技术资料

Activation of thermogenic beige adipocytes has recently emerged as a promising therapeutic target in obesity and diabetes. Relevant human models for beige adipocyte differentiation are essential to implement such therapeutic strategies. We report a straightforward and efficient protocol to generate functional human beige adipocytes from human induced pluripotent stem cells (hiPSCs). Without overexpression of exogenous adipogenic genes,our method recapitulates an adipogenic developmental pathway through successive mesodermal and adipogenic progenitor stages. hiPSC-derived adipocytes are insulin sensitive and display beige-specific markers and functional properties,including upregulation of thermogenic genes,increased mitochondrial content,and increased oxygen consumption upon activation with cAMP analogs. Engraftment of hiPSC-derived adipocytes in mice produces well-organized and vascularized adipose tissue,capable of β-adrenergic-responsive glucose uptake. Our model of human beige adipocyte development provides a new and scalable tool for disease modeling and therapeutic screening. View Publication

The pig is the large animal model of choice for study of nerve regeneration and wound repair. Availability of porcine sensory neural cells would conceptually allow for analogous cell-based peripheral nerve regeneration in porcine injuries of similar severity and size to those found in humans. After recently reporting that porcine (or pig) induced pluripotent stem cells (piPSCs) differentiate into neural rosette (NR) structures similar to human NRs,here we demonstrate that pig NR cells could differentiate into neural crest cells and other peripheral nervous system-relevant cell types. Treatment with either bone morphogenetic protein 4 or fetal bovine serum led to differentiation into BRN3A-positive sensory cells and increased expression of sensory neuron TRK receptor gene family: TRKA,TRKB,and TRKC. Porcine sensory neural cells would allow determination of parallels between human and porcine cells in response to noxious stimuli,analgesics,and reparative mechanisms. In vitro differentiation of pig sensory neurons provides a novel model system for neural cell subtype specification and would provide a novel platform for the study of regenerative therapeutics by elucidating the requirements for innervation following injury and axonal survival. View Publication

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation,migration,and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme,a highly aggressive brain cancer,suggesting that ion channel expression may be perturbed in this population. However,little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing,we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance,expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally,genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes,gene mutations,survival outcomes,regional tumor expression,and experimental responses to loss-of-function. Together,the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance. 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

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable,synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells,including impaired neural rosette formation,increased susceptibility to growth factor withdrawal,and deficits in mitochondrial respiration,are rescued in isogenic controls. Importantly,using genome-wide expression analysis,we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines,suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities,and the importance of isogenic controls for disease modeling using hiPSCs. View Publication

BACKGROUND Neuroblastoma (NB),a tumor of the primitive neural crest,despite aggressive treatment portends a poor long-term survival for patients with advanced high stage NB. New treatment strategies are required. METHODS We investigated coordinated targeting of essential homeostatic regulatory factors involved in cancer progression,histone deacetylases (HDACs) and carbonic anhydrases (CAs). RESULTS We evaluated the antitumor potential of the HDAC inhibitor (HDACi),pyridylmethyl-N-4-[(2-aminophenyl)-carbamoyl]-benzyl-carbamate (MS-275) in combination with a pan CA inhibitor,acetazolamide (AZ) on NB SH-SY5Y,SK-N-SH and SK-N-BE(2) cells. The key observation was that the combination AZ + MS-275 significantly inhibited growth,induced cell cycle arrest and apoptosis,and reduced migration capacity of NB cell line SH-SY5Y. In addition,this combination significantly inhibited tumor growth in vivo,in a pre-clinical xenograft model. Evidence was obtained for a marked reduction in tumorigenicity and in the expression of mitotic,proliferative,HIF-1α and CAIX. NB xenografts of SH-SY5Y showed a significant increase in apoptosis. CONCLUSION MS-275 alone at nanomolar concentrations significantly reduced the putative cancer stem cell (CSC) fraction of NB cell lines,SH-SY5Y and SK-N-BE(2),in reference to NT2/D1,a teratocarcinoma cell line,exhibiting a strong stem cell like phenotype in vitro. Whereas stemness genes (OCT4,SOX2 and Nanog) were found to be significantly downregulated after MS-275 treatment,this was further enhanced by AZ co-treatment. The significant reduction in initial tumorigenicity and subsequent abrogation upon serial xenografting suggests potential elimination of the NB CSC fraction. The significant potentiation of MS-275 by AZ is a promising therapeutic approach and one amenable for administration to patients given their current clinical utility. View Publication

