25 results on '"Rick I. Cohen"'
Search Results
2. Peptide-Based Scaffolds for the Culture and Transplantation of Human Dopaminergic Neurons
- Author
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Prabhas V. Moghe, Janace J. Gifford, Hannah R. Calvelli, Astha Saini, Nanxia Zhao, Rick I. Cohen, Zhiping P. Pang, Nicola L. Francis, and George C. Wagner
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Induced Pluripotent Stem Cells ,0206 medical engineering ,Biomedical Engineering ,Biocompatible Materials ,Bioengineering ,02 engineering and technology ,Striatum ,Biochemistry ,Biomaterials ,03 medical and health sciences ,In vivo ,medicine ,Humans ,Induced pluripotent stem cell ,Cell encapsulation ,030304 developmental biology ,0303 health sciences ,Tissue Scaffolds ,Chemistry ,Dopaminergic Neurons ,Dopaminergic ,Hydrogels ,Original Articles ,020601 biomedical engineering ,In vitro ,Cell biology ,Transplantation ,medicine.anatomical_structure ,nervous system ,Neuron ,Peptides ,Stem Cell Transplantation - Abstract
Cell replacement therapy is a promising treatment strategy for Parkinson's disease (PD); however, the poor survival rate of transplanted neurons is a critical barrier to functional recovery. In this study, we used self-assembling peptide nanofiber scaffolds (SAPNS) based on the peptide RADA16-I to support the in vitro maturation and in vivo post-transplantation survival of encapsulated human dopaminergic (DA) neurons derived from induced pluripotent stem cells. Neurons encapsulated within the SAPNS expressed mature neuronal and midbrain DA markers and demonstrated in vitro functional activity similar to neurons cultured in two dimensions. A microfluidic droplet generation method was used to encapsulate cells within monodisperse SAPNS microspheres, which were subsequently used to transplant adherent, functional networks of DA neurons into the striatum of a 6-hydroxydopamine-lesioned PD mouse model. SAPNS microspheres significantly increased the in vivo survival of encapsulated neurons compared with neurons transplanted in suspension, and they enabled significant recovery in motor function compared with control lesioned mice using approximately an order of magnitude fewer neurons than have been previously needed to demonstrate behavioral recovery. These results indicate that such biomaterial scaffolds can be used as neuronal transplantation vehicles to successfully improve the outcome of cell replacement therapies for PD. IMPACT STATEMENT: Transplantation of dopaminergic (DA) neurons holds potential as a treatment for Parkinson's disease (PD), but low survival rates of transplanted neurons is a barrier to successfully improving motor function. In this study, we used hydrogel scaffolds to transplant DA neurons into PD model mice. The hydrogel scaffolds enhanced survival of the transplanted neurons compared with neurons that were transplanted in a conventional manner, and they also improved recovery of motor function by using significantly fewer neurons than have typically been transplanted to see functional benefits. This cell transplantation technology has the capability to improve the outcome of neuron transplantation therapies.
- Published
- 2020
3. Cell adhesion upregulates the cortical tension and actin cortex thickness during the adhesion process
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Seyedsajad Moazzeni, Rick I. Cohen, David I. Shreiber, Jeffrey Zahn, Zheng Shi, and Hao Lin
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Biophysics - Published
- 2022
4. Elastin-like polypeptides: A strategic fusion partner for biologics
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Charles P. Rabolli, Rick I. Cohen, Martin L. Yarmush, Francois Berthiaume, and Agnes Yeboah
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0106 biological sciences ,0301 basic medicine ,Fusion ,Tropoelastin ,biology ,Chemistry ,Bioengineering ,Nanotechnology ,Biocompatible material ,01 natural sciences ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Biopharmaceutical ,Targeted drug delivery ,010608 biotechnology ,Protein purification ,biology.protein ,Elastin like polypeptides ,Target protein ,Biotechnology - Abstract
Elastin-like peptides (ELPs) are derivatives of tropoelastin with a unique property that allows them to stay soluble below a certain critical temperature but reversibly form aggregates above that temperature. Since they are derived from tropoelastin, ELPs are biocompatible, non-toxic, and non-immunogenic. The unique properties of ELPs have made them a desirable class of fusion tags used in several biomedical applications including targeted drug delivery and enhancing the half-life of protein drugs. The most significant property of an ELP is that when fused to other proteins, the phase transition property of the ELP is maintained, and the target protein can be purified using the thermally driven property of the ELP. The ELP tag purification process is simple and inexpensive, and involves cycling the protein above and below the transition temperature of the ELP fusion followed by centrifugation to obtain the desired protein, without any need for chromatography. Consequently, using ELPs as a purification tag should be potentially interesting to biopharmaceutical companies who spend a significant percentage of their manufacturing costs on expensive protein purification techniques such as chromatography and filtration. However, ELP tags have not yet been used for commercial protein purification due to some challenges of translating this technique, which has been demonstrated mostly in academic laboratories, to a biotechnology manufacturing environment. The article first reviews the state-of-the-art in protein "ELPylation," and discusses some applications which have benefitted from using ELP as a fusion tag. Then, the article discusses the main advantages of using ELP as a purification tag, and evaluates the remaining hurdles for its implementation in industrial protein production. Biotechnol. Bioeng. 2016;113: 1617-1627. © 2016 Wiley Periodicals, Inc.
