技术资料

DPF2,also named ubi-d4/requiem (REQU),interacts with a protein complex containing OCT4. This paper provides data in support of the research article entitled DPF2 regulates OCT4 protein level and nuclear distribution". The highlights include: (1) Denature-immunoprecipitation assay revealed ubiquitination of OCT4 in pluripotent H9 cells� View Publication

The fluorescence ubiquitination cell cycle indicator (FUCCI) system provides a powerful method to evaluate cell cycle mechanisms associated with stem cell self-renewal and cell fate specification. By integrating the FUCCI system into human pluripotent stem cells (hPSCs) it is possible to isolate homogeneous fractions of viable cells representative of all cell cycle phases. This method avoids problems associated with traditional tools used for cell cycle analysis such as synchronizing drugs,elutriation and temperature sensitive mutants. Importantly,FUCCI reporters allow cell cycle events in dynamic systems,such as differentiation,to be evaluated. Initial reports on the FUCCI system focused on its strengths in reporting spatio-temporal aspects of cell cycle events in living cells and developmental models. In this report,we describe approaches that broaden the application of FUCCI reporters in PSCs through incorporation of FACS. This approach allows molecular analysis of the cell cycle in stem cell systems that were not previously possible. View Publication

Retinal pigment epithelium-specific 65 kDa (RPE65)-associated Leber congenital amaurosis is an autosomal recessive disease that results in reduced visual acuity and night blindness beginning at birth. It is one of the few retinal degenerative disorders for which promising clinical gene transfer trials are currently underway. However,the ability to enroll patients in a gene augmentation trial is dependent on the identification of 2 bona fide disease-causing mutations,and there are some patients with the phenotype of RPE65-associated disease who might benefit from gene transfer but are ineligible because 2 disease-causing genetic variations have not yet been identified. Some such patients have novel mutations in RPE65 for which pathogenicity is difficult to confirm. The goal of this study was to determine if an intronic mutation identified in a 2-year-old patient with presumed RPE65-associated disease was truly pathogenic and grounds for inclusion in a clinical gene augmentation trial. Sequencing of the RPE65 gene revealed 2 mutations: (1) a previously identified disease-causing exonic leucine-to-proline mutation (L408P) and (2) a novel single point mutation in intron 3 (IVS3-11) resulting in an AtextgreaterG change. RT-PCR analysis using RNA extracted from control human donor eye-derived primary RPE,control iPSC-RPE cells,and proband iPSC-RPE cells revealed that the identified IVS3-11 variation caused a splicing defect that resulted in a frameshift and insertion of a premature stop codon. In this study,we demonstrate how patient-specific iPSCs can be used to confirm pathogenicity of unknown mutations,which can enable positive clinical outcomes. View Publication

Summary The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14,when expressed in tumors,causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor,rather than host,is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia,thereby extending lifespan and improving quality of life for cancer patients. View Publication

This paper proposes a bio-driven algorithm that detects cell regions automatically in the human embryonic stem cell (hESC) images obtained using a phase contrast microscope. The algorithm uses both statistical intensity distributions of foreground/hESCs and background/substrate as well as cell property for cell region detection. The intensity distributions of foreground/hESCs and background/substrate are modeled as a mixture of two Gaussians. The cell property is translated into local spatial information. The algorithm is optimized by parameters of the modeled distributions and cell regions evolve with the local cell property. The paper validates the method with various videos acquired using different microscope objectives. In comparison with the state-of-the-art methods,the proposed method is able to detect the entire cell region instead of fragmented cell regions. It also yields high marks on measures such as Jacard similarity,Dice coefficient,sensitivity and specificity. Automated detection by the proposed method has the potential to enable fast quantifiable analysis of hESCs using large data sets which are needed to understand dynamic cell behaviors. View Publication

