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Mol Reprod Dev ; 47 — Molecular characterization of spontaneous mesenchymal stem cell transformation. PloS ONE ; 3 :e Spontaneous human adult stem cell transformation. Cancer Res ; 65 — Mesenchymal stem cells and their use as a cell replacement therapy and disease modelling tool. J Cell Mol Med ; 12 :1— Leukemia ; 22 — Genetic stability of human embryonic stem cells: a first-step toward the development of potential hESC-based systems for modelling childhood leukemia. Leukemia Res ; doi: Conventional and molecular cytogenetic diagnostic methods in stem cell research: a concise review.
Cell Biol Int ; 31 — Whole-blastocyst culture followed by laser drilling technology enhances the efficiency of inner cell mass isolation and embryonic stem cell derivation from good- and poor-quality mouse embryos: new insights for derivation of human embryonic stem cell lines. Stem Cells Dev ; 17 — Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?
Mol Cancer ; 7 — Download references. We are indebted to Prof. Peter Andrews University of Sheffield and Prof. You can also search for this author in PubMed Google Scholar. Correspondence to Clara Bueno or Pablo Menendez. Reprints and Permissions. Montes, R. Cell Res 19, — Download citation.
Received : 05 October Revised : 28 October Accepted : 30 December Published : 24 March Issue Date : June Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Bioprocess and Biosystems Engineering Reproductive Biology and Endocrinology Advanced search. Skip to main content Thank you for visiting nature.
Download PDF. Introduction Human embryonic stem cells hESCs are pluripotent stem cells derived from human blastocyst-stage embryos 1. Figure 1. Full size image. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Discussion Many efforts have been made to develop hESC culture systems based on human feeders 8 , 9 , 19 , 20 , 22 , 23 , Conventional karyotyping G-banding studies were performed as described previously 31 , 32 , 33 , View author publications.
Rights and permissions Reprints and Permissions. Our results suggest that cell viability would be necessary for improving differentiation efficiency using the cytokine-based approach. This study identified a practical iPSC differentiation approach to generating TM-like cells that is more feasible for clinic usage. We first defined two distinct stages of iPSC differentiation and selected highly expressed receptors at each stage of differentiation, and we have developed a new type of differentiation using the corresponding recombinant cytokines.
Disclosure: W. Wang, None; Y. Wang, None; S. Wu, None; Q. Cao, None; H. Duan, None; X. Qi, None; Q. Zhou, None; X. Pan, None; J. Zhang, None; X. Chen, None; Y. Han, None; N. Wang, None; M. Kuehn, None; W. Zhu, Qingdao Haier Biotech Co. Johnson M. What controls aqueous humour outflow resistance. Exp Eye Res. Quigley HA. The Lancet.
The number of people with glaucoma worldwide in and Br J Ophthalmol. Castro A, Du Y. Curr Ophthalmol Rep. J Ocul Pharmacol Ther. Intraocular pressure reduction and neuroprotection conferred by bone marrow-derived mesenchymal stem cells in an animal model of glaucoma. Stem Cell Res Ther. Stem cells from trabecular meshwork home to TM tissue in vivo. Invest Ophthalmol Vis Sci. Sci Rep. Stem Cells. Adv Biosyst. Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser-induced paracrine factor secretion and progenitor cell recruitment.
Induction of trabecular meshwork cells from induced pluripotent stem cells. Front Cell Dev Biol. Consensus recommendations for trabecular meshwork cell isolation, characterization and culture. Passage-dependent accumulation of somatic mutations in mesenchymal stromal cells during in vitro culture revealed by whole genome sequencing. Changes in mesenchymal stem cells following long-term culture in vitro. Mol Med Rep. Generating a non-integrating human induced pluripotent stem cell bank from urine-derived cells.
PloS One. Establishment of alternative culture method for spermatogonial stem cells using knockout serum replacement. PloS one. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture. Mol Hum Reprod. AutoSOME: a clustering method for identifying gene expression modules without prior knowledge of cluster number.
