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Although human induced pluripotent stem cells (hiPSCs) hold great potential for the study of human diseases affecting disparate cell types,they have been underutilized in seeking mechanistic insights into the pathogenesis of congenital craniofacial disorders. Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in EFNB1 and characterized by craniofacial,skeletal,and neurological anomalies. Heterozygous females are more severely affected than hemizygous males,a phenomenon termed cellular interference that involves mosaicism for EPHRIN-B1 function. Although the mechanistic basis for cellular interference in CFNS has been hypothesized to involve Eph/ephrin-mediated cell segregation,no direct evidence for this has been demonstrated. Here,by generating hiPSCs from CFNS patients,we demonstrate that mosaicism for EPHRIN-B1 expression induced by random X inactivation in heterozygous females results in robust cell segregation in human neuroepithelial cells,thus supplying experimental evidence that Eph/ephrin-mediated cell segregation is relevant to pathogenesis in human CFNS patients. View Publication

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches,we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis,the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture,accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development,we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach,we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases. The ability to differentiate human pluripotent stem cells (hPSCs) into insulin producing cells holds potential for diabetes treatments,but many of these approaches lack the complexity needed for in vitro disease modeling. Here they develop an hPSC-derived islet spheroid system,offering an experimental model to study pancreatic budding and islet morphogenesis with human cells. View Publication

Hematopoietic stem cells (HSCs) react to various stress conditions. However,it is unclear whether and how HSCs respond to severe anemia. Here,we demonstrate that upon induction of acute anemia,HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia,lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs,transcription levels of lipid metabolism-related genes,such as very low-density lipoprotein receptor ( Vldlr ),are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential,whereas HSCs in Apoe knockout mice do not respond to anemia induction. Vldlr high HSCs show higher erythroid potential,which is enhanced after acute anemia induction. Vldlr high HSCs are epigenetically distinct because of their low chromatin accessibility,and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions. Subject terms: Haematopoietic stem cells,Fat metabolism,Chromatin,Anaemia View Publication

The ten‐eleven translocation family of enzymes (TET1/2/3) promotes DNA demethylation and is essential for hematopoiesis. While the roles of TET1 and TET2 are well‐studied in hematopoiesis,the requirement of TET3 in embryonic and adult hematopoiesis is less investigated. In this study,by characterizing embryonic and adult hematopoiesis in Tie2 +/cre ; Tet3 f/f mice,we have established a requirement for TET3 in regulating hematopoietic stem cells (HSCs; CD150 + CD48 – ). We found that loss of TET3 in the fetal liver and adult bone marrow causes a reduction in the percent of long‐term HSCs (LT‐HSCs; CD150 + CD48 – CD34 – ). This was accompanied by reduced colony forming capacity of TET3‐deficient HSCs in vitro and reduced contribution of HSCs after a competitive bone marrow transplantation in vivo. TET3 deficiency increased DNA methylation at several cell cycle regulator genes leading to their down regulation. This is consistent with,and likely underpins,the reduced number of quiescent HSCs in TET3‐deficient bone marrow. These findings uncover a new role for TET3 in HSC homeostasis during embryonic and adult hematopoiesis. View Publication

The impact of exogenous stressors,such as cancer chemotherapies,on the genomic integrity and clonal dynamics of normal hematopoiesis is not well defined. We conducted whole-genome sequencing on 1,276 single-cell-derived hematopoietic stem and progenitor cell (HSPC) colonies from ten patients with multiple myeloma treated with chemotherapies and six normal donors. Melphalan treatment significantly increased the mutational burden,producing a distinctive mutation signature,whereas other chemotherapeutic agents had minimal effects. Consequently,the clonal diversity and architecture of post-treatment HSPCs resemble those observed in normal elderly individuals,particularly through the progression of oligoclonal hematopoiesis,thereby suggesting that chemotherapy accelerates clonal aging. Integrated phylogenetic analysis of matched therapy-related myeloid neoplasm samples traced their clonal origin to a single-HSPC clone among multiple competing clones,supporting a model of oligoclonal to monoclonal transformation. These findings underscore the need for further systematic research on the long-term hematological consequences of cancer chemotherapy. Subject terms: Genetics research,Acute myeloid leukaemia View Publication

BACKGROUND The conducting airway epithelium is repaired by tissue specific stem cells (TSC). In response to mild/moderate injury,each TSC repairs a discrete area of the epithelium. In contrast,severe epithelial injury stimulates TSC migration and expands the stem cell's reparative domain. Lung transplantation (LTx) can cause a moderate/severe airway injury and the remodeled airway contains a chimeric mixture of donor and recipient cells. These studies supported the hypothesis,LTx stimulates TSC migration resulting in epithelial chimerism. We tested this hypothesis in cystic fibrosis (CF) LTx patients. METHODS Airway mucosal injury was quantified using bronchoscopic imaging and a novel grading system. Bronchial brushing was used to recover TSC from 10 sites in the recipient and allograft airways. TSC chimerism was quantified by short tandem repeat analysis. TSC self-renewal and differentiation potential were assayed using the clone forming cell frequency and air-liquid-interface methods. Electrophysiology was used to determine if TSC chimerism altered epithelial ion channel activity. RESULTS LTx caused a mild to moderate airway mucosal injury. Donor and recipient TSC were identified in 91{\%} of anastomotic sites and 93{\%} of bronchial airways. TSC chimerism did not alter stem cell self-renewal or differentiation potential. The frequency of recipient TSC was proportional to CF Transmembrane Conductance Regulator (CFTR)-dependent ion channel activity and 33{\%} of allograft regions were at risk for abnormal CFTR activity. CONCLUSIONS LTx in CF patients stimulates bidirectional TSC migration across the anastomoses. TSC chimerism may alter ion homeostasis and compromise the host defense capability of the allograft airway epithelium. View Publication

