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Since the discovery of induced pluripotent stem cells (iPSCs),numerous approaches have been explored to improve the original protocol,which is based on a two-dimensional (2D) cell-culture system. Surprisingly,nothing is known about the effect of a more biologically faithful 3D environment on somatic-cell reprogramming. Here,we report a systematic analysis of how reprogramming of somatic cells occurs within engineered 3D extracellular matrices. By modulating microenvironmental stiffness,degradability and biochemical composition,we have identified a previously unknown role for biophysical effectors in the promotion of iPSC generation. We find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodelling. We conclude that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity. View Publication

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited self-renewal and can generate nearly all cells in the body. Changes induced by different LSD1 activities on the regulation of hiPSC self-renewal and differentiation and the mechanism underlying such changes were determined. We used two different LSD1 inhibitors (phenelzine sulfate and tranylcypromine) and RNAi technique to inhibit LSD1 activity,and we obtained hiPSCs showing 71.3%,53.28%,and 31.33% of the LSD1 activity in normal hiPSCs. The cells still maintained satisfactory self-renewal capacity when LSD1 activity was at 71.3%. The growth rate of hiPSCs decreased and cells differentiated when LSD1 activity was at approximately 53.28%. The hiPSCs were mainly arrested in the G0/G1 phase and simultaneously differentiated into endodermal tissue when LSD1 activity was at 31.33%. Teratoma experiments showed that the downregulation of LSD1 resulted in low teratoma volume. When LSD1 activity was below 50%,pluripotency of hiPSCs was impaired,and the teratomas mainly comprised endodermal and mesodermal tissues. This phenomenon was achieved by regulating the critical balance between histone methylation and demethylation at regulatory regions of several key pluripotent and developmental genes. View Publication

Elevated levels of the proinflammatory cytokine IL6 are associated with poor survival outcomes in many cancers. Antibodies targeting IL6 and its receptor have been developed for chronic inflammatory disease,but they have not yet been shown to clearly benefit cancer patients,possibly due to antibody potency or the settings in which they have been tested. In this study,we describe the development of a novel high-affinity anti-IL6 antibody,MEDI5117,which features an extended half-life and potent inhibitory effects on IL6 biologic activity. MEDI5117 inhibited IL6-mediated activation of STAT3,suppressing the growth of several tumor types driven by IL6 autocrine signaling. In the same models,MEDI5117 displayed superior preclinical activity relative to a previously developed anti-IL6 antibody. Consistent with roles for IL6 in promoting tumor angiogenesis,we found that MEDI5117 inhibited the growth of endothelial cells,which can produce IL6 and support tumorigenesis. Notably,in tumor xenograft assays in mice,we documented the ability of MEDI5117 to enhance the antitumor activities of chemotherapy or gefitinib in combination treatment regimens. MEDI5117 also displayed robust activity on its own against trastuzumab-resistant HER2(+) tumor cells by targeting the CD44(+)CD24(-) cancer stem cell population. Collectively,our findings extend the evidence of important pleiotropic roles of IL6 in tumorigenesis and drug resistance,and offer a preclinical proof of concept for the use of IL6 antibodies in combination regimens to heighten therapeutic responses and overcome drug resistance. View Publication

BACKGROUND The incidence of neurological complications and fatalities associated with Hand,Foot & Mouth disease has increased over recent years,due to emergence of newly-evolved strains of Enterovirus 71 (EV71). In the search for new antiviral therapeutics against EV71,accurate and sensitive in vitro cellular models for preliminary studies of EV71 pathogenesis is an essential prerequisite,before progressing to expensive and time-consuming live animal studies and clinical trials. METHODS This study thus investigated whether neural lineages derived from pluripotent human embryonic stem cells (hESC) can fulfil this purpose. EV71 infection of hESC-derived neural stem cells (NSC) and mature neurons (MN) was carried out in vitro,in comparison with RD and SH-SY5Y cell lines. RESULTS Upon assessment of post-infection survivability and EV71 production by the various types,it was observed that NSC were significantly more susceptible to EV71 infection compared to MN,RD (rhabdomyosarcoma) and SH-SY5Y cells,which was consistent with previous studies on mice. The SP81 peptide had significantly greater inhibitory effect on EV71 production by NSC and MN compared to the cancer-derived RD and SH-SY5Y cell lines. CONCLUSIONS Hence,this study demonstrates that hESC-derived neural lineages can be utilized as in vitro models for studying EV71 pathogenesis and for screening of antiviral therapeutics. View Publication

MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes,as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation,a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 overexpression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion,we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. Stem Cells 2016;34:1284-1296. View Publication

Mutations in SCN1A,the gene encoding the α subunit of Nav1.1 channel,can cause epilepsies with wide ranges of clinical phenotypes,which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project,CRISPR/Cas9- and TALEN-mediated genome-editing techniques were applied to induced pluripotent stem cell (iPSC)-based-disease model to explore the mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9,we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G,which led to no current of Nav1.1 in exogenously transfected system,influenced the properties of not only Nav current amount,but also Nav activation in Nav1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons,and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in the patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly,when the frequencies of both sIPSCs and sEPSCs were further analyzed,we found the whole postsynaptic activity transferred from the inhibition-dominated state to excitation in patient-derived neuronal networks,suggesting that changes in sIPSCs alone were sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity. In summary,our findings fill the gap of our knowledge regarding the relationship between SCN1A mutation effect recorded on exogenously transfected cells and on Nav1.1-expressing neurons,and reveal the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation. View Publication

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR,cyclic enrichment strategy gave ˜100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that,when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways. View Publication

Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene,which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. View Publication

Chromatin structure affects the extent of DNA damage and repair. Thus,it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here,we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC,heterochromatin is confined to distinct regions,while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives,normal human fibroblasts; and one cancer cell line. At the same time,the number of DNA repair foci were not statistically different among these cells. We showed that in hESC,DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells,and to a lesser extent in IMR90 normal fibroblasts,but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC. View Publication

Human adipose-derived stem cells (hASCs) become an appealing source for regenerative medicine. However,with the multi-passage or cryopreservation for large-scale growth procedures in terms of preclinical and clinical purposes,hASCs often reveal defective cell viability,which is a major obstacle for cell therapy. In our study,the effects of induced pluripotent stem cells-derived conditioned medium (iPS-CM) on the proliferation and anti-apoptosis in hASCs were investigated. hASCs at passage 1 were identified by the analysis of typical surface antigens with flow cytometry assay and adipogenic and osteogenic differentiation. The effect of iPS-CM on the proliferation in hASCs was analyzed by cell cycle assay and Ki67/P27 quantitative polymerase chain reaction analysis. The effect of iPS-CM on the anti-apoptosis of hASCs irradiated by 468 J/m(2) of ultraviolet C was investigated by annexin v/propidium iodide analysis,mitochondrial membrane potential assay,intracellular reactive oxygen species assay,Western blotting and caspase activity assays. The effect of iPS-CM on the surface antigen expressions of hASCs was analyzed using flow cytometry assay. The levels of Activin A and bFGF in culture supernatant of hASCs with different treatments were also detected by enzyme-linked immunosorbent assay. iPS-CM promoted proliferation and inhibited apoptosis of hASCs. This discovery demonstrates that iPS-CM might be used as one of the available means to overcome the propagation obstacle for hASCs and make for large-scale growth procedures in terms of preclinical and clinical purposes. View Publication

Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However,the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1(+)-ventralized anterior foregut endoderm cells (VAFECs),we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM(+) VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore,the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT,a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a 9 + 2" microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine." View Publication

Limited data are available on the effects of stem cells in non-ischemic dilated cardiomyopathy (DCM). Since the diffuse nature of the disease calls for a broad distribution of cells,this study investigated the scaffold-based delivery of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) in a mouse model of DCM. Nanofibrous scaffolds were produced using a clinical grade atelocollagen which was electrospun and cross-linked under different conditions. As assessed by scanning electron microscopy and shearwave elastography,the optimum crosslinking conditions for hiPS-CM colonization proved to be a 10% concentration of citric acid crosslinking agent and 150 min of post-electrospinning baking. Acellular collagen scaffolds were first implanted in both healthy mice and those with induced DCM by a cardiac-specific invalidation of serum response factor (SRF). Seven and fourteen days after implantation,the safety of the scaffold was demonstrated by echocardiography and histological assessments. The subsequent step of implantation of the scaffolds seeded with hiPS-CM in DCM induced mice,using cell-free scaffolds as controls,revealed that after fourteen days heart function decreased in controls while it remained stable in the treated mice. This pattern was associated with an increased number of endothelial cells,in line with the greater vascularity of the scaffold. Moreover,a lesser degree of fibrosis consistent with the upregulation of several genes involved in extracellular matrix remodeling was observed. These results support the interest of the proposed hiPS-CM seeded electrospun scaffold for the stabilization of the DCM outcome with potential for its clinical use in the future. View Publication