技术资料

Acute inflammatory bowel disease (AIBD) is a wide clinical entity including severe gastrointestinal pathologies with common histopathological basis. Epidemiologically increasing diseases,such as necrotizing enterocolitis (NEC),gastrointestinal graft versus host disease (GVHD),and the primary acute phase of chronic inflammatory bowel disease (CIBD),exhibit a high necessity for new therapeutic strategies. Mesenchymal stem cell (MSC) cellular therapy represents a promising option for the treatment of these diseases. In our study,we comparatively assess the efficacy of human MSCs derived from bone marrow (BM),umbilical cord blood (UCB),human embryonic stem cells (ESCs),or human-induced pluripotent stem cells (iPSCs) in a mouse model of chemically induced acute enterocolitis. The laboratory animals were provided ad libitum potable dextrane sulfate sodium solution (DSS) in order to reproduce an AIBD model and then individually exposed intraperitoneally to MSCs derived from BM (BM-MSCs),UCB (UCB-MSCs),ESCs (ESC-MSCs),or iPSCs (iPSC-MSCs). The parameters used to evaluate the cellular treatment efficacy were the animal survival prolongation and the histopathological-macroscopic picture of bowel sections. Although all categories of mesenchymal stem cells led to statistically significant survival prolongation compared to the control group,significant clinical and histopathological improvement was observed only in mice receiving BM-MSCs and UCB-MSCs. Our results demonstrated that the in vivo anti-inflammatory effect of ESC-MSCs and iPSC-MSCs was inferior to that of UCB-MSCs and BM-MSCs. Further investigation will clarify the potential of ESCs and iPSC-derived MSCs in AIBD treatment. View Publication

Deregulation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is found in cancer with STAT5A/B controlling leukemic cell survival and disease progression. As mutations in STAT5B,but not STAT5A,have been frequently described in hematopoietic tumors,we used BCR/ABL as model systems to investigate the contribution of STAT5A or STAT5B for leukemogenesis. The absence of STAT5A decreased cell survival and colony formation. Even more drastic effects were observed in the absence of STAT5B. STAT5B-deficient cells formed BCR/ABL+ colonies or stable cell lines at low frequency. The rarely evolving Stat5b-/- cell lines expressed enhanced levels of BCR/ABL oncoprotein compared to wild-type cells. In line,Stat5b-/- leukemic cells induced leukemia with a significantly prolonged disease onset,whereas Stat5a-/- cells rapidly caused a fatal disease superimposable to wild-type cells. RNA-sequencing (RNA-seq) profiling revealed a marked enhancement of interferon (IFN)-alpha and IFN-gamma signatures in Stat5b-/- cells. Inhibition of IFN responses rescued BCR/ABL+ colony formation of Stat5b-/--deficient cells. A downregulated IFN response was also observed in patients suffering from leukemia carrying STAT5B mutations. Our data define STAT5B as major STAT5 isoform driving BCR/ABL+ leukemia. STAT5B enables transformation by suppressing IFN-alpha/gamma,thereby facilitating leukemogenesis. Our findings might help explain the high frequency of STAT5B mutations in hematopoietic tumors. View Publication

Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety,however,is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1,but none has quantitatively defined the safety level of transplant therapies. Here,using genome-engineering strategies,we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore,we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here,we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic. View Publication

