技术资料

Faithful execution of developmental gene expression programs occurs at multiple levels and involves many different components such as transcription factors,histone-modification enzymes,and mRNA processing proteins. Recent evidence suggests that nucleoporins,well known components that control nucleo-cytoplasmic trafficking,have wide-ranging functions in developmental gene regulation that potentially extend beyond their role in nuclear transport. Whether the unexpected role of nuclear pore proteins in transcription regulation,which initially has been described in fungi and flies,also applies to human cells is unknown. Here we show at a genome-wide level that the nuclear pore protein NUP98 associates with developmentally regulated genes active during human embryonic stem cell differentiation. Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes. In addition,we identify two modes of developmental gene regulation by NUP98 that are differentiated by the spatial localization of NUP98 target genes. Genes in the initial stage of developmental induction can associate with NUP98 that is embedded in the nuclear pores at the nuclear periphery. Alternatively,genes that are highly induced can interact with NUP98 in the nuclear interior,away from the nuclear pores. This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation,revealing a role of a nuclear pore protein in regulating developmental gene expression programs. View Publication

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver,a preleukemic condition termed transient myeloproliferative disorder,and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins,human embryonic stem (hES),and induced pluripotent stem (iPS) cells,were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation,we isolated disomic and trisomic subclones from the same parental iPS line,thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like,γ-globin expressing,definitive hematopoiesis,we found that trisomic cells of hES,iPS,or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells,accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. View Publication

Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes,yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified,homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here,we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized,and defined growth factors were used to generate KDR+ EC progenitors. Magnetic purification of a KDR+ progenitor subpopulation resulted in an expanding,homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders. STEM Cells2013;31:92–103 View Publication

This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol,passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization,centrifugation or drug treatment. It also allows for higher throughput,requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation,colony expansion,cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion,and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages,this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium. View Publication

FTIR micro-spectroscopy is a sensitive,non-destructive and label-free method offering diffraction-limited resolution with high signal-to-noise ratios when combined with a synchrotron radiation source. The vibrational signature of individual cells was used to validate an alternative strategy for reprogramming induced pluripotent stem cells generated from amniocytes. The iPSC lines PB09 and PB10,were reprogrammed from the same amniocyte cell line using respectively the Oct54,Sox2,Lin28,and Nanog and the Oct4 and Sox2 transcription factor cocktail. We show that cells reprogrammed by the two different sets of transfection factors have similar spectral signatures after reprogramming,except for a small subpopulation of cells in one of the cell lines. Mapping HeLa cells at subcellular resolution,we show that the Golgi apparatus,the cytoplasm and the nucleus have a specific spectral signature. The CH(3):CH(2) ratio is the highest in the nucleus and the lowest in the Golgi apparatus/endoplasmic reticulum,in agreement with the membrane composition of these organelles. This is confirmed by specific staining of the organelles with fluorescent dyes. Subcellular differentiation of cell compartments is also demonstrated in living cells. View Publication

AIM: Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this,we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. MATERIALS & METHODS: Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. RESULTS & CONCLUSION: Two models were defined to predict cell yield and cell recovery rate postpassage,in terms of the predictor variables of media volume,cell seeding density,media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm,and to build regulatory confidence in cell therapy manufacturing processes. View Publication

BACKGROUND: Fragile X syndrome (FXS) is a common form of inherited intellectual disability caused by an expansion of CGG repeats located in the 5' untranslated region (UTR) of the FMR1 gene,which leads to hypermethylation and silencing of this locus. Although a dramatic increase in DNA methylation of the FMR1 full mutation allele is well documented,the extent to which these changes affect DNA methylation throughout the rest of the genome has gone unexplored. METHODS: Here we examined genome-wide methylation in both peripheral blood (N = 62) and induced pluripotent stem cells (iPSCs; N = 10) from FXS individuals and controls. RESULTS: We not only found the expected significant DNA methylation differences in the FMR1 promoter and 5' UTR,we also saw that these changes inverse in the FMR1 gene body. Importantly,we found no other differentially methylated loci throughout the remainder of the genome,indicating the aberrant methylation of FMR1 in FXS is locus-specific. CONCLUSIONS: This study provides a comprehensive methylation profile of FXS and helps refine our understanding of the mechanisms behind FMR1 silencing. View Publication

Human somatic cells can be reprogrammed to the pluripotent state to become human-induced pluripotent stem cells (hiPSC). This reprogramming is achieved by activating signaling pathways that are expressed during early development. These pathways can be induced by ectopic expression of four transcription factors—Oct4,Sox2,Klf4,and c-Myc. Although there are many ways to deliver these transcription factors into the somatic cells,this chapter will provide protocols that can be used to generate hiPSC from lentiviruses. View Publication

Patterning of bioactive enzymes with subcellular resolution is achieved by dispensing droplets of dextran (DEX) onto polyethylene glycol (PEG)-covered cells though a glass capillary needle connected to a pneumatic pump. This technique is applied to purify colonies of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblast (MEF) feeder cultures and inefficiently induced iPSC colonies by selectively dissociating the iPSCs with proteases. View Publication

The family of calcium activated potassium channels of low and intermediate conductance,known as SK channels,consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes,such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date,the role of SK channels in developmental processes has been merely a marginal focus of investigation,although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently,several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein,we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells,neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly,we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis. ?? 2013 Elsevier GmbH. View Publication

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus,hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks,however,conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology,retained a normal karyotype,and expressed characteristic hESC markers (OCT4,SSEA3,SSEA4 and TRA-1-60). Moreover,the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus,the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells. View Publication

Induced pluripotent stem cells (iPSC) have successfully been derived from somatic fibroblasts through transfection of synthetic modified mRNA encoding transcription factors. This technique obviates the use of recombinant DNA and viral vectors in cellular reprogramming. The present study derived iPSC from adipose-derived mesenchymal stem cells (of a 50-year-old female patient) by utilizing a similar technique,but with defined culture medium without feeder cells,during both reprogramming and propagation. Clonal selection was performed to yield 12 putative iPSC lines from individual colonies of nascent reprogrammed cells,starting from 150,000 cells. However,only seven lines maintained their undifferentiated state after 10 continuous serial passages. These seven lines were then subjected to a rigorous battery of analyses to confirm their identity as iPSC. These tests included immunostaining,flow cytometry,qRT-PCR,in vitro differentiation assay,and teratoma formation assay within SCID mice. Positive results were consistently observed in all analyses,thus verifying the cells as fully reprogrammed iPSC. While all 7 iPSC lines displayed normal karyogram up to passage 13,chromosomal anomalies occurred in 4 of 7 lines with extended in vitro culture beyond 24 serial passages. Only three lines retained normal karyotype of 46,XX. The remaining four lines displayed mosaicism of normal and abnormal karyotypes. Hence,this study successfully derived iPSC from abundant and easily accessible adipose tissues of a middle-aged patient; utilizing a mRNA-based integration-free technique under feeder-free conditions. This is a step forward in translating iPSC into personalized regenerative medicine within the clinic. ?? 2013 Elsevier Inc. View Publication