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Spinal cord injury (SCI) remains a significant clinical challenge and poses a dramatic threat to the life quality of patients due to limited neural regeneration and detrimental post-injury alternations in tissue microenvironment. We developed a therapeutic approach by transplanting spinal neural progenitor cells (spNPGs),derived from human induced pluripotent stem cell (iPSC)-generated neuromesodermal progenitors,into a contusive SCI model in NOD-SCID mice. Single-cell RNA sequencing mapped the in vitro differentiation of iPSC-spNPGs,confirming their specification into spinal neuronal lineages. Single-nucleus transcriptomics at 1 week post-transplantation showed that the grafted cells differentiated in vivo into motor neurons and two interneuron subtypes (V2 and dI4). Additionally,spNPGs integrated into host neural circuits,enhancing synaptic connectivity,while simultaneously modulating the injury microenvironment by shifting microglia and astrocyte polarization toward anti-inflammatory and neuroprotective phenotypes. This dual mechanism promoted axonal regrowth,remyelination,and significant sensorimotor recovery,as evidenced by improved locomotor scores. Our findings highlight the therapeutic potential of human iPSC-spNPGs in reconstructing neural networks and mitigating secondary damage,providing compelling preclinical evidence for advancing stem cell-based SCI therapies. Subject terms: Stem-cell differentiation,Spinal cord injury View Publication

Constitutive activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in human cancers is often associated with mutational activation of BRAF or RAS. MAPK/ERK kinase 1/2 kinases lie downstream of RAS and BRAF and are the only acknowledged activators of ERK1/2,making them attractive targets for therapeutic intervention. AZD6244 (ARRY-142886) is a potent,selective,and ATP-uncompetitive inhibitor of MAPK/ERK kinase 1/2. In vitro cell viability inhibition screening of a tumor cell line panel found that lines harboring BRAF or RAS mutations were more likely to be sensitive to AZD6244. The in vivo mechanisms by which AZD6244 inhibits tumor growth were investigated. Chronic dosing with 25 mg/kg AZD6244 bd resulted in suppression of growth of Colo-205,Calu-6,and SW-620 xenografts,whereas an acute dose resulted in significant inhibition of ERK1/2 phosphorylation. Increased cleaved caspase-3,a marker of apoptosis,was detected in Colo-205 and Calu-6 but not in SW-620 tumors where a significant decrease in cell proliferation was detected. Chronic dosing of AZD6244 induced a morphologic change in SW-620 tumors to a more differentiated phenotype. The potential of AZD6244 in combination with cytotoxic drugs was evaluated in mice bearing SW-620 xenografts. Treatment with tolerated doses of AZD6244 and either irinotecan or docetaxel resulted in significantly enhanced antitumor efficacy relative to that of either agent alone. These results indicate that AZD6244 has potential to inhibit proliferation and induce apoptosis and differentiation,but the response varies between different xenografts. Moreover,enhanced antitumor efficacy can be obtained by combining AZD6244 with the cytotoxic drugs irinotecan or docetaxel. View Publication

Human interferon (IFN)-alpha is the standard therapy for chronic hepatitis C to prevent its progression to liver cirrhosis and hepatocellular carcinoma. Thrombocytopenia is one of the major adverse effects of IFN-alpha and often leads to dose reduction or treatment discontinuation. However,there is little information on how IFN-alpha inhibits human megakaryopoiesis. In this study,we demonstrated that IFN-alpha did not inhibit colony formation of megakaryocytes from human CD34(+) hematopoietic stem cells. IFN-alpha did not inhibit endomitosis but did inhibit cytoplasmic maturation of megakaryocytes and platelet production in vitro. IFN-alpha suppressed the expression of transcription factors regulating late-stage megakaryopoiesis,such as GATA-1,p45(NF-E2),MafG. IFN-alpha also significantly reduced the number of human platelets but not megakaryocytes,and did not inhibit endomitosis of human megakaryocytes in immunodeficient NOD/Shi-scid/IL-2R gamma(null) (NOG) mice transplanted with human CD34(+) cells (hu-NOG). We also demonstrated that a novel thrombopoietin mimetic,NIP-004,was effective for treating IFN-alpha-induced thrombocytopenia in hu-NOG mice. From ultrastructural study,IFN-alpha inhibited the maturation of demarcation membranes in megakaryocytes,although NIP-004 prevented the inhibitory effects of IFN-alpha. These results defined the pathogenesis of IFN-alpha-induced thrombocytopenia and suggested possible future clinical applications for thrombopoietin mimetics. View Publication

Peripheral blood was collected from a clinically diagnosed 60-year old female patient with multiple schwannoma. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers,and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for further pathological studies of multiple schwannoma. View Publication