- Published
- 2016
5. The development and characterization of SDF1α-elastin-like-peptide nanoparticles for wound healing
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Renea Faulknor, Rick I. Cohen, Agnes Yeboah, Rene S. Schloss, Francois Berthiaume, and Martin L. Yarmush
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0301 basic medicine ,Receptors, CXCR4 ,Proteases ,Pathology ,medicine.medical_specialty ,Angiogenesis ,Recombinant Fusion Proteins ,Pharmaceutical Science ,HL-60 Cells ,Mice, Transgenic ,Pharmacology ,Article ,Neovascularization ,030207 dermatology & venereal diseases ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,Diabetes Mellitus ,medicine ,Animals ,Humans ,Receptor ,Skin ,Wound Healing ,integumentary system ,biology ,business.industry ,Fusion protein ,Chemokine CXCL12 ,Elastin ,030104 developmental biology ,biology.protein ,Nanoparticles ,medicine.symptom ,Peptides ,Wound healing ,business - Abstract
Chronic skin wounds are characterized by poor re-epithelialization, angiogenesis and granulation. Previous work has demonstrated that topical stromal cell-derived growth factor-1 (SDF1) promotes neovascularization, resulting in faster re-epithelialization of skin wounds in diabetic mice. However, the clinical usefulness of such bioactive peptides is limited because they are rapidly degraded in the wound environment due to high levels of proteases. Here, we describe the development of a recombinant fusion protein comprised of SDF1 and an elastin-like peptide that confers the ability to self-assemble into nanoparticles. The fusion protein and recombinant human SDF1 showed similar binding characteristics, as indicated by the measured equilibrium dissociation constant (Kd) for the binding of free SDF1 or the fusion protein to the CXCR4 receptor. The biological activity of SDF1-ELP, as measured by intracellular calcium release in HL60 cells was dose dependent, and also very similar to that of free SDF1. In contrast, the biological activity of SDF1-ELP in vivo was significantly superior to that of free SDF1. When applied to full thickness skin wounds in diabetic mice, wounds treated with SDF1-ELP nanoparticles were 95% closed by day 21, and fully closed by day 28, while wounds treated with free SDF1, ELP alone, or vehicle were only 80% closed by day 21, and took 42days to fully close. In addition, the SDF1-ELP nanoparticles significantly increased the epidermal and dermal layer of the healed wound, as compared to the other groups. These results indicate that SDF1-ELP fusion protein nanoparticles are promising agents for the treatment of chronic skin wounds.
- Published
- 2016
6. Microfibrous substrate geometry as a critical trigger for organization, self‐renewal, and differentiation of human embryonic stem cells within synthetic 3‐dimensional microenvironments
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Aaron L. Carlson, Joseph J. Kim, Thomas Neubauer, Rick I. Cohen, Joachim Kohn, Jennifer C. Moore, Prabhas V. Moghe, Charles A. Florek, and Martin Grumet
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Cellular differentiation ,Cell Culture Techniques ,Nanotechnology ,Biochemistry ,Research Communications ,Extracellular matrix ,Biopolymers ,Directed differentiation ,Tissue engineering ,Cell Adhesion ,Genetics ,Humans ,Polylysine ,Cell adhesion ,Molecular Biology ,Embryonic Stem Cells ,Cell Proliferation ,Matrigel ,Tissue Scaffolds ,Chemistry ,Cell Differentiation ,Stereoisomerism ,Embryonic stem cell ,Cell biology ,Transplantation ,Drug Combinations ,Tyrosine ,Proteoglycans ,Collagen ,Laminin ,Biotechnology - Abstract
Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature, with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries, including planar 2-D films, macroporous 3-D sponges, and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self-renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3-D microgeometries can support hESC colony formation, self-renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions.—Carlson, A. L., Florek, C. A., Kim, J. J., Neubauer, T., Moore, J. C., Cohen, R. I., Kohn, J., Grumet, M., Moghe, P. V. Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.
- Published
- 2012
7. E-Cadherin-Expressing Feeder Cells Promote Neural Lineage Restriction of Human Embryonic Stem Cells
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Jocie F. Cherry, Martin Grumet, Rebecca N. Moore, Prabhas V. Moghe, Vani Mathur, and Rick I. Cohen
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Cellular differentiation ,Cell Culture Techniques ,Gene Expression ,Nerve Tissue Proteins ,Biology ,Mice ,Neurosphere ,Animals ,Humans ,Cell Lineage ,Progenitor cell ,Cell Shape ,Embryonic Stem Cells ,Neurons ,Feeder Cells ,Membrane Proteins ,Cell Differentiation ,Cell Biology ,Hematology ,Fibroblasts ,Cadherins ,Antigens, Differentiation ,Molecular biology ,Coculture Techniques ,Neural stem cell ,Cell biology ,Neuroepithelial cell ,Cytoskeletal Proteins ,embryonic structures ,Neural cell adhesion molecule ,Stem cell ,Developmental Biology ,Adult stem cell - Abstract
Human embryonic stem cells (hESCs) represent a promising source of tissues of different cell lineages because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. In this article, we report on a new approach to differentiate hESCs into neural stem cells that can be differentiated further into neuronal restricted cells. We have rapidly and efficiently differentiated hESCs into neural stem cells by presenting the cell adhesion molecule, E-cadherin, to undifferentiated hESCs via E-cadherin transfected fibroblast monolayers. The neural restricted progenitor cells rapidly express nestin and beta-III-tubulin, but not glial fibrillary acidic protein (GFAP) during the 1-week E-cadherin induction phase, suggesting that E-cadherin promotes rapid neuronal differentiation. Further, these cells are able to achieve enhanced neuronal differentiation with the addition of exogenous growth factors. Cadherin-induced hESCs show a loss in Oct4 and nestin expression associated with positive staining for vimentin, neurofilament, and neural cell adhesion molecule. Moreover, blocking by functional E-cadherin antibody and failure of paracrine stimulation suggested that direct E-cadherin engagement is necessary to induce neural restriction. By providing hESCs with molecular cues to promote differentiation, we are able to utilize a specific cell-cell adhesion molecule, E-cadherin, to influence the nature and degree of neural specialization.
- Published
- 2012
8. A Model to Evaluate the Cytotoxicity of the Fungal Volatile Organic Compound 1-octen-3-ol in Human Embryonic Stem Cells
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Rick I. Cohen, Arati A. Inamdar, Joan W. Bennett, and Jennifer C. Moore
- Subjects
Octanols ,Volatile Organic Compounds ,Chromatography ,Cell Survival ,Veterinary (miscellaneous) ,Bacterial growth ,Applied Microbiology and Biotechnology ,Microbiology ,Embryonic stem cell ,In vitro ,Calcein ,Toxicology ,Inhibitory Concentration 50 ,chemistry.chemical_compound ,chemistry ,1-Octen-3-ol ,Humans ,Enantiomer ,Cytotoxicity ,Agronomy and Crop Science ,IC50 ,Cells, Cultured ,Embryonic Stem Cells - Abstract
Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol ("mushroom alcohol"), a major fungal volatile organic compound (VOC) associated with mushroom and mold odors. Using an airborne exposure technique, human embryonic stem cells were exposed for 1 h to different concentrations (0-1,000 ppm) of racemic 1-octen-3-ol and its enantiomers, (R)-(-)-1-octen-3-ol and (S)-(+)-1-octen-3-ol. Cytotoxicity was assayed using both the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and the fluorescently tagged Calcein AM-mediated "live and dead" assay. Racemic 1-octen-3-ol and (S)-(+)-1-octen-3-ol exhibited greater cytotoxicity to the undifferentiated human cell line H1 than did (R)-(-)-1-octen-3-ol. The inhibition concentration 50 (IC(50)) values assessed by the MTS assay for racemic 1-octen-3-ol, (S)-(+)-1-octen-3-ol and (R)-(-)-1-octen-3-ol were, respectively, 109, 98, and 258 ppm. These IC(50) values were 40-80-fold lower than that of vapor phase toluene, an industrial chemical used as a positive control in this study. Our report pioneers the modeling of human embryonic stem cells as an in vitro approach to screen the potential toxicity of fungal VOCs. Human embryonic stem cells exposed to 1-octen-3-ol, and its enantiomers in the vapor phase showed more cytotoxicity than those exposed to toluene.
