3'-UTR Sequence of Exosomal NANOGP8 DNA as an Extracellular Vesicle-Localization Signal.

Extracellular vesicles (EVs) are garnering attention as a safe and efficient biomolecule delivery system. EVs intrinsically play a crucial role in intercellular communication and pathophysiology by transporting functionally active DNA molecules. The internalized DNA pleiotropically affects the recipient cells. Considering these salient features, an intentional incorporation of specific DNA gene cassettes into EVs and their subsequent delivery to the target cells has potential applications in genetic engineering. Moreover, efficient ways to insert the DNA into EVs during their biogenesis is valuable. Our current research is a step in the development of this technology. As such, cancer cells are known to secrete exosomes containing increased amounts of double-stranded DNA than normal cells. The clonal analysis in our previously published data revealed that exosomes released from various cancer cells contained a significantly larger population of NANOGP8 DNA with a 22-base pair insertion in the 3'-untranslated region (UTR) compared to those secreted by normal cells. This finding led us to hypothesize that the 22-base pair insertion may act as a signal to facilitate the incorporation of NANOGP8 DNA into the exosomes. To test this hypothesis, we compared the EV localization of an Enhanced Green Fluorescent Protein (EGFP) gene fused with the NANOGP8 3'-UTR, with and without the 22-base pair insertion. The quantitative PCR analysis showed a significantly higher EGFP DNA accumulation in exosomes released from cells transfected with the gene cassette containing the 3'-UTR with the 22-base pair insertion. The discovery of a DNA localization signal in exosomal DNA's 3'-UTR could pave the way for the development of an EV-based DNA delivery system. This technology will open new possibilities in genetic engineering and innovative therapies using nucleic acid medicine.

Neural Differentiation of Induced Pluripotent Stem Cells for a Xenogeneic Material-Free 3D Neurological Disease Model Neurulation from Pluripotent Cells Using a Human Hydrogel.

Alzheimer's Disease (AD) is characterized by synapse and neuronal loss and the accumulation of neurofibrillary tangles and Amyloid ß plaques. Despite significant research efforts to understand the late stages of the disease, its etiology remains largely unknown. This is in part because of the imprecise AD models in current use. In addition, little attention has been paid to neural stem cells (NSC), which are the cells responsible for the development and maintenance of brain tissue during an individual's lifespan. Thus, an in vitro 3D human brain tissue model using induced pluripotent stem (iPS) cell-derived neural cells in human physiological conditions may be an excellent alternative to standard models to investigate AD pathology. Following the differentiation process mimicking development, iPS cells can be turned into NSCs and, ultimately, neural cells. During differentiation, the traditionally used xenogeneic products may alter the cells' physiology and prevent accurate disease pathology modeling. Hence, establishing a xenogeneic material-free cell culture and differentiation protocol is essential. This study investigated the differentiation of iPS cells to neural cells using a novel extracellular matrix derived from human platelet lysates (PL Matrix). We compared the stemness properties and differentiation efficacies of iPS cells in a PL matrix against those in a conventional 3D scaffold made of an oncogenic murine-matrix. Using well-defined conditions without xenogeneic material, we successfully expanded and differentiated iPS cells into NSCs via dual-SMAD inhibition, which regulates the BMP and TGF signaling cascades in a manner closer to human conditions. This in vitro, 3D, xenogeneic-free scaffold will enhance the quality of disease modeling for neurodegenerative disease research, and the knowledge produced could be used in developing more effective translational medicine.

Amelioration of Motor Performance and Nigrostriatal Dopamine Cell Volume Using a Novel Far-Infrared Ceramic Blanket in an A53T Alpha-Synuclein Transgenic Parkinson's Disease Mouse Model.