Interactions between stem cells and their microenvironment are critical for regulation and maintenance of stem cell function. To elucidate the molecular interactions within the human limbal epithelial stem/progenitor cell (LEPC) niche,which is essential for maintaining corneal transparency and vision,we performed a comprehensive expression analysis of cell adhesion molecules (CAMs) using custom-made quantitative real-time polymerase chain reaction (qRT-PCR) arrays and laser capture-microdissected LEPC clusters,comprising LEPCs,melanocytes,mesenchymal cells,and transmigrating immune cells. We show that LEPCs are anchored to their supporting basement membrane by the laminin receptors $\$3$\$1 and $\$6$\$4 integrin and the dystroglycan complex,while intercellular contacts between LEPCs and melanocytes are mediated by N-,P-,and E-cadherin together with L1-CAM,a member of the immunoglobulin superfamily (Ig)CAMs. In addition to the LEPC-associated heparan sulfate proteoglycans syndecan-2,glypican-3,and glypican-4,the IgCAM members ICAM-1 and VCAM-1 were found to be variably expressed on LEPCs and associated niche cells and to be dynamically regulated in response to chemokines such as interferon-$\$ enhance interactions with immune cells. Moreover,junctional adhesion molecule JAM-C accumulating in the subepithelial limbal matrix,appeared to be involved in recruitment of immune cells,while mesenchymal stromal cells appeared to use the nephronectin receptor integrin $\$8 for approaching the limbal basement membrane. In summary,we identified a novel combination of cell surface receptors that may regulate both stable and dynamic cell-matrix and cell-cell interactions within the limbal niche. The findings provide a solid foundation for further functional studies and for advancement of our current therapeutic strategies for ocular surface reconstruction. View Publication

Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup),carrying different duplication sizes,to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition,using multi-electrodes arrays,we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level,we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor,NCH-51,was validated as a potential clinical candidate. Interestingly,this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders. View Publication

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus,they are increasingly employed as models for cancer and drug research,as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures,and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore,SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells,providing a pathway to enable our understanding of morphogenesis in 3D cultures. View Publication

Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs),but little is known about SSEA-5 expression in other primitive tissues (e.g.,human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors,cell lines,teratomas,and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors,such as embryonal carcinoma. In tumor cell lines,SSEA-5 was highly immunoreactive in Capan-1 cells,while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs,with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use. View Publication

Chronic granulomatous disease (CGD) is an inherited immunodeficiency,caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD),which is due to mutations in the CYBB (gp91phox) gene,a component of NADPH oxidase,accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function,we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both,BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion,we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general. View Publication

Significant efforts have been invested into the differentiation of stem cells into functional hepatocyte-like cells that can be used for cell therapy,disease modeling and drug screening. Most of these efforts have been concentrated on the use of growth factors to recapitulate developmental signals under in vitro conditions. Using small molecules instead of growth factors would provide an attractive alternative since small molecules are cell-permeable and cheaper than growth factors. We have developed a protocol for the differentiation of human embryonic stem cells into hepatocyte-like cells using a predominantly small molecule-based approach (SM-Hep). This 3 step differentiation strategy involves the use of optimized concentrations of LY294002 and bromo-indirubin-3'-oxime (BIO) for the generation of definitive endoderm; sodium butyrate and dimethyl sulfoxide (DMSO) for the generation of hepatoblasts and SB431542 for differentiation into hepatocyte-like cells. Activin A is the only growth factor required in this protocol. Our results showed that SM-Hep were morphologically and functionally similar or better compared to the hepatocytes derived from the growth-factor induced differentiation (GF-Hep) in terms of expression of hepatic markers,urea and albumin production and cytochrome P450 (CYP1A2 and CYP3A4) activities. Cell viability assays following treatment with paradigm hepatotoxicants Acetaminophen,Chlorpromazine,Diclofenac,Digoxin,Quinidine and Troglitazone showed that their sensitivity to these drugs was similar to human primary hepatocytes (PHHs). Using SM-Hep would result in 67% and 81% cost reduction compared to GF-Hep and PHHs respectively. Therefore,SM-Hep can serve as a robust and cost effective replacement for PHHs for drug screening and development. View Publication

Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely defined xeno-free systems for large-scale cell expansion and differentiation. Previously,we demonstrated that microcarriers (MCs) coated with mouse laminin-111 (LN111) and positively charged poly-l-lysine (PLL) critically enable the formation and evolution of cells/MC aggregates with high cell yields obtained under agitated conditions. In this article,we further improved the MC system into a defined xeno-free MC one in which the MCs are coated with recombinant human laminin-521 (LN521) alone without additional positive charge. The high binding affinity of the LN521 to cell integrins enables efficient initial HES-3 cell attachment (87%) and spreading (85%),which leads to generation of cells/MC aggregates (400 $\$ in size) and high cell yields (2.4-3.5×10(6) cells/mL) within 7 days in agitated plate and scalable spinner cultures. The universality of the system was demonstrated by propagation of an induced pluripotent cells line in this defined MC system. Long-term pluripotent (textgreater90% expression Tra-1-60) cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover,tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved. The new LN521-based MC system offers a defined,xeno-free,GMP-compatible,and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on MCs enabled a 34% savings in matrix and media costs over monolayer cultures to produce 10(8) cells. View Publication