参考文献

Manipulation of genes in human embryonic stem cells (hESCs) is imperative for their highly potential applications; however,the transduction efficiency remains very low. Although existing evidence revealed the type,size,and zeta potential of vector affect gene transfection efficiency in cells,the systematic study in hESCs is scarce. In this study,using poly(amidoamine) (PAMAM) dendrimers ended with amine,hydroxyl,or carboxyl as model,we tested the influences of size and surface group as well as cytotoxicity and endocytosis on hESC gene transfection. We found that in culture medium of mTeSR the particle sizes of G5,G7,G4.5COOH,and G5OH were around 5 nm and G1 had a smaller size of 3.14 nm. G5 and G7 had a slight and significant positive zeta potential,respectively,whereas G1 was slightly negative,and G4.5COOH and G5OH were significantly negative. We demonstrated that only amine-terminated dendrimers accomplished gene transfection in hESCs,which is greater than that from Lipofectamine 2000 transfection. Ten micromolar G5 had the greatest efficiency and was better than 1000 μM G1. Only a low concentration (0.5 and 1 μM) of G7 realized gene delivery. Amine-ended dendrimers,especially with higher generations,were detrimental to the growth and pluripotent maintenance of hESCs. In contrast,similarly sized hydroxyl- and carboxyl-terminated dendrimers exerted much lower cytotoxicity,in which carboxyl-terminated dendrimer maintained pluripotency of hESCs. We also confirmed the endocytosis into and significant exocytosis from hESCs using FITC-labeled G5 dendrimer. These results suggested that careful considerations of size,concentration,and zeta potential,particularly the identity and position of groups,as well as minimized exocytosis in the design of a vector for hESC gene delivery are necessary,which helps to better design an effective vector in hESC gene transduction. View Publication

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung,liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells,hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2,SOX17,HNF1B,members of the GATA family,and the surface receptor CXCR4. However,differentiation protocols are rarely 100% efficient. Here,we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations. View Publication

We present a simple microfluidic system that generates water-in-water,aqueous two phase system (ATPS) droplets,by passive flow focusing. ATPS droplet formation is achieved by applying weak hydrostatic pressures,with liquid-filled pipette tips as fluid columns at the inlets,to introduce low speed flows to the flow focusing junction. To control the size of the droplets,we systematically vary the interfacial tension and viscosity of the ATPS fluids and adjust the fluid column height at the fluid inlets. The size of the droplets scales with a power law of the ratio of viscous stresses in the two ATPS phases. Overall,we find a drop size coefficient of variation (CV; i.e.,polydispersity) of about 10%. We also find that when drops form very close to the flow focusing junction,the drops have a CV of less than 1%. Our droplet generation method is easily scalable: we demonstrate a parallel system that generates droplets simultaneously and improves the droplet production rate by up to one order of magnitude. Finally,we show the potential application of our system for encapsulating cells in water-in-water emulsions by encapsulating microparticles and cells. To the best of our knowledge,our microfluidic technique is the first that forms low interfacial tension ATPS droplets without applying external perturbations. We anticipate that this simple approach will find utility in drug and cell delivery applications because of the all-biocompatible nature of the water-in-water ATPS environment. View Publication

Multiple sclerosis (MS) is caused by T cells that are reactive for brain antigens. In experimental autoimmune encephalomyelitis,the animal model for MS,myelin-reactive T cells initiate the autoimmune process when entering the nervous tissue and become reactivated upon local encounter of their cognate CNS antigen. Thereby,the strength of the T-cellular reactivation process within the CNS tissue is crucial for the manifestation and the severity of the clinical disease. Recently,B cells were found to participate in the pathogenesis of CNS autoimmunity,with several diverse underlying mechanisms being under discussion. We here report that B cells play an important role in promoting the initiation process of CNS autoimmunity. Myelin-specific antibodies produced by autoreactive B cells after activation in the periphery diffused into the CNS together with the first invading pathogenic T cells. The antibodies accumulated in resident antigen-presenting phagocytes and significantly enhanced the activation of the incoming effector T cells. The ensuing strong blood-brain barrier disruption and immune cell recruitment resulted in rapid manifestation of clinical disease. Therefore,myelin oligodendrocyte glycoprotein (MOG)-specific autoantibodies can initiate disease bouts by cooperating with the autoreactive T cells in helping them to recognize their autoantigen and become efficiently reactivated within the immune-deprived nervous tissue. View Publication

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4high cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation,efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved,while retaining their pluripotency. When added during the reprogramming process,CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus,CD30-LV may serve as novel tool for the selective gene transfer into pluripotent stem cells with broad applications in basic and therapeutic research. View Publication

The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However,the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis,the non-canonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a two-day window showed significantly enhanced osteogenic yield. Mechanistically,rWNT5a supplementation up-regulated PKC,CamKII and JNK activity while antagonizing the key effector of canonical Wnt signaling: beta-catenin. Conversely,when recombinant WNT3a (rWNT3a) or other positive regulators of ?-catenin were employed during this same time-window there was a decrease in osteogenic marker expression. However,if rWNT3a was supplemented during a time-window following rWNT5a treatment,osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine. View Publication

Coronary arteriogenesis is a central step in cardiogenesis,requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present,it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro,and contribute extensively to coronary-like vessels in vivo,forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates,and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo. View Publication

Defining immunological mechanisms underlying NK cell biology is crucial for the treatment and prevention of immune deficiency and malignancy. The limited availability of human biological specimens presents a challenge to the study of human immunobiology. The use of high throughput,multi-parametric assays will not only aid in the definition and diagnosis of complex human immune disorders affecting NK cell function but also advance NK cell biology through population-based assessment of molecular signaling. In an effort to garner the most information from limited numbers of human cells,we designed a quantitative method to study NK cell function using imaging flow cytometry (IFC),which combines multiparametric flow cytometry and fluorescence microscopy. Specifically,we developed IFC as a tool to measure polarization and secretion of lytic granules at the immunological synapse formed between an NK cell and a susceptible target. We have further validated our approach through quantitative comparison with high-resolution confocal microscopy. We show that IFC can be used as a quantitative,high throughput measure of NK cell biological function possessing greater dimensionality than standard flow cytometry. View Publication

Insulin resistance,a critical component of type 2 diabetes (T2D),precedes and predicts T2D onset. T2D is also associated with mitochondrial dysfunction. To define the cause-effect relationship between insulin resistance and mitochondrial dysfunction,we compared mitochondrial metabolism in induced pluripotent stem cells (iPSC) from 5 healthy individuals and 4 patients with genetic insulin resistance due to insulin receptor mutations. Insulin-resistant iPSC had increased mitochondrial number and decreased mitochondrial size. Mitochondrial oxidative function was impaired,with decreased citrate synthase activity and spare respiratory capacity. Simultaneously,expression of multiple glycolytic enzymes was decreased,while lactate production increased 80%. These perturbations were accompanied by an increase in ADP/ATP ratio and 3-fold increase in AMPK activity,indicating energetic stress. Insulin-resistant iPSC also showed reduced catalase activity and increased susceptibility to oxidative stress. Thus,insulin resistance can lead to mitochondrial dysfunction with reduced mitochondrial size,oxidative activity,and energy production. View Publication

Conventional generation of stem cells from human blastocysts produces a developmentally advanced,or primed,stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However,whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here,we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration,global gene expression,and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. View Publication