技术资料

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells,it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system,aerobic glycolysis,and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells,while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass,increased proton leak,and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference,and highlight the importance of further research concerning energy metabolism of stem cells. View Publication

Overexpression of high mobility group AT-hook 2 (HMGA2) is found in a number of benign and malignant tumors,including the clonal PIGA(-) cells in 2 cases of paroxysmal nocturnal hemoglobinuria (PNH) and some myeloproliferative neoplasms (MPNs),and recently in hematopoietic cell clones resulting from gene therapy procedures. In nearly all these cases overexpression is because of deletions or translocations that remove the 3' untranslated region (UTR) which contains binding sites for the regulatory micro RNA let-7. We were therefore interested in the effect of HMGA2 overexpression in hematopoietic tissues in transgenic mice (ΔHmga2 mice) carrying a 3'UTR-truncated Hmga2 cDNA. ΔHmga2 mice expressed increased levels of HMGA2 protein in various tissues including hematopoietic cells and showed proliferative hematopoiesis with increased numbers in all lineages of peripheral blood cells,hypercellular bone marrow (BM),splenomegaly with extramedullary erythropoiesis and erythropoietin-independent erythroid colony formation. ΔHmga2-derived BM cells had a growth advantage over wild-type cells in competitive repopulation and serial transplantation experiments. Thus overexpression of HMGA2 leads to proliferative hematopoiesis with clonal expansion at the stem cell and progenitor levels and may account for the clonal expansion in PNH and MPNs and in gene therapy patients after vector insertion disrupts the HMGA2 locus. View Publication

Utilizing a fast 31P magnetic resonance spectroscopy (MRS) 2-dimensional chemical shift imaging (2D-CSI) method,this study examined the heterogeneity of creatine kinase (CK) forward flux rate of hearts with postinfarction left ventricular (LV) remodeling. Immunosuppressed Yorkshire pigs were assigned to 4 groups: 1) A sham-operated normal group (SHAM,n = 6); 2) A 60 minutes distal left anterior descending coronary artery ligation and reperfusion (MI,n = 6); 3) Open patch group; ligation injury plus open fibrin patch over the site of injury (Patch,n = 6); and 4) Cell group,hiPSCs-cardiomyocytes,-endothelial cells,and -smooth muscle cells (2 million,each) were injected into the injured myocardium pass through a fibrin patch (Cell+Patch,n = 5). At 4 weeks,the creatine phosphate (PCr)/ATP ratio,CK forward flux rate (Flux PCr→ATP),and k constant of CK forward flux rate (kPCr→ATP) were severely decreased at border zone myocardium (BZ) adjacent to MI. Cell treatment results in significantly increase of PCr/ATP ratio and improve the value of kPCr→ATP and Flux PCr→ATP in BZ myocardium. Moreover,the BZ myocardial CK total activity and protein expression of CK mitochondria isozyme and CK myocardial isozyme were significantly reduced,but recovered in response to cell treatment. Thus,cell therapy results in improvement of BZ bioenergetic abnormality in hearts with postinfarction LV remodeling,which is accompanied by significantly improvements in BZ CK activity and CK isozyme expression. The fast 2D 31P MR CSI mapping can reliably measure the heterogeneity of bioenergetics in hearts with post infarction LV remodeling. View Publication

The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine,including individualized,patient-specific stem cell-based treatments. There are,however,few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues,ideally comprising direct-write printing of cells for encapsulation,proliferation,and differentiation. Here,such a method,employing a clinically amenable polysaccharide-based bioink,is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically,There are extrusion printed the bioink including iPSCs,alginate (Al; 5% weight/volume [w/v]),carboxymethyl-chitosan (5% w/v),and agarose (Ag; 1.5% w/v),crosslinked the bioink in calcium chloride for a stable and porous construct,proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm,ectoderm,and mesoderm,or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined,scalable,and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine. View Publication

No treatments exist to effectively treat many retinal diseases. Retinal pigmented epithelium (RPE) and neural retina can be generated from human embryonic stem cells/induced pluripotent stem cells (hESCs/hiPSCs). The efficacy of current protocols is,however,limited. It was hypothesised that generation of laminated neural retina and/or RPE from hiPSCs/hESCs could be enhanced by three dimensional (3D) culture in hydrogels. hiPSC- and hESC-derived embryoid bodies (EBs) were encapsulated in 0.5% RGD-alginate; 1% RGD-alginate; hyaluronic acid (HA) or HA/gelatin hydrogels and maintained until day 45. Compared with controls (no gel),0.5% RGD-alginate increased: the percentage of EBs with pigmented RPE foci; the percentage EBs with optic vesicles (OVs) and pigmented RPE simultaneously; the area covered by RPE; frequency of RPE cells (CRALBP+); expression of RPE markers (TYR and RPE65) and the retinal ganglion cell marker,MATH5. Furthermore,0.5% RGD-alginate hydrogel encapsulation did not adversely affect the expression of other neural retina markers (PROX1,CRX,RCVRN,AP2α or VSX2) as determined by qRT-PCR,or the percentage of VSX2 positive cells as determined by flow cytometry. 1% RGD-alginate increased the percentage of EBs with OVs and/or RPE,but did not significantly influence any other measures of retinal differentiation. HA-based hydrogels had no significant effect on retinal tissue development. The results indicated that derivation of retinal tissue from hESCs/hiPSCs can be enhanced by culture in 0.5% RGD-alginate hydrogel. This RGD-alginate scaffold may be useful for derivation,transport and transplantation of neural retina and RPE,and may also enhance formation of other pigmented,neural or epithelial tissue. STATEMENT OF SIGNIFICANCE The burden of retinal disease is ever growing with the increasing age of the world-wide population. Transplantation of retinal tissue derived from human pluripotent stem cells (PSCs) is considered a promising treatment. However,derivation of retinal tissue from PSCs using defined media is a lengthy process and often variable between different cell lines. This study indicated that alginate hydrogels enhanced retinal tissue development from PSCs,whereas hyaluronic acid-based hydrogels did not. This is the first study to show that 3D culture with a biomaterial scaffold can improve retinal tissue derivation from PSCs. These findings indicate potential for the clinical application of alginate hydrogels for the derivation and subsequent transplantation retinal tissue. This work may also have implications for the derivation of other pigmented,neural or epithelial tissue. View Publication

