGA, induced neural stem cells (iNSCs) could survive and differentiate for at least 2 weeks, but the neurites failed to extend in an aligned way. Fibroblast graft are useful in many indications, such as in skin burns. Fibroblasts generally grew better on the tested materials than did the neural cells, and fibroblasts could grow directionally on the aligned fibrin and scattered around on the alveolate collagen. The study provided information which may be used to further optimize the materials to support culture of each type of cells.

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 ß-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 ß-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 ß-catenin.

Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017).

Cell therapy has been shown to be a key clinical therapeutic option for central nervous system diseases or damage. Standardization of clinical cell therapy procedures is an important task for professional associations devoted to cell therapy. The Chinese Branch of the International Association of Neurorestoratology (IANR) completed the first set of guidelines governing the clinical application of neurorestoration in 2011. The IANR and the Chinese Association of Neurorestoratology (CANR) collaborated to propose the current version "Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017)". The IANR council board members and CANR committee members approved this proposal on September 1, 2016, and recommend it to clinical practitioners of cellular therapy. These guidelines include items of cell type nomenclature, cell quality control, minimal suggested cell doses, patient-informed consent, indications for undergoing cell therapy, contraindications for undergoing cell therapy, documentation of procedure and therapy, safety evaluation, efficacy evaluation, policy of repeated treatments, do not charge patients for unproven therapies, basic principles of cell therapy, and publishing responsibility.

Neuronal activity pattern defects in the striatum in awake mouse model of Parkinson's disease.

Previous studies showed the loss of dopaminergic neurons directly leads to both changes in firing rate and neuronal synchrony in the striatum by pharmacogenetic approach, but physiological observation of striatal neurons in awake animal is rare up to now due to the limitation of recording methods. We use multichannel in vivo recording system, to record the activity pattern of both medium spiny projecting neurons (MSNs) and fast spiking interneurons (FSIs) in awake mouse model of Parkinson's disease (PD), created by injection of 6-hydroxyl-dopamine (6-OHDA) into dorsolateral striatum bilaterally and unilaterally. The abnormal discharge of neurons, including oscillations, burst activity and firing rate were systematically observed, and we used these index together to comprehensively analyse the functional change of striatal neurons in PD mouse model. We found that PD mouse model exhibited elevated synchronized oscillatory activity in ß frequency band and decreased firing rate of FSIs during movement. The firing rate and burst activity of MSNs clearly reduced during movement after bilateral dopamine depletion. The present study has novelly shown the firing pattern changes of the MSNs and FSIs in DL striatum in awake PD mouse model, by combination of electrophysiology with molecular biological technology. Our results may help to reveal a new circuitry mechanism of movement disorders in PD.

Axon guidance pathways served as common targets for human speech/language evolution and related disorders.

Human and several nonhuman species share the rare ability of modifying acoustic and/or syntactic features of sounds produced, i.e. vocal learning, which is the important neurobiological and behavioral substrate of human speech/language. This convergent trait was suggested to be associated with significant genomic convergence and best manifested at the ROBO-SLIT axon guidance pathway. Here we verified the significance of such genomic convergence and assessed its functional relevance to human speech/language using human genetic variation data. In normal human populations, we found the affected amino acid sites were well fixed and accompanied with significantly more associated protein-coding SNPs in the same genes than the rest genes. Diseased individuals with speech/language disorders have significant more low frequency protein coding SNPs but they preferentially occurred outside the affected genes. Such patients' SNPs were enriched in several functional categories including two axon guidance pathways (mediated by netrin and semaphorin) that interact with ROBO-SLITs. Four of the six patients have homozygous missense SNPs on PRAME gene family, one youngest gene family in human lineage, which possibly acts upon retinoic acid receptor signaling, similarly as FOXP2, to modulate axon guidance. Taken together, we suggest the axon guidance pathways (e.g. ROBO-SLIT, PRAME gene family) served as common targets for human speech/language evolution and related disorders.

Recombinant insulin-like growth factor binding protein-4 inhibits proliferation and promotes differentiation of neural progenitor cells.