The flow cytometric crossmatch (FCXM) assay,which detects the presence of donor specific HLA antibodies in patient sera,is a cornerstone of HLA compatibility testing. Since relatively long FCXM assay turnaround times may contribute to transplant delays and increased graft ischemia time,we developed and validated two modified crossmatch procedures,namely the Halifax and Halifaster FCXM protocols. These protocols reduce FCXM assay time >60{\%} and simplify their set-up without compromising quality or sensitivity. Optimization of the FCXM (the Halifax protocol) includes a 96-well tray platform,reduced wash times,increased serum to cell suspension volume ratio,shortened incubations and higher incubation temperature. The Halifaster protocol is a further modification,employing methods that improve lymphocyte purity compared to density gradient centrifugation (96 ± 2.63{\%} vs 69 ± 19.06{\%}),reduce cell isolation time (by ∼40{\%}) and conserve FCXM assay reagents. Importantly,linear regression analysis of the median channel fluorescence shift (MCFS) values revealed excellent concordance (R2 of 0.98-0.99) among all three FCXM protocols (standard vs Halifax vs Halifaster). Finally,a retrospective review of 2013 crossmatches performed using the Halifax protocol demonstrated excellent correlation with the virtual crossmatch (95.7{\%} and 96.8{\%} specificity and sensitivity,respectively) regarding the identification of donor specific antibodies (HLA-A/B/DR) assigned based on the single antigen bead (SAB) assay testing with a 2000 mean fluorescence intensity (MFI) cutoff. Implementation of the Halifax or Halifaster protocols will expedite pre-transplantation work-up and improve patient care. View Publication

Antagonists of the 5-hydroxytryptamine (serotonin) 3 receptor (5-HT3R) have anti-inflammatory and anti-apoptotic activities,but the detailed,underlying mechanisms are not well understood. We focused on anti-apoptotic activities via 5-HT3R signaling to clarify the underlying mechanisms. Mice were administered 5-fluorouracil (5-FU),which induced apoptosis in intestinal epithelial cells. Coadministration with 5-HT3R antagonists or agonists tended to decrease or increase the number of apoptotic cells,respectively. In serotonin 3A receptor (5-HT3AR) null (HTR3A-/-) mice,the number of apoptotic cells induced by 5-FU was decreased compared with that in wild-type (WT) mice. Bone marrow (BM) transplantation was performed to determine if BM-derived immune cells regulated 5-FU-induced apoptosis,but they were found to be unrelated to this process. Data from 5-HT3AR/enhanced green fluorescent protein reporter mice revealed that 50{\%} of enterochromaffin (EC) cells expressed 5-HT3AR,but the number of apoptotic cells induced by 5-FU in the intestinal crypt organoids of HTR3A-/- mice was not altered compared with WT mice. In contrast,plasma 5-HT concentrations in WT mice but not in HTR3A-/- mice administered 5-FU were increased significantly. In conclusion,5-HT3R signaling may enhance 5-HT release,possibly from EC cells intravascularly,or paracrine,resulting in increases in plasma 5-HT concentration,which in turn,enhances apoptotic activities induced by 5-FU.-Mikawa,S.,Kondo,M.,Kaji,N.,Mihara,T.,Yoshitake,R.,Nakagawa,T.,Takamoto,M.,Nishimura,R.,Shimada,S.,Ozaki,H.,Hori,M. Serotonin 3 receptor signaling regulates 5-fluorouracil-mediated apoptosis indirectly via TNF-alpha production by enhancing serotonin release from enterochromaffin cells. View Publication

Mesenchymal stromal cells (MSCs) are multipotent stem cells that participate in tissue repair and posses considerable immunomodulatory potential. MSCs have been shown to promote allograft survival,yet the mechanisms behind this phenomenon have not been fully defined. Here,we investigate the capacity of MSCs to suppress the allogeneic immune response by secreting the pleiotropic molecule hepatocyte growth factor (HGF). Using an in vivo mouse model of corneal transplantation,we report that MSCs promote graft survival in an HGF-dependent manner. Moreover,our data indicate that topically administered recombinant HGF (1) suppresses antigen-presenting cell maturation in draining lymphoid tissue,(2) limits T-helper type-1 cell generation,(3) decreases inflammatory cell infiltration into grafted tissue,and (4) is itself sufficient to promote transplant survival. These findings have potential translational implications for the development of HGF-based therapeutics. Stem Cells Translational Medicine 2019. View Publication

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS),but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight,we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin beta7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore,phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFalpha secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFalpha production,while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms,but not markers of immune activation,decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation,but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms. View Publication