We had attended a Parkinson's Disease (PD) patient for a non-healing wound who reported a marked decrease in his hand tremor and freezing of gait when his wound was exposed to a ceramic far-field infrared (cFIR) blanket. PD is the most frequent motor disorder and the second most frequent neurodegenerative disease after Alzheimer's Disease (AD). The tremor, rigidity, and slowness of movement associated with Parkinson's disease (PD) affect up to 10 million people throughout the world, and the major contributing factor to the pathogenesis of PD is the accumulation and propagation of pathological α-synuclein (α-Syn) and the death of dopaminergic cells in the Nigrostriatal system. Efforts to slow or stop its spreading have resulted in the development and use of dopaminergic drug replacement therapy. Unfortunately, there is a loss of about 70-80% of substantia nigral dopaminergic neurons in patients by the time they are diagnosed with PD, and various dopaminergic drugs provide only temporary relief of their motor symptoms. There are limitations in treating PD with many conventional medications, necessitating a combination of pharmaceutical and non-pharmacological therapy as an essential adjunct to better address the health and welfare of PD patients. We used male adult A53T alpha-synuclein transgenic mice exposed to a ceramic far-infrared blanket. Motor activity was assessed using the rotarod apparatus, and mouse brains were examined to quantify the fluorescence intensities of the immunostained samples. A53T alpha-synuclein transgenic mice had a significantly shorter time stay on the rotating bar than the wild-type mice (B6C3H). The rotarod performance was significantly improved in A53T alpha-synuclein transgenic mice exposed to cFIR as well as B6C3H healthy wild mice exposed to cFIR. There was a significant statistical and substantive increase in the cellular composition of the Striatum and substantia nigra of cFIR-treated mice. Improvement in motor performance is seen in PD mice and wild mice and is associated with increases in cell volume in the substantia nigra and striatum after treatment.

Coculture with Neural Stem Cells May Shift the Transcription Profile of Glioblastoma Multiforme towards Cancer-Specific Stemness.

Glioblastoma multiforme (GBM) possesses a small but significant population of cancer stem cells (CSCs) thought to play a role in its invasiveness, recurrence, and metastasis. The CSCs display transcriptional profiles for multipotency, self-renewal, tumorigenesis, and therapy resistance. There are two possible theories regarding the origin of CSCs in the context of neural stem cells (NSCs); i.e., NSCs modify cancer cells by conferring them with cancer-specific stemness, or NSCs themselves are transformed into CSCs due to the tumor environment created by cancer cells. To test the theories and to investigate the transcriptional regulation of the genes involved in CSC formation, we cocultured NSC and GBM cell lines together. Where genes related to cancer stemness, drug efflux, and DNA modification were upregulated in GBM, they were downregulated in NSCs upon coculture. These results indicate that cancer cells shift the transcriptional profile towards stemness and drug resistance in the presence of NSCs. Concurrently, GBM triggers NSCs differentiation. Because the cell lines were separated by a membrane (0.4 µm pore size) to prevent direct contact between GBM and NSCs, cell-secreted signaling molecules and extracellular vesicles (EVs) are likely involved in reciprocal communication between NSCs and GBM, causing transcription modification. Understanding the mechanism of CSC creation will aid in the identification of precise molecular targets within the CSCs to exterminate them, which, in turn, will increase the efficacy of chemo-radiation treatment.

Stem Cell Therapies for Neurodegenerative Diseases.

Stem cell therapies have been proposed as a treatment option for neurodegenerative diseases, but the best stem cell source and therapeutic efficacy for neuroregeneration remain uncertain. Embryonic stem cells (ESCs) and neural stem cells (NSCs), which can efficiently generate neural cells, could be good candidates but they pose ethical and practical issues. Not only difficult to find the good source of those cells but also they alway pose immunorejection problem since they may not be an autologous cells. Even if we overcome the immunorejection problem, it has also been reported that transplantation of ESCs develop teratoma. Although adult stem cells are more accessible, they have a limited developmental potential. We developed technologies to increase potency of mesenchymal stem cells, which allow them to develop into neural cells, by over expression of the ESC gene, nanog. We also developed a small molecule compound, which significantly increases endogenous NSCs by peripheral administration, eliminating even the necessity of stem cell injection to the brain. These novel technologies may offer neuroregenerative therapies for Alzheimers disease (AD). However, we found that AD pathological condition prevent neurogenesis from NSCs. This chapter discusses how to overcome the problem associated stem cell therapy under AD pathology and introduces exosome as a tool to improve the modification of adult stem cells. These new technologies may open a door for the new era for AD therapy.

