技术资料

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here,we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging. View Publication

Background: Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder,which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD,existing pharmaceutical can only relieve its symptoms. Results: Here,induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene,and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro,as evidenced by mutant huntingtin protein aggregation,increased number of lysosomes/autophagosomes,nuclear indentations,and enhanced neuronal death during cell aging. Moreover,store-operated channel (SOC) currents were detected in the differentiated neurons,and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally,the quinazoline derivative,EVP4593,reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging. Conclusions: Our data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks,providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug. [ABSTRACT FROM AUTHOR] View Publication

Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However,how it mediates Ca2+ signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here,we aimed to determine the role of P2Rs in mediating Ca2+ mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP3Rs) was analyzed by quantitative/RT-PCR. IP3R3 knockdown (KD) or IP3R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations,respectively. Confocal imaging revealed that Ca2+ responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently,the gene expression levels of most P2YRs except P2Y1 were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca2+ transients in hESCs but only partially inhibited those in CVPCs. Moreover,the P2Y1 receptor-specific antagonist MRS2279 abolished most ATP-induced Ca2+ signals in hESCs but not in CVPCs. P2Y1 receptor-specific agonist MRS2365 induced Ca2+ transients only in hESCs but not in CVPCs. Furthermore,IP3R2KO but not IP3R3KD decreased the proportion of hESCs responding to MRS2365. In contrast,both IP3R2 and IP3R3 contributed to UTP-induced Ca2+ responses while ATP-induced Ca2+ responses were more dependent on IP3R2 in the CVPCs. In conclusion,a predominant role of P2Y1 receptors in hESCs and a transition of P2Y-IP3R coupling in derived CVPCs are responsible for the differential Ca2+ mobilization between these cells. View Publication

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome,a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction,but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14,the ribosomal protein gene deleted in the 5q-syndrome,or RPS19,the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect,whereas nutlin-3,a compound that activates p53 through inhibition of HDM2,selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome,we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53,providing a basis for the failure of erythropoiesis in the 5q-syndrome,DBA,and perhaps other bone marrow failure syndromes. 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

SPARC is an extracellular matrix protein that exerts pleiotropic effects on extracellular matrix organization,growth factor availability,cell adhesion,differentiation,and immunity in cancer. Chronic myelogenous leukemia (CML) cells resistant to the BCR-ABL inhibitor imatinib (IM-R cells) were found to overexpress SPARC mRNA. In this study,we show that imatinib triggers SPARC accumulation in a variety of tyrosine kinase inhibitor (TKI)-resistant CML cell lines. SPARC silencing in IM-R cells restored imatinib sensitivity,whereas enforced SPARC expression in imatinib-sensitive cells promoted viability as well as protection against imatinib-mediated apoptosis. Notably,we found that the protective effect of SPARC required intracellular retention inside cells. Accordingly,SPARC was not secreted into the culture medium of IM-R cells. Increased SPARC expression was intimately linked to persistent activation of the Fyn/ERK kinase signaling axis. Pharmacologic inhibition of this pathway or siRNA-mediated knockdown of Fyn kinase resensitized IM-R cells to imatinib. In support of our findings,increased levels of SPARC mRNA were documented in blood cells from CML patients after 1 year of imatinib therapy compared with initial diagnosis. Taken together,our results highlight an important role for the Fyn/ERK signaling pathway in imatinib-resistant cells that is driven by accumulation of intracellular SPARC. View Publication

Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However,little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here,we used aldehyde dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin-) led to tumors in 13 (out of 15) mice,whereas 10,000 noncancer stem cells (ALDH-CD44-Lin-) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a subpopulation of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin- cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-μm radius) of blood vessels in human tumors,suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC,as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared with controls. Notably,selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively,these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck CSC. View Publication

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However,growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis,in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular,we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently,the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion,our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood. View Publication

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However,this is a slow and inefficient process,depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover,once cell reprogramming is accomplished,these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However,no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus,which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes,deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore,interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus,this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research. View Publication

Maintaining a steady pool of self-renewing hematopoietic stem cells (HSCs) is critical for sustained production of multiple blood lineages. Many transcription factors and molecules involved in chromatin and epigenetic modifications have been found to be critical for HSC self-renewal and differentiation; however,their interplay is less understood. The transcription factor GA binding protein (GABP),consisting of DNA-binding subunit GABPα and transactivating subunit GABPβ,is essential for lymphopoiesis as shown in our previous studies. Here we demonstrate cell-intrinsic,absolute dependence on GABPα for maintenance and differentiation of hematopoietic stem/progenitor cells. Through genome-wide mapping of GABPα binding and transcriptomic analysis of GABPα-deficient HSCs,we identified Zfx and Etv6 transcription factors and prosurvival Bcl-2 family members including Bcl-2,Bcl-X(L),and Mcl-1 as direct GABP target genes,underlying its pivotal role in HSC survival. GABP also directly regulates Foxo3 and Pten and hence sustains HSC quiescence. Furthermore,GABP activates transcription of DNA methyltransferases and histone acetylases including p300,contributing to regulation of HSC self-renewal and differentiation. These systematic analyses revealed a GABP-controlled gene regulatory module that programs multiple aspects of HSC biology. Our studies thus constitute a critical first step in decoding how transcription factors are orchestrated to regulate maintenance and multipotency of HSCs. View Publication