���ϳԹ�

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease, as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture, differentiation, isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 d), isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes, respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs. View Publication

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.

Identification of cancer stem cells is crucial for advancing cancer biology and therapy. Several markers including CD24, CD44, CD117, CD133, the G subfamily of ATP-binding cassette transporters (ABCG), epithelial specific antigen (ESA) and aldehyde dehydrogenase (ALDH) are used to identify and investigate human epithelial cancer stem cells in the literature. We have now systemically analyzed and compared the expression of these markers in fresh ovarian epithelial carcinomas. Although the expression levels of these markers were unexpectedly variable and partially overlapping in fresh ovarian cancer cells from different donors, we reliably detected important levels of CD133 and ALDH in the majority of fresh ovarian cancer. Furthermore, most of these stem cell markers including CD133 and ALDH were gradually lost following in vitro passage of primary tumor cells. However, the expression of ALDH and CD133, but not CD24, CD44 and CD117, could be partially rescued by the in vitro serum-free and sphere cultures and by the in vivo passage in the immune-deficient xenografts. ALDH+ and CD133+ cells formed three-dimensional spheres more efficiently than their negative counterparts. These sphere-forming cells expressed high levels of stem cell core gene transcripts and could be expanded and form additional spheres in long-term culture. ALDH+ , CD133+ and ALDH+ CD133+ cells from fresh tumors developed larger tumors more rapidly than their negative counterparts. This property was preserved in the xenografted tumors. Altogether, the data suggest that ALDH+ and CD133+ cells are enriched with ovarian cancer-initiating (stem) cells and that ALDH and CD133 may be widely used as reliable markers to investigate ovarian cancer stem cell biology.

The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.

The notion that gliomas could originate from mutated glial precursor cells highlights the possibility of modulating the proliferative and migratory behaviour of glioma cells by acting on the molecular mechanisms operative during the development of the Central Nervous System (CNS), but absent in the normal adult brain. We show that the GL15 glioblastoma derived human cell line displays a high expression of nestin which, combined with the previously demonstrated high expression of vimentin, constitutes a characteristic of astrocyte restricted precursors. We also show that, in analogy with some leukaemia cells, GL15 cells display the constitutively phosphorylated form of Janus kinase 2 (JAK2), a tyrosine kinase expressed during CNS development but undetectable in the normal adult brain. The constitutive activation of JAK2 does not result from chromosomal aberrations involving the JAK2 gene, but most probably from abnormally activated transduction systems operative in glioblastoma cells. We then investigated the effects of tyrphostin AG490, an inhibitor of JAK2 autophosphorylation, on GL15 cell growth. In the absence of exogenous growth factors and cytokines, 10 microM tyrphostin AG490 induces an S phase arrest, combined with a partial impairment of the G2 phase of the cell cycle. The abnormally activated JAK2 could then potentially represent a target for a selective pharmacological approach in glioblastoma cells in which a combination of glial precursor characteristics and genetic alterations occurs.

Human embryonic stem cell line BJNhem20-OCIAD1-Tet-On was generated using non-viral method. The constructs pCAG-Tet-On and pTRE-Tight vector driving OCIAD1 expression were transfected using microporation procedure. pCAG-Tet-On cells can be used for inducible expression of any coding sequence cloned into pTRE-Tight vector. For example, in human embryonic stem cells, Tet-On system has been used to generate SOX2 overexpression cell line (Adachi et al., 2010).

Previous reports have shown that antidepressants increase neuronal cell proliferation and enhance neuroplasticity both in vivo and in vitro. This study investigated the direct effects of one such antidepressant, fluoxetine , on cell proliferation and on the production of neurotrophic factors in neuronal precursors derived from human embryonic stem cells (hESCs; H9). Fluoxetine induced the differentiation of neuronal precursors, strongly enhancing neuronal characteristics. The rate of proliferation was higher in fluoxetine -treated cells than in control cells, as determined by MTT [3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. The CPDL (cumulative population doubling level) of the fluoxetine-treated cells was significantly increased in comparison to that of control cells (ptextless.001). Bromodeoxyuridine incorporation and staurosporine-induced apoptosis assays were elevated in fluoxetine-treated cells. Quantitative RT-PCR analysis revealed no significant differences in the expression of neurotrophic factors, brain-derived neurotrophic factor (BDNF);glial-derived neurotrophic factor (GDNF) and cAMP-responsive element-binding protein (CREB) between cells treated with fluoxetine for two weeks and their untreated counterparts. These results may help elucidate the mechanism of action of fluoxetine as a therapeutic drug for the treatment of depression. Data presented herein provide more evidence that, in addition to having a direct chemical effect on serotonin levels, fluoxetine can influence hESC-derived neuronal cells by increasing cell proliferation, while allowing them to maintain their neuronal characteristics.

INTRODUCTION: The induced pluripotent stem cell (iPSC) technology allows generation of patient-specific pluripotent stem cells, thereby providing a novel cell-therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming. METHODS: We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and peripheral blood mononuclear cells (PBMCs) and tested the pluripotency of derived iPS clones. RESULTS: Ectopic expression of OCT4, SOX2, KLF4, and c-MYC in HPCs and PBMCs resulted in rapid iPSC derivation. Long-term time-lapse imaging revealed efficient iPSC growth under serum- and feeder-free conditions with frequent mitotic events. HPC- and PBMC-derived iPS cells expressed pluripotency-associated markers, including SSEA-4, TRA-1-60, and NANOG. The global gene-expression profiles demonstrated the induction of endogenous pluripotent genes, such as LIN28, TERT, DPPA4, and PODXL, in derived iPSCs. iPSC clones from blood and other cell sources showed similar ultrastructural morphologies and genome-wide gene-expression profiles. On spontaneous and guided differentiation, HPC- and PBMC-derived iPSCs were differentiated into cells of three germ layers, including insulin-producing cells through endodermal lineage, verifying the pluripotency of the blood-derived iPSC clones. CONCLUSIONS: Because the use of blood cells allows minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming, mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation, especially from diabetes patients complicated by slow-healing wounds.

SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB, it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study, we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly, fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation, suggesting that although BCR and FcγRIIB can both recruit SHIP, this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation, but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP, whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk, as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP, but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling.

The ability of human embryonic stem cells (hESCs) to differentiate into essentially all somatic cell types has made them a valuable tool for studying human development and has positioned them for broad applications in toxicology, regenerative medicine, and drug discovery. This unit describes a protocol for the large-scale expansion and maintenance of hESCs in vitro. hESC cultures must maintain a balance between the cellular states of pluripotency and differentiation; thus, researchers must use care when growing these technically demanding cells. The culture system is based largely on the use of a proprietary serum-replacement product and basic fibroblast growth factor (bFGF), with mouse embryonic fibroblasts as a feeder layer. These conditions provide the basis for relatively inexpensive maintenance and expansion of hESCs, as well as their engineered counterparts, human induced pluripotent stem cells (hiPSCs).

Airway epithelial cells are of great interest for research on lung development, regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation, and full maturation of the cells in air-liquid interface cultures occurs in textless5 weeks. This protocol can be used for drug discovery, tissue regeneration or disease modeling.