BACKGROUND Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential,a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation,the medium for 3D microcarriers was directly switched to EB medium. RESULTS hESCs on 3D microcarriers maintained pluripotency and formed EBs,which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating,EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore,this 3D system can also be adapted to human induced pluripotent stem cells,which generate functional hemangioblasts with high efficiency. CONCLUSION This 3D serum- and stromal-free microcarrier system is important for future clinical applications,with the potential of developing to a GMP-compatible scalable system. View Publication

Three-dimensional (3D) printing is advantageous over conventional technologies for the fabrication of sophisticated structures such as 3D micro-channels for future applications in tissue engineering and drug screening. We aimed to apply this technology to cell-based assays using polydimethylsiloxane (PDMS),the most commonly used material for fabrication of micro-channels used for cell culture experiments. Useful properties of PDMS include biocompatibility,gas permeability and transparency. We developed a simple and robust protocol to generate PDMS-based devices using a soft lithography mold produced by 3D printing. 3D chemical gradients were then generated to stimulate cells confined to a micro-channel. We demonstrate that concentration gradients of growth factors,important regulators of cell/tissue functions in vivo,influence the survival and growth of human embryonic stem cells. Thus,this approach for generation of 3D concentration gradients could have strong implications for tissue engineering and drug screening. View Publication

RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome wide in mouse and human cells,and it is required to limit enhancer-RNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA-damage signaling. 7SK physically interacts with the BAF chromatin-remodeling complex,recruits BAF to enhancers and inhibits enhancer transcription by modulating chromatin structure. In turn,7SK occupancy at enhancers coincides with that of Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus,7SK uses distinct mechanisms to counteract the diverse consequences of pervasive transcription that distinguish super enhancers,enhancers and promoters. View Publication

Realizing the potential of human pluripotent stem cell (hPSC)-based therapy requires the development of defined scalable culture systems with efficient expansion,differentiation and isolation protocols. We report an engineered 3D microfiber system that efficiently supports long-term hPSCs self-renewal under chemically defined conditions. The unique feature of this system lies in the application of a 3D ECM-like environment in which cells are embedded,that affords: (i) uniform high cell loading density in individual cell-laden constructs (∼10 7 cells/ml); (ii) quick recovery of encapsulated cells (textless10min at 37°C) with excellent preservation of cell viability and 3D multicellular structure; (iii) direct cryopreservation of the encapsulated cells in situ in the microfibers with textgreater17-fold higher cell viability compared to those cultured on Matrigel surface; (iv) long-term hPSC propagation under chemically defined conditions. Four hPSC lines propagated in the microfibrous scaffold for 10 consecutive passages were capable of maintaining an undifferentiated phenotype as demonstrated by the expression of stem cell markers and stable karyotype invitro and the ability to form derivatives of the three germ layers both invitro and invivo. Our 3D microfibrous system has the potential for large-scale cultivation of transplantable hESCs and derivatives for clinical applications. textcopyright 2011 Elsevier Ltd. View Publication

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However,scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard,suspension cultures are a viable alternative,because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However,the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here,we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production. ?? 2014 The Authors. View Publication

The advance in stem cell research relies largely on the efficiency and biocompatibility of technologies used to manipulate stem cells. In our previous study,we had designed an amphipathic peptide RV24 that can deliver proteins into cancer cell lines efficiently without significant side effects. Encouraged by this observation,we moved forward to test whether RV24 could be used to deliver proteins into human embryonic stem cells and human induced pluripotent stem cells. RV24 successfully mediated protein delivery into these pluripotent stem cells,as well as their derivatives including neural stem cells and dendritic cells. Based on NMR studies and particle surface charge measurements,we proposed that hydrophobic domain of RV24 interacts with ??-sheet structures of the proteins,followed by formation of peptide cage" to facilitate delivery across cellular membrane. These findings suggest the feasibility of using amphipathic peptide to deliver functional proteins intracellularly for stem cell research. ?? 2012 Elsevier Inc." View Publication