技术资料

On the basis of our previous report verifying that CXCR2 ligands in human placenta-conditioned medium (hPCCM) support human pluripotent stem cell (hPSC) propagation without exogenous bFGF,this study was designed to identify the effect of CXCR2 manipulation on the fate of hPSCs and the underlying mechanism,which had not been previously determined. We observed that CXCR2 inhibition in hPSCs induces predominant differentiation to mesoderm and endoderm with concomitant loss of hPSC characteristics and accompanying decreased expression of mTOR,beta-catenin,and hTERT. These phenomena are recapitulated in hPSCs propagated in conventional culture conditions including bFGF as well as those in hPCCM without exogenous bFGF,suggesting that the action of CXCR2 on hPSCs might not be associated with a bFGF-related mechanism. In addition,the specific CXCR2 ligand GROalpha markedly increased the expression of ectodermal markers in differentiation-committed embryoid bodies derived from hPSCs. This finding suggests that CXCR2 inhibition in hPSCs prohibits the propagation of hPSCs and leads to predominant differentiation to mesoderm and endoderm owing to the blockage of ectodermal differentiation. Taken together,our results indicate that CXCR2 preferentially supports the maintenance of hPSC characteristics as well as facilitates ectodermal differentiation after the commitment to differentiation,and that the mechanism might be associated with mTOR,beta-catenin,and hTERT activities. View Publication

The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1$$ (HIF-1$$) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1$$ in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study,we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1$$ and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1$$. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats,DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone,leading to bone regeneration. Collectively,the results indicate that DMOG,via activation of the PI3K/Akt pathway,promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited as a potential therapeutic tool in bone regeneration. View Publication

Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies,the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case,the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity. View Publication

In this study,we examined whether bacterial pathogen-associated molecular patterns recognized by toll-like receptors (TLRs) can modify the CCR7-dependent migration of human monocytes. MonoMac-1 (MM-1) cells and freshly isolated human monocytes were cultivated in the presence of agonists for TLR4 (which senses lipopolysaccharides from gram-negative bacteria),TLR1/2 (which senses peptidoglycan from gram-positive bacteria),and TLR9 (which recognizes bacterial DNA rich in unmethylated CpG DNA). CCR7 mRNA transcription was measured using quantitative reverse transcription polymerase chain reaction and protein expression was examined using flow cytometry. CCR7 function was monitored using migration and transmigration assays in response to CCL19/CCL21,which are natural ligands for CCR7. Our results show that TLR4 strongly increases monocyte migratory capacity in response to CCL19 in chemotaxis and transmigration assays in a model that mimics the human blood-brain barrier,whereas TLR1/2 and 9 have no effect. Examination of monocyte migration in response to TLRs that are activated by bacterial components would contribute to understanding the excessive monocyte migration that characterizes the pathogenesis of bacterial infections and/or neuroinflammatory diseases. View Publication

The fact that Parkinson's disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis,we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro,we generated neurons harboring disease-causing mutations from patient-specific,induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons,reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro,including oxidative stress vulnerability and altered neuronal activity. Notably,PD neurons show that elevated RBFOX1,a gene previously linked to neurodevelopmental diseases,underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes. View Publication

CD8(+) T cells develop increased sensitivity following Ag experience,and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that textgreater50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation,compared with textless20% in central memory T cells (TCM). Immediately proximal to Lck signaling,we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore,we observed superior cytotoxic effector function in TEM compared with TCM,and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase,and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM,as well as increased cytotoxic effector function in TCM Collectively,this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity,and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications. View Publication

Th2-eosinophil immune responses are well known for mediating host defence against helminths. Herein we describe a function of Th2-eosinophil responses in counteracting the development of arthritis. In two independent models of arthritis,Nippostrongylus brasiliensis infection leads to Th2 and eosinophil accumulation in the joints associated with robust inhibition of arthritis and protection from bone loss. Mechanistically,this protective effect is dependent on IL-4/IL-13-induced STAT6 pathway. Furthermore,we show that eosinophils play a central role in the modulation of arthritis probably through the increase of anti-inflammatory macrophages into arthritic joints. The presence of these pathways in human disease is confirmed by detection of GATA3-positive cells and eosinophils in the joints of rheumatoid arthritis patients. Taken together,these results demonstrate that eosinophils and helminth-induced activation of the Th2 pathway axis effectively mitigate the course of inflammatory arthritis. View Publication

