技术资料

The generation of hematopoietic cells from human embryonic stem cells (hESC) has raised the possibility of using hESC as an alternative donor source for transplantation. However,functional defects identified in hESC-derived cells limit their use for full lymphohematopoietic reconstitution. The purpose of the present study was to define and quantitate key functional and molecular differences between CD34(+) hematopoietic progenitor subsets derived from hESC and CD34(+) subsets from umbilical cord blood (UCB) representing definitive hematopoiesis. Two distinct sub-populations were generated following mesodermal differentiation from hESC,a CD34(bright) (hematoendothelial) and CD34(dim) (hematopoietic-restricted) subset. Limiting dilution analysis revealed profound defects in clonal proliferation relative to UCB particularly in B lymphoid conditions. Transcription factors normally expressed at specific commitment stages during B lymphoid development from UCB-CD34(+) cells were aberrantly expressed in hESC-derived CD34(+) cells. Moreover,strong negative regulators of lymphopoiesis such as the adaptor protein LNK and CCAAT/enhancer-binding protein-α (CEBPα),were exclusively expressed in hESC-CD34(+) subsets. Knockdown of LNK lead to an increase in hematopoietic progenitors generated from hESCs. The aberrant molecular profile seen in hESC-CD34(+) cells represents persistence of transcripts first expressed in undifferentiated hESC and/or CD326-CD56(+) mesoderm progenitors,and may contribute to the block in definitive hematopoiesis from hESC. View Publication

The developmental potential of human embryonic stem cells (hESCs) holds great promise to provide a source of human hepatocytes for use in drug discovery,toxicology,hepatitis research,and extracorporeal bioartificial liver support. There are,however,limitations to induce fully functional hepatocytes on conventional two-dimensional (2D) static culture. It had been shown that dynamic three-dimensional (3D) perfusion culture is superior to induce maturation in fetal hepatocytes and prolong hepatic functions of primary adult hepatocytes. We investigated the potential of using a four-compartment 3D perfusion culture to induce hepatic differentiation in hESC. Undifferentiated hESC were inoculated into hollow fiber-based 3D perfusion bioreactors with integral oxygenation. Hepatic differentiation was induced with a multistep growth factor cocktail protocol. Parallel controls were operated under equal perfusion conditions without the growth factor supplementations to allow for spontaneous differentiation,as well as in conventional 2D static conditions using growth factors. Metabolism,hepatocyte-specific gene expression,protein expression,and hepatic function were evaluated after 20 days. Significantly upregulated hepatic gene expression was observed in the hepatic differentiation 3D culture group. Ammonia metabolism activity and albumin production was observed in the 3D directed differentiation culture. Drug-induced cytochrome P450 gene expression was increased with rifampicin induction. Using flow cytometry analysis the mature hepatocyte marker asialoglycoprotein receptor was found on up to 30% of the cells in the 3D system with directed hepatic differentiation. Histological and immunohistochemical analysis revealed structural formation of hepatic and biliary marker-positive cells. In contrast to 2D culture,the 3D perfusion culture induced more functional maturation in hESC-derived hepatic cells. 3D perfusion bioreactor technologies may be useful for further studies on generating hESC-derived hepatic cells. View Publication

BACKGROUND: RNAs can be physically classified into poly(A)+ or poly(A)- transcripts according to the presence or absence of a poly(A) tail at their 3' ends. Current deep sequencing approaches largely depend on the enrichment of transcripts with a poly(A) tail,and therefore offer little insight into the nature and expression of transcripts that lack poly(A) tails. RESULTS: We have used deep sequencing to explore the repertoire of both poly(A)+ and poly(A)- RNAs from HeLa cells and H9 human embryonic stem cells (hESCs). Using stringent criteria,we found that while the majority of transcripts are poly(A)+,a significant portion of transcripts are either poly(A)- or bimorphic,being found in both the poly(A)+ and poly(A)- populations. Further analyses revealed that many mRNAs may not contain classical long poly(A) tails and such messages are overrepresented in specific functional categories. In addition,we surprisingly found that a few excised introns accumulate in cells and thus constitute a new class of non-polyadenylated long non-coding RNAs. Finally,we have identified a specific subset of poly(A)- histone mRNAs,including two histone H1 variants,that are expressed in undifferentiated hESCs and are rapidly diminished upon differentiation; further,these same histone genes are induced upon reprogramming of fibroblasts to induced pluripotent stem cells. CONCLUSIONS: We offer a rich source of data that allows a deeper exploration of the poly(A)- landscape of the eukaryotic transcriptome. The approach we present here also applies to the analysis of the poly(A)- transcriptomes of other organisms. View Publication

Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines,and we describe their pluripotent phenotype and ability to differentiate into erythroid cells,monocytes,and endothelial cells. More significantly,however,when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells,we observed the formation of pre-B cells. These cells were CD45(+)CD19(+)CD10(+) and were positive for transcripts Pax5,IL7αR,λ-like,and VpreB receptor. Although they were negative for surface IgM and CD5 expression,iPS-derived CD45(+)CD19(+) cells also exhibited multiple genomic D-J(H) rearrangements,which supports a pre-B-cell identity. We therefore have been able to demonstrate,for the first time,that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage. View Publication

