MAB353 Sigma-AldrichAnti-Nestin Antibody, clone rat-401

In primary brain tumors, oncogenes are frequently amplified and maintained on extrachromosomal DNA as double minutes (DM), but the underlying mechanisms remain poorly understood. We have generated a mouse model of malignant glioma based on knock-in of a mutant PDGF receptor α (PDGFRα) that is expressed in oligodendrocyte precursor cells (OPCs) after activation by a Cre recombinase. In the tumor suppressor INK4/Arf(-/-) background, mutant animals frequently developed brain tumors resembling anaplastic human gliomas (WHO grade III). Besides brain tumors, most animals also developed aggressive fibrosarcomas, likely triggered by Cre activation of mutant PDGFRα in fibroblastic cell lineages. Importantly, in the brain tumors and cell lines derived from brain tumor tissues, we identified a high prevalence of DM Pdgfra gene amplification, suggesting its occurrence as an early mutational event contributing to the malignant transformation of OPCs. Amplicons extended beyond the Pdgfra locus and included in some cases neighboring genes Kit and Kdr. Our genetically defined mouse brain tumor model therefore supports OPC as a cell of origin for malignant glioma and offers an example of a defined temporal sequence of mutational events, thus providing an entry point for a mechanistic understanding of DM gene amplification and its functionality in gliomagenesis.

Large doses of recombinant growth factors formulated in solution form directly injected into the body is usual clinical practice in treating second-degree scald injuries, with promising results, but this approach creates side effects; furthermore, it may not allow appropriate levels of the factor to be sensed by the target injured tissue/organ in the specific time frame, owing to complications arising from regeneration. In this research, two delivery methods (infusion pumping and local topical application) were applied to deliver recombinant human erythropoietin (rHuEPO) for skin regeneration. First, rHuEPO was given in deep second-degree scald injury sites in mice by infusion pump. Vascularization was remarkably higher in the rHuEPO pumping group than in controls. Second, local topical application of rHuEPO gel was given in deep second-degree scald injury sites in rats. Histological analysis showed that epithelialization rate was significantly higher in the rHuEPO gel-treated group than in controls. Immunohistochemical studies showed that the rHuEPO gel-treated group showed remarkably higher expression of skin regeneration makers than the control group. An accurate method for visualization and quantification of blood vessel networks in target areas has still not been developed up to this point, because of technical difficulties in detecting such thin blood vessels. A method which utilizes a series of steps to enhance the image, removes noise from image background, and tracks the vessels edges for vessel segmentation and quantification has been used in this study. Using image analysis methods, we were able to detect the microvascular networks of newly formed blood vessels (less than 500 μm thickness), which participate in the healing process, providing not only nutrition and oxygen to grow tissues but also necessary growth factors to grow tissue cells for complete skin regeneration. The rHuEPO-treated group showed higher expression of stem cell markers (CD 31, CD 90, CD 71, and nestin), which actively contribute to in-wound-healing processes for new hair follicle generation as well as skin regeneration. Collectively, both rHuEPO group pumping into the systemic circulation system, and injection into the local injury area, prompted mice and rats to form new blood vessel networks in scald injury sites, which significantly participate in the scald healing process. These results may lead to the development of novel treatments for scald wounds.

Neural stem cell (NSC) proliferation and differentiation are required to replace neurons damaged or lost after hypoxic-ischemic events and recover brain function. Periostin (POSTN), a novel matricellular protein, plays pivotal roles in the survival, migration, and regeneration of various cell types, but its function in NSCs of neonatal rodent brain is still unknown. The purpose of this study was to investigate the role of POSTN in NSCs following hypoxia-ischemia (HI). We found that POSTN mRNA levels significantly increased in differentiating NSCs. The proliferation and differentiation of NSCs in the hippocampus is compromised in POSTN knockout mice. Moreover, NSC proliferation and differentiation into neurons and astrocytes significantly increased in cultured NSCs treated with recombinant POSTN. Consistently, injection of POSTN into neonatal hypoxic-ischemic rat brains stimulated NSC proliferation and differentiation in the subventricular and subgranular zones after 7 and 14 days of brain injury. Lastly, POSTN treatment significantly improved the spatial learning deficits of rats subjected to HI. These results suggest that POSTN significantly enhances NSC proliferation and differentiation after HI, and provides new insights into therapeutic strategies for the treatment of hypoxic-ischemic encephalopathy.

Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4-dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c-Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS-treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA-positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c-Kit- and TMEM16A/ANO1-positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS-induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.

Periventricular leukomalacia (PVL) is a common ischemic brain injury in premature infants for which there is no effective treatment. The objective of this study was to determine whether transplanted mouse oligodendrocyte progenitor cells (OPCs) have neuroprotective effects in a rat model of PVL. Hypoxia-ischemia (HI) was induced in 3-day-old rat pups by left carotid artery ligation, followed by exposure to 6% oxygen for 2.5 h. Animals were assigned to OPC transplantation or sham control groups and injected with OPCs or PBS, respectively, and sacrificed up to 6 weeks later for immunohistochemical analysis to investigate the survival and differentiation of transplanted OPCs. Apoptosis was evaluated by double immunolabeling of brain sections for caspase-3 and neuronal nuclei (NeuN), while proliferation was assessed using a combination of anti-Nestin and -bromodeoxyuridine antibodies. The expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 was examined 7 days after OPC transplantation. The Morris water maze was used to test spatial learning and memory. The results showed that transplanted OPCs survived and formed a myelin sheath, and stimulated BDNF and Bcl-2 expression and the proliferation of neural stem cells (NSC), while inhibiting HI-induced neuronal apoptosis relative to control animals. Moreover, deficits in spatial learning and memory resulting from HI were improved by OPC transplantation. These results demonstrate an important neuroprotective role for OPCs that can potentially be exploited in cell-based therapeutic approaches to minimize HI-induced brain injury.