Oct4 and Sox2 are pluripotent stem cell factors but the interplay between them in tumorigenesis is unclear. The aim of the present study was to investigate the roles of Oct4 and Sox2 in the reprogramming of oral cancer stem cells. One or both Oct4 and Sox2 were overexpressed in immortalized oral epithelial (hTERT+-OME) cells by lentivirus transduction. In addition,Oct4 and Sox2 proteins in two oral squamous cell carcinoma cell (OSCC) lines (Cal27 and primary cultured OSCC from a T2N2M0 patient) were individually or combinedly knocked down by shRNA. The results showed that the doubly transduced (Oct4+Sox2+) cells could trigger neoplasms in immunodeficient mice after lentivirus transduction,but single transduced (Oct4+ or Sox2+) cells had no tumor formation ability. The knockdown Sox2low and knockdown Oct4lowSox2low cells resulted in decreased tumor size in the immunodeficient mice but the single knockdown Oct4low cancer cells acquired more aggressive xenografts. Our findings suggest that Oct4+Sox2+ cells may be reprogrammed cancer stem cells inducing oral carcinogenesis. View Publication

Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body,including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system,provides a platform for the screening of chemical libraries that affect these processes,and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However,defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner,with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells,exhibit spontaneous electrical activity,and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months,even without astrocyte feeder layers. View Publication

BACKGOUND The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson's disease (PD). METHODS The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD,using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study,patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson's Disease Rating Scale (UPDRS) to measure clinical symptoms. RESULTS The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC,consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; ptextless0.05 for all values) in the PD group treated with NAC,and no measurable changes in the control group. UPDRS scores were also significantly improved in the NAC group (mean improvement of 12.9%,p = 0.01). CONCLUSIONS The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients,and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted. TRIAL REGISTRATION ClinicalTrials.gov NCT02445651. View Publication

Graphical Abstract Highlights d SMAD activity is active in suprabasal cells but is weaker in basal epithelial cells d SMAD signaling activity correlates with mucociliary differentiation in the airway d Dual TGFb/BMP inhibition prevents spontaneous differentiation in culture d Dual TGFb/BMP inhibition allows prolonged culture of diverse epithelial basal cells Correspondence jrajagopal@partners.org In Brief Mou et al. show that small-molecule-mediated SMAD signaling inhibition allows prolonged feeder-free culture of diverse functional epithelial basal stem cells in a 2D format. This methodology provides a facile patient-specific epithelial disease modeling platform,as shown by the expansion of airway epithelium from non-invasively obtained specimens from cystic fibrosis patients. View Publication

Haplo-cord stem cell transplantation combines the infusion of CD34 selected hematopoietic progenitors from a haplo-identical donor with an umbilical cord blood (UCB) graft from an unrelated donor and allows faster count recovery,with low rates of disease recurrence and chronic graft-versus-host disease (GVHD). But the contribution of the umbilical cord blood graft to long-term transplant outcome remains unclear. We analyzed 39 recipients of haplo-cord transplants with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS),engrafted and in remission at 2 months. Median age was 66 (18-72) and all had intermediate,high,or very-high risk disease. Less than 20% UCB chimerism in the CD33 lineage was associated with an increased rate of disease recurrence (54% versus 11% p textless 0.0001) and decrease in one year progression-free (20% versus 55%,p = 0.004) and overall survival (30% versus 62%,p = 0.02). Less than 100% UCB chimerism in the CD3 lineage was associated with increase rate of disease recurrence (46% versus 12%,p = 0.007). Persistent haplo-chimerism in the CD3 lineage was associated with an increased rate of disease recurrence (40% versus 15%,p = 0.009) Chimerism did not predict for treatment related mortality. The cumulative incidence of acute GVHD by day 100 was 43%. The cumulative incidence of moderate/severe chronic GVHD was only 5%. Engraftment of the umbilical cord blood grafts provides powerful graft-versus-leukemia (GVL) effects which protect against disease recurrence and is associated with low risk of chronic GVHD. Engraftment of CD34 selected haplo-identical cells can lead to rapid development of circulating T-cells,but when these cells dominate,GVL-effects are limited and rates of disease recurrence are high. View Publication