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INTRODUCTION Hirschsprung's disease is characterized by a developmental arrest of neural crest cell migration,causing distal aganglionosis. Transplanted cells derived from the neural crest may regenerate enteric ganglia in this condition. We investigated the potential of skin-derived precursor cells (SKPs) to engraft and to differentiate into enteric ganglia in aganglionic rat intestine in vivo. METHODS Adult Lewis rat jejunal segments were separated from intestinal continuity and treated with benzalkonium chloride to induce aganglionosis. Ganglia were identified via immunohistochemical stains for S100 and β-III tubulin (TUJ1). SKPs were procured from neonatal Lewis rats expressing enhanced green fluorescent protein (GFP) and cultured in neuroglial-selective media. SKP cell line expansion was quantified,and immunophenotypes were assessed by immunocytochemistry. Aganglionic segments underwent SKP transplantation 21-79days after benzalkonium chloride treatment. The presence of GFP+cells,mature neurons,and mature glia was evaluated at posttransplant days 1,6,and 9. RESULTS Benzalkonium chloride-induced aganglionosis persisted for at least 85days. Prior to differentiation,SKPs expressed S100,denoting neural crest lineage,and nestin,a marker of neuronal precursors. Differentiated SKPs in vitro expressed GFAP,a marker of glial differentiation,as well as TUJ1 and several enteric neurotransmitters. After transplantation,GFP+structures resembling ganglia were identified between longitudinal and circular smooth muscle layers. CONCLUSION SKPs are capable of engraftment,migration,and differentiation within aganglionic rodent intestine in vivo. Differentiated SKPs generate structures that resemble enteric ganglia. Our observations suggest that SKPs represent a potential gangliogenic therapeutic agent for Hirschsprung's disease. View Publication

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies characterized by an intense tumor stroma with hypoperfused regions,a significant inflammatory response and pronounced therapy resistance. New therapeutic agents are urgently needed. The plant-derived agent triptolide also known as thunder god vine" has a long history in traditional Chinese medicine for treatment of rheumatoid arthritis and cancer and is now in a clinical phase II trial for establishing the efficacy against a placebo. The authors mimicked the situation in patient tumors by induction of hypoxia in experimental models of pancreatic cancer stem cells (CSCs) and evaluated the therapeutic effect of triptolide. Hypoxia led to induction of colony and spheroid formation aldehyde dehydrogenase 1 (ALDH1) and NF-κB activity migratory potential and a switch in morphology to a fibroblastoid phenotype as well as stem cell- and epithelial-mesenchymal transition-associated protein expression. Triptolide efficiently inhibited hypoxia-induced transcriptional signaling and downregulated epithelial-mesenchymal transition (EMT) and CSC features in established highly malignant cell lines whereas sensitive cancer cells or nonmalignant cells were less affected. In vivo triptolide inhibited tumor take and tumor growth. In primary CSCs isolated from patient tumors triptolide downregulated markers of CSCs proliferation and mesenchymal cells along with upregulation of markers for apoptosis and epithelial cells. This study is the first to show that triptolide reverses EMT and CSC characteristics and therefore may be superior to current chemotherapeutics for treatment of PDA. View Publication

Adult hippocampal neurogenesis is stimulated by chronic administration of antidepressants (ADs) and by voluntary exercise. Neural progenitor cells (NPCs) in the dentate gyrus (DG) that are capable of continuous proliferation and neuronal differentiation are the source of such structural plasticity. Here we report that mice lacking the receptor tyrosine kinase TrkB in hippocampal NPCs have impaired proliferation and neurogenesis. When exposed to chronic ADs or wheel-running,no increase in proliferation or neurogenesis is observed. Ablation of TrkB also renders these mice behaviorally insensitive to antidepressive treatment in depression- and anxiety-like paradigms. In contrast,mice lacking TrkB only in differentiated DG neurons display typical neurogenesis and respond normally to chronic ADs. Thus,our data establish an essential cell-autonomous role for TrkB in regulating hippocampal neurogenesis and behavioral sensitivity to antidepressive treatments,and support the notion that impairment of the neurogenic niche is an etiological factor for refractory responses to an antidepressive regimen. View Publication

