技术资料

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state,instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date,only mouse iPSC lines are known to be truly pluripotent. However,initial mouse iPSC lines failed to form chimeric offspring,but did generate teratomas and differentiated embryoid bodies,and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore,there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1,SOX2,NANOG,KLF4,LIN28,and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high,85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies,genetic engineering,and other aspects of stem cell and developmental biology. View Publication

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34(+) cells. This pure population of immature erythroblasts can be expanded to obtain 4x10(8) erythroblasts from 1x10(8) PBMC after 13-14 days in culture. Upon synchronized differentiation,high levels of enucleation (80-90%) and low levels of cell death (textless10%) are achieved. We compared the yield of erythroblasts obtained from PBMC,CD34(+) cells or PBMC depleted of CD34(+) cells and show that CD34(-) cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available. View Publication

Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter,technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple,effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple,but important step results in remarkable protein deposition efficiencies often exceeding 50%,whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays,and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions,as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies. View Publication

Human amnion epithelial cells (hAECs) are a heterologous population positive for stem cell markers; they display multilineage differentiation potential,differentiating into cells of the endoderm (liver,lung epithelium),mesoderm (bone,fat),and ectoderm (neural cells). They have a low immunogenic profile and possess potent immunosuppressive properties. Hence,hAECs may be a valuable source of cells for cell therapy. This unit describes an efficient and effective method of hAEC isolation,culture,and cryopreservation that is animal product-free and in accordance with current guidelines on preparation of cells for clinical use. Cells isolated using this method were characterized after 5 passages by analysis of karyotype,cell cycle distribution,and changes in telomere length. The differentiation potential of hAECs isolated using this animal product-free method was demonstrated by differentiation into lineages of the three primary germ layers and expression of lineage-specific markers analyzed by PCR,immunocytochemistry,and histology. View Publication

Numerous publications have described the importance of bone morphogenetic protein (BMP) signaling in the specification of hematopoietic tissue in developing embryos. Here we investigate the full role of canonical BMP signaling in both adult and fetal liver hematopoiesis using conditional knockout strategies because conventional disruption of components of the BMP signaling pathway result in early death of the embryo. By targeting both Smad1 and Smad5,we have generated a double-knockout mouse with complete disruption of canonical BMP signaling. Interestingly,concurrent deletion of Smad1 and Smad5 results in death because of extrahematopoietic pathologic changes in the colon. However,Smad1/Smad5-deficient bone marrow cells can compete normally with wild-type cells and display unaffected self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Moreover,although BMP receptor expression is increased in fetal liver,fetal liver cells deficient in both Smad1 and Smad5 remain competent to long-term reconstitute lethally irradiated recipients in a multilineage manner. In conclusion,canonical BMP signaling is not required to maintain either adult or fetal liver hematopoiesis,despite its crucial role in the initial patterning of hematopoiesis in early embryonic development. View Publication

Whether garcinol,the active component of Garcinia indica,can modulate the sensitivity of cancer cells to TRAIL,a cytokine currently in phase II clinical trial,was investigated. We found that garcinol potentiated TRAIL-induced apoptosis of cancer cells as indicated by intracellular esterase activity,DNA strand breaks,accumulation of the membrane phospholipid phosphatidylserine,mitochondrial activity,and activation of caspase-8,-9,and -3. We found that garcinol,independent of the cell type,induced both of the TRAIL receptors,death receptor 4 (DR4) and DR5. Garcinol neither induced the receptors on normal cells nor sensitized them to TRAIL. Deletion of DR5 or DR4 by small interfering RNA significantly reduced the apoptosis induced by TRAIL and garcinol. In addition,garcinol downregulated various cell survival proteins including survivin,bcl-2,XIAP,and cFLIP,and induced bid cleavage,bax,and cytochrome c release. Induction of death receptors by garcinol was found to be independent of modulation of CCAAT/enhancer-binding protein-homologous protein,p53,bax,extracellular signal-regulated kinase,or c-Jun-NH(2)-kinase. The effect of garcinol was mediated through the generation of reactive oxygen species,in as much as induction of both death receptors,modulation of antiapoptotic and proapoptotic proteins,and potentiation of TRAIL-induced apoptosis were abolished by N-acetyl cysteine and glutathione. Interestingly,garcinol also converted TRAIL-resistant cells into TRAIL-sensitive cells. Overall,our results indicate that garcinol can potentiate TRAIL-induced apoptosis through upregulation of death receptors and downregulation of antiapoptotic proteins. Mol Cancer Ther; 9(4); 856-68. (c)2010 AACR. View Publication