- Published
- 2011
9. A non‐transformed oligodendrocyte precursor cell line, OL‐1, facilitates studies of insulin‐like growth factor‐I signaling during oligodendrocyte development
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Robert W. Benjamin, William H. Lagarde, A. Joseph D'Ercole, Ping Ye, Rick I. Cohen, Billie M. Moats-Staats, and Ann T Heerens
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Central Nervous System ,Proteolipid protein 1 ,Cell Survival ,Cellular differentiation ,medicine.medical_treatment ,Cell Count ,Article ,Cell Line ,Myelin ,Developmental Neuroscience ,medicine ,Animals ,RNA, Messenger ,Antigens ,Enzyme Inhibitors ,Insulin-Like Growth Factor I ,Myelin Proteolipid Protein ,Cell Shape ,Cells, Cultured ,biology ,Stem Cells ,Growth factor ,Cell Differentiation ,Myelin Basic Protein ,Caspase Inhibitors ,Molecular biology ,Oligodendrocyte ,Rats ,Myelin proteolipid protein ,Myelin basic protein ,Oligodendroglia ,medicine.anatomical_structure ,Animals, Newborn ,Cell culture ,Caspases ,Culture Media, Conditioned ,Antigens, Surface ,biology.protein ,Proteoglycans ,Receptors, Atrial Natriuretic Factor ,Signal Transduction ,Developmental Biology - Abstract
The process by which oligodendrocyte progenitors differentiate into mature oligodendrocytes is complex and incompletely understood in part because of the paucity of oligodendrocyte precursors cell lines that can be studied in culture. We have developed a non-immortalized rat oligodendrocyte precursor line, called OL-1, which behaves in a fashion consistent with developing oligodendrocytes in vivo. This OL-1 line provides a model for the study of oligodendrocyte development and offers an alternative to the CG-4 cell line. When OL-1 cells are propagated in conditioned growth media, they have morphology consistent with immature oligodendrocytes and exhibit A2B5 antigen positive and myelin basic protein-negative immunoreactivity. Withdrawal of conditioned growth media and culture in serum-free medium results in OL-1 cell maturation, manifested by a shift to myelin basic protein-positive immunoreactivity, A2B5 antigen-negative immunoreactivity, decreased NG2 mRNA expression, increased expression of proteolipid protein mRNA, and increased expression of CNP protein. In addition, the expression of proteolipid protein and its splicing variant DM-20 exhibit a pattern that is similar to brain proteolipid protein expression during development. When OL-1 cells are exposed to Insulin-like growth factor-I, there are significant increases in proteolipid protein mRNA expression ( p < 0.05), the number of cell processes ( p < 0.05), and cell number ( p < 0.05). Treatment with the caspase inhibitors Z-DEVD-FMK and Z-VAD-FMK (inhibitors of caspases 3, 6, 7, 8, 10 and 1, 3, 4, respectively), Insulin-like growth factor-I, or both, results in a similar increase in cell number. Because Insulin-like growth factor-I does not substantially increase the BrdU labeling of OL-1 cells, these data collectively indicate that Insulin-like growth factor-I increases OL-1 cell number predominately by promoting survival, rather than stimulating proliferation. This non-immortalized oligodendrocyte precursor cell line, therefore, exhibits behavior consistent with the in vivo development of oligodendrocytes and provides an excellent model for the study of developing oligodendrocytes.
- Published
- 2007
10. Elastin-like polypeptides: A strategic fusion partner for biologics
- Author
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Agnes, Yeboah, Rick I, Cohen, Charles, Rabolli, Martin L, Yarmush, and Francois, Berthiaume
- Subjects
Drug Delivery Systems ,Recombinant Fusion Proteins ,Peptides ,Elastin - Abstract
Elastin-like peptides (ELPs) are derivatives of tropoelastin with a unique property that allows them to stay soluble below a certain critical temperature but reversibly form aggregates above that temperature. Since they are derived from tropoelastin, ELPs are biocompatible, non-toxic, and non-immunogenic. The unique properties of ELPs have made them a desirable class of fusion tags used in several biomedical applications including targeted drug delivery and enhancing the half-life of protein drugs. The most significant property of an ELP is that when fused to other proteins, the phase transition property of the ELP is maintained, and the target protein can be purified using the thermally driven property of the ELP. The ELP tag purification process is simple and inexpensive, and involves cycling the protein above and below the transition temperature of the ELP fusion followed by centrifugation to obtain the desired protein, without any need for chromatography. Consequently, using ELPs as a purification tag should be potentially interesting to biopharmaceutical companies who spend a significant percentage of their manufacturing costs on expensive protein purification techniques such as chromatography and filtration. However, ELP tags have not yet been used for commercial protein purification due to some challenges of translating this technique, which has been demonstrated mostly in academic laboratories, to a biotechnology manufacturing environment. The article first reviews the state-of-the-art in protein "ELPylation," and discusses some applications which have benefitted from using ELP as a fusion tag. Then, the article discusses the main advantages of using ELP as a purification tag, and evaluates the remaining hurdles for its implementation in industrial protein production. Biotechnol. Bioeng. 2016;113: 1617-1627. © 2016 Wiley Periodicals, Inc.