CD4 T cells from HIV-1 infected patients die at excessive rates compared to those from uninfected patients,causing immunodeficiency. We previously identified a dominant negative ligand that antagonizes the TRAIL-dependent pathway of cell death,which we called TRAILshort. Because the TRAIL pathway has been implicated in CD4 T cell death occurring during HIV-1 infection,we used short hairpin RNA knockdown,CRISPR deletion,or Abs specific for TRAILshort to determine the effect of inhibiting TRAILshort on the outcome of experimental acute HIV infection in vitro. Strikingly,all three approaches to TRAILshort deletion/inhibition enhanced HIV-induced death of both infected and uninfected human CD4 T cells. Thus,TRAILshort impacts T cell dynamics during HIV infection,and inhibiting TRAILshort causes more HIV-infected and uninfected bystander cells to die. TRAILshort is,therefore,a host-derived,host-adaptive mechanism to limit the effects of TRAIL-induced cell death. Further studies on the effects of TRAILshort in other disease states are warranted. View Publication

Background Cancer has been considered a serious global health problem and a leading cause of morbidity and mortality worldwide. Despite recent advances in cancer therapy,treatments of advance stage cancers are mostly ineffective resulting in poor survival of patients. Recent evidences suggest that multipotent human mesenchymal stem cells (hMSCs) play important roles in growth and metastasis of several cancers by enhancing their engraftment and inducing tumor neovascularization. However,the effect of hMSCs on cancer cells is still controversial because there are also evidences demonstrating that hMSCs inhibited growth and metastasis of some cancers. Methods In this study,we investigated the effects of bioactive molecules released from bone marrow and gestational tissue-derived hMSCs on the proliferation of various human cancer cells,including C3A,HT29,A549,Saos-2,and U251. We also characterized the hMSC-derived factors that inhibit cancer cell proliferation by protein fractionation and mass spectrometry analysis. Results We herein make a direct comparison and show that the effects of hMSCs on cancer cell proliferation and migration depend on both hMSC sources and cancer cell types and cancer-derived bioactive molecules did not affect the cancer suppressive capacity of hMSCs. Moreover,hMSCs use distinct combination of bioactive molecules to suppress the proliferation of human hepatoblastoma and colorectal cancer cells. Using protein fractionation and mass spectrometry analysis,we have identified several novel hMSC-derived factors that might be able to suppress cancer cell proliferation. Conclusion We believe that the procedure developed in this study could be used to discover other therapeutically useful molecules released by various hMSC sources for a future in vivo study. View Publication

Hematopoiesis provides an accessible system for studying the principles underlying cell-fate decisions in stem cells. Proposed models of hematopoiesis suggest that quantitative changes in lineage-specific transcription factors (LS-TFs) underlie cell-fate decisions. However,evidence for such models is lacking as TF levels are typically measured via RNA expression rather than by analyzing temporal changes in protein abundance. Here,we used single-cell mass cytometry and absolute quantification by mass spectrometry to capture the temporal dynamics of TF protein expression in individual cells during human erythropoiesis. We found that LS-TFs from alternate lineages are co-expressed,as proteins,in individual early progenitor cells and quantitative changes of LS-TFs occur gradually rather than abruptly to direct cell-fate decisions. Importantly,upregulation of a megakaryocytic TF in early progenitors is sufficient to deviate cells from an erythroid to a megakaryocyte trajectory,showing that quantitative changes in protein abundance of LS-TFs in progenitors can determine alternate cell fates. View Publication

Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation,generating self-renewing leukemic stem cells (LSCs). Here,we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function,including the tumor necrosis factor receptor Tnfrsf2,whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly,YTHDF2 is not essential for normal HSC function,with YTHDF2 deficiency actually enhancing HSC activity. Thus,we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion. View Publication

MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-kappaB activation. We discovered nanomolar,selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580,locking the protease in an inactive conformation. Interestingly,we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability,reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency,we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein,the most potent of the allosteric inhibitors rescued NF-kappaB and JNK signaling in patient lymphocytes. Following compound washout,MALT1 substrate cleavage was partly recovered. Thus,a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue,inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies. View Publication