Toxic shock syndrome (TSS) is caused by staphylococcal and streptococcal superantigens (SAgs) that provoke a swift hyperinflammatory response typified by a cytokine storm. The precipitous decline in the host's clinical status and the lack of targeted therapies for TSS emphasize the need to identify key players of the storm's initial wave. Using a humanized mouse model of TSS and human cells,we herein demonstrate that SAgs elicit in vitro and in vivo IL-17A responses within hours. SAg-triggered human IL-17A production was characterized by remarkably high mRNA stability for this cytokine. A distinct subpopulation of CD4+ effector memory T (TEM) cells that secrete IL-17A,but not IFN-$\gamma$,was responsible for early IL-17A production. We found mouse TEM-17" cells to be enriched within the intestinal epithelium and among lamina propria lymphocytes. Furthermore interfering with IL-17A receptor signaling in human PBMCs attenuated the expression of numerous inflammatory mediators implicated in the TSS-associated cytokine storm. IL-17A receptor blockade also abrogated the secondary effect of SAg-stimulated PBMCs on human dermal fibroblasts as judged by C/EBP $\delta$ expression. Finally the early IL-17A response to SAgs was pathogenic because in vivo neutralization of IL-17A in humanized mice ameliorated hepatic and intestinal damage and reduced mortality. Together our findings identify CD4+ TEM cells as a key effector of TSS and reveal a novel role for IL-17A in TSS immunopathogenesis. Our work thus elucidates a pathogenic as opposed to protective role for IL-17A during Gram-positive bacterial infections. Accordingly the IL-17-IL-17R axis may provide an attractive target for the management of SAg-mediated illnesses." View Publication

Targeting of myeloid-dendritic cell receptor DC-SIGN by numerous chronic infectious agents,including Porphyromonas gingivalis,is shown to drive-differentiation of monocytes into dysfunctional mDCs. These mDCs exhibit alterations of their fine-tuned homeostatic function and contribute to dysregulated immune-responses. Here,we utilize P. gingivalis mutant strains to show that pathogen-differentiated mDCs from primary human-monocytes display anti-apoptotic profile,exhibited by elevated phosphorylated-Foxo1,phosphorylated-Akt1,and decreased Bim-expression. This results in an overall inhibition of DC-apoptosis. Direct stimulation of complex component CD40 on DCs leads to activation of Akt1,suggesting CD40 involvement in anti-apoptotic effects observed. Further,these DCs drove dampened CD8(+) T-cell and Th1/Th17 effector-responses while inducing CD25(+)Foxp3(+)CD127(-) Tregs. In vitro Treg induction was mediated by DC expression of indoleamine 2,3-dioxygenase,and was confirmed in IDO-KO mouse model. Pathogen-infected &CMFDA-labeled MoDCs long-lasting survival was confirmed in a huMoDC reconstituted humanized mice. In conclusion,our data implicate PDDCs as an important target for resolution of chronic infection. View Publication

The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers,they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei,and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs,resulting in efficient sequential generation of nonneuronal ectoderm,preplacodal ectoderm,early prosensory ONPs,late ONPs,and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage,thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs,advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. textcopyright Stem Cells Translational Medicine 2017;6:923-936. View Publication

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/β-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 β-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 β-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. View Publication

BACKGROUND Neonatal hypoxia-ischemia induces massive brain damage during the perinatal period,resulting in long-term consequences to central nervous system structural and functional maturation. Although neural progenitor cells (NPCs) migrate through the parenchyma and home in to injury sites in the rodent brain,the molecular mechanisms are unknown. We examined the role of chemokines in mediating NPC migration after neonatal hypoxic-ischemic brain injury. METHODS Nine-day-old mice were exposed to a 120-minute hypoxia following unilateral carotid occlusion. Chemokine levels were quantified in mouse brain extract. Migration and proliferation assays were performed using embryonic and infant mouse NPCs. RESULTS The neonatal hypoxic-ischemic brain injury resulted in an ipsilateral lesion,which was extended to the cortical and striatal areas. NPCs migrated toward an injured area,where a marked increase of CC chemokines was detected. In vitro studies showed that incubation of NPCs with recombinant mouse CCL11 promoted migration and proliferation. These effects were partly inhibited by a CCR3 antagonist,SB297006. CONCLUSIONS Our data implicate an important effect of CCL11 for mouse NPCs. The effective activation of NPCs may offer a promising strategy for neuroregeneration in neonatal hypoxic-ischemic brain injury. View Publication

The development of targeted therapeutics for rare neurodevelopmental disorders (NDDs) faces significant challenges due to the scarcity of subjects and the difficulty of obtaining human neural cells. Here,we illustrate a rapid,simple protocol by which patient derived cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using an episomal vector and differentiated into neurons. Using this platform enables patient somatic cells to be converted to physiologically active neurons in less than two months with minimal labor. This platform includes a method to combine somatic cell reprogramming with CRISPR/Cas9 gene editing at single cell resolution,which enables the concurrent development of clonal knockout or knock-in models that can be used as isogenic control lines. This platform reduces the logistical barrier for using iPSC technology,allows for the development of appropriate control lines for use in rare neurodevelopmental disease research,and establishes a fundamental component to targeted therapeutics and precision medicine. Stem Cells Translational Medicine 2017;6:886-896. View Publication