- Published
- 2015
11. A role for semaphorins and neuropilins in oligodendrocyte guidance
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Jeffery L. Barker, Rick I. Cohen, Daniele M. Rottkamp, Dragan Maric, and Lynn D. Hudson
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Neuropilins ,Biology ,Biochemistry ,Oligodendrocyte ,Cellular and Molecular Neuroscience ,medicine.anatomical_structure ,nervous system ,Semaphorin ,embryonic structures ,medicine ,Neuropilin ,Neuroglia ,Progenitor cell ,Growth cone ,Neuroscience ,Progenitor - Abstract
Oligodendrocytes develop in defined CNS regions as progenitor cells, which migrate to their final destinations, encountering soluble and membrane-bound signals that influence their differentiation and potential to myelinate axonal projections. To identify the regulatory genes that may be involved in this process, microarray analysis of developing oligodendroglia was performed. Several neural guidance genes, including members of the neuropilin (NP) and semaphorin families were detected. These findings were verified and expanded upon using RT-PCR with RNA from fluorescent activated cell sorted A2B5+ oligodendrocyte progenitors and O4+ pro-oligodendrocytes isolated from in vitro and in vivo sources. RT-PCR, western and immunocytochemical analyses revealed that oligodendrocytes expressed NP1, several alternatively spliced isoforms of NP2, and a broad spectrum of both soluble (Class 3), membrane-spanning (Class 4-6), and membrane-tethered (Class 7) semaphorin ligands. Class 3 semaphorins, in a modified stripe assay, caused the collapse of oligodendrocyte progenitor growth cones, redirection of processes, and altered progenitor migration. Our data support a role for neuropilins and semaphorins in orchestrating the migration patterns of developing oligodendrocytes in the CNS.
- Published
- 2003
12. Standardized cryopreservation of pluripotent stem cells
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Rick I. Cohen, Maria L. Thompson, Brian Schryver, and Rolf O. Ehrhardt
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Cryopreservation ,Pluripotent Stem Cells ,Cell type ,business.industry ,Cell ,Cold storage ,Cell Biology ,General Medicine ,Biology ,Reference Standards ,Embryonic stem cell ,Cell biology ,Biotechnology ,Cell Line ,medicine.anatomical_structure ,Freezing ,medicine ,Humans ,Stem cell line ,Viability assay ,Induced pluripotent stem cell ,business ,Developmental Biology - Abstract
The successful exploitation of human cells for research, translational, therapeutic, and commercial purposes requires that effective and simple cryopreservation methods be applied for storage in local and master cell banks. Of all the cell types utilized in modern research, human embryonic stem cells and their more recent relatives, induced pluripotent stem cells, are two of the most sensitive to cryopreservation. It is frequently observed that the lack of quality control and proper processing techniques yield poor recovery of pluripotent stem cells. The procedures in this unit have been optimized for handling some of the most recalcitrant stem cell lines, and provide a method for controlled-rate freezing, using minimal equipment that affords levels of cell viability comparable to expensive controlled-rate freezers. The protocol also eliminates the requirement for isopropanol, avoiding the hazards, on-going cost, and inconsistencies associated with its use and disposal. It provides a clinically relevant, inexpensive, reliable, and user-friendly method that successfully prepares cells for long-term cold storage and ensures maximum levels of cell viability post thaw.
- Published
- 2014
13. Fibroblast growth factor-9 modulates the expression of myelin related proteins and multiple fibroblast growth factor receptors in developing oligodendrocytes
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Rick I. Cohen and Karen J. Chandross
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musculoskeletal diseases ,animal structures ,Fibroblast growth factor receptor 2 ,Fibroblast growth factor receptor 1 ,Fibroblast growth factor receptor 4 ,Fibroblast growth factor receptor 3 ,Biology ,Fibroblast growth factor ,Molecular biology ,Oligodendrocyte ,Cellular and Molecular Neuroscience ,Myelin ,medicine.anatomical_structure ,Fibroblast growth factor receptor ,embryonic structures ,medicine ,biological phenomena, cell phenomena, and immunity - Abstract
The effect of fibroblast growth factor (FGF)-9 on the expression of FGF receptors (FGFR) and the major myelin proteins was examined in cultures of developing rat brain oligodendrocytes (OLs), using immunological techniques. FGFR-1, -3, and -4 were expressed at all developmental stages but were not present in isolated myelin fractions. By contrast, FGFR-2 protein was predominantly localized to differentiating cells and myelin. FGF-9 altered FGFR and myelin protein levels during OL differentiation; there was increased expression of FGFR-1 and decreased levels of both FGFR-2 and myelin proteins. Further, FGF-9 stimulated mitogen-associated protein kinase (MAPK) phosphorylation. The effect of FGF-9 on MAPK, however, was transient and less robust in progenitor cells than in differentiated oligodendrocytes. The effects of FGF-9 and FGF-2 on FGFR and myelin protein levels were comparable; both up-regulated FGFR-1, and down-regulated FGFR-2, CNP, PLP and MBP. These findings suggest that FGF-9 may be important for glial cell development. J. Neurosci. Res. 61:273–287, 2000. Published 2000 Wiley-Liss, Inc.
- Published
- 2000
14. Cyclic AMP Regulates PDGF-stimulated signal transduction and differentiation of an immortalized optic-nerve-derived cell line
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Ronald D.G. McKay, Rick I. Cohen, and Guillermina Almazan
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Platelet-derived growth factor ,Physiology ,Inositol Phosphates ,Cellular differentiation ,Aquatic Science ,chemistry.chemical_compound ,medicine ,Animals ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Cell Line, Transformed ,Platelet-Derived Growth Factor ,Forskolin ,biology ,Colforsin ,Cell Differentiation ,Optic Nerve ,Protein-Tyrosine Kinases ,Molecular biology ,Oligodendrocyte ,Rats ,Cell biology ,Oligodendroglia ,Phenotype ,medicine.anatomical_structure ,Bucladesine ,chemistry ,Cell culture ,Insect Science ,biology.protein ,Calcium ,Animal Science and Zoology ,Signal transduction ,Platelet-derived growth factor receptor ,Intracellular ,Signal Transduction - Abstract
To facilitate the study of the molecular events underlying the development of optic-nerve-derived oligodendrocytes and their growth-factor-related signal transduction events, we immortalized perinatal rat optic nerve cells with a temperature-sensitive simian virus 40 large T-antigen, carrying the tsA58 and U19 mutations, via a retrovirus vector. The line, tsU19-9, was selected on the basis of the expression of the neural precursor marker nestin. At the permissive temperature, 33 °C, tsU19-9 cells had a flat epithelial morphology. In contrast, following exposure to platelet-derived growth factor (PDGF), a factor important in the lineage progression of oligodendrocytes, or in the presence of dibutyryl cyclic AMP at 39 °C (the non-permissive temperature), the cells underwent morphological and antigenic differentiation to cells characteristic of the oligodendrocyte lineage. We used this cell line to investigate the binding characteristics of PDGF and related signalling cascades. Competition binding, phosphoinositide hydrolysis and intracellular Ca2+ mobilization assays all demonstrated that the three different isoforms of PDGF (AA, AB and BB) bound to and acted on the cell line. Overnight exposure to forskolin, a treatment that initiated morphological and phenotypic progression into an oligodendrocyte lineage, decreased PDGF-BB-induced intracellular Ca2+ mobilization and inhibited basal and PDGF-stimulated [3H]thymidine incorporation. Our results demonstrate that tsU19-9 may serve as a resource to study early optic-nerve oligodendrocyte development.
