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Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterized patient with autism spectrum disorder (ASD). The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The pluripotency of transgene-free iPSCs was verified by immunocytochemistry for pluripotency markers and by spontaneous in vitro differentiation towards the 3 germ layers. Furthermore,the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of ASD,also for drug testing,early biomarker discovery and gene therapy studies. View Publication

The laminin receptor integrin alpha6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. We have studied its role for homing of stem and progenitor cells to mouse hematopoietic tissues in vivo. A function-blocking anti-integrin alpha6 antibody significantly reduced progenitor cell homing to bone marrow (BM) of lethally irradiated mice,with a corresponding retention of progenitors in blood. Remarkably,the anti-integrin alpha6 antibody profoundly inhibited BM homing of long-term multilineage engrafting stem cells,studied by competitive repopulation assay and analysis of donor-derived lymphocytes and myeloid cells in blood 16 weeks after transplantation. A similar profound inhibition of long-term stem cell homing was obtained by using a function-blocking antibody against alpha4 integrin,studied in parallel. Furthermore,the anti-integrin alpha6 and alpha4 antibodies synergistically inhibited homing of short-term repopulating stem cells. Intravenous injection of anti-integrin alpha6 antibodies,in contrast to antibodies against alpha4 integrin,did not mobilize progenitors or enhance cytokine-induced mobilization by G-CSF. Our results provide the first evidence for a distinct functional role of integrin alpha6 receptor during hematopoietic stem and progenitor cell homing and collaboration of alpha6 integrin with alpha4 integrin receptors during homing of short-term stem cells. View Publication

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However,clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody,we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A. Here,we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells,lymphocytes,or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors,suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely,2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs,melphalan,or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore,administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma. View Publication

Long-term in vitro culture of undifferentiated human embryonic stem cells (hESCs) traditionally requires a fibroblast feeder cell layer. Using feeder cells in hESC cultures is highly laborious and limits large-scale hESC production for potential application in regenerative medicine. Replacing feeder cells with defined human extracellular matrix (ECM) components or synthetic biomaterials would be ideal for large-scale production of clinical-grade hESCs. We tested and compared different feeder cell-free hESC culture methods based on different human ECM proteins,human and animal sera matrices,and a Matrigel matrix. Also selected biomaterials were tested for feeder cell-free propagation of undifferentiated hESCs. The matrices were tested together with conventional and modified hESC culture media,human foreskin fibroblast-conditioned culture medium,chemically defined medium,TeSR1,and modified TeSR1 media. The results showed the undefined,xenogeneic Matrigel to be a superior matrix for hESC culture compared with the purified human ECM proteins,serum matrices,and the biomaterials tested. A long-term,feeder cell-free culture system was successful on Matrigel in combination with mTeSR1 culture medium,but a xeno-free,fully defined,and reproducible feeder cell-free hESC culture method still remains to be developed. View Publication

Cancer stem cells are known for their inherent resistance to therapy. Here we investigated whether normal stem cells with acquired resistance to stress can be used to identify novel markers of cancer stem cells. For this,we generated a human embryonic stem cell line resistant to Trichostatin A and analyzed changes in its gene expression. The resistant cells over-expressed various genes associated with tumor aggressiveness,many of which are also expressed in the CD133+ glioma cancer stem cells. These findings suggest that stress-resistant stem cells generated in vitro may be useful for the discovery of novel markers of cancer stem cells. View Publication

The demonstration of the brain's ability to initiate repair in response to disease or injury has sparked considerable interest in therapeutic strategies to stimulate adult neurogenesis. In this study we examined the effect of a progressive neurodegenerative condition on neural precursor activity in the subventricular zone (SVZ) and hippocampus of the R6/1 transgenic mouse model of Huntington's disease (HD). Our results revealed an age-related decline in SVZ precursor numbers in both wild-type (WT) and HD mice. Interestingly,hippocampal precursor numbers declined with age in WT mice,although we observed maintenance in hippocampal precursor number in the HD animals in response to advancement of the disease. This maintenance was consistent with activation of a recently identified latent hippocampal precursor population. We found that the small latent stem cell population was also maintained in the HD hippocampus at 33 weeks,whereas it was not present in the WT. Our findings demonstrate that,despite a loss of neurogenesis in the HD hippocampus in vivo,there is a unique maintenance of the precursor and stem cells,which may potentially be activated to ameliorate disease symptoms. View Publication