Verteporfin (VP),a benzoporphyrin derivative,is clinically used in photodynamic therapy for neovascular macular degeneration. Recent studies indicate that VP may inhibit growth of hepatoma cells without photoactivation through inhibition of YAP-TEAD complex. In this study,we examined the effects of VP without light activation on human retinoblastoma cell lines. Verteporfin but not vehicle control inhibited the growth,proliferation and viability of human retinoblastoma cell lines (Y79 and WERI) in a dose-dependent manner and was associated with downregulation of YAP-TEAD associated downstream proto-oncogenes such as c-myc,Axl,and surviving. In addition VP affected signals involved in cell migration and angiogenesis such as CTGF,cyr61,and VEGF-A but was not associated with significant effect on the mTOR/autophagy pathway. Of interest the pluripotency marker Oct4 were downregulated by Verteporfin treatment. Our results indicate that the clinically used photosensitizer VP is a potent inhibitor of cell growth in retinoblastoma cells,disrupting YAP-TEAD signaling and pluripotential marker OCT4. This study highlights for the first time the role of the YAP-TEAD pathway in Retinoblastoma and suggests that VP may be a useful adjuvant therapeutic tool in treating Rb patients. View Publication

Recent evidence indicates that mouse and human embryonic stem cells (ESCs) are fixed at different developmental stages,with the former positioned earlier. We show that a narrow concentration of the naturally occurring short-chain fatty acid,sodium butyrate,supports the extensive self-renewal of mouse and human ESCs,while promoting their convergence toward an intermediate stem cell state. In response to butyrate,human ESCs regress to an earlier developmental stage characterized by a gene expression profile resembling that of mouse ESCs,preventing precocious Xist expression while retaining the ability to form complex teratomas in vivo. Other histone deacetylase inhibitors (HDACi) also support human ESC self-renewal. Our results indicate that HDACi can promote ESC self-renewal across species,and demonstrate that ESCs can toggle between alternative states in response to environmental factors. View Publication

BACKGROUND The incidence and number of circulating tumor cells (CTCs) in the peripheral blood of colorectal cancer patients are lower than in other cancer types,which may point to a particular biology of colorectal cancer affecting CTC detection. METHODS We detected CTCs in the peripheral and mesenteric blood of colorectal cancer patients by use of 2 independent technologies on the basis of different biological properties of colon cancer cells. Seventy-five patients diagnosed with localized (M0,n = 60) and metastatic (M1,n = 15) colorectal cancer were included. Peripheral and mesenteric blood samples were collected before tumor resection. We performed CTC enumeration with an EpCAM-independent enrichment method followed by the Epispot assay that detected only viable CK19-releasing CTCs. In parallel,we used the FDA-cleared EpCAM-dependent CellSearch® as the reference method. RESULTS The enumeration of CK19-releasing cells by the CK19-Epispot assay revealed viable CTCs in 27 of 41 (65.9%) and 41 of 74 (55.4%) (P = 0.04) patients in mesenteric and peripheral blood,respectively,whereas CellSearch detected CTCs in 19 of 34 (55.9%) and 20 of 69 (29.0%) (P = 0.0046) patients. In mesenteric blood,medians of 4 (range 0-247) and 2.7 CTCs (range 0-286) were found with Epispot and CellSearch (P = 0.2),respectively,whereas in peripheral blood,Epispot and CellSearch detected a median of 1.2 (range 0-92) and 0 CTCs (range 0-147) (P = 0.002). CONCLUSIONS A considerable portion of viable CTCs detectable by the Epispot assay are trapped in the liver as the first filter organ in CRC patients. View Publication

The inner ear contains sensory epithelia that detect head movements,gravity and sound. It is unclear how to develop these sensory epithelia from pluripotent stem cells,a process that will be critical for modelling inner ear disorders or developing cell-based therapies for profound hearing loss and balance disorders. So far,attempts to derive inner ear mechanosensitive hair cells and sensory neurons have resulted in inefficient or incomplete phenotypic conversion of stem cells into inner-ear-like cells. A key insight lacking from these previous studies is the importance of the non-neural and preplacodal ectoderm,two critical precursors during inner ear development. Here we report the stepwise differentiation of inner ear sensory epithelia from mouse embryonic stem cells (ESCs) in three-dimensional culture. We show that by recapitulating in vivo development with precise temporal control of signalling pathways,ESC aggregates transform sequentially into non-neural,preplacodal and otic-placode-like epithelia. Notably,in a self-organized process that mimics normal development,vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia bundles and a kinocilium. Moreover,these stem-cell-derived hair cells exhibit functional properties of native mechanosensitive hair cells and form specialized synapses with sensory neurons that have also arisen from ESCs in the culture. Finally,we demonstrate how these vesicles are structurally and biochemically comparable to developing vestibular end organs. Our data thus establish a new in vitro model of inner ear differentiation that can be used to gain deeper insight into inner ear development and disorder. View Publication

Without effective therapy,chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized,but biologically poorly characterized,accelerated phase (AP). Here,we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts,which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML,we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML,including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients. View Publication