Secreted type of amyloid precursor protein induces glial differentiation by stimulating the BMP/Smad signaling pathway.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases leading to dementia. Although cytotoxicity of amyloid ß peptides has been intensively studied within pathophysiology of AD, the physiological function of amyloid precursor protein (APP) still remains unclarified. We have shown previously that secreted APPα (sAPPα) is associated with glial differentiation of neural stem cells. To elucidate specific mechanisms underlying sAPPα-induced gliogenesis, we examined the potential involvement of bone morphogenic proteins (BMPs). BMPs are one of the factors involved in glial differentiation of neural progenitor cells. When expressions of BMP-2, -4, and -7 were examined, upregulation of BMP-4 expression was solely observed as a result of treatment with sAPPα in a time and dose-dependent manner. Furthermore, the treatment of sAPPα promoted phosphorylation of Smad1/5/8, a downstream signaling mediator of BMP receptors. Interestingly, N-terminal domain of APP (1-205) was sufficient to elevate BMP4 expression, resulting in an increase of glial fibrillary acidic protein (GFAP) expression and phosphorylation of Smad1/5/8. However, the application of APP neutralizing antibody and anti-BMP4 antibody significantly suppressed expression of BMP-4 as well as phosphorylation of Smad1/5/8. Thus, our results indicate that sAPPα-induced gliogenesis is in part mediated by the BMP-4 signaling pathway. We also observed upregulation of BMP-4 and phosphorylation of Smad1/5/8 in APP transgenic mice. It is imperative to unravel the mechanisms underlying the role of BMP-4 during APPα-induced glial differentiation in hope of providing novel prevention or treatment for AD.

Suppression of NANOG Expression Reduces Drug Resistance of Cancer Stem Cells in Glioblastoma.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that harbors therapy-resistant cancer stem cells (CSCs). Due to the limited effectiveness of conventional chemotherapies and radiation treatments against CSCs, there is a critical need for the development of innovative therapeutic approaches. Our previous research revealed the significant expression of embryonic stemness genes, NANOG and OCT4, in CSCs, suggesting their role in enhancing cancer-specific stemness and drug resistance. In our current study, we employed RNA interference (RNAi) to suppress the expression of these genes and observed an increased susceptibility of CSCs to the anticancer drug, temozolomide (TMZ). Suppression of NANOG expression induced cell cycle arrest in CSCs, specifically in the G0 phase, and it concomitantly decreased the expression of PDK1. Since PDK1 activates the PI3K/AKT pathway to promote cell proliferation and survival, our findings suggest that NANOG contributes to chemotherapy resistance in CSCs through PI3K/AKT pathway activation. Therefore, the combination of TMZ treatment with RNAi targeting NANOG holds promise as a therapeutic strategy for GBM.

Analysis of regulatory sequences in exosomal DNA of NANOGP8.

Exosomes participate in intercellular communication by transporting functionally active molecules. Such cargo from the original cells comprising proteins, micro-RNA, mRNA, single-stranded (ssDNA) and double-stranded DNA (dsDNA) molecules pleiotropically transforms the target cells. Although cancer cells secrete exosomes carrying a significant level of DNA capable of modulating oncogene expression in a recipient cell, the regulatory mechanism is unknown. We have previously reported that cancer cells produce exosomes containing NANOGP8 DNA. NANOGP8 is an oncogenic paralog of embryonic stem cell transcription factor NANOG and does not express in cells since it is a pseudogene. However, in this study, we evaluated NANOGP8 expression in glioblastoma multiforme (GBM) tissue from a surgically removed brain tumor of a patient. Significantly higher NANOGP8 transcription was observed in GBM cancer stem cells (CSCs) than in GBM cancer cells or neural stem cells (NSCs), despite identical sequences of NANOGP8-upstream genomic region in all the cell lines. This finding suggests that upstream genomic sequences of NANOGP8 may have environment-dependent promoter activity. We also found that the regulatory sequences upstream of exosomal NANOGP8 GBM DNA contain multiple core promoter elements, transcription factor binding sites, and segments of human viruses known for their oncogenic role. The exosomal sequence of NANOGP8-upstream GBM DNA is different from corresponding genomic sequences in CSCs, cancer cells, and NSCs as well as from the sequences reported by NCBI. These sequence dissimilarities suggest that exosomal NANOGP8 GBM DNA may not be a part of the genomic DNA. Exosomes possibly acquire this DNA from other sources where it is synthesized by an unknown mechanism. The significance of exosome-bestowed regulatory elements in the transcription of promoter-less retrogene such as NANOGP8 remains to be determined.

Methods and Protocols for Using Extracellular Vesicles as Delivery Vehicles in Neuronal Research.

Extracellular vesicles (EVs) transport nucleic acids, proteins, and lipid molecules for intercellular communication. The biomolecular cargo from EVs can modify the recipient cell genetically, physiologically, and pathologically. This innate ability of EVs can be harnessed to deliver the cargo of interest to a specific organ or a cell type. Importantly, due to their ability to cross the blood-brain barrier (BBB), the EVs can be used as delivery vehicles to transport therapeutic drugs and other macromolecules to inaccessible organs such as the brain. Therefore, the current chapter includes laboratory techniques and protocols focusing on the customization of EVs for neuronal research.

