技术资料

BACKGROUND AIMS: Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently,stem cell culture has been carried out using feeder cells,and culture media,that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells,essential conditions for the use of stem cells for clinical purposes. To date,however,there has been limited success in achieving this aim. METHODS,RESULTS AND CONCLUSIONS: Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntington's disease and myotonic dystrophy 1) genomically stable hES cell lines,and their adaptation to feeder-free culture,all under xeno-free conditions. View Publication

PURPOSE: To investigate the distribution of CD44(+)/CD24(-) cells in breast cancers in relation to tumor size before and after the administration of neoadjuvant chemotherapy. METHODS: CD44(+)/CD24(-) tumor cells obtained from breast cancer specimens were characterized in vivo and in vitro using tumor formation assays and mammosphere generation assays,respectively. The distribution of CD44+/CD24- tumor cells in 78 breast cancer specimens following administration of neoadjuvant chemotherapy was also evaluated using immunofluorescence assays,and this distribution was compared with the extent of tumor invasion predicted by Response Evaluation Criteria in Solid Tumours (RECIST). RESULTS: In 27/78 cases,complete remission (CR) was identified using RECIST. However,18 of these CR cases were associated with a scattered distribution of tumor stem cells in the outline of the original tumor prior to neoadjuvant chemotherapy. After neoadjuvant chemotherapy,24 cases involved cancer cells that were confined to the tumor outline,and 21 cases had tumor cells or tumor stem cells overlapping the tumor outline. In addition,there were 6 patients who were insensitive to chemotherapy,and in these cases,both cancer cells and stem cells were detected outside the contours of the tumor volume imaged prior to chemotherapy. CONCLUSION: CD44+/CD24- tumor cells may be an additional parameter to evaluate when determining the extent of breast cancer invasion. View Publication

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however,methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible,floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors,we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system,we insert the myogenic regulator,Myf5,into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate. View Publication

Human pluripotent stem cells will likely be a significant part of the regenerative medicine-driven healthcare revolution. In order to realize this potential,culture processes must be standardized,scalable and able to produce clinically relevant cell numbers,whilst maintaining critical biological functionality. This review comprises a broad overview of important bioprocess considerations,referencing the development of biopharmaceutical processes in an effort to learn from current best practice in the field. Particular focus is given to the recent efforts to grow human pluripotent stem cells in microcarrier or aggregate suspension culture,which would allow geometric expansion of productive capacity were it to be fully realized. The potential of these approaches is compared with automation of traditional T-flask culture,which may provide a cost-effective platform for low-dose,low-incidence conditions or autologous therapies. This represents the first step in defining the full extent of the challenges facing bioprocess engineers in the exploitation of large-scale human pluripotent stem cell manufacture. View Publication

Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely,while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential,the long reprogramming process (up to 1. month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study,we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells,using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4,Sox2,Klf4,and L-Myc. Under optimized conditions,we observed human embryonic stem (ES)-like cells as early as 6. days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate,morphology,pluripotency-associated markers,global gene expression patterns,genome-wide DNA methylation states,and the ability to differentiate into all three of the germ layers,both in vitro and in vivo. Our method,when combined with chemical inhibitors under conditions of physiological hypoxia,offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research. textcopyright 2011 Elsevier Inc. View Publication

Intercellular interactions in the cell microenvironment play a critical role in determining cell fate,but the effects of these interactions on pathways governing human embryonic stem cell (hESC) behavior have not been fully elucidated. We and others have previously reported that 3-D culture of hESCs affects cell fates,including self-renewal and differentiation to a variety of lineages. Here we have used a microwell culture system that produces 3-D colonies of uniform size and shape to provide insight into the effect of modulating cell-cell contact on canonical Wnt/??-catenin signaling in hESCs. Canonical Wnt signaling has been implicated in both self-renewal and differentiation of hESCs,and competition for ??-catenin between the Wnt pathway and cadherin-mediated cell-cell interactions impacts various developmental processes,including the epithelial-mesenchymal transition. Our results showed that hESCs cultured in 3-D microwells exhibited higher E-cadherin expression than cells on 2-D substrates. The increase in E-cadherin expression in microwells was accompanied by a downregulation of Wnt signaling,as evidenced by the lack of nuclear ??-catenin and downregulation of Wnt target genes. Despite this reduction in Wnt signaling in microwell cultures,embryoid bodies (EBs) formed from hESCs cultured in microwells exhibited higher levels of Wnt signaling than EBs from hESCs cultured on 2-D substrates. Furthermore,the Wnt-positive cells within EBs showed upregulation of genes associated with cardiogenesis. These results demonstrate that modulation of intercellular interactions impacts Wnt/??-catenin signaling in hESCs. ?? 2011 Elsevier Ltd. View Publication