- Published
- 1999
15. Identification and Characterization of Early Glial Progenitors Using a Transgenic Selection Strategy
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Peter Paras, Peter E. Braun, Karen J. Chandross, Rick I. Cohen, Michel Gravel, and Lynn D. Hudson
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Genetically modified mouse ,Cell type ,Recombinant Fusion Proteins ,Transgene ,Blotting, Western ,Drug Resistance ,Mice, Transgenic ,Biology ,Article ,Mice ,Genes, Reporter ,Culture Techniques ,medicine ,Animals ,Selection, Genetic ,Progenitor cell ,Kanamycin Kinase ,Microglia ,Reverse Transcriptase Polymerase Chain Reaction ,Stem Cells ,General Neuroscience ,Neural crest ,Neomycin ,DNA ,beta-Galactosidase ,Immunohistochemistry ,Embryonic stem cell ,Anti-Bacterial Agents ,Cell biology ,Oligodendroglia ,medicine.anatomical_structure ,nervous system ,3',5'-Cyclic-AMP Phosphodiesterases ,Schwann Cells ,Sciatic nerve ,Neuroglia ,Neuroscience - Abstract
To define the spatiotemporal development of and simultaneously select for oligodendrocytes (OLs) and Schwann cells (SCs), transgenic mice were generated that expressed a bacterial β-galactosidase (β-gal) and neomycin phosphotransferase fusion protein (βgeo) under the control of murine 2′3′-cyclic nucleotide 3′-phosphodiesterase (muCNP) promoters I and II. Transgenic β-gal activity was detected at embryonic day 12.5 in the ventral region of the rhombencephalon and spinal cord and in the neural crest. When cells from the rhombencephalon were cultured in the presence of G418, surviving cells differentiated into OLs, indicating that during development this brain region provides one source of OL progenitors. Postnatally, robust β-gal activity was localized to OLs throughout the brain and was absent from astrocytes, neurons, and microglia or monocytes. In the sciatic nerve β-gal activity was localized exclusively to SCs. Cultures from postnatal day 10 brain or sciatic nerve were grown in the presence of G418, and within 8–9 d exposure to antibiotic, 99% of all surviving cells were β-gal-positive OLs or SCs. These studies demonstrate that the muCNP-βgeo transgenic mice are useful for identifying OLs and SCs beginning at early stages of the glial cell lineage and throughout their development. This novel approach definitively establishes that the β-gal-positive cells identifiedin vivoare glial progenitors, as defined by their ability to survive antibiotic selection and differentiate into OLs or SCsin vitro. Moreover, this experimental paradigm facilitates the rapid and efficient selection of pure populations of mouse OLs and SCs and further underscores the use of cell-specific promoters in the purification of distinct cell types.
- Published
- 1999
16. Analysis of human embryonic stem cells with regulatable expression of the cell adhesion molecule l1 in regeneration after spinal cord injury
- Author
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Gunho Anthony Lee, Melitta Schachner, Christopher S Park, Myungsik Yoo, and Rick I. Cohen
- Subjects
Neurogenesis ,Central nervous system ,Neural Cell Adhesion Molecule L1 ,Biology ,Cell Line ,Mice ,medicine ,Animals ,Humans ,Spinal cord injury ,Embryonic Stem Cells ,Spinal Cord Injuries ,Doxycycline ,Regeneration (biology) ,Recovery of Function ,Original Articles ,medicine.disease ,Embryonic stem cell ,Cell biology ,Nerve Regeneration ,medicine.anatomical_structure ,Cell culture ,Immunology ,Neurology (clinical) ,Stem cell ,medicine.drug - Abstract
Cell replacement therapy is one potential avenue for central nervous system (CNS) repair. However, transplanted stem cells may not contribute to long-term recovery of the damaged CNS unless they are engineered for functional advantage. To fine tune regenerative capabilities, we developed a human neural cell line expressing L1, a regeneration-conducive adhesion molecule, under the control of a doxycycline regulatable Tet-off promoter. Controlled expression of L1 is desired because overexpression after regenerative events may lead to adverse consequences. The regulated system was tested in several cell lines, where doxycycline completely eliminated green fluorescent protein or L1 expression by 3–5 days in vitro. Increased colony formation as well as decreased proliferation were observed in H9NSCs without doxycycline (hL1-on). To test the role of L1 in vivo after acute compression spinal cord injury of immunosuppressed mice, quantum dot labeled hL1-on or hL1-off cells were injected at three sites: lesion; proximal; and caudal. Mice transplanted with hL1-on cells showed a better Basso Mouse Scale score, when compared to those with hL1-off cells. As compared to the hL1-off versus hL1-on cell transplanted mice 6 weeks post-transplantation, expression levels of L1, migration of transplanted cells, and immunoreactivity for tyrosine hydroxylase were higher, whereas expression of chondroitin sulfate proteoglycans was lower. Results indicate that L1 expression is regulatable in human stem cells by doxycycline in a nonviral engineering approach. Regulatable expression in a prospective nonleaky Tet-off system could hold promise for therapy, based on the multifunctional roles of L1, including neuronal migration and survival, neuritogenesis, myelination, and synaptic plasticity.
