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Genome instability is a potential limitation to the research and therapeutic application of induced pluripotent stem cells (iPSCs). Observed genomic variations reflect the combined activities of DNA damage,cellular DNA damage response (DDR),and selection pressure in culture. To understand the contribution of DDR on the distribution of copy number variations (CNVs) in iPSCs,we mapped CNVs of iPSCs with mutations in the central DDR gene ATM onto genome organization landscapes defined by genome-wide replication timing profiles. We show that following reprogramming the early and late replicating genome is differentially affected by CNVs in ATM deficient iPSCs relative to wild type iPSCs. Specifically,the early replicating regions had increased CNV losses during retroviral reprogramming. This differential CNV distribution was not present after later passage or after episomal reprogramming. Comparison of different reprogramming methods in the setting of defective DNA damage response reveals unique vulnerability of early replicating open chromatin to retroviral vectors. View Publication

Although natural killer (NK) cells are well known for their ability to kill tumors,few studies have addressed the interactions between resting (nonactivated) NK cells and freshly isolated human tumors. Here,we show that human leukocyte antigen class I(low) tumor cells isolated directly from patients with advanced ovarian carcinoma trigger degranulation by resting allogeneic NK cells. This was paralleled by induction of granzyme B and caspase-6 activities in the tumor cells and significant tumor cell lysis. Ovarian carcinoma cells displayed ubiquitous expression of the DNAX accessory molecule-1 (DNAM-1) ligand PVR and sparse/heterogeneous expression of the NKG2D ligands MICA/MICB and ULBP1,ULBP2,and ULBP3. In line with the NK receptor ligand expression profiles,antibody-mediated blockade of activating receptor pathways revealed a dominant role for DNAM-1 and a complementary contribution of NKG2D signaling in tumor cell recognition. These results show that resting NK cells are capable of directly recognizing freshly isolated human tumor cells and identify ovarian carcinoma as a potential target for adoptive NK cell-based immunotherapy. View Publication

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation,we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells,cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid,B-lymphoid,and erythroid lineages,but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization,which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice. View Publication

Oxidative stress contributes to tissue injury and cell death during the development of various diseases. The present study aims at investigating whether oxidative stress triggered by the exposure to hydrogen peroxide (H2O2) can induce apoptosis of induced pluripotent stem cells (iPS cells) in a mechanism mediated by insulin-like growth factor (IGF-1) and microRNA-1 (miR-1). iPS cells treated with H2O2 showed increases in miR-1 expression,mitochondria dysfunction,cytochrome-c release and apoptosis,Addition of IGF-1 into the iPS cell cultures reduced the H2O2 cytotoxicity. Prediction algorithms showed that 3'-untranslated regions of IGF-1 gene as a target of miR-1. Moreover,miR-1 mimic,but not miR-1 mimic negative control,diminished the protective effect of IGF-1 on H2O2-induced mitochondrial dysfunction,cytochrome-c release and apoptosis in iPS cells. In conclusion,IGF-1 inhibits H2O2-induced mitochondrial dysfunction,cytochrome-c release and apoptosis. IGF-1's effect is,at least partially,regulated by miR-1 in iPS cells. ?? 2012 Elsevier Inc. View Publication

Identification of prevalent specific markers is crucial to stem/progenitor cell purification. Determinants such as the surface antigens CD34 and CD38 are traditionally used to analyze and purify hematopoietic stem/progenitor cells (HSCs/HPCs). However,the variable expression of these membrane antigens poses some limitations to their use in HSC/HPC purification. Techniques based on drug/stain efflux through the ATP-binding cassette (ABC)G2 pump (side population [SP] phenotype) or on detection of aldehyde dehydrogenase (ALDH) activity have been independently developed and distinguish the SP and ALDH(Bright) (ALDH(Br)) cell subsets for their phenotype and proliferative capability. In this study,we developed a multiparametric flow cytometric method associating both SP and ALDH activities on human lineage negative (Lin(-)) bone marrow cells and sorted different cell fractions according to their SP/ALDH activity level. We find that Lin(-)CD34(+)CD38(Low/-) cells are found throughout the spectrum of ALDH expression and are enriched especially in ALDH(Br) cells when associated with SP functionality (SP/ALDH(Br) fraction). Furthermore,the SP marker identified G(0) cells in all ALDH fractions,allowing us to sort quiescent cells regardless of ALDH activity. Moreover,we show that,within the Lin(-)CD34(+)CD38(-)ALDH(Br) population,the SP marker identifies cells with higher primitive characteristics,in terms of stemness-related gene expression and in vitro and in vivo proliferative potential,than the Lin(-)CD34(+) CD38(-)ALDH(Br) main population cells. In conclusion,our study shows that the coexpression of SP and ALDH markers refines the Lin(-)CD34(+)CD38(-) hematopoietic compartment and identifies an SP/ALDH(Br) cell subset enriched in quiescent primitive HSCs/HPCs. View Publication

