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Ring chromosomes are structural aberrations commonly associated with birth defects,mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome,and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes,no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division,ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations,enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of /`chromosome therapy/' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition,our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control,which is of critical relevance to human development and disease. View Publication

The concept that bone marrow (BM)-derived cells participate in neural regeneration remains highly controversial and the identity of the specific cell type(s) involved remains unknown. We recently reported that the BM contains a highly mobile population of CXCR4+ cells that express mRNA for various markers of early tissue-committed stem cells (TCSCs),including neural TCSCs. Here,we report that these cells not only express neural lineage markers (beta-III-tubulin,Nestin,NeuN,and GFAP),but more importantly form neurospheres in vitro. These neural TCSCs are present in significant amounts in BM harvested from young mice but their abundance and responsiveness to gradients of motomorphogens,such as SDF-1,HGF,and LIF,decreases with age. FACS analysis,combined with analysis of neural markers at the mRNA and protein levels,revealed that these cells reside in the nonhematopoietic CXCR4+/Sca-1+/lin-/CD45 BM mononuclear cell fraction. Neural TCSCs are mobilized into the peripheral-blood following stroke and chemoattracted to the damaged neural tissue in an SDF-1-CXCR4-,HGF-c-Met-,and LIF-LIF-R-dependent manner. Based on these data,we hypothesize that the postnatal BM harbors a nonhematopoietic population of cells that express markers of neural TCSCs that may account for the beneficial effects of BM-derived cells in neural regeneration. View Publication

Transplantation of human neural progenitor cells (NPCs) into the brain or spinal cord to replace lost cells,modulate the injury environment,or create a permissive milieu to protect and regenerate host neurons is a promising therapeutic strategy for neurological diseases. Deriving NPCs from human fetal tissue is feasible,although problematic issues include limited sources and ethical concerns. Here we describe a new and abundant source of NPCs derived from human induced pluripotent stem cells (iPSCs). A novel chopping technique was used to transform adherent iPSCs into free-floating spheres that were easy to maintain and were expandable (EZ spheres) (Ebert et al. [2013] Stem Cell Res 10:417–427). These EZ spheres could be differentiated towards NPC spheres with a spinal cord phenotype using a combination of all-trans retinoic acid (RA) and epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) mitogens. Suspension cultures of NPCs derived from human iPSCs or fetal tissue have similar characteristics,although they were not similar when grown as adherent cells. In addition,iPSC-derived NPCs (iNPCs) survived grafting into the spinal cord of athymic nude rats with no signs of overgrowth and with a very similar profile to human fetal-derived NPCs (fNPCs). These results suggest that human iNPCs behave like fNPCs and could thus be a valuable alternative for cellular regenerative therapies of neurological diseases. J. Comp. Neurol. 522:2707–2728,2014. textcopyright 2014 Wiley Periodicals,Inc. View Publication

Human pluripotent stem cells (hPSCs) possess great value in the aspect of cellular therapies due to its self-renewal and potential to differentiate into all somatic cell types. A few defined synthetic surfaces such as polymers and adhesive biological materials conjugated substrata were established for the self-renewal of hPSCs. However,none of them was effective in the generation of human induced pluripotent stem cells (hiPSCs) and long-term maintenance of multiple hPSCs,and most of them required complicated manufacturing processes. Polydopamine has good biocompatibility,is able to form a stable film on nearly all solid substrates surface,and can immobilize adhesive biomolecules. In this manuscript,a polydopamine-mediated surface was developed,which not only supported the reprogramming of human somatic cells into hiPSCs under defined conditions,but also sustained the growth of hiPSCs on diverse substrates. Moreover,the proliferation and pluripotency of hPSCs cultured on the surface were comparable to Matrigel for more than 20 passages. Besides,hPSCs were able to differentiate to cardiomyocytes and neural cells on the surface. This polydopamine-based synthetic surface represents a chemically-defined surface extensively applicable both for fundamental research and cell therapies of hPSCs. View Publication

BACKGROUND Transplantation of enteric neural stem cells (ENSC) holds promise as a potential therapy for enteric neuropathies,including Hirschsprung disease. Delivery of transplantable cells via laparotomy has been described,but we propose a novel,minimally invasive endoscopic method of cell delivery. METHODS Enteric neural stem cells for transplantation were cultured from dissociated gut of postnatal donor mice. Twelve recipient mice,including Ednrb(-/-) mice with distal colonic aganglionosis,underwent colonoscopic injection of ENSC under direct vision using a 30-gauge Hamilton needle passed through a rigid cystoureteroscope. Cell engraftment,survival,and neuroglial differentiation were studied 1-4 weeks after the procedure. KEY RESULTS All recipient mice tolerated the procedure without complications and survived to sacrifice. Transplanted cells were found within the colonic wall in 9 of 12 recipient mice with differentiation into enteric neurons and glia. CONCLUSIONS & INFERENCES Endoscopic injection of ENSC is a safe and reliable method for cell delivery,and can be used to deliver a large number of cells to a specific area of disease. This minimally invasive endoscopic approach may prove beneficial to future human applications of cell therapy for neurointestinal disease. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease,and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly,as well as scFvs that target the CD133-positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv,and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium. Taken together,these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library,which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. View Publication