Botulinum neurotoxins (BoNTs) inhibit cholinergic synaptic transmission by specifically cleaving proteins that are crucial for neurotransmitter exocytosis. Due to the lethality of these toxins,there are elevated concerns regarding their possible use as bioterrorism agents. Moreover,their widespread use for cosmetic purposes,and as medical treatments,has increased the potential risk of accidental overdosing and environmental exposure. Hence,there is an urgent need to develop novel modalities to counter BoNT intoxication. Mammalian motoneurons are the main target of BoNTs; however,due to the difficulty and poor efficiency of the procedures required to isolate the cells,they are not suitable for high-throughput drug screening assays. Here,we explored the suitability of embryonic stem (ES) cell-derived motoneurons as a renewable,reproducible,and physiologically relevant system for BoNT studies. We found that the sensitivity of ES-derived motoneurons to BoNT/A intoxication is comparable to that of primary mouse spinal motoneurons. Additionally,we demonstrated that several BoNT/A inhibitors protected SNAP-25,the BoNT/A substrate,in the ES-derived motoneuron system. Furthermore,this system is compatible with immunofluorescence-based high-throughput studies. These data suggest that ES-derived motoneurons provide a highly sensitive system that is amenable to large-scale screenings to rapidly identify and evaluate the biological efficacies of novel therapeutics. View Publication

Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However,little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis,and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4,Sox2,C-myc,and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2 × 10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3,STRA8,and germ cell marker-VASA,and haploid marker-FE-J1,PRM1,indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after induction. Approximately 34.88% (30/86) embryos showed cleavage and four embryos were cultured on murine fibroblast feeder and formed small embryonic stem like colonies. However,most stalled at four-cell stage after intracytoplasmic sperm injection (ICSI) of these cells. Transplantation of DAPI labeled mGSCs into the seminiferous tubules of busulfan-treated mice,and showed that mGSCs can colonize,self-renew,and differentiate into germ cells. Thus,we have established a goat GSC cell line and these cells could be differentiated into sperm-like cells in vivo and sperms in vitro,providing a promising platform for generation of transgenic goat for production of specific humanized proteins. View Publication

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons,muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood,pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres,a hallmark of dyskeratosis congenita,impair tissue stem cell function in mouse models,indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state,iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT,the telomerase reverse transcriptase,a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast,mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53),telomerase catalytic activity is unperturbed,yet the ability of telomerase to lengthen telomeres is abrogated,because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal,indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics. View Publication

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells,which also expressed Oct4,SSEA-3 and SSEA-4. We also found another population expressing SSEA-4,but without Nanog,Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog,Oct4,SSEA-3,SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block,the cell cycle of hESC normalised,but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition,the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle,which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4. View Publication

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media,attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component,as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces,we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives,and should be applicable to other reprogramming methods. View Publication

The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II. Med1 (also known as PBP or TRAP220) is a key component of Mediator that interacts with nuclear hormone receptors and GATA transcription factors. Here,we show dynamic recruitment of GATA-1,TFIIB,Mediator,and RNA polymerase II to the β-globin locus in induced mouse erythroid leukemia cells and in an erythropoietin-inducible hematopoietic progenitor cell line. Using Med1 conditional knockout mice,we demonstrate a specific block in erythroid development but not in myeloid or lymphoid development,highlighted by the complete absence of β-globin gene expression. Thus,Mediator subunit Med1 plays a pivotal role in erythroid development and in β-globin gene activation. View Publication

Mesenchymal stem cells (MSCs) possess tumor-tropic properties and consequently have been used to deliver therapeutic agents for cancer treatment. Their potential in cancer therapy highlights the need for a consistent and renewable source for the production of uniform human MSCs suitable for clinical applications. In this study,we seek to investigate whether human embryonic stem cells can be used as a cell source to fulfill this goal. We generated MSC-like cells from two human embryonic stem cell lines,HuES9 and H1,and observed that MSC-like cells derived from human embryonic stem cells were able to migrate into human glioma intracranial xenografts after being injected into the cerebral hemisphere contralateral to the tumor inoculation site. We engineered these cells with baculoviral and lentiviral vectors,respectively,for transient and stable expression of the herpes simplex virus thymidine kinase gene. In tumor-bearing mice the engineered MSC-like cells were capable of inhibiting tumor growth and prolonging survival in the presence of ganciclovir after they were injected either directly into the xenografts or into the opposite hemisphere. Our findings suggest that human embryonic stem cell-derived MSCs may be a viable and attractive alternative for large-scale derivation of targeting vehicles for cancer therapy. View Publication

We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD),a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91(phox) deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production,reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs,with subsequent selection and full characterization to ensure no off-target changes,holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs." View Publication