技术资料

Human embryonic stem cell (hESC) line was derived from abnormal blastocyst donated by Marfan syndrome patient after preimpantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that the hESC line carried the heterozygous deletion mutation,c.3536delA,of FBN1 gene. Characteristic tests proved that the hESC line presented typicalmarkers of pluripotency and had the capability to formthe three germlayers both in vitro and in vivo. View Publication

The neutralization of integrin α4 (Itga4) is currently used as treatment in multiple sclerosis. Although most studies have focused on its function on lymphocyte migration to the CNS,we have uncovered the importance of Itga4 for the generation of regulatory B cells in peripheral immune organs and their control of pathogenic T cell response and CNS pathology. Our study underscores the importance of looking at the dual role of B cells in CNS autoimmunity and provides important perspectives regarding the efficacy and side effects associated with Itga4 neutralization and other B cell-targeting therapies. View Publication

Under defined differentiation conditions,human embryonic stem cells (hESCs) can be directed toward a mesendoderm (ME) or neuroectoderm (NE) fate,the first decision during hESC differentiation. Coupled with lineage-specific G1 lengthening,a divergent ciliation pattern emerged within the first 24 hr of induced lineage specification,and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2,increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2 and thereby relieved transcriptional activation of OCT4 and NANOG. Nrf2 binds directly to upstream regions of these pluripotency genes to promote their expression and repress NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events had been initiated did neural precursor markers get expressed at day 4. Thus,we have identified a primary cilium-autophagy-Nrf2 (PAN) control axis coupled to cell-cycle progression that directs hESCs toward NE. View Publication

Efficient cryopreservation of human pluripotent stem cells (hPSCs) in chemically defined,xeno-free conditions is highly desirable for medical research and clinical applications such as cell-based therapies. Here we present a simple and effective slow freezing-rapid thawing protocol for the cryopreservation of feeder-free,single hPSCs. This cryopreservation protocol involves the supplementation of 10 % dimethyl sulfoxide (DMSO) and 10 $$M Rho-associated kinase inhibitor Y-27632 into two types of xeno-free,defined media supplements (Knockout Serum Replacement and TeSR2). High post-thaw cell recovery (˜90 %) and cell expansion (˜70 %) can be achieved using this protocol. The cryopreserved single cells retain the morphological characteristics of hPSCs and differentiation capabilities of pluripotent stem cells. View Publication

We demonstrate a highly efficient method for gene delivery into clinically relevant human cell types,such as induced pluripotent stem cells (iPSCs) and fibroblasts,reducing the protocol time by one full day. To preserve cell physiology during gene transfer,we designed a microfluidic strategy,which facilitates significant gene delivery in a short transfection time (textless1 min) for several human cell types. This fast,optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. View Publication

BACKGROUND AND OBJECTIVES Our post-thaw cell recovery rates differed substantially in interlaboratory comparisons of identical samples,potentially due to different temperatures during cell staining. MATERIALS AND METHODS Viable CD34(+) cells and leucocyte (WBC) subtypes were quantified by multiparameter single-platform flow cytometry in leucapheresis products collected from 30 adult lymphoma and myeloma patients,and from 10 paediatric patients. After thawing,cells were prepared for analysis within 30 min between thawing and acquisition,at either 4°C or at room temperature. RESULTS For cell products cryopreserved in conventional freezing medium (10% final DMSO),viable cell recovery was clearly lower after staining at 4°C than at RT. Of all WBC subtypes analysed,CD4(+) T cells showed the lowest median recovery of 4% (4°C) vs. 25% (RT),followed by CD3,CD34 and CD8 cells. The recovery was highest for CD3γδ cells with 44% (4°C) vs. 71% (RT). In the 10 samples cryopreserved in synthetic freezing medium (5% final DMSO),median recovery rates were 89% for viable CD34 (both at 4°C and RT) and 79% (4°C) vs 68% (RT) for WBC. CONCLUSIONS The post-thaw environment and,potentially,the cryoprotectant impact the outcome of cell enumeration,and results from the analysis tube may not be representative of the cells infused into a patient. View Publication

