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DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway,which promotes error-free repair of DNA double-strand breaks,is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus,cells from Fanconi anemia patients,which lack this critical pathway,fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study,we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6,but not E7,recovers FA iPSC colony formation and,furthermore,that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase,NANOG,and Tra-1-60,indicating that they were fully reprogrammed into pluripotent cells. However,FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression,whereas their FA-complemented counterparts grew efficiently. Thus,we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. IMPORTANCE A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53. View Publication

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes,with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations,leading to behavioural pathologies in humans,remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome,we narrowed this cellular phenotype to a single gene candidate,frizzled 9 (FZD9). At the neuronal stage,layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites,increased numbers of spines and synapses,aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain. View Publication

Stem cell therapies hold great promise for repairing tissues damaged due to disease or injury. However,a major obstacle facing this field is the difficulty in identifying cells of a desired phenotype from the heterogeneous population that arises during stem cell differentiation. Conventional fluorescence flow cytometry and magnetic cell purification require exogenous labeling of cell surface markers which can interfere with the performance of the cells of interest. Here,we describe a non-genetic,label-free cell cytometry method based on electrophysiological response to stimulus. As many of the cell types relevant for regenerative medicine are electrically-excitable (e.g. cardiomyocytes,neurons,smooth muscle cells),this technology is well-suited for identifying cells from heterogeneous stem cell progeny without the risk and expense associated with molecular labeling or genetic modification. Our label-free cell cytometer is capable of distinguishing clusters of undifferentiated human induced pluripotent stem cells (iPSC) from iPSC-derived cardiomyocyte (iPSC-CM) clusters. The system utilizes a microfluidic device with integrated electrodes for both electrical stimulation and recording of extracellular field potential (FP) signals from suspended cells in flow. The unique electrode configuration provides excellent rejection of field stimulus artifact while enabling sensitive detection of FPs with a noise floor of 2 $$V(rms). Cells are self-aligned to the recording electrodes via hydrodynamic flow focusing. Based on automated analysis of these extracellular signals,the system distinguishes cardiomyocytes from non-cardiomyocytes. This is an entirely new approach to cell cytometry,in which a cell's functionality is assessed rather than its expression profile or physical characteristics. View Publication

Since the 1960s,the stem cells have been extensively studied including embryonic stem cells,neural stem cells,bone marrow hematopoietic stem cells,and mesenchymal stem cells. In the recent years,several stem cells have been initially used in the treatment of diseases,such as in bone marrow transplant. At the same time,isolation and culture experimental technologies for stem cell research have been widely developed in recent years. In addition,molecular imaging technologies including optical molecular imaging,positron emission tomography,single-photon emission computed tomography,and computed tomography have been developed rapidly in recent the 10 years and have also been used in the research on disease mechanism and evaluation of treatment of disease related with stem cells. This paper will focus on recent typical isolation,culture,and observation techniques of stem cells followed by a concise introduction. Finally,the current challenges and the future applications of the new technologies in stem cells are given according to the understanding of the authors,and the paper is then concluded. View Publication

We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct,allowing its regulated expression (and,at the same time,deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes,miRNA-based RNAi (including two shRNA constructs at once),and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture,efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny,and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo. View Publication

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

OBJECTIVE: Ionizing radiation (IR) and busulfan (BU) are commonly used as preconditioning regimens for bone marrow transplantation (BMT). We examined whether induction of apoptosis in murine bone marrow (BM) hematopoietic cells contributes to IR- and BU-induced suppression of their hematopoietic function. METHODS: The hematopoietic functions of hematopoietic stem cells (HSCs) and progenitors were analyzed by the cobblestone area-forming cell (CAFC) assay. Apoptosis was determined by measuring 3,3'-dihexyloxacarbocyanine iodide (DiCO6) uptake,annexin V staining,and/or sub-G(0/1) cells. Four cell types were studied: murine BM mononuclear cells (BM-MNCs),linage-negative hematopoietic cells (Lin-) cells),Lin- Scal+ c-kit+ cells,and Lin- Scal- c-kit+ cells by flow cytometry. RESULTS: Exposure of BM-MNCs to IR (4 Gy) or incubation of the cells with BU (30 microM) resulted in a significant reduction in CAFC frequency (ptextless0.001). The survival fractions of various day-types of CAFC for the irradiated cells were less than 10%,while that for BU-treated cells was 71.3% on day 7 and progressively declined to 5.3% on day 35. Interestingly,IR significantly induced apoptosis in BM-MNCs,Lin- cells,HSCs,and progenitors,whereas BU failed to increase apoptosis in these cells. In addition,preincubation of BM-MNCs with z-Val-Ala-Asp (OCH3)-fluoromethylketone,methyl ester (z-VAD) attenuated IR-induced reduction in CAFC but not that induced by BU. CONCLUSION: IR and BU differentially suppress the hematopoietic function of HSCs and progenitors by fundamentally different mechanisms. IR inhibits the function primarily by the induction of HSC and progenitor apoptosis. In contrast,BU suppresses HSC and progenitor function via an apoptosis-independent mechanism. View Publication