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We reconstituted type I collagen nanofibers prepared by electrospin technology and examined the morphology,growth,adhesion,cell motility,and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) on three nano-sized diameters (50-200,200-500,and 500-1,000 nm). Results from scanning electron microscopy showed that cells on the nanofibers had a more polygonal and flattened cell morphology. MTS (3-[4,5-dimethythiazol-2-yl]-5-[3-carboxy-methoxyphenyl]-2-[4-sul-fophenyl]-2H-tetrazolium compound) assay demonstrated that the MSCs grown on 500-1,000-nm nanofibers had significantly higher cell viability than the tissue culture polystyrene control. A decreased amount of focal adhesion formation was apparent in which quantifiable staining area of the cytoplasmic protein vinculin for the 200-500-nm nanofibers was 39% less compared with control,whereas the area of quantifiable vinculin staining was 45% less for both the 200-500-nm and 500-1,000-nm nanofibers. The distances of cell migration were quantified on green fluorescent protein-nucleofected cells and was 56.7%,37.3%,and 46.3% for 50-200,200-500,and 500-1,000 nm,respectively,compared with those on the control. Alkaline phosphatase activity demonstrated no differences after 12 days of osteogenic differentiation,and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed comparable osteogenic gene expression of osteocalcin,osteonectin,and ostepontin between cells differentiated on polystyrene and nanofiber surfaces. Moreover,single-cell RT-PCR of type I collagen gene expression demonstrated higher expression on cells seeded on the nanofibers. Therefore,type I collagen nanofibers support the growth of MSCs without compromising their osteogenic differentiation capability and can be used as a scaffold for bone tissue engineering to facilitate intramembranous bone formation. Further efforts are necessary to enhance their biomimetic properties. View Publication

Pluripotent stem cells have the potential to become most of the cell types that make up an organism. However,the signals that trigger these cells to turn into neurons rather than lung cells or muscle cells,for example,are not fully understood. Proteins called growth factors are known to have a role in this process,as are transcription factors,but it is not clear if other factors are also involved. In an attempt to identify additional mechanisms that could contribute to the formation of neurons,Sun et al. screened more than 2,000 small molecules for their ability to transform mouse pluripotent stem cells into neurons in cell culture. Surprisingly,they found that a compound called selamectin,which is used to treat parasitic flatworm infections,also triggered stem cells to turn into neurons. Selamectin works by blocking a particular type of ion channel in flatworms,but this ion channel is not found in vertebrates,which means that selamectin must be promoting the formation of neurons in mice via a different mechanism. Given that a drug related to selamectin is known to act on a subtype of receptors for the neurotransmitter GABA,Sun et al. wondered whether these receptors—known as GABAA receptors—might also underlie the effects of selamectin. Consistent with this idea,drugs that increased GABAA activity stimulated the formation of neurons,whereas drugs that reduced GABAA function blocked the effects of selamectin. In addition,Sun et al. showed that selamectin triggers human embryonic stem cells to become neurons,and that it also promotes the formation of new neurons in developing zebrafish in vivo. As well as revealing an additional mechanism for the formation of neurons from stem cells,the screening technique introduced by Sun et al. could help to identify further pro-neuronal molecules,which could aid the treatment of neurodevelopmental and neurodegenerative disorders. DOI: [http://dx.doi.org/10.7554/eLife.00508.002][1] [1]: /lookup/doi/10.7554/eLife.00508.002 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

Induction of pluripotent stem cells from human fibroblasts has been achieved by the ectopic expression of two different sets of four genes. However,the mechanism of the pluripotent stem cell induction has not been elucidated. Here we identified a marked heterogeneity in colonies generated by the four-gene (Oct3/4,Sox2,c-Myc,and Klf4) transduction method in human neonatal skin-derived cells. The four-gene transduction gave a higher probability of induction for archetypal pluripotent stem cell marker genes (Nanog,TDGF,and Dnmt3b) than for marker genes that are less specific for pluripotent stem cells (CYP26A1 and TERT) in primary induction culture. This tendency may reflect the molecular mechanism underlying the induction of human skin-derived cells into pluripotent stem cells. Among the colonies induced by the four-gene transduction,small cells with a high nucleus-to-cytoplasm ratio could be established by repeated cloning. Subsequently established cell lines were similar to human embryonic stem cells as well as human induced pluripotent stem (iPS) cells derived from adult tissue in morphology,gene expression,long-term self-renewal ability,and teratoma formation. Genome-wide single-nucleotide polymorphism array analysis of the human iPS cell line indicates that the induction process did not induce DNA mutation. ?? 2008 Elsevier B.V. All rights reserved. View Publication