- Published
- 2013
17. Nerve Growth Factor and Neurotrophin-3 Differentially Regulate the Proliferation and Survival of Developing Rat Brain Oligodendrocytes
- Author
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William T. Norton, Rick I. Cohen, Ronen Marmur, John A. Kessler, and Mark F. Mehler
- Subjects
medicine.medical_specialty ,Cell Survival ,medicine.medical_treatment ,Tropomyosin receptor kinase B ,Neurotrophin-3 ,Tropomyosin receptor kinase A ,Tropomyosin receptor kinase C ,Internal medicine ,Cell Adhesion ,medicine ,Animals ,Nerve Growth Factors ,Dose-Response Relationship, Drug ,biology ,General Neuroscience ,Growth factor ,Brain ,Articles ,Immunohistochemistry ,Rats ,Cell biology ,Oligodendroglia ,stomatognathic diseases ,Nerve growth factor ,Endocrinology ,nervous system ,biology.protein ,Tyrosine kinase ,Neurotrophin - Abstract
There is increasing evidence that the neurotrophins, particularly nerve growth factor (NGF) and neurotrophin-3 (NT-3), play a role in the regulation of glial development in the CNS. Recent studies have shown that the proliferation of optic nerve-derived O2A progenitors (OLPs) is potentiated by NT-3 in combination with platelet-derived growth factor, whereas NT-3 alone supports the survival of their differentiated progeny (Barres et al., 1994). In this study, we have examined the expression of the high-affinity neurotrophin receptors (trks) and the low-affinity nerve growth factor receptor p75 in developing oligodendrocytes (OLs). In addition, we have examined the effects of NGF and NT-3 on proliferation and survival of OLPs and OLs, respectively. TrkC, the high-affinity NT-3 receptor, and trkA, the high-affinity NGF receptor, are both expressed from the early OLP through the mature OL stage. The truncated form of trkB, lacking the tyrosine kinase domain, and the low-affinity neurotrophin receptor p75 are expressed at low levels in OLPs and are upregulated in mature OLs. NGF and NT-3 both induced the phosphorylation of mitogen-activated protein kinase (MAPK) in OLPs and in OLs. In both OLPs and OLs, NT-3 sustained the activation of MAPK more than NGF. NT-3 enhanced the proliferation of OLPs and supported the survival of OLs. By contrast, unless coadministered with FGF-2, NGF did not exhibit mitogenic effects on OLPs but did enhance the survival of differentiated OLs. Our data demonstrate the presence of functional trkA and trkC in developing OLs and indicate that both NGF and NT-3 have a broad spectrum of developmental actions on cells of the OL lineage.
- Published
- 1996
18. Altered Connexin Expression after Peripheral Nerve Injury
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Phyllis Bieri, Karen J. Chandross, Rolf Dermietzel, Rick I. Cohen, John A. Kessler, David C. Spray, and Eva Simburger
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Pathology ,medicine.medical_specialty ,Nerve Crush ,Neural Conduction ,Schwann cell ,Connexin ,Nerve Tissue Proteins ,In situ hybridization ,Biology ,Connexins ,Rats, Sprague-Dawley ,Cellular and Molecular Neuroscience ,Peripheral Nerve Injuries ,medicine ,Animals ,Peripheral Nerves ,RNA, Messenger ,Molecular Biology ,Cells, Cultured ,In Situ Hybridization ,Regulation of gene expression ,Messenger RNA ,Macrophages ,Cell Biology ,Anatomy ,Fibroblasts ,medicine.disease ,Sciatic Nerve ,Nerve Regeneration ,Rats ,medicine.anatomical_structure ,Gene Expression Regulation ,nervous system ,Connexin 43 ,Nerve Degeneration ,Peripheral nerve injury ,Crush injury ,Schwann Cells ,Sciatic nerve - Abstract
The identification of connexin32 (Cx32) in myelinating Schwann cells and the association of Cx32 mutations with peripheral neuropathies suggest a functional role for gap junction proteins in the nerve. However, after nerve crush injury, Cx32 expression dramatically decreases in Schwann cells in the degenerating region, returning to control levels at newly formed nodes of Ranvier and Schmidt-Lantermann incisures by 30 days. The present study examined increases in expression of other connexins that occur after peripheral nerve injury. A 56/58-kDa connexin46 (Cx46) protein species was detected in adult rat sciatic nerve, along with very low levels of Cx46 mRNA. However, by 3 days after crush injury, coincident with changes in Schwann cell phenotype, Cx46 mRNA rapidly increased in the degenerating regions. Additionally, the 56/58-kDa Cx46 protein species present in adult nerve decreased and a 53-kDa Cx46 species, which was also present in cultured Schwann cells, became apparent. Connexin43 (Cx43) mRNA and protein, which was localized to perineurial cells in adult nerve, dramatically increased in endoneurial fibroblasts in the crush and distal regions by 3 days, coincident with macrophage infiltration. By 12 days after injury, Cx43 decreased and was comparable to normal nerve. These results suggest that enhanced expression of Cx46 and Cx43, by nonneuronal cells, may be important for the injury and regenerative responses of peripheral nerves.
- Published
- 1996
19. Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles
- Author
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Dorian M. Hall, Mahlet G. Tadesse, Biagio Saitta, Kenro Kusumi, Vitali Y. Lounev, Eric F. Rappaport, Rick I. Cohen, Dilusha A. William, Arlene Carlton, Jay Leonard, and Vladimir Markov
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Stromal cell ,Cellular differentiation ,Population ,Cell Culture Techniques ,Cell Separation ,Biology ,Cell morphology ,Immunophenotyping ,medicine ,Humans ,education ,Oligonucleotide Array Sequence Analysis ,education.field_of_study ,Gene Expression Profiling ,Mesenchymal stem cell ,Cell Differentiation ,Mesenchymal Stem Cells ,Cell Biology ,Hematology ,Fetal Blood ,Molecular biology ,Up-Regulation ,Gene expression profiling ,Adult Stem Cells ,Kinetics ,Hepatocyte growth factor ,Stromal Cells ,Developmental Biology ,Adult stem cell ,medicine.drug - Abstract
Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct isogenic MSC populations isolated from umbilical cord blood (UCB1 and UCB2). The use of isogenic populations eliminated differences contributed by genetic background. We characterized these UCB MSCs for cell morphology, growth kinetics, immunophenotype, and potential for differentiation. UCB1 displayed faster growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2. However, osteogenic differentiation was stronger for the UCB2 population. To identify MSC-specific genes and developmental genes associated with observed phenotypic differences, we performed expression analysis using Affymetrix microarrays and compared them to bone marrow (BM) MSCs. We compared UCB1, UCB2, and BM and identified distinct gene expression patterns. Selected clusters were analyzed demonstrating that genes of multiple developmental pathways, such as transforming growth factor-beta (TGF-beta) and wnt genes, and markers of early embryonic stages and mesodermal differentiation displayed significant differences among the MSC populations. In undifferentiated UCB1 cells, multiple genes were significantly up-regulated (p < 0.0001): peroxisome proliferation activated receptor gamma (PPARG), which correlated with adipogenic differentiation capacities, hepatocyte growth factor (HGF), and stromal-derived factor 1 (SDF1/CXCL12), which could both potentially contribute to the higher growth kinetics observed in UCB1 cells. Overall, the results confirmed the presence of two distinct isogenic UCB-derived cell populations, identified gene profiles useful to distinguish MSC types with different lineage differentiation potentials, and helped clarify the heterogeneity observed in these cells.