PURPOSE: Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells,but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. METHODS: The seven osteosarcoma cell lines Cal72,SJSA-1,Saos-2,SK-ES-1,U2OS,143.98.2,and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). RESULTS: Although proliferation often was relatively low in serum-free media (X-vivo 10,X-vivo 15,X-vivo 20,Stem Span SFEM),some cell lines (Cal72,KHOS-32IH,Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However,all cell lines proliferated well in Stem Span with FCS,and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS),and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72,SJSA-1),and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1. CONCLUSIONS: Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines,but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation,cytokine release); and (iii) whether coculture experiments are included. View Publication

AIM: The recently published REPAIR-AMI and ASTAMI trial showed differences in contractile recovery of left ventricular function after infusion of bone marrow-derived cells in acute myocardial infarction. Since the trials used different protocols for cell isolation and storage (REPAIR-AMI: Ficoll,storage in X-vivo 10 medium plus serum; ASTAMI: Lymphoprep,storage in NaCl plus plasma),we compared the functional activity of BMC isolated by the two different protocols. METHODS AND RESULTS: The recovery of total cell number,colony-forming units (CFU),and the number of mesenchymal stem cells were significantly reduced to 77 +/- 4%,83 +/- 16%,and 65 +/- 15%,respectively,when using the ASTAMI protocol compared with the REPAIR protocol. The capacity of the isolated BMC to migrate in response to stromal cell-derived factor 1 (SDF-1) was profoundly reduced when using the ASTAMI cell isolation procedure (42 +/- 8% and 78 +/- 3% reduction in healthy and CAD-patient cells,respectively). Finally,infusion of BMC into a hindlimb ischaemia model demonstrated a significantly blunted blood-flow-recovery by BMC isolated with the ASTAMI protocol (54 +/- 6% of the effect obtained by REPAIR cells). Comparison of the individual steps identified the use of NaCl and plasma for cell storage as major factors for functional impairment of the BMC. CONCLUSION: Cell isolation protocols have a major impact on the functional activity of bone marrow-derived progenitor cells. The assessment of cell number and viability may not entirely reflect the functional capacity of cells in vivo. Additional functional testing appears to be mandatory to assure proper cell function before embarking on clinical cell therapy trials. View Publication

Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy,although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore,in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells,cetuximab concentration,and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets,respectively,in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore,supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level,cetuximab concentration,and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P textless 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a,and secreted significantly (P textless 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab mediated clinical responses. Cellular and secreted immune profiles and FcgammaR genotypes from patients' lymphocytes may provide clinically useful biomarkers of immune activation in cetuximab treated patients. View Publication

Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source,potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein (Fluc- eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n = 35) or skeletal muscle (n = 35) injection of 1 × 102,1 × 103,1 × 104,1 × 105 or 1 × 106 DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after 8 weeks were followed out to day 365 to confirm tumor absence. Significantly,in this study,a minimum of 1 × 105 ES cells in the myocardium and 1 × 104 cells in the skeletal muscle was observed to be requisite for teratoma development,suggesting that human ES cell number may be a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic. Keywords View Publication

Previous studies from this laboratory have demonstrated that fibroblast growth factor 1 together with a number of co-activator molecules (dopamine,TPA,IBMX/forskolin),will induce the expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in 10% of human neurons (hNTs) derived from the NT2 cell line [10]. In the present study,we found that TH induction was increased to nearly 75% in hNTs when cells were permitted to age 2 weeks in culture prior to treatment with the differentiation cocktail. This high level of TH expression was sustained 7 days after removal of the differentiating agents from the media. Moreover,the induced TH present in these cells was enzymatically active,resulting in the production of low levels of dopamine (DA) and its metabolite DOPAC. These findings suggest that hNTs may provide an important tissue culture model for the study of factors regulating TH gene expression in human neurons. Moreover,hNTs may serve,in vivo,as a source of human DA neurons for use in transplantation therapies. View Publication

The development of dengue antivirals and vaccine has been hampered by the incomplete understanding of molecular mechanisms of dengue virus (DENV) infection and pathology,partly due to the limited suitable cell culture or animal models that can capture the comprehensive cellular changes induced by DENV. In this study,we differentiated human pluripotent stem cells (hPSCs) into hepatocytes,one of the target cells of DENV,to investigate various aspects of DENV-hepatocyte interaction. hPSC-derived hepatocyte-like cells (HLCs) supported persistent and productive DENV infection. The activation of interferon pathways by DENV protected bystander cells from infection and protected the infected cells from massive apoptosis. Furthermore,DENV infection activated the NF-$$B pathway,which led to production of proinflammatory cytokines and downregulated many liver-specific genes such as albumin and coagulation factor V. Our study demonstrates the utility of hPSC-derived hepatocytes as an in vitro model for DENV infection and reveals important aspects of DENV-host interactions. View Publication