BMC Bioinform. Comparative transcriptome analysis by RNAseq of necrotic enteritis Clostridium perfringens during in vivo colonization and in vitro conditions. BMC Microbiol. Glucocorticoid-induced formation of cross-linked actin networks in cultured human trabecular meshwork cells. Cross-linked actin networks CLANs in bovine trabecular meshwork cells. A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis. Mol Cell. Directed network wiring identifies a key protein interaction in embryonic stem cell differentiation.
Metabolic regulation by the mitochondrial phosphatase PTPMT1 is required for hematopoietic stem cell differentiation. Cell Stem Cell. Development of a model of elevated intraocular pressure in rats by gene transfer of bone morphogenetic protein 2. Primary cilia signaling mediates intraocular pressure sensation. Immunohistochemical localization of transforming growth factor-beta 1, -beta 2, and -beta 3 latency-associated peptide in human cornea.
Effect of elevated intracellular cAMP levels on actomyosin contraction in bovine trabecular meshwork cells. Lysosomal enzyme and inhibitor levels in the human trabecular meshwork. TGF-beta induces phosphorylation of phosphatase and tensin homolog: implications for fibrosis of the trabecular meshwork tissue in glaucoma.
Targeting the vascular-specific phosphatase PTPRB protects against retinal ganglion cell loss in a pre-clinical model of glaucoma. Presence of an established calcification marker in trabecular meshwork tissue of glaucoma donors. Zaiden M, Beit-Yannai E. MicroRNAa controls angiogenesis and functional recovery of ischemic tissues in mice.
Wandzioch E, Zaret KS. Dynamic signaling network for the specification of embryonic pancreas and liver progenitors. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol. Adv Funct Mater. Agarwal P, Agarwal R. Expert Opin Ther Targets. Changes in aqueous humor dynamics with age and glaucoma. Prog Retin Eye Res. J Cell Physiol. Tamm ER. Myocilin and glaucoma: facts and ideas. Functional role of peroxiredoxin 6 in the eye. Free Radic Biol Med. Disparate differentiation in hemopoietic colonies derived from human paired progenitors.
Single-cell origin of human mixed hemopoietic colonies expressing various combinations of cell lineages. Cellular regulation of hemoglobin switching: evidence for inverse relationship between fetal hemoglobin synthesis and degree of maturity of human erythroid cells.
Self-renewal of hemopoietic stem cells during mixed colony formation in vitro. Disparate differentiation in mouse hemopoietic colonies derived from paired progenitors. Future possibilities in glaucoma therapy. Reactive oxygen species regulate hematopoietic stem cell self-renewal, migration and development, as well as their bone marrow microenvironment. Antioxid Redox Signal. We have doubtless only scratched the surface in terms of the potential for technical improvements to RNA-based reprogramming.
Size-based purification of synthetic mRNA may further reduce activation of cellular antiviral responses and any associated priming of senescence pathways, while possibly obviating the need for an interferon inhibitor 33 , 34 , Numerous candidate RFs have been identified which might yield additional performance gains if incorporated into mRNA cocktails 36 , 37 , 38 , 39 , 40 , while the advantage realized by substituting M 3 O for wild-type Oct4 offers fresh support for the application of engineered chimeric factors to cell-fate direction.
Acceleration of the reprogramming process could likewise come about through optimization of factor stoichiometries 41 , and we note that the mRNA method opens up the possibility of fine-tuning cocktails to address early and late phases of iPSC induction independently. The methodology we describe already represents a major advance over current protocols. It cuts mRNA reprogramming time by as much as a half with even greater reductions in hands-on time and material costs, while removing troublesome steps from the procedure and allowing mRNA to be handled as a media supplement with almost the same facility as growth factors and cytokines.