- Published
- 2007
20. A role for semaphorins and neuropilins in oligodendrocyte guidance
- Author
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Rick I, Cohen, Daniele M, Rottkamp, Dragan, Maric, Jeffery L, Barker, and Lynn D, Hudson
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Rats, Sprague-Dawley ,Alternative Splicing ,Oligodendroglia ,Cell Movement ,Multigene Family ,Animals ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Neuropilins ,Semaphorins ,Cells, Cultured ,Oligonucleotide Array Sequence Analysis ,Rats - Abstract
Oligodendrocytes develop in defined CNS regions as progenitor cells, which migrate to their final destinations, encountering soluble and membrane-bound signals that influence their differentiation and potential to myelinate axonal projections. To identify the regulatory genes that may be involved in this process, microarray analysis of developing oligodendroglia was performed. Several neural guidance genes, including members of the neuropilin (NP) and semaphorin families were detected. These findings were verified and expanded upon using RT-PCR with RNA from fluorescent activated cell sorted A2B5+ oligodendrocyte progenitors and O4+ pro-oligodendrocytes isolated from in vitro and in vivo sources. RT-PCR, western and immunocytochemical analyses revealed that oligodendrocytes expressed NP1, several alternatively spliced isoforms of NP2, and a broad spectrum of both soluble (Class 3), membrane-spanning (Class 4-6), and membrane-tethered (Class 7) semaphorin ligands. Class 3 semaphorins, in a modified stripe assay, caused the collapse of oligodendrocyte progenitor growth cones, redirection of processes, and altered progenitor migration. Our data support a role for neuropilins and semaphorins in orchestrating the migration patterns of developing oligodendrocytes in the CNS.
- Published
- 2003
21. Carbachol stimulates c-fos expression and proliferation in oligodendrocyte progenitors
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Guillermina Almazan, Rick I Cohen, and Eduardo Molina-Holgado
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medicine.medical_specialty ,Carbachol ,Gene Expression ,Biology ,Rats, Sprague-Dawley ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Internal medicine ,Muscarinic acetylcholine receptor ,medicine ,Animals ,Molecular Biology ,Protein kinase C ,Acetylcholine receptor ,Dose-Response Relationship, Drug ,Muscarinic antagonist ,Immunohistochemistry ,Oligodendrocyte ,Cell biology ,Rats ,Oligodendroglia ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Proto-Oncogene Proteins c-fos ,Acetylcholine ,Bromodeoxyuridine ,Cell Division ,medicine.drug - Abstract
To determine if muscarinic receptor-activation plays a role in oligodendrocyte development, the effect of carbachol, a stable acetylcholine analog, on gene expression and proliferation was investigated. Using Northern blot analysis we showed that carbachol caused a time and concentration-dependent increase in c-fos mRNA. This effect was blocked by atropine, a non-selective muscarinic antagonist. In addition, the muscarinic-stimulated c-fos increase was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C (PKC), but not by N-2-(p-bromocinnamylamino)-ethyl-5-isoquinoline-sulfonamide (H-89), a potent inhibitor of protein kinase A, suggesting the involvement of PKC in mediating the response. Down-regulation of PKC by overnight pre-treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) blocked only the phorbol ester-stimulated c-fos accumulation while no effect was observed in the carbachol-induced response. These results suggested that carbachol stimulated an H-7 sensitive PKC pathway which may be different than that activated by TPA. Further evidence for two separate mechanisms of proto-oncogene induction was provided by the additive effect of carbachol and TPA. Induction of c-fos mRNA by carbachol was dependent on both influx of extracellular Ca2+ and release from intracellular stores, as both EDTA and BAPTA blocked the response. Since activation of muscarinic receptors can affect cell division in other cellular systems, the effect of carbachol on [ 3 H ]thymidine and bromodeoxyuridine incorporation into oligodendrocyte DNA was measured. Carbachol stimulated DNA synthesis in oligodendrocyte progenitors. This effect was mediated by muscarinic receptors as [ 3 H ]thymidine incorporation was prevented or significantly reduced by the addition of atropine. In conclusion, the present findings suggest that, the neurotransmitter, acetylcholine may act as a trophic factor in developing oligodendrocytes, regulating their growth and development in the central nervous system.
- Published
- 1996
22. TNF alpha inhibits Schwann cell proliferation, connexin46 expression, and gap junctional communication
- Author
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Karen J. Chandross, David C. Spray, Rolf Dermietzel, Rick I. Cohen, Nalin M. Kumar, Marian Kremer, and John A. Kessler
- Subjects
Cell division ,medicine.medical_treatment ,Schwann cell ,Nerve Tissue Proteins ,Cell Communication ,Polymerase Chain Reaction ,Connexins ,Schwann cell proliferation ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Glial Fibrillary Acidic Protein ,medicine ,Animals ,RNA, Messenger ,Molecular Biology ,Cells, Cultured ,Lucifer yellow ,Glial fibrillary acidic protein ,biology ,Tumor Necrosis Factor-alpha ,Gap junction ,Gap Junctions ,Cell Biology ,Sciatic Nerve ,Cell biology ,Rats ,medicine.anatomical_structure ,Cytokine ,nervous system ,chemistry ,Animals, Newborn ,Gene Expression Regulation ,biology.protein ,Tumor necrosis factor alpha ,Schwann Cells ,Myelin P0 Protein ,Cell Division - Abstract
Schwann cell responses to nerve injury are stimulated, in part, by inflammatory cytokines. This study compares changes in the phenotype of cultured Schwann cells after exposure to the cytokine tumor necrosis factor (TNF)-alpha or the mitogen neu differentiation factor (NDF)-beta. TNF alpha inhibited proliferation in a dose-dependent manner without altering Schwann cell survival. TNF alpha also reduced both gap junctional conductance and Lucifer yellow dye coupling between Schwann cells. Moreover, both Po and glial fibrillary acidic protein (GFAP) immunoreactivity were reduced. By contrast, NDF beta initially had little effect on cell division although it reduced junctional coupling within 8 h. However, by 48 h, NDF beta stimulated proliferation with a concomitant increase in coupling. Dividing Schwann cells (BrdU+) were preferentially dye coupled compared to nondividing cells, indicating an association between proliferation and coupling. Moreover, cultured Schwann cells expressed connexin46 mRNA and protein, and changes in the levels of the protein correlated with the degree of proliferation and coupling. The data thus provide evidence for cytokine-induced modulation of Schwann cell antigenic phenotype, proliferation, and gap junction properties. These observations suggest that enhanced gap junctional communication among Schwann cells after nerve injury could help to coordinate cellular responses to the injury, and that TNF alpha may be a signal which terminates proliferation as well as junctional communication.
