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Hematotoxicity is a significant issue for drug safety and can result from direct cytotoxicity toward circulating mature blood cell types as well as targeting of immature blood-forming stem cells/progenitor cells in the bone marrow. In vitro models for understanding and investigating the hematotoxicity potential of new test items/drugs are critical in early preclinical drug development. The traditional method,colony forming unit (CFU) assay,is commonly used and has been validated as a method for hematotoxicity screening. The CFU assay has multiple limitations for its application in investigative work. In this paper,we describe a detailed protocol for a liquid-culture,microplate-based in vitro hematotoxicity assay to evaluate lineage-specific (myeloid,erythroid,and megakaryocytic) hematotoxicity at different stages of differentiation. This assay has multiple advantages over the traditional CFU assay,including being suitable for high-throughput screening and flexible enough to allow inclusion of additional endpoints for mechanistic studies. Therefore,it is an extremely useful tool for scientists in pharmaceutical discovery and development. {\textcopyright} 2018 by John Wiley & Sons,Inc. View Publication

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) isolated from the whole blood of a 43-year-old male with hypertrophic cardiomyopathy (HCM) who carries the pathogenic variant p.Val698Ala in beta-myosin heavy chain (MYH7). Patient-derived PBMCs were reprogrammed using non-integrative episomal vectors containing reprogramming factors OCT4,SOX2,LIN28,KLF4 and L-MYC. iPSCs were shown to express pluripotent markers,have trilineage differentiation potential,carry the pathogenic MYH7 variant p.Val698Ala,have a normal karyotype and no longer carry the episomal reprogramming vector. This line is useful for studying the link between variants in MYH7 and the pathogenesis of HCM. View Publication

Background: Local ischemia is the main pathological performance in osteonecrosis of the femoral head (ONFH). There is currently no effective therapy to promote angiogenesis in the femoral head. Recent studies revealed that exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSC-Exos) have great therapeutic potential in ischemic tissues,but whether they could promote angiogenesis in ONFH has not been reported,and little is known regarding the underlying mechanism. Methods: iPS-MSC-Exos were intravenously injected to a steroid-induced rat osteonecrosis model. Samples of the femoral head were obtained 3 weeks after all the injections. The effects were assessed by measuring local angiogenesis and bone loss through histological and immunohistochemical (IHC) staining,micro-CT and three-dimensional microangiography. The effects of exosomes on endothelial cells were studied through evaluations of proliferation,migration and tube-forming analyses. The expression levels of angiogenic related PI3K/Akt signaling pathway of endothelial cells were evaluated following stimulation of iPS-MSC-Exos. The promoting effects of exosomes were re-evaluated following blockade of PI3K/Akt. Results: The in vivo study revealed that administration of iPS-MSC-Exos significantly prevented bone loss,and increased microvessel density in the femoral head compared with control group. We found that iPS-MSC-Exos significantly enhanced the proliferation,migration and tube-forming capacities of endothelial cells in vitro. iPS-MSC-Exos could activate PI3K/Akt signaling pathway in endothelial cells. Moreover,the promoting effects of iPS-MSC-Exos were abolished after blockade of PI3K/Akt on endothelial cells. Conclusions: Our findings suggest that transplantation of iPS-MSC-Exos exerts a preventative effect on ONFH by promoting local angiogenesis and preventing bone loss. The promoting effect might be attributed to activation of the PI3K/Akt signaling pathway on endothelial cells. The data provide the first evidence for the potential of iPS-MSC-Exos in treating ONFH. View Publication

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a low-grade B-cell neoplasm and ∼2{\%} to 9{\%} patients develop an aggressive lymphoma,most commonly diffuse large B-cell lymphoma (Richter transformation,DLBCL-RT). Programmed death-1 (PD-1) pathway plays a crucial role in tumor host immunity evasion and its blockade has emerged as an effective anti-cancer immunotherapy. PD-L1 and PD-1 expression has shown predictive value in anti-PD cancer immunotherapy; however,it has not been well documented in CLL/SLL and DLBCL-RT. We evaluated PD-1 and PD-L1 expression by immunohistochemistry in 39 CLL/SLL,15 DLBCL-RT,and 26 other DLBCL. In CLL/SLL,neoplastic B-cell PD-1 expression was weak and restricted to prolymphocytes/paraimmunoblasts within proliferation centers (PCs) and accentuated PCs of all sizes. Neoplastic B-cell PD-1 expression was highly prevalent and demonstrated increased intensity in DLBCL-RT,but in contrast was only rarely seen in other DLBCL (12/15 vs. 1/26; P{\textless}0.0001). An excellent correlation (90{\%} concordance) was observed between neoplastic B-cell PD-1 immunohistochemistry positivity and molecularly defined CLL/SLL clonal relatedness in DLBCL-RT. PD-L1 expression was observed on the neoplastic B cells in rare DLBCL-RT and other DLBCL cases (1/15 vs. 1/26; P{\textgreater}0.05) as well as background histiocytes and dendritic cells. Overall survival of DLBCL-RT was significantly inferior to that of the other DLBCL (median,16.9 vs. 106.1 mo; P=0.002). Our findings suggest a biological continuum from prolymphocytes/paraimmunoblasts in CLL/SLL PCs to the neoplastic B-cells in DLBCL-RT. The characteristic PD-1 expression in DLBCL-RT makes it a potential surrogate marker for determining clonal relatedness to CLL/SLL,which may have important prognostic and therapeutic implications. View Publication