The classical view of mammalian cortical development suggests that pyramidal neurons are generated in a temporal sequence, with all radial glial cells (RGCs) contributing to both lower and upper neocortical layers. A recent opposing proposal suggests there is a subgroup of fate-restricted RGCs in the early neocortex, which generates only upper-layer neurons. Little is known about the existence of fate restriction of homologous progenitors in other vertebrate species. We investigated the lineage of selected Emx2+ [vertebrate homeobox gene related to Drosophila empty spiracles (ems)] RGCs in mouse neocortex and chick forebrain and found evidence for both sequential and fate-restricted programs only in mouse, indicating that these complementary populations coexist in the developing mammalian but not avian brain. Among a large population of sequentially programmed RGCs in the mouse brain, a subset of self-renewing progenitors lack neurogenic potential during the earliest phase of corticogenesis. After a considerable delay, these progenitors generate callosal upper-layer neurons and glia. On the other hand, we found no homologous delayed population in any sectors of the chick forebrain. This finding suggests that neurogenic delay of selected RGCs may be unique to mammals and possibly associated with the evolution of the corpus callosum.

The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown.In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria.Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.

Increasing evidence suggests that three-dimensional (3D) cultures provide more appropriate microenvironments to control stem cell response compared with traditional two-dimensional (2D) cultures. However, the molecular mechanism involved in 3D cultured stem cells is not well known. Several microRNAs whose target genes involved in the regulation of self-renewal and differentiation of stem cells were found to be downregulated in 3D cultured PA-1 cells. Among them, miR-7 was predicted to target Kruppel-like factor 4 (Klf4), a key gene for self-renewal of neural stem cells (NSCs). We showed that the differentiation of NSCs was inhibited in 3D collagen scaffolds compared with 2D cultured cells. The quantitative real-time PCR (qPCR) analysis indicated that the expression of miR-7 and Klf4 changed significantly in 2D cultures, whereas the expression stability of miR-7 and Klf4 was detected in 3D cultures. Using luciferase assay and western blot, Klf4 was identified as a target of miR-7 indicating that miR-7 plays a critical role in maintaining the self-renewal capacity through a Klf4-dependent mechanism in 3D cultured cells. Thus, the collagen scaffold-based 3D cell cultures may provide a platform to reveal the regulatory mechanism of cell regulators, which are difficult to find in traditional 2D cell cultures.

Stem cell transplantation represents a promising strategy for the repair of spinal cord injury (SCI). However, the low survival rate of the grafted cells is a major obstacle hindering clinical success because of ongoing secondary injury processes, which includes excitotoxicity, inflammation and oxidative stress. Previous studies have shown that 17b-estradiol (E2) protects several cell types against cytotoxicity. Thus, we examined the effects of E2 on the viability of human eyelid adipose-derived stem cells (hEASCs) in vitro with hydrogen peroxide (H₂O₂)-induced cell model and in vivo within a rat SCI model. Our results showed that E2 protected hEASCs against H₂O₂-induced cell death in vitro, and enhanced the survival of grafted hEASCs in vivo by reducing apoptosis. Additionally, E2 also enhanced the secretion of growth factors by hEASCs, thereby making the local microenvironment more conducive for tissue regeneration. Overall, E2 administration enhanced the therapeutic efficacy of hEASCs transplantation and facilitated motor function recovery after SCI. Hence, E2 administration may be an intervention of choice for enhancing survival of transplanted hEASCs after SCI.

Apoptosis regulatory proteins, such as p53, play a pivotal role in neural differentiation, through mechanisms independent of cell death. In addition, p53 has been identified as an important regulator of mitochondrial survival response, maintaining mitochondrial DNA (mtDNA) integrity and oxidative protection. The aim of this study was to determine the role of mitochondrial p53 in organelle damage and neural differentiation.Our results show that mitochondrial apoptotic events such as reactive oxygen species production, mitochondrial membrane permeabilization, and cytochrome c release are typical of early-stage mouse neural stem cell differentiation, which occurs 3-18 h after induction of differentiation, with no evidence of cell death. In addition, decreased mtDNA content, lipidated LC3 (LC3-II), colocalization of mitochondria and LC3-II puncta, and mitochondria-associated Parkin are consistent with activation of mitophagy. Importantly, at early stages of neural differentiation, p53 was actively translocated to mitochondria and attenuated mitochondrial oxidative stress, cytochrome c release, and mitophagy. Forced mitochondrial translocation of p53 increased neurogenic potential and neurite outgrowth.In conclusion, our results reveal a novel role for mitochondrial p53, which modulates mitochondrial damage and apoptosis-related events in the context of neural differentiation, thus enhancing neuronal fate.