Insulin-like growth factor (IGF) is involved in regulating many processes during neural development, and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g., via inhibiting Wnt/ß-catenin signaling). In the present study, neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and ß-catenin. Phosphorylation levels of Akt, Erk1, 2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of ß-catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1, 2 and p38 were not significantly changed in IGFBP4-treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway.

Long-term protective effects of AAV9-mesencephalic astrocyte-derived neurotrophic factor gene transfer in parkinsonian rats.

Intrastriatal injection of mesencephalic astrocyte-derived neurotrophic factor (MANF) protein has been shown to provide neuroprotective and neurorestorative effects in a 6-hydroxydopamine (6-OHDA) - lesioned rat model of Parkinson's disease. Here, we used an adeno-associated virus serotype 9 (AAV9) vector to deliver the human MANF (hMANF) gene into the rat striatum 10days after a 6-OHDA lesion to examine long-term effects of hMANF on nigral dopaminergic neurons and mechanisms underlying MANF neuroprotection. Intrastriatal injection of AAV9-hMANF vectors led to a robust and widespread expression of the hMANF gene in the injected striatum up to 24weeks. Increased levels of hMANF protein were also detected in the ipsilateral substantia nigra. The hMANF gene transfer promoted the survival of nigral dopaminergic neurons, regeneration of striatal dopaminergic fibers and an upregulation of striatal dopamine levels, resulting in a long-term improvement of rotational behavior up to 16weeks after viral injections. By using SH-SY5Y cells, we found that intra- and extracellular application of MANF protected cells against 6-OHDA-induced toxicity via inhibiting the endoplasmic reticulum stress and activating the PI3K/Akt/mTOR pathway. Our results suggest that AAV9-mediated hMANF gene delivery into the striatum exerts long-term neuroprotective and neuroregenerative effects on the nigrostriatal dopaminergic system in parkinsonian rats, and provide insights into mechanisms responsible for MANF neuroprotection.

Fusion of Ssm6a with a protein scaffold retains selectivity on NaV 1.7 and improves its therapeutic potential against chronic pain.

Voltage-gated sodium channel NaV 1.7 serves as an attractive target for chronic pain treatment. Several venom peptides were found to selectively inhibit NaV 1.7 but with intrinsic problems. Among them, Ssm6a, a recently discovered centipede venom peptide, shows the greatest selectivity against NaV 1.7, but dissociates from the target too fast and loses bioactivity in synthetic forms. As a disulfide-rich venom peptide, it is difficult to optimize Ssm6a by artificial mutagenesis and produce the peptide with common industrial manufacturing methods. Here, we developed a novel protein scaffold fusion strategy to address these concerns. Instead of directly mutating Ssm6a, we genetically fused Ssm6a with a protein scaffold engineered from human muscle fatty acid-binding protein. The resultant fusion protein, SP-TOX, maintained the selectivity and potency of Ssm6a upon NaV 1.7 but dissociated from target at least 10 times more slowly. SP-TOX dramatically reduced inflammatory pain in a rat model through DRG-targeted delivery. Importantly, SP-TOX can be expressed cytosolically in Escherichia coli and purified in a cost-effective way. In summary, our study provided the first example of cytosolically expressed fusion protein with high potency and selectivity on NaV 1.7. Our protein scaffold fusion approach may have its broad application in optimizing disulfide-rich venom peptides for therapeutic usage.

Angiogenic microspheres promote neural regeneration and motor function recovery after spinal cord injury in rats.