The MAPK pathway is one of the most important pathways for novel anticancer drug development. We performed high-throughput screening for compounds that induce expression of p15INK4b,and identified JTP-74057 (GSK1120212),which is being evaluated in ongoing phase I,II and III clinical trials. We characterized its antitumor activities in vitro and in vivo. JTP-74057 strongly inhibited MEK1/2 kinase activities,but did not inhibit another 98 kinase activities. Treatment by JTP-74057 resulted in growth inhibition accompanied with upregulation of p15INK4b and/or p27KIP1 in most of the colorectal cancer cell lines tested. Daily oral administration of JTP-74057 for 14 days suppressed tumor growth of HT-29 and COLO205 xenografts in nude mice. Notably,tumor regression was observed only in COLO205 xenografts,and COLO205 was much more sensitive to JTP-74057-induced apoptosis than HT-29 in vitro. Treatment with an Akt inhibitor enhanced the JTP-74057-induced apoptosis in HT-29 cells. Finally,JTP-74057 exhibited an additive or a synergistic effect in combination with the standard-of-care agents,5-fluorouracil,oxaliplatin or SN-38. JTP-74057,a highly specific and potent MEK1/2 inhibitor,exerts favorable antitumor activities in vitro and in vivo. Sensitivity to JTP-74057-induced apoptosis may be an important factor for the estimation of in vivo efficacy,and sensitivity was enhanced by an Akt inhibitor. These results suggest the usefulness of JTP-74057 in therapeutic applications for colorectal cancer patients. View Publication

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with $$-thalassemia ($$-thal) with the aim to generate trangene-free $$-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4,Klf4,Sox2,cMyc,and Lin28 resulted in formation of five iPSC colonies,from which three were picked up and expanded in $$-thal-iPSC lines. After 10 serial passages in vitro,$$-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs,whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%,but with a decreased hematopoietic colony-forming capability. In conclusion,we report herein the generation of transgene-free $$-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover,it was demonstrated that the mRNA-based reprogramming method,used mainly in fibroblasts,is also suitable for reprogramming of human BM-MSCs. View Publication

Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors,but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned medium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay,we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts,perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells,specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs),including a clonogenic potential of ˜21.5% and expression of pluripotency associated genes like Oct-4,Nanog,c-Myc and Klf-4. Similar to CASCs,migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and Imatinib Mesylate,a PDGF receptor inhibitor,suggested a role for the PDGF-AA/PDGF receptor $$ axis in enhancing the migration process of CASCs. In conclusion,our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors,like PDGF-AA,can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury. View Publication

ETV6::RUNX1,the most common oncogenic fusion in pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL),induces a clinically silent preleukemic state that can persist in carriers for over a decade and may progress to overt leukemia upon acquisition of secondary lesions. The mechanisms contributing to quiescence of ETV6::RUNX1+ preleukemic cells still remain elusive. In this study,we identify linker histone H1-0 as a critical mediator of the ETV6::RUNX1+ preleukemic state by employing human -induced pluripotent stem cell (hiPSC) models engineered by using CRISPR/Cas9 gene editing. Global gene expression analysis revealed upregulation of H1-0 in ETV6::RUNX1+ hiPSCs that was preserved upon hematopoietic differentiation. Moreover,whole transcriptome data of 1,727 leukemia patient samples showed significantly elevated H1-0 levels in ETV6::RUNX1+ BCP-ALL compared to other leukemia entities. Using dual-luciferase promoter assays,we show that ETV6::RUNX1 induces H1-0 promoter activity. We further demonstrate that depletion of H1-0 specifically inhibits ETV6::RUNX1 signature genes,including RAG1 and EPOR. Single-cell sequencing showed that H1-0 is highly expressed in quiescent hematopoietic cells. Importantly,H1-0 protein levels correspond to susceptibility of BCP-ALL cells towards histone deacetylase inhibitors (HDACis) and combinatorial treatment using the H1-0-inducing HDACi Quisinostat showed promising synergism with established chemotherapeutic drugs. Taken together,our data identify H1-0 as a key regulator of the ETV6::RUNX1+ transcriptome and indicate that the addition of Quisinostat may be beneficial to target non-responsive or relapsing ETV6::RUNX1+ BCP-ALL. View Publication