Reelin regulates differentiation of neural stem cells by activation of notch signaling through Disabled-1 tyrosine phosphorylation.

We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin-stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development.

Exosome mediated Tom40 delivery protects against hydrogen peroxide-induced oxidative stress by regulating mitochondrial function.

Mitochondrial dysfunction is a hallmark of neurodegeneration. The expression level of Tom40, a crucial mitochondrial membrane protein, is significantly reduced in neurodegenerative disease subjects. Tom40 overexpression studies have shown to protect the neurons against oxidative stress by improving mitochondrial function. Thus, successful delivery of Tom40 protein to the brain could lead to a novel therapy for neurodegenerative diseases. However, delivering protein to the cell may be difficult. Especially the blood-brain barrier (BBB) is a big hurdle to clear in order to deliver the protein to the brain. In the current study, we engineered exosomes, which are the extracellular vesicles of endosomal origin, and able to cross BBB as delivery vehicles packing human Tom40. We found Tom40 protein delivery by the exosome successfully protected the cells against hydrogen peroxide-induced oxidative stress. This result suggests that exosome-mediated delivery of Tom40 may potentially be useful in restoring mitochondrial functions and alleviating oxidative stress in neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases.

Exposure to a Pathological Condition May Be Required for the Cells to Secrete Exosomes Containing mtDNA Aberration.

Exosomes, nanovesicles secreted by all cells, carry out intercellular communication by transmitting biologically active cargo comprising DNA, RNA, and proteins. These biomolecules reflect the status of their parent cells and can be altered by pathological conditions. Therefore, the researchers have been investigating differential sequences and quantities of DNA associated with exosomes as valuable biomarkers of diseases. Exosomes carry different types of DNA molecules, including genomic, cytoplasmic, and mitochondrial (mtDNA). The mtDNA aberrations are reported to be a hallmark of diseases involving oxidative stress, such as cancer and neurodegenerative diseases. Establishing robust in vitro models comprising appropriate cell lineages is the first step towards investigating disease-specific anomalies and testing therapeutics. Induced pluripotent stem (iPS) cells from patients with diseases have been used for this purpose since they can differentiate into various cells. The current study investigated mtDNA aberrations in exosomes secreted by primary cancer cells and neural stem cells (NSCs) differentiated from iPS cells. The primary cancer cells were isolated from surgically removed glioblastoma multiforme (GBM) tissue, and the iPS cells were produced from control and Alzheimer's disease (AD) subjects' B lymphocytes. We detected aberrations in mtDNA associated with exosomes secreted from GBM cells but not from the NSCs. This result indicates that the cells may not secrete exosomes carrying mtDNA aberration without exposure to a pathological condition. Thus, we may need to consider this fact when we use iPS cell-derived cells as an in vitro disease model.

Homologous Use of Allogeneic Umbilical Cord Tissue to Reduce Knee Pain and Improve Knee Function.

To determine if knee pain subjects who received cryopreserved umbilical cord tissue (UCT) injected into knee joints experience less knee pain, better function, decreased physical limitations, and reduction of medications (opiates, NSAIDs, and acetaminophen) over a 24 week period, Visual Analog Scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and medication usage data were recorded for 30 consenting human knee pain subjects receiving UCT at a single site in the United States. Subject profile information was gathered and analyzed to gain insight into the effects of age, sex, and BMI on improvement over time. Mean resting VAS scores and mean VAS scores with activity improved over 24 weeks (from 1.95 to 0.83 and from 6.28 to 2.87, respectively, p < 0.001). There was no strong evidence of a correlation between sex and VAS scores. There were statistically significant correlations for BMI vs. pre-injection VAS with activity scores and Age vs. pre-injection VAS with activity scores (r = 0.402, p = 0.028 and r = 0.434, p = 0.017, respectively). Mean WOMAC scores improved from 44.7 to 18.5 over 24 weeks (p < 0.001). 77.8% of patients who used medications at the beginning of the study reduced or eliminated medication use. The analysis demonstrates that injections with UCT decrease pain, improve physical function, and allow for less medication use for at least 24 weeks.

Methods for the Detection of Circulating Pseudogenes and Their Use as Cancer Biomarkers.