This study examined sustained co-delivery of vascular endothelial growth factor (VEGF), angiopoietin-1 and basic fibroblast growth factor (bFGF) encapsulated in angiogenic microspheres. These spheres were delivered to sites of spinal cord contusion injury in rats, and their ability to induce vessel formation, neural regeneration and improve hindlimb motor function was assessed. At 2-8 weeks after spinal cord injury, ELISA-determined levels of VEGF, angiopoietin-1, and bFGF were significantly higher in spinal cord tissues in rats that received angiogenic microspheres than in those that received empty microspheres. Sites of injury in animals that received angiogenic microspheres also contained greater numbers of isolectin B4-binding vessels and cells positive for nestin or ß III-tubulin (P < 0.01), significantly more NF-positive and serotonergic fibers, and more MBP-positive mature oligodendrocytes. Animals receiving angiogenic microspheres also suffered significantly less loss of white matter volume. At 10 weeks after injury, open field tests showed that animals that received angiogenic microspheres scored significantly higher on the Basso-Beattie-Bresnahan scale than control animals (P < 0.01). Our results suggest that biodegradable, biocompatible PLGA microspheres can release angiogenic factors in a sustained fashion into sites of spinal cord injury and markedly stimulate angiogenesis and neurogenesis, accelerating recovery of neurologic function.

RNAi-mediated silencing of HLA A2 suppressed acute rejection against human fibroblast xenografts in the striatum of 6-OHDA lesioned rats.

Major histocompatibility complex class l (MHC I) molecules play a role in determining whether transplanted cells will be accepted or rejected, and masking of MHC I on donor cells has been found useful for immunoprotection of neural xenografts. In the present study, primary human embryonic lung fibroblasts (HELF), HELF treated with lentivirus-mediated small interfering RNAs (siRNAs) targeting human leukocyte antigen A2 (HLA A2, MHC I in humans) (siHELF), and rat embryonic lung fibroblasts (RELF) were stereotaxically grafted into the striatum of 6-hydroxydopamine lesioned rats to explore whether knockdown of HLA A2 could reduce host immune responses against xenografts. Before lentiviral infection, the cells were transduced with retroviruses harboring tyrosine hydroxylase cDNA. Knockdown of HLA A2 protein was examined by Western blotting. The immune responses (the number of CD4 and CD8 T-cells in the brain and peripheral blood), glial reaction, and survival of human fibroblasts were quantitatively evaluated by flow cytometry and immunohistochemistry at 4d, 2w, and 6w post-graft. Animal behaviors were assessed by counting apomorphine-induced rotations pre- and post-grafts. It was shown that a lower level of HLA A2 was observed in siHELF grafts than in HELF grafts, and knockdown of HLA A2 decreased rat immune responses, as indicated by less remarkable increases in the number of CD8 and CD4 T-cells in the brain and the ratio of CD4:CD8 T-cells in the peripheral blood in rats grafted with siHELF. Rats grafted with siHELF exhibited a significant improvement in motor asymmetry post-transplantation and a better survival of human fibroblasts at 2w. The increasing number of activated microglia and the decreasing number of astrocytes were found in three groups of rats post-implantation. These data suggested that RNAi-mediated knockdown of HLA A2 could suppress acute rejection against xenogeneic human cell transplants in the rat brain.

Conversion of adult human peripheral blood mononuclear cells into induced neural stem cell by using episomal vectors.

Human neural stem cells (NSCs) hold great promise for research and therapy in neural diseases. Many studies have shown direct induction of NSCs from human fibroblasts, which require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Peripheral blood (PB) is routinely used in medical diagnoses, and represents a noninvasive and easily accessible source of cells. Here we show direct derivation of NSCs from adult human PB mononuclear cells (PB-MNCs) by employing episomal vectors for transgene delivery. These induced NSCs (iNSCs) can expand more than 60 passages, can exhibit NSC morphology, gene expression, differentiation potential, and self-renewing capability and can give rise to multiple functional neural subtypes and glial cells in vitro. Furthermore, the iNSCs carry a specific regional identity and have electrophysiological activity upon differentiation. Our findings provide an easily accessible approach for generating human iNSCs which will facilitate disease modeling, drug screening, and possibly regenerative medicine.

Malignant transformation of bone marrow stromal cells induced by the brain glioma niche in rats.

