参考文献

The fabrication of high-density polymer microarray is described,allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance. View Publication

BACKGROUND: Gene delivery can potentially be used as a therapeutic for treating genetic diseases,including neurodegenerative diseases,as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts.backslashnbackslashnMETHODS: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.backslashnbackslashnRESULTS: 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents,including Lipofectamine® 2000,FuGENE® HD,and 25 kDa branched polyethylenimine,for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa,and enabled coexpression of exogenously delivered genes,as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation,but not by poly(beta-amino ester) reprogramming,could be differentiated toward the neuronal lineage,specifically pseudostratified optic cups.backslashnbackslashnCONCLUSION: This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming. View Publication

The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks,we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells,we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes,which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions,which can then be employed to direct cellular differentiation down desired pathways. View Publication

High telomerase activity is a characteristic of human embryonic stem cells (hESCs),however,the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion,but stabilized at later passages. We report that the core unit of telomerase,hTERT,was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore,this was regulated in a Wnt-β-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation,demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation,suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines,we were able to establish that telomere length remained relatively stable at 12.02±1.01 kb during later passages (15-95). In contrast,telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary,we propose that correct,stable telomere length may serve as a potential biomarker for genetically stable hESCs. View Publication

Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors,but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned medium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay,we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts,perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells,specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs),including a clonogenic potential of ˜21.5% and expression of pluripotency associated genes like Oct-4,Nanog,c-Myc and Klf-4. Similar to CASCs,migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and Imatinib Mesylate,a PDGF receptor inhibitor,suggested a role for the PDGF-AA/PDGF receptor $$ axis in enhancing the migration process of CASCs. In conclusion,our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors,like PDGF-AA,can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury. View Publication

The superiority of dendritic cells (DCs) as antigen-presenting cells has been exploited in numerous clinical trials,where generally monocyte-derived DCs (Mo-DCs) are injected to induce immunity in patients with cancer or infectious diseases. Despite promising expansion of antigen-specific T cells,the clinical responses following vaccination have been limited,indicating that further improvements of DC vaccine potency are necessary. Pre-clinical studies suggest that vaccination with combination of primary DC subsets,such as myeloid and plasmacytoid blood DCs (mDCs and pDCs,respectively),may result in stronger clinical responses. However,it is a challenge to obtain high enough numbers of primary DCs for immunotherapy,since their frequency in blood is very low. We therefore explored the possibility to generate them from hematopoietic progenitor cells (HPCs). Here,we show that by inhibiting the aryl hydrocarbon receptor with its antagonist StemRegenin 1 (SR1),clinical-scale numbers of functional BDCA2(+)BDCA4(+) pDCs,BDCA1(+) mDCs,and BDCA3(+)DNGR1(+) mDCs can be efficiently generated from human CD34(+) HPCs. The ex vivo-generated DCs were phenotypically and functionally comparable to peripheral blood DCs. They secreted high levels of pro-inflammatory cytokines such as interferon (IFN)-α,interleukin (IL)-12,and tumor necrosis factor (TNF)-α and upregulated co-stimulatory molecules and maturation markers following stimulation with Toll-like receptor (TLR) ligands. Further,they induced potent allogeneic T-cell responses and activated antigen-experienced T cells. These findings demonstrate that SR1 can be exploited to generate high numbers of functional pDCs and mDCs from CD34(+) HPCs,providing an alternative option to Mo-DCs for immunotherapy of patients with cancer or infections. View Publication

Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers,and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells,to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells,we examined the effect of varying initial cell seeding density from 1.3 x 104 cells/cm2 to 5.3 x 104 cells/cm2 followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1,PTF1a,NGN3,ARX,and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin,glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression,followed by pancreatic endocrine specification marker expression (BRN4,PAX4,ARX,NEUROD1,NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin,glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment,which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols. View Publication

Many solid cancers display cellular hierarchies with self-renewing,tumorigenic stemlike cells,or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies,they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs,we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays,but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines,including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion,and targeting IL-17A signaling impaired CIC growth. Notably,IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer. View Publication