The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting,X-chromosome inactivation and genomic architecture. In this study,we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes,including NANOG,SOX2,cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation,but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs,CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors,such as Cohesin,differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly,the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology. View Publication

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had textgreater1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization,two iPSC lines from each subject were selected,differentiated into postmitotic neurons,and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs,iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover,repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism. View Publication

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways,however,it remains unclear as to what extent these pathways can be exploited,either individually or in combination,in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro,but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways,we developed a high-density microbioreactor array (HDMA) - a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt,Hedgehog,IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation,inducing cell cycle activity marked by Ki67,and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration. View Publication

Induced pluripotent stem cells hold great potential in regenerative medicine as it enables to generate pluripotent stem cells from any available cell types. Ectopic expression of four transcription factors (Oct4,Sox2,Klf4,and c-Myc) can reprogram fibroblasts directly to pluripotency as shown in multiple species. Here,we describe detailed protocols for generation of iPSCs from adult canine fibroblasts. Robust canine iPSCs will provide powerful tools not only to study human diseases,but also for the development of therapeutic approaches. View Publication

Tissue engineering provides a new paradigm for periodontal tissue regeneration in which proper stem cells and effective cellular factors are very important. The objective of this study was,for the first time,to investigate the capabilities and advantages of periodontal tissue regeneration using induced pluripotent stem (iPS) cells and enamel matrix derivatives (EMD). In this study the effect of EMD gel on iPS cells in vitro was first determined,and then tissue engineering technique was performed to repair periodontal defects in three groups: silk scaffold only; silk scaffold + EMD; and silk scaffold + EMD + iPS cells. EMD greatly enhanced the mRNA expression of Runx2 but inhibited the mRNA expression of OC and mineralization nodule formation in vitro. Transplantation of iPS cells showed higher expression levels of OC,Osx,and Runx2 genes,both 12 and 24 days postsurgery. At 24 days postsurgery in the iPS cell group,histological analysis showed much more new alveolar bone and cementum formation with regenerated periodontal ligament between them. The results showed the commitment role that EMD contributes in mesenchymal progenitors to early cells in the osteogenic lineage. iPS cells combined with EMD provide a valuable tool for periodontal tissue engineering,by promoting the formation of new cementum,alveolar bone,and normal periodontal ligament. View Publication

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine,disease modeling,and drug screening. However,their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research,only the best,competent clones should be used. The standard assays for pluripotency are based on genomic approaches,which take up to 1 week to perform and incur significant cost. Therefore,there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here,we describe a novel multiplexed,high-throughput,and sensitive peptide-based multiple reaction monitoring mass spectrometry assay,allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests. View Publication

The characterisation of stem cells is of vital importance to regenerative medicine. Failure to separate out all stem cells from differentiated cells before therapies can result in teratomas - tumours of multiple cell types. Typically,characterisation is performed in a destructive manner with fluorescent assays. A truly non-invasive method of characterisation would be a major breakthrough in stem cell-based therapies. Raman spectroscopy has revealed that DNA and RNA levels drop when a stem cell differentiates into other cell types,which we link to a change in the relative sizes of the nucleus and cytoplasm. We also used Raman spectroscopy to investigate the biochemistry within an early embryo,or blastocyst,which differs greatly from colonies of embryonic stem cells. Certain cell types that differentiate from stem cells can be identified by directly imaging the biochemistry with CARS microscopy; examples presented are hydroxyapatite - a precursor to bone,and lipids in adipocytes. View Publication