技术资料

textlessptextgreater Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases,thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics,but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover,the protooncogene textlessitalictextgreatermyctextless/italictextgreater showed the strongest expression in HEP-iPSC,compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as textlessitalictextgreatermyctextless/italictextgreater might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases. textless/ptextgreater View Publication

Encapsulation of donor islets using a hydrogel material is a well-studied strategy for islet transplantation,which protects donor islets from the host immune response. Replacement of donor islets by human embryonic stem cell (hESC) derived islets will also require a means of immune-isolating hESCs by encapsulation. However,a critical consideration of hESC differentiation is the effect of surrounding biophysical environment,in this case capsule biophysical properties,on differentiation. The objective of this study,thus,was to evaluate the effect of capsule properties on growth,viability,and differentiation of encapsulated hESCs throughout pancreatic induction. It was observed that even in the presence of soluble chemical cues for pancreatic induction,substrate properties can significantly modulate pancreatic differentiation,hence necessitating careful tuning of capsule properties. Capsules in the range of 4-7. kPa supported cell growth and viability,whereas capsules of higher stiffness suppressed cell growth. While an increase in capsule stiffness enhanced differentiation at the intermediate definitive endoderm (DE) stage,increased stiffness strongly suppressed pancreatic progenitor (PP) induction. Signaling pathway analysis indicated an increase in pSMAD/pAKT levels with substrate stiffness likely the cause of enhancement of DE differentiation. In contrast,sonic hedgehog inhibition was more efficient under softer gel conditions,which is necessary for successful PP differentiation. Statement of Significance: Cell replacement therapy for type 1 diabetes (T1D),affecting millions of people worldwide,requires the immunoisolation of insulin-producing islets by encapsulation with a semi-impermeable material. Due to the shortage of donor islets,human pluripotent stem cell (hPSC) derived islets are an attractive alternative. However,properties of the encapsulating substrate are known to influence hPSC cell fate. In this work,we determine the effect of substrate stiffness on growth and pancreatic fate of encapsulated hPSCs. We precisely identify the range of substrate properties conducive for pancreatic cell fate,and also the mechanism by which substrate properties modify the cell signaling pathways and hence cell fate. Such information will be critical in driving regenerative cell therapy for long term treatment of T1D. View Publication

Background & Aims Hepatocytes differentiated from human embryonic stem cells (hESCs) have the potential to overcome the shortage of primary hepatocytes for clinical use and drug development. Many strategies for this process have been reported,but the functionality of the resulting cells is incomplete. We hypothesize that the functionality of hPSC-derived hepatocytes might be improved by making the differentiation method more similar to normal in vivo hepatic development. Methods We tested combinations of growth factors and small molecules targeting candidate signaling pathways culled from the literature to identify optimal conditions for differentiation of hESCs to hepatocytes,using qRT-PCR for stage-specific markers to identify the best conditions. Immunocytochemistry was then used to validate the selected conditions. Finally,induction of expression of metabolic enzymes in terminally differentiated cells was used to assess the functionality of the hESC-derived hepatocytes. Results Optimal differentiation of hESCs was attained using a 5-stage protocol. After initial induction of definitive endoderm (stage 1),we showed that inhibition of the WNT/??-catenin pathway during the 2nd and 3rd stages of differentiation was required to specify first posterior foregut,and then hepatic gut cells. In contrast,during the 4th stage of differentiation,we found that activation of the WNT/??-catenin pathway allowed generation of proliferative bipotent hepatoblasts,which then were efficiently differentiated into hepatocytes in the 5th stage by dual inhibition of TGF-?? and NOTCH signaling. Conclusion Here,we show that stage-specific regulation of the WNT/??-catenin pathway results in improved differentiation of hESCs to functional hepatocytes. 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

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

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

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

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

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function,developmental pathways,and disease mechanisms. Here,we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi,in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain,can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors,cardiomyocytes,and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn),CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types,dissect developmental pathways,and model disease. View Publication

Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis,while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX,indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus,increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown. View Publication

mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here,we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels,particularly when oxidative phosphorylation is impaired,such as in neurons treated with mitochondrial inhibitors,or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly,in mitochondrially defective neurons,but not neuroprogenitor cells,ribosomal S6 and S6 kinase phosphorylation increased over time,despite activation of AMPK,which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis,a major energy-consuming process,may account for its ATP-saving effect. We propose that a mild reduction in protein synthesis may have the potential to treat mitochondria-related neurodegeneration. View Publication

Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells,but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation,the process by which pathogen Ags are internalized,degraded,and presented by MHC class I,is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article,we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs,dendritic cells and macrophages,and CD4 T cells,three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities,reducing NADPH oxidase 2 activation,and lowering phagolysosomal reactive oxygen species production and pH,which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell-mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification,to our knowledge,of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV. View Publication