BACKGROUND: Abnormal activation of endochondral bone formation in soft tissues causes significant medical diseases associated with disability and pain. Hyperactive mutations in the bone morphogenetic protein (BMP) type 1 receptor ACVR1 lead to fibrodysplasia ossificans progressiva (FOP),a rare genetic disorder characterized by progressive ossification in soft tissues. However,the specific cellular mechanisms are unclear. In addition,the difficulty obtaining tissue samples from FOP patients and the limitations in mouse models of FOP hamper our ability to dissect the pathogenesis of FOP.backslashnbackslashnMETHODS: To address these challenges and develop a disease model in a dish"� View Publication

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4,MEF2C,and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However,GMT alone appears insufficient in human fibroblasts,at least in vitro. Here,we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells,fetal heart,and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming,including sarcomere formation,calcium transients,and action potentials,although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore,we found that transforming growth factor β signaling was important for,and improved the efficiency of,human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage,and lay the foundation for future refinements in vitro and in vivo. textcopyright 2013 The Authors. View Publication

Breast Cancer Stem (BCS) cells play critical roles in self-renewal,Multi Drug Resistance (MDR),differentiation and generation of secondary tumors. Conventional chemotherapy may efficiently kill the bulk of differentiated drug sensitive breast cancer cells,but not the MDR self-renewable BCS cells,leading to enrichment of the MDR BCS cells. In order to target the MDR BCS cells,we have isolated: 1) BCS cells from either breast cancer cell lines or fresh breast cancer specimens; 2) ATP binding cassette (ABC) transporter group G number 2 (ABCG2)-specific aptamers; and 3) BCS cell-binding aptamers. Interestingly,ABCG2-specific aptamers labeled the membrane surface of the ABCG2-expressing baby hamster kidney (BHK) cells,but stained whole cells of the BCS cells derived from mammospheres,implying that BCS cells might have much higher rate of endocytosis than the ABCG2-expressing BHK cells. In addition,5D3,a monoclonal antibody that recognizes the extracellular loops of ABCG2 protein,also stained whole BCS cells. Furthermore,BCS cell-binding aptamers stained whole BCS cells,but not the differentiated breast cancer MCF-7 cells. All these results support above conclusion that BCS cells might have high rate of endocytosis. Further experiments performed with aptamers and human transferrin or lactosylceramide showed that BCS cells do have much higher endocytosis rate than the differentiated breast cancer cells. Interestingly,clathrin dependent endocytosis inhibitors,such as monodansylcadaverine or sucrose,or caveolin-dependent endocytosis inhibitors,such as methyl-$$-cyclodextrin or genistein,can inhibit the internalization of transferrin or lactosylceramide into the differentiated breast cancer cells,but cannot block the internalization of these compounds into the BCS cells,suggesting that BCS cells undergo clathrin-independent and caveolin-independent endocytosis. Taken together,our data suggest that BCS cells have high rate of endocytosis and open the possibilities for delivering therapeutic agents directly into the MDR BCS cells with aptamer-coated liposomes. View Publication

It has been recently reported that the regulatory circuitry formed by OCT4,miR-302,and NR2F2 controls both pluripotency and neural differentiation of human embryonic stem cells (hESCs). We show here that JMJD1C,a histone 3 lysine 9 (H3K9) demethylase expressed in hESCs,directly interacts with this circuitry. hESCs with stable knockdown of JMJD1C remain pluripotent while having reduced miR-302 expression,decreased BMP signaling,and enhanced TGF$\$ JMJD1C binds to the miR-302 promoter and reduces H3K9 methylation. Withdrawal of basic fibroblast growth factor (bFGF) from the culture induces neural differentiation of the knockdown,but not the control,cells within 3 days,accompanied by elevated NR2F2 expression. This can be attenuated with miR-302 mimics or an H3K9 methytransferase inhibitor. Together,our findings suggest that JMJD1C represses neural differentiation of hESCs at least partially by epigenetically sustaining miR-302 expression and that JMJD1C knockdown is sufficient to trigger neural differentiation upon withdrawal of exogenous bFGF. View Publication