Pseudogenes, once considered the "junk remnants of genes," are found to significantly affect the regulatory network of healthy and cancer cells, as well as to be highly specific markers of cancer cell identity. Qualitative and quantitative analysis of pseudogenes has a diagnostic and prognostic value in cancer research via the detection of cell-free pseudogenic DNA circulating throughout the body. Exosomes, nanoparticles with a lipid membrane secreted by almost all types of cells, carry cellular-blueprint molecules, including pseudogenic DNA, as cancer-specific cargo. Therefore, it is vital to develop better laboratory techniques and protocols to identify exosome-associated pseudogenes.

Accelerated Wound Healing Using a Novel Far-Infrared Ceramic Blanket.

INTRODUCTION: Wounds are associated with ranges of simple to complex disruption or damage to anatomical structure and function. They are also associated with enormous economic and social costs, increasing yearly, resulting in a severe impact on the wellbeing of individuals and society. Technology that might accelerate wound healing is associated with many benefits to injured people. METHODS: BALBc mice underwent symmetrical excisional wounds through the panniculus carnosus. They were divided into a treatment group placed on an autonomous ceramic far-field infrared blanket (cIFRB) and a control group maintained under standard conditions. We also expanded and cultured adipose tissue-derived mesenchymal stem cells (MSCs) on cIFRB and compared them to standard conditions subjected to a scratch injury to compare survival, proliferation, and wound healing. RESULTS: The wound healing of the cIRFB treatment group was significantly faster than the control group of mice. The wound-healing effect of mesenchymal stem cells on cIRFB was also increased and associated with significant migration to the wound area. CONCLUSIONS: Wound healing is improved in a mouse model exposed to cFIRB. The ceramic blanket also promotes survival, proliferation, increased migration, and wound healing of MSCs without affecting their survival and proliferation. The utilization of cFIRB in cellular biology and medical applications may be promising in many situations currently explored in animal and human models. This technology needs no direct or battery power source and is entirely autonomous and noninvasive, making its application possible in any environment.

The Pupillary Light Reflex as a Biomarker of Concussion.

The size of our pupils changes continuously in response to variations in ambient light levels, a process known as the pupillary light reflex (PLR). The PLR is not a simple reflex as its function is modulated by cognitive brain function and any long-term changes in brain function secondary to injury should cause a change in the parameters of the PLR. We performed a retrospective clinical review of the PLR of our patients using the BrightLamp Reflex iPhone app. The PLR variables of latency, maximum pupil diameter (MaxPD), minimum pupil diameter (MinPD), maximum constriction velocity (MCV), and the 75% recovery time (75% PRT) were associated with significant differences between subjects who had suffered a concussion and those that had not. There were also significant differences in PLR metrics over the life span and between genders and those subjects with and without symptoms. The differences in PLR metrics are modulated not only by concussion history but also by gender and whether or not the person has symptoms associated with a head injury. A concussive injury to the brain is associated with changes in the PLR that persist over the life span, representing biomarkers that might be used in clinical diagnosis, treatment, and decision making.

DNA Associated with Circulating Exosomes as a Biomarker for Glioma.

Cancerous and non-cancerous cells secrete exosomes, a type of nanovesicle known to carry the molecular signature of the parent for intercellular communications. Exosomes secreted by tumor cells carry abnormal DNA, RNA, and protein molecules that reflect the cancerous status. DNA is the master molecule that ultimately affects the function of RNA and proteins. Aberrations in DNA can potentially lead a cell to malignancy. Deviant quantities and the differential sequences of exosomal DNA are useful characteristics as cancer biomarkers. Since these alterations are either associated with specific stages of cancer or caused due to a clinical treatment, exosomal DNA is valuable as a diagnostic, prognostic, predictive, and therapeutic-intervention response biomarker. Notably, the exosomes can cross an intact blood-brain barrier and anatomical compartments by transcytosis. As such, the cancer-specific trademark molecules can be detected in systemic blood circulation and other body fluids, including cerebrospinal fluid, with non-invasive or minimally invasive procedures. This comprehensive review highlights the cancer-specific modulations of DNA associated with circulating exosomes that are beneficial as glioma biomarkers.

Differential sequences and single nucleotide polymorphism of exosomal SOX2 DNA in cancer.