BackgroundNatural killer (NK) cells are key effector cells of antitumor immunity. However,tumors can acquire resistance programs to escape NK cell-mediated immunosurveillance. Identifying mechanisms that mediate this resistance enables us to define approaches to improve immune-mediate antitumor activity. In previous studies from our group,a genome-wide CRISPR-Cas9 screen identified Charged Multivesicular Body Protein 2A (CHMP2A) as a novel mechanism that mediates tumor intrinsic resistance to NK cell activity.MethodsHere,we use an immunocompetent mouse model to demonstrate that CHMP2A serves as a targetable regulator of not only NK cell-mediated immunity but also other immune cell populations. Using the recently characterized murine 4MOSC model system,a syngeneic,tobacco-signature murine head and neck squamous cell carcinoma model,we deleted mCHMP2A using CRISPR/Cas9-mediated knock-out (KO),following orthotopic transplantation into immunocompetent hosts.ResultsWe found that mCHMP2A KO in 4MOSC1 cells leads to more potent NK-mediated tumor cell killing in vitro in these tumor cells. Moreover,following orthotopic transplantation,KO of mCHMP2A in 4MOSC1 cells,but not the more immune-resistant 4MOSC2 cells enables both T cells and NK cells to better mediate antitumor activity compared with wild type (WT) tumors. However,there was no difference in tumor development between WT and mCHMP2A KO 4MOSC1 or 4MOSC2 tumors when implanted in immunodeficient mice. Mechanistically,we find that mCHMP2A KO 4MOSC1 tumors transplanted into the immunocompetent mice had significantly increased CD4+T cells,CD8+T cells. NK cell,as well as fewer myeloid-derived suppressor cells (MDSC).ConclusionsTogether,these studies demonstrate that CHMP2A is a targetable inhibitor of cellular antitumor immunity. View Publication

Tumor patients often exhibit limited responses to immunotherapy owing to the low immunogenicity and immunosuppressive environment of tumors. Our previous studies revealed that cryo-thermal therapy caused tumor cell rupture due to mechanical compression,notably causing the release of a substantial amount of DAMPs (danger-associated molecular patterns),such as heat shock protein 70,calreticulin and high-mobility group box protein 1; the release of these DAMPs increased myeloid cell maturation,thereby reshaping the systemic immune environment and ultimately inducing durable CD4+ T helper type 1 (Th1) cell-dominated antitumor immunity. In fact,under conditions of mechanical stress and rapid temperature changes,the disruption of tumor cells caused by cryo-thermal therapy results in extensive deoxyribonucleic acid (DNA) damage and the rapid release of substantial amounts of DNA. Consequently,tumor-derived DNA,which potently activates innate immunity by engaging multiple DNA sensors,plays a pivotal role in orchestrating antitumor immunity. We hypothesized that cryo-thermal therapy induces the transient release of high levels of DNA,which modulates CD11b+ myeloid cell function,subsequently influencing CD4+ Th1-cell dominated antitumor immune responses. In this study,a B16F10 melanoma model was established,and DNA concentrations were measured at different time points after cryo-thermal therapy. Deoxyribonuclease I (DNase I) was subsequently administered immediately following cryo-thermal therapy to deplete extracellular DNA,allowing an investigation of the role of DNA in regulating CD11b+ myeloid cell function and CD4+ T cell differentiation. The phenotype and function of CD11b+ myeloid cells and CD4+ T cells were assessed by flow cytometry,RNA sequencing,and cell culture in vitro. Our studies confirmed that cryo-thermal therapy triggered a transient release of high levels of DNA,which was internalized by CD11b+ myeloid cells via C-type lectin receptors and subsequently sensed by inflammasomes. Then,the intracellular sensing of DNA induced the production of the mature form of interleukin (IL)-18,ultimately promoting the Th1 differentiation of CD4+ T cells. This study highlights the pivotal role of DNA release after cryo-thermal therapy in driving CD4+ Th1 cell-dominant antitumor immunity. View Publication