Background: B cells play an important role in the development and maintenance of rheumatoid arthritis (RA). Although IL-10-producing B cells represent a major subset of regulatory B cells (Bregs) able to suppress autoimmune and inflammatory responses,recent reports showed that B cell-mediated immune suppression may also occur independent of IL-10. For instance,B cells can modulate T cell immune responses through the expression of regulatory molecules such as PD-L1. So far,PD-L1-expressing B cells have not been analyzed in RA patients. Objective: To analyze the frequency of PD-L1-expressing B cells in the peripheral blood of RA patients compared to healthy controls (HC) matched for sex and age,their function on T cell response and their changes in response to therapy. Methods: Fresh peripheral blood B cells from RA patients and HC were characterized by flow cytometry and their functionality assessed in a co-culture system with autologous T cells. Results: The frequencies of CD19+PD-L1+ B cells,CD24hiCD38-PD-L1+ and CD24hiCD38hiPD-L1+ B cells were significantly lower in untreated RA patients than in HC. In a follow-up study,the frequencies of PD-L1+ B cells (CD19+PD-L1+ B cells,CD24hiCD38-PD-L1+ and CD24hiCD38hiPD-L1+ B cells) increased significantly after treatment in good responder patients,although the frequency of total CD24hiCD38hi B cells decreased. CD19+ B cells from untreated RA patients and HC upregulated PD-L1 expression similarly upon stimulation with CpG plus IL-2 and were able to suppress,in vitro,CD8+ T cell proliferation and cytokine production in a PD-L1-dependent manner. Conclusions: Our results show that PD-L1+ B cells exhibiting T cell suppressive capacity are significantly decreased in untreated RA patients but increase in response to successful treatment. PD-L1 expression on B cells from RA patients can be modulated in vitro and PD-L1+ B cells could thus provide new perspectives for future treatment strategies. View Publication

We examined the unique contributions of the cytokines IL-21 and IL-4 on germinal center (GC) B cell initiation and subsequent maturation in a murine model system. Similar to other reports,we found T follicular helper cell expression of IL-21 begins prior to T follicular helper cell migration into the B cell follicle and precedes that of IL-4. Consistent with this timing,IL-21 signaling has a greater influence on the perifollicular pre-GC B cell transition to the intrafollicular stage. Notably,Bcl6hi B cells can form in the combined absence of IL-21R- and STAT6-derived signals; however,these nascent GC B cells cease to proliferate and are more prone to apoptosis. When B cells lack either IL-21R or STAT6,aberrant GCs form atypical centroblasts and centrocytes that differ in their phenotypic maturation and costimulatory molecule expression. Thus,IL-4 and IL-21 play nonredundant roles in the phased progression of GC B cell development that can initiate in the combined absence of these cytokine signals. View Publication

Chronic hepatitis C virus (HCV) infection causes generalized CD8+ T cell impairment,not limited to HCV-specific CD8+ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver,including CD8+ T-cells. MDMs can be polarized into M1 (classically activated) and M2a,M2b,and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions,respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics,such as increased CD86 expression and IL-10 secretion,compared to uninfected controls. In contrast,M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-gamma. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8+ T-cells from controls,M1 macrophages alone significantly increased CD8+ T cell IFN-gamma expression in a cytokine-independent and cell-contact-dependent manner. However,M1 macrophages from HCV-infected individuals significantly decreased IFN-gamma expression in CD8+ T-cells. Therefore,altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8+ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV+ individuals,and aid in the mitigation of associated negative clinical outcomes. View Publication

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function,though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mphis) directs HSC platelet-bias. Mphis from the marrow of aged mice and humans exhibited an activated phenotype,with increased expression of inflammatory signals. Aged marrow Mphis also displayed decreased phagocytic function. Senescent neutrophils,typically cleared by marrow Mphis,were markedly increased in aged mice,consistent with functional defects in Mphi phagocytosis and efferocytosis. In aged mice,Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity,which can process pro-IL1B,was increased in marrow Mphis and neutrophils. Mechanistically,IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice,depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mphis induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mphis and IL1B in the age-associated lineage-skewing of HSCs,and reveals the therapeutic potential of their manipulation as antigeronic targets. View Publication

Emerging evidence suggests an immunosuppressive role of altered tumor glycosylation due to downregulation of innate immune responses via immunoregulatory Siglecs. In contrast,human T cells,a major anticancer effector cell,only rarely express Siglecs. However,here,we report that the majority of intratumoral,but not peripheral blood,cytotoxic CD8+ T cells expressed Siglec-9 in melanoma. We identified Siglec-9+ CD8+ T cells as a subset of effector memory cells with high functional capacity and signatures of clonal expansion. This cytotoxic T-cell subset was functionally inhibited in the presence of Siglec-9 ligands or by Siglec-9 engagement by specific antibodies. TCR signaling pathways and key effector functions (cytotoxicity,cytokine production) of CD8+ T cells were suppressed by Siglec-9 engagement,which was associated with the phosphorylation of the inhibitory protein tyrosine phosphatase SHP-1,but not SHP-2. Expression of cognate Siglec-9 ligands was observed on the majority of tumor cells in primary and metastatic melanoma specimens. Targeting the tumor-restricted,glycosylation-dependent Siglec-9 axis may unleash this intratumoral T-cell subset,while confining T-cell activation to the tumor microenvironment. View Publication

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

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