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological,genetic,and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells,much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM),epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly,despite its greater biological activity than wild-type EGFR (wtEGFR),individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population,and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes,we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues,glioma cell lines,glioma stem cells,and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130,which in turn activates wtEGFR in neighboring cells,leading to enhanced rates of tumor growth. Ablating IL-6,LIF,or gp130 uncouples this cellular cross-talk,and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass,and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention. View Publication

Engineered neural stem cells (NSCs) are a promising approach to treating glioblastoma (GBM). The ideal NSC drug carrier for clinical use should be easily isolated and autologous to avoid immune rejection. We transdifferentiated (TD) human fibroblasts into tumor-homing early-stage induced NSCs (h-iNSC(TE)),engineered them to express optical reporters and different therapeutic gene products,and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSC(TE) in patient-derived GBM models of surgical and nonsurgical disease. Molecular and functional analysis revealed that our single-factor SOX2 TD strategy converted human skin fibroblasts into h-iNSC(TE) that were nestin(+) and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSC(TE) rapidly migrated to human GBM cells and penetrated human GBM spheroids,a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSC(TE) delivery of the proapoptotic agent tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) reduced the size of solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. Additionally,h-iNSC(TE) thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSC(TE)-TK) reduced the size of patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical NSC therapy,h-iNSC(TE)-TK therapy delivered into the postoperative surgical resection cavity delayed the regrowth of residual GBMs threefold and prolonged survival from 46 to 60 days. These results suggest that TD of human skin into h-iNSC(TE) is a platform for creating tumor-homing cytotoxic cell therapies for cancer,where the potential to avoid carrier rejection could maximize treatment durability in human trials. View Publication

The relatively new field of stem cell biology is hampered by a lack of sufficient means to accurately determine the phenotype of cells. Cell-type-specific markers,such as cell surface proteins used for flow cytometry or fluorescence-activated cell sorting,are limited and often recognize multiple members of a stem cell lineage. We sought to develop a complementary approach that would be less dependent on the identification of particular markers for the subpopulations of cells and would instead measure their overall character. We tested whether a microfluidic system using dielectrophoresis (DEP),which induces a frequency-dependent dipole in cells,would be useful for characterizing stem cells and their differentiated progeny. We found that populations of mouse neural stem/precursor cells (NSPCs),differentiated neurons,and differentiated astrocytes had different dielectric properties revealed by DEP. By isolating NSPCs from developmental ages at which they are more likely to generate neurons,or astrocytes,we were able to show that a shift in dielectric property reflecting their fate bias precedes detectable marker expression in these cells and identifies specific progenitor populations. In addition,experimental data and mathematical modeling suggest that DEP curve parameters can indicate cell heterogeneity in mixed cultures. These findings provide evidence for a whole cell property that reflects stem cell fate bias and establish DEP as a tool with unique capabilities for interrogating,characterizing,and sorting stem cells. View Publication

Schizophrenia has been considered a devastating clinical syndrome rather than a single disease. Nevertheless,the mechanisms behind the onset of schizophrenia have been only partially elucidated. Several studies propose that levels of trace elements are abnormal in schizophrenia; however,conflicting data generated from different biological sources prevent conclusions being drawn. In this work,we used synchrotron radiation X-ray microfluorescence spectroscopy to compare trace element levels in neural progenitor cells (NPCs) derived from two clones of induced pluripotent stem cell lines of a clozapine-resistant schizophrenic patient and two controls. Our data reveal the presence of elevated levels of potassium and zinc in schizophrenic NPCs. Neural cells treated with valproate,an adjunctive medication for schizophrenia,brought potassium and zinc content back to control levels. These results expand the understanding of atomic element imbalance related to schizophrenia and may provide novel insights for the screening of drugs to treat mental disorders. ?? 2014 Elsevier B.V. View Publication