Glioblastoma multiforme (GBM) is the most common form of brain cancer, with an average life expectancy of fewer than two years post-diagnosis. We have previously reported that cancer cell originated exosomes, including GBM, have NANOG and NANOGP8 DNA associated with them. The exosomal NANOG DNA has certain differences as compared to its normal counterpart that are of immense importance as a potential cancer biomarker. NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. Similarly, SOX2 is another stemness gene highly expressed in cancer stem cells with an intimate involvement in GBM progression and metastasis as well as promotion of tumorigenicity in Neuroblastoma (NB). Since exosomes are critical in intercellular communication with a role in dissipating hallmark biomolecules responsible for cancer, we conducted a detailed analysis of the association of the SOX2 gene with exosomes whose sequence modulations with further research and appropriate sample size can help to identify diagnostic markers for cancer. We have detected SOX2 DNA associated with exosomes and have identified some of the SNPs and nucleotide variations in the sequences from a GBM and SH-SY5Y sample. Although a further systematic investigation of exosomal DNA from GBM and NB patient's blood is needed, finding of SOX2 DNA in exosomes in the current study may have value in clinical research. SOX2 is known to be misregulated in cancer cells by changes in miRNA function, such as SNPs in the binding sites. Our finding of cancer-specific SNPs in exosomal SOX2 DNA sequence may reflect those changes in the cancer stem cells as well as cancer cells. A series of our study on embryonic stem cell gene analysis in exosomal DNA may lead to a minimally invasive exosome-based diagnosis, and give us a key in understanding the mechanisms of cancer formation, progression, and metastasis.

Xeno- and transgene-free reprogramming of mesenchymal stem cells toward the cells expressing neural markers using exosome treatments.

Neural stem cells (NSCs), capable of self-renew and differentiate into neural cells, hold promise for use in studies and treatments for neurological diseases. However, current approaches to obtain NSCs from a live brain are risky and invasive, since NSCs reside in the subventricular zone and the in the hippocampus dentate gyrus. Alternatively, mesenchymal stem cells (MSCs) could be a more available cell source due to their abundance in tissues and easier to access. However, MSCs are committed to producing mesenchymal tissue and are not capable of spontaneously differentiating into neural cells. Thus, the process of reprogramming of MSCs into neural cells to use in clinical and scientific settings has significantly impacted the advancement of regenerative medicine. Previously, our laboratory reported trans-differentiation of MSCs to neural cells through the induced pluripotent stem (iPS) cells state, which was produced by overexpression of the embryonic stem cell gene NANOG. In the current study, we demonstrate that treatment with exosomes derived from NSCs makes MSCs capable of expressing neural cell markers bypassing the generation of iPS cells. An epigenetic modifier, decitabine (5-aza-2'-deoxycytidine), enhanced the process. This novel Xeno and transgene-free trans-differentiation technology eliminates the issues associated with iPS cells, such as tumorigenesis. Thus, it may accelerate the development of neurodegenerative therapies and in vitro neurological disorder models for personalized medicine.

MCP-1-induced migration of NT2 neuroprogenitor cells involving APP signaling.

Neuroprogenitor cells are an important resource because of their great potential to replace damaged cells in the brain caused by trauma and disease. Studies have shown that when neuroprogenitor cells are transplanted into the brain they migrate towards damaged areas, suggesting that these areas express factors that recruit migrating cells. Generally, after neuronal injury, there is a neuroinflammatory response that results in increased chemokine production. In this present study, we demonstrate that monocyte chemoattractant protein-1 (MCP-1) significantly induces the migration of NT2 neuroprogenitor cells. Activation of intracellular cyclic adenosine monophosphate or protein kinase C with forskolin and phorbol 12-myristate 13-acetate, respectively, was able to completely abolish the MCP-1-induced migration. Contrarily, neither extracellular signal-regulated kinase nor p38 mitogen-activated protein kinase was required for NT2 cells to respond to MCP-1. Previously, we showed that amyloid precursor protein (APP) activity increases MCP-1 expression in NT2 cells. We now demonstrate that NT2 cells expressing APP can induce migration of other neuroprogenitor cells. Utilizing a MCP-1 neutralizing antibody, we discovered that APP-induced migration was not caused solely by increased MCP-1 production. Interestingly, APP-increased expression of several C-C chemokines: MCP-1, regulated upon activation, normal T-cell expressed, and secreted (RANTES), and macrophage inflammatory protein alpha (MIP-1 alpha). This demonstrates the unique role APP has in regulating chemokine production, which directly affects cell migration. Taken together, these data provides greater detail of the chemotactic factors and intracellular signaling that direct neuroprogenitor cell migration, allowing for better understanding of cell migration during transplantation.