At the same time, the feeder-free protocol meets a key requirement for moving iPSCs from laboratory to clinic by eliminating potential sources of non-human contamination from the derivation process. If this fast, economical footprint-free iPSC origination technique can be combined with advances in high-throughput propagation and quality assurance of pluripotent clones 45 , the remaining barriers to large-scale, cost-effective production of therapeutically-qualified patient-specific iPSC lines should be few indeed.
The T sequence was introduced by use of a tailed reverse primer. Cocktails were assembled by pooling preps representing the various RFs in the desired stoichiometric ratios. The fraction of each RF used took into account the predicted molecular weight of the respective transcript, all RFs being equimolar except for Oct4 and its derivatives, which were included at 3X molar concentration.
The feeder cells used were G irradiated neonatal human foreskin fibroblasts GlobalStem and FibroGRO mitomycin C-inactivated xeno-free human neonatal fibroblasts Millipore. Cell passaging steps pertaining to xeno-free feeder-based and feeder-free reprogramming trials were conducted using TrypLE Select Gibco , an animal product-free cell dissociation reagent.
All reprogramming experiments described were performed in 6-well tissue culture plates coated with CELLstart Gibco xeno-free substrate in accordance with the manufacturer's directions. In some of the later feeder-based trials the seeding density was increased and feeders were supplemented ad hoc during medium changes in an effort to sustain near-confluent feeder layers in response to the higher attrition rates encountered using novel RF cocktails.
Xeno-free feeders were plated in Pluriton xeno-free medium Stemgent without serum. Media was replaced daily during and after reprogramming, B18R supplementation being discontinued the day after the final transfection. For hour transfections, dose ramping was achieved by delivering 0. Colony picking, expansion, and subsequent immunostaining and trilineage differentiation assays for molecular and functional validation of pluripotency were carried out by the Sanford Burnham Medical Research Institute.
Ectodermal differentiation was performed by breaking up Dispase-detached colonies into chunks and culturing them in neural progenitor cell NPC media within low-attachment plates for 6 days, after which the resulting NPCs were transferred to a tissue-culture plate coated with Fibronectin Sigma.
The plated cells were stained after 2—3 days using primary antibodies against Nestin and Pax6 Abcam diluted LW and JW planned the project. YN and XG participated in discussion. XG provided funding and support. LW conducted the majority of the experiments. JW and YN performed certain plasmid construction work.
We thank Drs. A patent has been filed on the reported technologies. Sci Rep. Published online Sep Author information Article notes Copyright and License information Disclaimer. Received May 25; Accepted Aug All rights reserved. This article has been cited by other articles in PMC. Supplementary Information Supplementary Movie 1. Supplementary Information Supplementary Movie 2. Abstract The therapeutic promise of induced pluripotent stem cells iPSCs has spurred efforts to circumvent genome alteration when reprogramming somatic cells to pluripotency.
Results Our experiments initially focused on the question of whether mRNA reprogramming could be enhanced using engineered variants of Oct4 incorporating an N-terminal MyoD transactivation domain 20 , 21 or a C-terminal triple repeat of the VP16 transactivation domain 22 , or by augmenting the cocktail with transcripts for two additional factors, Rarg and Lrh-1 Open in a separate window. Figure 1. Figure 2. Figure 3. Discussion We have doubtless only scratched the surface in terms of the potential for technical improvements to RNA-based reprogramming.
Reprogramming of human fibroblasts All reprogramming experiments described were performed in 6-well tissue culture plates coated with CELLstart Gibco xeno-free substrate in accordance with the manufacturer's directions. Supplementary Information: Supplementary Movie 1. Click here to view. Supplementary Information: Supplementary Movie 2. Acknowledgments We thank Drs. References Takahashi K. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.
Cell , —76 Reprogramming of murine and human somatic cells using a single polycistronic vector. Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells 27 , —9 Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells 28 , —40 Nature , —70 Virus-free induction of pluripotency and subsequent excision of reprogramming factors.
Nature , —5 Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods 6 , —9 Generation of mouse induced pluripotent stem cells without viral vectors. Science , —53 Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.
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