- Published
- 1996
23. Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors
- Author
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Ronald P. Hart, Jonathan Davila, Loyal A. Goff, Hao Wu, Mavis R. Swerdel, Yi E. Sun, Rick I. Cohen, and Jennifer C. Moore
- Subjects
RISC complex ,Cellular differentiation ,Cell Biology/Developmental Molecular Mechanisms ,Eukaryotic Initiation Factor-2 ,lcsh:Medicine ,Computational biology ,Biology ,Polymerase Chain Reaction ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,microRNA ,Humans ,Immunoprecipitation ,lcsh:Science ,Induced pluripotent stem cell ,Cell Biology/Gene Expression ,Embryonic Stem Cells ,Oligonucleotide Array Sequence Analysis ,030304 developmental biology ,Neurons ,Regulation of gene expression ,Genetics ,0303 health sciences ,Genome ,Multidisciplinary ,Gene Expression Profiling ,lcsh:R ,Cell Differentiation ,Genetics and Genomics/Bioinformatics ,Argonaute ,Neuroscience/Neurodevelopment ,Developmental Biology/Stem Cells ,Chromatin ,Gene expression profiling ,MicroRNAs ,Gene Expression Regulation ,Karyotyping ,030220 oncology & carcinogenesis ,Molecular Biology/Post-Translational Regulation of Gene Expression ,Argonaute Proteins ,RNA ,Genetics and Genomics/Gene Discovery ,lcsh:Q ,Research Article ,Computational Biology/Genomics - Abstract
Background: MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. Methodology/Principal Findings: SOLiD ultra-deep sequencing identified .10 7 unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. Conclusions/Significance: Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.
- Published
- 2009
24. Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction
- Author
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Michael C. Naski, Michael Weinstein, David M. Ornitz, Rick I. Cohen, Xiaoling Xu, Cuiling Li, Chu-Xia Deng, and Philip Leder
- Subjects
musculoskeletal diseases ,Male ,medicine.medical_specialty ,Mesoderm ,Fibroblast Growth Factor 8 ,Ectoderm ,Biology ,Fibroblast growth factor ,Polymerase Chain Reaction ,Limb bud ,Mice ,FGF8 ,Pregnancy ,Internal medicine ,medicine ,Limb development ,Animals ,Receptor, Fibroblast Growth Factor, Type 2 ,Growth Substances ,Molecular Biology ,DNA Primers ,Sequence Deletion ,Embryonic Induction ,Mice, Knockout ,FGF10 ,Base Sequence ,Fibroblast growth factor receptor 2 ,Gene Expression Regulation, Developmental ,Receptor Protein-Tyrosine Kinases ,Extremities ,Receptors, Fibroblast Growth Factor ,Placentation ,Cell biology ,Fibroblast Growth Factors ,stomatognathic diseases ,Endocrinology ,medicine.anatomical_structure ,Phenotype ,Ear, Inner ,embryonic structures ,Mutation ,Female ,Fibroblast Growth Factor 10 ,Developmental Biology ,Signal Transduction - Abstract
FGFR2 is a membrane-spanning tyrosine kinase that serves as a high affinity receptor for several members of the fibroblast growth factor (FGF) family. To explore functions of FGF/FGFR2 signals in development, we have mutated FGFR2 by deleting the entire immunoglobin-like domain III of the receptor. We showed that murine FGFR2 is essential for chorioallantoic fusion and placenta trophoblast cell proliferation. Fgfr2ΔgIII/ΔIgIII embryos displayed two distinct defects that resulted in failures in formation of a functional placenta. About one third of the mutants failed to form the chorioallantoic fusion junction and the remaining mutants did not have the labyrinthine portion of the placenta. Consequently, all mutants died at 10-11 days of gestation. Interestingly, Fgfr2ΔgIII/ΔIgIII embryos do not form limb buds. Consistent with this defect, the expression of Fgf8, an apical ectodermal factor, is absent in the mutant presumptive limb ectoderm, and the expression of Fgf10, a mesenchymally expressed limb bud initiator, is down regulated in the underlying mesoderm. These findings provide direct genetic evidence that FGF/FGFR2 signals are absolutely required for vertebrate limb induction and that an FGFR2 signal is essential for the reciprocal regulation loop between FGF8 and FGF10 during limb induction.
25. Efficient, high-throughput transfection of human embryonic stem cells
- Author
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Kevin Thompson, Christopher L. Ricupero, Jennifer C. Moore, Alana J. Toro-Ramos, Cynthia Camarillo, Rick I. Cohen, Ronald P. Hart, Kristin Atze, Mark A. Brenneman, and Percy Luk Yeung
- Subjects
Pluripotent Stem Cells ,animal structures ,Cell Survival ,viruses ,Genetic Vectors ,Green Fluorescent Proteins ,Medicine (miscellaneous) ,Biology ,Transfection ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,03 medical and health sciences ,0302 clinical medicine ,RNA interference ,Humans ,Viability assay ,RNA, Small Interfering ,Cells, Cultured ,Embryonic Stem Cells ,030304 developmental biology ,0303 health sciences ,Gene knockdown ,Oligonucleotide ,Research ,Electroporation ,fungi ,Cell Differentiation ,DNA ,Cell Biology ,Flow Cytometry ,Embryonic stem cell ,Cell biology ,030220 oncology & carcinogenesis ,embryonic structures ,Molecular Medicine ,RNA Interference ,Stem cell - Abstract
Introduction Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. Methods We investigated the ability of two commercially available electroporation systems, the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. Results Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. Conclusions Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC.
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