We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only ∼ 44% of the seeded cells were rescued,but an optimized heat shock treatment combined with Ri significantly increased cell survival to ∼ 60%. Mechanistically,our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2,hES3) that were used at higher passages (textgreater 86). In contrast,rapid down regulation of Oct4,Tra-1-60,and SSEA4 was observed for ESI049,a clinically compliant line,used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless,our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines. textcopyright 2010 Elsevier B.V. All rights reserved. View Publication

Recently,it has been shown that certain combinations of TLR ligands act in synergy to induce the release of IL-12 by DCs. In this study,we sought to define the critical parameters underlying TLR synergy. Our data show that TLR ligands act synergistically if MyD88- and TRIF-dependent ligands are combined. TLR4 uses both of these adaptor molecules,thus activation via TLR4 proved to be a synergistic event on its own. TLR synergy did not affect all aspects of DC activation but enhanced primarily the release of certain cytokines,particularly IL-12,whereas the expression of costimulatory molecules remained unchanged. Consequently,synergistic activation of DC did not affect their ability to induce T cell proliferation but resulted in T(H)1-biased responses in vitro and in vivo. Furthermore,we examined the impact of TLR ligand combinations on primary DC in vitro but observed only modest effects with a combination of CpG + Poly (I:C). However,noticeable synergy in terms of IL-12 production by DCs was detectable in vivo after systemic administration of CpG + Poly (I:C). Finally,we show that synergy is partially dependent on IFNAR signaling but does not require the release of IFNs to the enviroment,suggesting an autocrine action of type I IFNs. View Publication

More recently,reprogramming of somatic cells to an embryonic stem cell-like state presents a milestone in the realm of stem cells,making it possible to derive all cell types from any patients bearing specific genetic mutations. With the development of induced pluripotent stem (iPS) cells,we are now able to use the derivatives of iPS cells to study the mechanisms of disease and to perform drug screening and toxicology testing. In addition,differentiated iPS cells are now close to be used in clinical practice. Here we review the progress of iPS technique and the possible application in the area of Parkinson's disease treatment. View Publication

Mesenchymal stem cells (MSCs) are highly useful in a variety of cell therapies owing to their multipotential differentiation capability. MSCs derived from umbilical cord blood are generally isolated by their plastic adherence without using specific cell surface markers and examined for their osteogenic,adipogenic,and chondrogenic differentiation properties retrospectively. Here,we report 2 subpopulations of MSCs,separated based on aldehyde dehydrogenase (ALDH) activity. MSCs with a high ALDH activity (Alde-High) proliferated more than those with a low ALDH activity (Alde-Low). Alde-High MSCs had a greater ability to differentiate than Alde-Low MSCs in in vitro culture. Transplantation of Alde-High MSCs into fractured mouse femurs enabled early repair of tissues and rapid bone substitution. Alde-High MSCs were also more responsive to hypoxia than Alde-Low MSCs,with the upregulation of Flt-1,CXCR4,and Angiopoietin-2. Thus,MSCs with a high ALDH activity might serve as an effective therapeutic tool for healing fractures within a short period of time. View Publication

Since the first report of derivation of human embryonic stem cell (hESC) lines in 1998,many progresses have been achieved to reliably and efficiently derive,maintain,and differentiate this therapeutically promising cell type. This chapter introduces some basic and widely recognized methods that we use in our hESC core laboratory. Specifically,it includes methods for (1) deriving hESC lines without using enzyme and antibody to isolate the inner cell mass; (2) sustaining hESC self-renewal under feeder-dependent,feeder-conditioned,and defined conditions as well as pluripotency validation and quality control assays; and (3) inducing hESC differentiation to trophoblast with BMP4. View Publication

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines,of which a significantly higher ALDH activity was present in the OS99-1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99-1 cells were grown in NOD/SCID mice,we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDH(br) cells) from the OS99-1 xenografts were much less frequent,averaging 3% of the entire tumor population,compared to those isolated directly from the OS99-1 cell line. ALDH(br) cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDH(lo) cells),generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDH(br) cells illustrated the stem cell characteristics of self-renewal,the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A,Nanog and Sox-2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma-initiating cells and may potentially have therapeutic implications for human osteosarcoma. View Publication