Innovative identification technologies for hematopoietic stem cells (HSCs) have expanded the scope of stem cell biology. Clinically,the functional quality of HSCs critically influences the safety and therapeutic efficacy of stem cell therapies. However,most analytical techniques capture only a single snapshot,disregarding the temporal context. A comprehensive understanding of the temporal heterogeneity of HSCs necessitates live-cell,real-time and non-invasive analysis. Here,we developed a prediction system for HSC diversity by integrating single-HSC ex vivo expansion technology with quantitative phase imaging (QPI)-driven machine learning. By analyzing the cellular kinetics of individual HSCs,we discovered previously undetectable diversity that snapshot analysis cannot resolve. The QPI-driven algorithm quantitatively evaluates stemness at the single-cell level and leverages temporal information to significantly improve prediction accuracy. This platform advances the field from snapshot-based identification of HSCs to dynamic,time-resolved prediction of their functional quality based on past cellular kinetics. Subject terms: Haematopoietic stem cells,Stem-cell differentiation,Self-renewal,Imaging View Publication

Ab-targeted vaccination involves targeting a receptor of choice expressed by dendritic cells (DCs) with Ag-coupled Abs. Currently,there is little consensus as to which criteria determine receptor selection to ensure superior Ag presentation and immunity. In this study,we investigated parameters of DC receptor internalization and determined how they impact Ag presentation outcomes. First,using mixed bone marrow chimeras,we established that Ag-targeted,but not nontargeted,DCs are responsible for Ag presentation in settings of Ab-targeted vaccination in vivo. Next,we analyzed parameters of DEC205 (CD205),Clec9A,CD11c,CD11b,and CD40 endocytosis and obtained quantitative measurements of internalization speed,surface turnover,and delivered Ag load. Exploiting these parameters in MHC class I (MHC I) and MHC class II (MHC II) Ag presentation assays,we showed that receptor expression level,proportion of surface turnover,or speed of receptor internalization did not impact MHC I or MHC II Ag presentation efficiency. Furthermore,the Ag load delivered to DCs did not correlate with the efficiency of MHC I or MHC II Ag presentation. In contrast,targeting Ag to CD8(+) or CD8(-) DCs enhanced MHC I or MHC II Ag presentation,respectively. Therefore,receptor expression levels,speed of internalization,and/or the amount of Ag delivered can be excluded as major determinants that dictate Ag presentation efficiency in setting of Ab-targeted vaccination. View Publication

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear,as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2,and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38,as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation,and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise,NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET,the positive regulator of IFN-gamma,and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC,and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively,our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC,and these effects are mediated via SMAD3. View Publication

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable,synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells,including impaired neural rosette formation,increased susceptibility to growth factor withdrawal,and deficits in mitochondrial respiration,are rescued in isogenic controls. Importantly,using genome-wide expression analysis,we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines,suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities,and the importance of isogenic controls for disease modeling using hiPSCs. View Publication

Articular cartilage,which is mainly composed of collagen II,enables smooth skeletal movement. Degeneration of collagen II can be caused by various events,such as injury,but degeneration especially increases over the course of normal aging. Unfortunately,the body does not fully repair itself from this type of degeneration,resulting in impaired movement. Microfracture,an articular cartilage repair surgical technique,has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However,the therapeutic outcomes of all these techniques vary in different patients depending on their age,health,lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage,both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone,or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs),which are able to self-renew and differentiate into multiple cell types,provides a potentially valuable cell resource for drug screening in a more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis." View Publication