Activation of neural stem/progenitor cells (NSPCs) is a potential therapeutic strategy of neurological disorders. In this study,NSPCs of subventricular zone were isolated and cultured from platelet-derived growth factor-β-receptor-knockout (PDGFR-β(-/-)) mice of postnatal day 1 (P1) and P28,and the roles of PDGFR-β were examined in these cells. In PDGFR-β-preserving control NSPCs,stem cell activities,such as numbers and diameters of secondary neurospheres,cell proliferation and survival rates,were significantly higher in P1 NSPCs than those in P28 NSPCs. In PDGFR-β(-/-) NSPCs,most of these parameters were decreased as compared with age-matched controls. Among them,the decrease of secondary neurosphere formation was most striking in P1 and P28 PDGFR-β(-/-) NSPCs and in P28 control NSPCs as compared with P1 control NSPCs. PCR-array and following quantitative real-time PCR (qRT-PCR) analyses demonstrated that expressions of fibroblast growth factor-2 (FGF2) and exons IV-IX of brain-derived neurotrophic factor (BDNF) were decreased,and noggin was increased in P1 PDGFR-β(-/-) as compared with P1 controls. Addition of BDNF rescued the number and diameter of secondary neurospheres in P1 PDGFR-β(-/-) NSPCs to similar levels as controls. The expressions of PDGFs and PDGFRs in control NSPCs were increased along with the differentiation-induction,where phosphorylated PDGFR-β was co-localized with neuronal and astrocyte differentiation markers. In controls,the neuronal differentiation was decreased,and the glial differentiation was increased from P1 to P28 NSPCs. Compared with P1 controls,neuronal differentiation was reduced in P1 PDGFR-β(-/-) NSPCs,whereas glial differentiation was comparable between the two genotypes. These results suggest that PDGFR-β signaling is important for the self-renewal and multipotency of NSPCs,particularly in neonatal NSPCs. BDNF,FGF2,and noggin may be involved in the effects of PDGFR-β signaling in these cells. Accordingly,the activation of PDGFR-β in NSPCs may be a novel therapeutic strategy of neurological diseases. View Publication

BIX01294 (Bix) is known to be a euchromatic histone-lysine N-methyltransferase 2 inhibitor and treatment with Bix suppresses cancer cell survival and proliferation. In the present study,it was observed that sequential treatment with low-dose Bix notably increases glioblastoma cell migration and metastasis. It was demonstrated that U251 cells sequentially treated with low-dose Bix exhibited induced characteristic changes in critical epithelial-mesenchymal transition (EMT) markers,including E-cadherin,N-cadherin,β-catenin and zinc finger protein SNAI2. Notably,sequential treatment with Bix also increased the expression of cancer stem cell-associated markers,including sex determining region Y-box 2,octamer-binding transcription factor 4 and cluster of differentiation 133. Neurosphere formation was significantly enhanced in cells sequentially treated with Bix,compared with control cells (control: P=0.011; single treatment of Bix,P=0.045). The results of the present study suggest that accumulation of low-dose Bix enhanced the migration and metastatic potential of glioblastoma cells by regulating EMT-associated gene expression,which may be the cause of the altered properties of glioblastoma stem cells. View Publication

Glycolipids are amphipathic molecules which are highly expressed on cell membranes in skin and brain where they mediate several key cellular processes. Neural stem cells are defined as undifferentiated,proliferative,multipotential cells with extensive self-renewal and are responsive to brain injury. Di-rhamnolipid: α-L-rhamnopyranosyl-(1-2)α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid,also referred to as di-rhamnolipid BAC-3,is a glycolipid isolated from the bacteria Pseudomonas aeruginosa. In the previous studies,di-rhamnolipid enhanced dermal tissue healing and regeneration. The present study provides the first assessment of di-rhamnolipid,and glycolipid biosurfactants in general,on the nervous system. Treatment of neural stem cells isolated from the lateral ventricle of adult mice and cultured in defined media containing growth factors at 0.5 and 1 μg/ml of di-rhamnolipid increased the number of neurospheres (2.7- and 2.8-fold,respectively) compared to controls and this effect remained even after passaging in the absence of di-rhamnolipid. In addition,neural stem cells treated with di-rhamnolipid at 50 and 100 μg/ml in defined media supplemented with fetal calf serum and without growth factors exhibited increased cell viability,indicating an interaction between di-rhamnolipid and serum components in the regulation of neural stem cells and neuroprogenitors. Intracerebroventricular administration of di-rhamnolipid at 300 and 120 ng/day increased the number of neurospheres (1.3- and 1.63-fold,respectively) that could be derived from the anterior lateral ventricles of adult mice. These results indicate that di-rhamnolipid stimulates proliferation of neural stem cells and increases their endogenous pools which may have therapeutic potential in managing neurodegenerative or neuropsychiatric disorders and promoting nervous tissue regeneration following injury. View Publication

Although human induced pluripotent stem cells (hiPSCs) hold great potential for the study of human diseases affecting disparate cell types,they have been underutilized in seeking mechanistic insights into the pathogenesis of congenital craniofacial disorders. Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in EFNB1 and characterized by craniofacial,skeletal,and neurological anomalies. Heterozygous females are more severely affected than hemizygous males,a phenomenon termed cellular interference that involves mosaicism for EPHRIN-B1 function. Although the mechanistic basis for cellular interference in CFNS has been hypothesized to involve Eph/ephrin-mediated cell segregation,no direct evidence for this has been demonstrated. Here,by generating hiPSCs from CFNS patients,we demonstrate that mosaicism for EPHRIN-B1 expression induced by random X inactivation in heterozygous females results in robust cell segregation in human neuroepithelial cells,thus supplying experimental evidence that Eph/ephrin-mediated cell segregation is relevant to pathogenesis in human CFNS patients. View Publication