Normal human embryonic stem cells (hESCs) can develop neoplastic cancer stem cell (CSC) properties after coculture with transformed hESCs in vitro. In the present study, the influence of the tumor microenvironment on malignant transformation of bone marrow stromal cells (BMSCs) was studied after allografting a mixture of enhanced green fluorescent protein (EGFP)-labeled BMSCs and C6 glioma cells into the rat brain to understand the influence of the cellular environment, especially the tumor environment, on the transformation of grafted BMSCs in the rat brain. We performed intracerebral transplantation in the rat brain using EGFP-labeled BMSCs coinjected with C6 tumor cells. After transplantation, the EGFP-labeled cells were isolated from the tumor using fluorescence-activated cell sorting, and the characteristics of the recovered cells were investigated. Glioma-specific biomarkers of the sorted cells and the biological characteristics of the tumors were analyzed. The BMSCs isolated from the cografts were transformed into glioma CSCs, as indicated by the marked expression of the glioma marker GFAP in glioma cells, and of Nestin and CD133 in neural stem cells and CSCs, as well as rapid cell growth, decreased level of the tumor suppressor gene p53, increased level of the oncogene murine double minute gene 2 (MDM2), and recapitulation of glioma tissues in the brain. These data suggest that BMSCs can be transformed into CSCs, which can be further directed toward glioma formation under certain conditions, supporting the notion that the tumor microenvironment is involved in transforming normal BMSCs into glial CSCs.

Spinal cord injury repair by implantation of structured hyaluronic acid scaffold with PLGA microspheres in the rat.

In order to create an optimal microenvironment for neural regeneration in the lesion area after spinal cord injury (SCI), we fabricated a novel scaffold composed of a hyaluronic acid (HA) hydrogel with a longitudinal multi-tubular conformation. The scaffold was modified by binding with an anti-Nogo receptor antibody (antiNgR) and mixed further with poly(lactic-co-glycolic acid) (PLGA) microspheres containing brain-derived neurotrophic factor and vascular endothelial growth factor (HA+PLGA). In the rat, after implantation of this composite into an injured area created by a dorsal hemisection at T9-10 of the spinal cord, favorable effects were seen with regard to the promotion of spinal repair, including excellent integration of the implants with host tissue, inhibition of inflammation, and gliosis. In particular, large numbers of new blood vessels and regenerated nerve fibers were found within and around the implants. Simultaneously, the implanted rats exhibited improved locomotor recovery. Thus, this novel composite material might provide a suitable microenvironment for neural regeneration following SCI.

Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1(Ser137) involvement in spindle formation and REC8 cleavage.

Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1(Ser137) and pPlk1(Thr210)) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1(Ser137) with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1(Thr210) was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1(Ser137) was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1(Thr210) was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1(Ser137) accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1(Ser137) and pPlk1(Thr210), as well as the subcellular distribution of pPlk1(Thr210), were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1(Ser137) is the action executor, in mouse oocytes during meiotic division.

The influence of GAP-43 on orientation of cell division through G proteins.

Recent studies have shown that GAP-43 is highly expressed in horizontally dividing neural progenitor cells, and G protein complex are required for proper mitotic-spindle orientation of those progenitors in the mammalian developing cortex. In order to verify the hypothesis that GAP-43 may influence the orientation of cell division through interacting with G proteins during neurogenesis, the GAP-43 RNA from adult C57 mouse was cloned into the pEGFP-N1 vector, which was then transfected into Madin-Darby Canine Kidney (MDCK) cells cultured in a three-dimensional (3D) cell culture system. The interaction of GAP-43 with Gαi was detected by co-immunoprecipitation (co-IP), while cystogenesis of 3D morphogenesis of MDCK cells and expression of GAP-43 and Gαi were determined by immunofluorescence and Western blotting. The results showed are as follows: After being transfected by pEGFP-N1-GAP-43, GAP-43 was localized on the cell membrane and co-localized with Gαi, and this dramatically induced a defective cystogenesis in 3D morphogenesis of MDCK cells. The functional interaction between GAP-43 and Gαi proteins was proven by the co-IP assay. It can be considered from the results that the GAP-43 is involved in the orientation of cell division by interacting with Gαi and this should be an important mechanism for neurogenesis in the mammalian brain.