Altering chromatin structure through histone posttranslational modifications has emerged as a key driver of transcriptional responses in cells. Modulation of these transcriptional responses by pharmacological inhibition of class I histone deacetylases (HDACs),a group of chromatin remodeling enzymes,has been successful in blocking the growth of some cancer cell types. These inhibitors also attenuate the pathogenesis of pathological cardiac remodeling by blunting and even reversing pathological hypertrophy. The mechanistic target of rapamycin (mTOR) is a critical sensor and regulator of cell growth that,as part of mTOR complex 1 (mTORC1),drives changes in protein synthesis and metabolism in both pathological and physiological hypertrophy. We demonstrated through pharmacological and genetic methods that inhibition of class I HDACs suppressed pathological cardiac hypertrophy through inhibition of mTOR activity. Mice genetically silenced for HDAC1 and HDAC2 had a reduced hypertrophic response to thoracic aortic constriction (TAC) and showed reduced mTOR activity. We determined that the abundance of tuberous sclerosis complex 2 (TSC2),an mTOR inhibitor,was increased through a transcriptional mechanism in cardiomyocytes when class I HDACs were inhibited. In neonatal rat cardiomyocytes,loss of TSC2 abolished HDAC-dependent inhibition of mTOR activity,and increased expression of TSC2 was sufficient to reduce hypertrophy in response to phenylephrine. These findings point to mTOR and TSC2-dependent control of mTOR as critical components of the mechanism by which HDAC inhibitors blunt pathological cardiac growth. These results also suggest a strategy to modulate mTOR activity and facilitate the translational exploitation of HDAC inhibitors in heart disease. View Publication

Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here,we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7),a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2,a receptor that recognizes NAP-2,abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration. View Publication

CD4 and CD8 T lymphocyte activation requires the generation of sufficient energy to support new biosynthetic demands. Following T cell receptor (TCR) engagement,these requirements are met by an increased glycolysis,due,at least in part,to induction of the Glut1 glucose transporter. As Glut1 is upregulated on tumor cells in response to hypoxia,we assessed whether surface Glut1 levels regulate the antigen responsiveness of human T lymphocytes in both hypoxic and atmospheric oxygen conditions. Notably,Glut1 upregulation in response to TCR stimulation was significantly higher in T lymphocytes activated under hypoxic as compared to atmospheric oxygen conditions. Furthermore,TCR-stimulated human T lymphocytes sorted on the basis of Glut1-Lo and Glut1-Hi profiles maintained distinct characteristics,irrespective of the oxygen tension. While T cells activated in hypoxia divided less than those activated in atmospheric oxygen,Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition,augmented proliferation,and an inverted CD4/CD8 ratio in both oxygen conditions. Moreover,Glut1-Hi T lymphocytes exhibited a significantly enhanced ability to produce IFN-γ and this secretion potential was completely dependent on continued glycolysis. Thus,Glut1 surface levels identify human T lymphocytes with distinct effector functions in both hypoxic and atmospheric oxygen tensions. View Publication

Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy,while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study,we generated TAP1- and TAPBP-deficient hESC lines,respectively,using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types,but maintained normal pluripotency,karyotypes,and differentiation ability. Thus,our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future. View Publication

BACKGROUND Neuritic degeneration is an important early pathological step in neurodegeneration. AIM The purpose of this study was to explore the mechanisms connecting neuritic degeneration to the functional and morphological remodeling of endoplasmic reticulum (ER) and mitochondria. METHODS Here,we set up neuritic degeneration models by neurite cutting-induced neural degeneration in human-induced pluripotent stem cell-derived neurons. RESULTS We found that neuritic ER becomes fragmented and forms complexes with mitochondria,which induces IP3R-dependent mitochondrial Ca(2+) elevation and dysfunction during neuritic degeneration. Furthermore,mitochondrial membrane potential is required for ER fragmentation and mitochondrial Ca(2+) elevation during neuritic degeneration. Mechanically,tightening of the ER-mitochondria associations by expression of a short synthetic linker" and ER Ca(2+) releasing together could promote mitochondrial Ca(2+) elevation� View Publication

Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody binding on B cells. However,we have observed unexpected positive results with pronase-treated T cells in human immunodeficiency virus (HIV)-infected patients. In this study,25 HIV-infected patients without HLA antibodies were tested with pronase-treated and nontreated cells. HIV-positive sera were pretreated with reducing agents and preabsorbed with pronase-treated and nontreated T or B cells before crossmatching. All patients displayed FCXM reactivity with pronase-treated T cells but not with nontreated T cells. None of the patients exhibited FCXM reactivity with pronase-treated and nontreated B cells. These patients displayed FCXM reactivity with pronase-treated CD4+ and CD8+ T cells but not with their nontreated counterparts. Preabsorption with pronase-treated T cells reduced the T cell FCXM reactivity. Preabsorption with pronase-treated B cells or nontreated T and B cells did not have any effect on the T cell FCXM reactivity. Pretreatment with reducing agents did not affect the T cell FCXM reactivity. 15 of 21 HIV-infected kidney allograft recipients with pronase-treated T cell FCXM reactivity display long-term graft survival (1193±631days). These data indicate that HIV-infected patients have nondeleterious autoantibodies recognizing cryptic epitopes exposed by pronase on T cells. View Publication