技术资料

Transforming growth factor (TGF)-beta superfamily proteins play a key role in the regulation of human embryonic stem cells (hESCs). Those of the TGFbeta/activin/nodal branch seem to support self-renewal and pluripotency,whereas those of the bone morphogenic protein (BMP) branch induce differentiation. In contrast to this generalization,we found that hESC remained undifferentiated after knockdown of SMAD4 with inducible short hairpin RNA interference,although the knockdown inhibited TGFbeta signaling and rendered the cells nonresponsive to BMP-induced differentiation. Moreover,the rapid differentiation of hESC after pharmacological inhibition of TGFbeta/activin/nodal receptor signaling was restricted after SMAD4 knockdown. These results suggest that TGFbeta/activin/nodal signaling supports the undifferentiated phenotype of hESC by suppressing BMP activity. During long-term culture,SMAD4 knockdown cell populations became less stable and more permissive to neural induction,a situation that was rescued by re-establishment of SMAD4 expression. These results suggest that SMAD4 is not required for maintenance of the undifferentiated state of hESC,but rather to stabilize that state. 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

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

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

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

Statin inhibitors,used to control hypercholesterolemia,trigger apoptosis of hematologic tumor cells and therefore have immediate potential as anticancer agents. Evaluations of statins in acute myelogenous leukemia and multiple myeloma have shown that statin efficacy is mixed,with only a subset of tumor cells being highly responsive. Our goal was to distinguish molecular features of statin-sensitive and -insensitive myeloma cells and gain insight into potential predictive markers. We show that dysregulation of the mevalonate pathway is a key determinant of sensitivity to statin-induced apoptosis in multiple myeloma. In sensitive cells,the classic feedback response to statin exposure is lost. This results in deficient up-regulation of 2 isoforms of hydroxymethylglutaryl coenzyme A reductase: the rate-limiting enzyme of the mevalonate pathway and hydroxymethylglutaryl coenzyme A synthase 1. To ascertain the clinical utility of these findings,we demonstrate that a subset of primary myeloma cells is sensitive to statins and that monitoring dysregulation of the mevalonate pathway may distinguish these cancers. We also show statins are highly effective and well tolerated in an orthotopic model of myeloma using cells harboring this dysregulation. This determinant of sensitivity further provides molecular rationale for the significant therapeutic index of statins on these tumor cells. View Publication

Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted,but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here,we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell-autonomous manner. Unexpectedly,loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly,null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation,thereby accelerating hematopoietic regeneration. Consistent with these findings,Puma is required for radiation-induced apoptosis in HSCs and HPCs,and Puma is selectively induced by irradiation in primitive hematopoietic cells,and this induction is impaired in Puma-heterozygous cells. Together,our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy. 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

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

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

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

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