Neurogenesis decreases during aging and following cranial radiotherapy,causing a progressive cognitive decline that is currently untreatable. However,functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover,we observed a marked increase in TGF-β1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. In co-cultures,irradiated brain endothelial cells induced the apoptosis of neural stem/progenitor cells via TGF-β/Smad3 signalling. Strikingly,the blockade of TGF-β signalling in vivo using a neutralizing antibody or the selective inhibitor SB-505124 significantly improved neurogenesis in aged and irradiated mice,prevented apoptosis and increased the proliferation of neural stem/progenitor cells. These findings suggest that anti-TGF-β-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging. View Publication

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited,stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently,the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition,the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However,the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation,we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice,hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling,increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. View Publication

BACKGROUND UBQLN2 mutations have recently been associated with familial forms of amyotrophic lateral sclerosis (ALS) and ALS-dementia. UBQLN2 encodes for ubiquilin-2,a member of the ubiquitin-like protein family which facilitates delivery of ubiquitinated proteins to the proteasome for degradation. To study the potential role of ubiquilin-2 in ALS,we used recombinant adeno-associated viral (rAAV) vectors to express UBQLN2 and three of the identified ALS-linked mutants (P497H,P497S,and P506T) in primary neuroglial cultures and in developing neonatal mouse brains. RESULTS In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates. Intracerebroventricular injection of FVB mice at post-natal day 0 with rAAV2/8 expressing wild type or mutant UBQLN2 resulted in widespread,sustained expression of ubiquilin-2 in brain. In contrast to wild type,mutant UBQLN2 expression induced significant pathology with large neuronal,cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil. Ubiquilin-2 inclusions co-localized with ubiquitin,p62/SQSTM,optineurin,and occasionally TDP-43,but were negative for α-synuclein,neurofilament,tau,and FUS. Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates. Mice expressing mutant forms of UBQLN2 variably developed a motor phenotype at 3-4 months,including nonspecific clasping and rotarod deficits. CONCLUSIONS These findings demonstrate that UBQLN2 mutants (P497H,P497S,and P506T) induce proteinopathy and cause behavioral deficits,supporting a toxic" gain-of-function which may contribute to ALS pathology. These data establish also that our rAAV model can be used to rapidly assess the pathological consequences of various UBQLN2 mutations and provides an agile system to further interrogate the molecular mechanisms of ubiquilins in neurodegeneration. View Publication

In the central nervous system,neural stem cells proliferate in the ventricular zone (VZ) and sequentially give rise to both neurons and glial cells in a temporally and spatially regulated manner,suggesting that stem cells may differ from one another in different brain regions and at different developmental stages. For the purpose of marking and purifying neural stem cells to ascertain whether such differences exist,we generated transgenic mice using promoters from Hes genes (pHes1 or pHes5) to drive expression of destabilized enhanced green fluorescent protein. In the developing brains of these transgenic mice,GFP expression was restricted to undifferentiated cells in the VZ,which could asymmetrically produce a Numb-positive neuronal daughter and a GFP-positive progenitor cell in clonal culture,indicating that they retain the capacity to self-renew. Our results suggest that pHes-EGFP transgenic mice can be used to explore similarities and differences among neural stem cells during development. View Publication

The WNT pathway plays multiple roles in neural development and is crucial for establishment of the embryonic cerebellum. In addition,WNT pathway mutations are associated with medulloblastoma,the most common malignant brain tumor in children. However,the cell types within the cerebellum that are responsive to WNT signaling remain unknown. Here we investigate the effects of canonical WNT signaling on two important classes of progenitors in the developing cerebellum: multipotent neural stem cells (NSCs) and granule neuron precursors (GNPs). We show that WNT pathway activation in vitro promotes proliferation of NSCs but not GNPs. Moreover,mice that express activated β-catenin in the cerebellar ventricular zone exhibit increased proliferation of NSCs in that region,whereas expression of the same protein in GNPs impairs proliferation. Although β-catenin-expressing NSCs proliferate they do not undergo prolonged expansion or neoplastic growth; rather,WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level,mutant NSCs exhibit increased expression of c-Myc,which might account for their transient proliferation,but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21,which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation. View Publication