Induced pluripotent stem (iPS) cells are being used increasingly to complement their embryonic counterparts to understand and develop the therapeutic potential of pluripotent cells. Our objectives were to identify an efficient cardiac differentiation protocol for human iPS cells as monolayers,and demonstrate that the resulting cardiac progenitors could provide a therapeutic benefit in a rodent model of myocardial infarction. Herein,we describe a 14-day protocol for efficient cardiac differentiation of human iPS cells as a monolayer,which routinely yielded a mixed population in which over 50% were cardiomyocytes,endothelium,or smooth muscle cells. When differentiating,cardiac progenitors from day 6 of this protocol were injected into the peri-infarct region of the rat heart; after coronary artery ligation and reperfusion,we were able to show that human iPS cell-derived cardiac progenitor cells engrafted,differentiated into cardiomyocytes and smooth muscle,and persisted for at least 10 weeks postinfarct. Hearts injected with iPS-derived cells showed a nonsignificant trend toward protection from decline in function after myocardial infarction,as assessed by magnetic resonance imaging at 10 weeks,such that the ejection fraction at 10 weeks in iPS treated hearts was 62%±4%,compared to that of control infarcted hearts at 45%±9% (Ptextless0.2). In conclusion,we demonstrated efficient cardiac differentiation of human iPS cells that gave rise to progenitors that were retained within the infarcted rat heart,and reduced remodeling of the heart after ischemic damage. View Publication

Achievements in tissue engineering using mesenchymal stem cells (MSC) demand a clinically acceptable off-the-shelf" cell therapy product. Efficacy of cryopreservation of human bone marrow-derived MSC in clinically safe animal product-free medium containing 2% 5% and 10% dimethyl sulfoxide (DMSO) was evaluated by measuring cell recovery viability apoptosis proliferation rate expression of a broad panel of MSC markers and osteogenic differentiation. Rate-controlled freezing in CryoStor media was performed in a programmable cell freezer. About 95% of frozen cells were recovered as live cells after freezing in CryoStor solutions with 5% and 10% DMSO followed by storage in liquid nitrogen for 1 month. Cell recovery after 5 months storage was 72% and 80% for 5% and 10% DMSO respectively. Measurements of caspase 3 activity demonstrated that 15.5% and 12.8% of cells after 1 month and 18.3% and 12.9% of cells after 5 months storage in 5% and 10% DMSO respectively were apoptotic. Proliferation of MSC recovered after cryopreservation was measured during 2 weeks post-plating. Proliferation rate was not compromised and was even enhanced. Cryopreservation did not alter expression of MSC markers. Quantitative analysis of alkaline phosphatase (ALP) activity ALP surface expression and Ca deposition in previously cryopreserved MSC and then differentiated for 3 weeks in osteogenic medium demonstrated the same degree of osteogenic differentiation as in unfrozen parallel cultures. Cell viability and functional parameters were analyzed in MSC after short-term storage at 4°C in HypoThermosol-FRS solution also free of animal products. Hypothermic storage for 2 and 4 days resulted in about 100% and 85% cell recovery respectively less than 10% of apoptotic cells and normal proliferation marker expression and osteogenic potential. Overall our results demonstrate that human MSC could be successfully cryopreserved for banking and clinical applications and delivered to the bedside in clinically safe protective reagents. View Publication

Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes,both in vitro and in vivo. Beyond uses in cell replacement therapy,patient-specific cardiomyocytes may find applications in drug testing,drug discovery,and disease modeling. Recently,approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs),adult heart-derived cardiac progenitor cells (CPCs),and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts,highlighting potential applications and future challenges. View Publication

Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count,and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation,using both microarray and sequence-based analyses,provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines,the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly,genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes,such as tumor suppressors and genetic disease genes,do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens,such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents. View Publication

Nuclear reprogramming enables patient-specific derivation of induced pluripotent stem (iPS) cells from adult tissue. Yet,iPS generation from patients with type 2 diabetes (T2D) has not been demonstrated. Here,we report reproducible iPS derivation of epidermal keratinocytes (HK) from elderly T2D patients. Transduced with human OCT4,SOX2,KLF4 and c-MYC stemness factors under serum-free and feeder-free conditions,reprogrammed cells underwent dedifferentiation with mitochondrial restructuring,induction of endogenous pluripotency genes - including NANOG,LIN28,and TERT,and down-regulation of cytoskeletal,MHC class I- and apoptosis-related genes. Notably,derived iPS clones acquired a rejuvenated state,characterized by elongated telomeres and suppressed senescence-related p15INK4b/p16INK4a gene expression and oxidative stress signaling. Stepwise guidance with lineage-specifying factors,including Indolactam V and GLP-1,redifferentiated HK-derived iPS clones into insulin-producing islet-like progeny. Thus,in elderly T2D patients,reprogramming of keratinocytes ensures a senescence-privileged status yielding iPS cells proficient for regenerative applications. View Publication

To identify early populations of committed progenitors derived from human embryonic stem cells (hESCs),we screened self-renewing,BMP4-treated and retinoic acid-treated cultures with >400 antibodies recognizing cell-surface antigens. Sorting of >30 subpopulations followed by transcriptional analysis of developmental genes identified four distinct candidate progenitor groups. Subsets detected in self-renewing cultures,including CXCR4(+) cells,expressed primitive endoderm genes. Expression of Cxcr4 in primitive endoderm was confirmed in visceral endoderm of mouse embryos. BMP4-induced progenitors exhibited gene signatures of mesoderm,trophoblast and vascular endothelium,suggesting correspondence to gastrulation-stage primitive streak,chorion and allantois precursors,respectively. Functional studies in vitro and in vivo confirmed that ROR2(+) cells produce mesoderm progeny,APA(+) cells generate syncytiotrophoblasts and CD87(+) cells give rise to vasculature. The same progenitor classes emerged during the differentiation of human induced pluripotent stem cells (hiPSCs). These markers and progenitors provide tools for purifying human tissue-regenerating progenitors and for studying the commitment of pluripotent stem cells to lineage progenitors. View Publication