High-Efficiency Transduction and Long-Term Gene Expression with a Murine Stem Cell Retroviral Vector Encoding the Green Fluorescent Protein in Human Marrow Stromal Cells

Objectives: Treatment of vocal fold scarring remains a therapeutic challenge. Our group previously reported the efficacy of treating injured vocal folds by implantation of bone marrow—derived stromal cells containing mesenchymal stem cells. Appropriate scaffolding is necessary for the stem cell implant to achieve optimal results. Terudermis is an atelocollagen sponge derived from calf dermis. It has large pores that permit cellular entry and is degraded in vivo. These characteristics suggest that this material may be a good candidate for use as scaffolding for implantation of cells. The present in vitro study investigated the feasibility of using Terudermis as such a scaffold. Methods: Bone marrow—derived stromal cells were obtained from GFP (green fluorescent protein) mouse femurs. The cells were seeded into Terudermis and incubated for 5 days. Their survival, proliferation, and expression of extracellular matrix were examined. Results: Bone marrow—derived stromal cells adhered to Terudermis and underwent significant proliferation. Immunohistochemical examination demonstrated that adherent cells were positive for expression of vimentin, desmin, fibronectin, and fsp1 and negative for beta III tubulin. These findings indicate that these cells were mesodermal cells and attached to the atelocollagen fibers biologically. Conclusions: The data suggest that Terudermis may have potential as stem cell implantation scaffolding for the treatment of scarred vocal folds.

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Bone Marrow Stromal Cells Promote Neuronal Restoration in Rats with Traumatic Brain Injury: Involvement of GDNF Regulating BAD and BAX Signaling

Cellular Physiology and Biochemistry ◽

10.1159/000443031 ◽

2016 ◽

Vol 38 (2) ◽

pp. 748-762 ◽

Cited By ~ 17

Author(s):

Qin Shen ◽

Yong Yin ◽

Qing-Jie Xia ◽

Na Lin ◽

You-Cui Wang ◽

...

Keyword(s):

Traumatic Brain Injury ◽

Bone Marrow ◽

Brain Injury ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Fluorescent Protein ◽

Neurological Function ◽

Marrow Stromal Cells ◽

Terminal Deoxynucleotidyl Transferase ◽

Green Fluorescent

Background/Aims: To investigate the effects of bone marrow stromal cells (BMSCs) and underlying mechanisms in traumatic brain injury (TBI). Methods: Cultured BMSCs from green fluorescent protein-transgenic mice were isolated and confirmed. Cultured BMSCs were immediately transplanted into the regions surrounding the injured-brain site to test their function in rat models of TBI. Neurological function was evaluated by a modified neurological severity score on the day before, and on days 7 and 14 after transplantation. After 2 weeks of BMSC transplantation, the brain tissue was harvested and analyzed by microarray assay. And the coronal brain sections were determined by immunohistochemistry with mouse anti-growth-associated protein-43 kDa (anti-GAP-43) and anti-synaptophysin to test the effects of transplanted cells on the axonal regeneration in the host brain. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and Western blot were used to detect the apoptosis and expression of BAX and BAD. Results: Microarray analysis showed that BMSCs expressed growth factors such as glial cell-line derived neurotrophic factor (GDNF). The cells migrated around the injury sites in rats with TBI. BMSC grafts resulted in an increased number of GAP-43-immunopositive fibers and synaptophysin-positive varicosity, with suppressed apoptosis. Furthermore, BMSC transplantation significantly downregulated the expression of BAX and BAD signaling. Moreover, cultured BMSC transplantation significantly improved rat neurological function and survival. Conclusion: Transplanted BMSCs could survive and improve neuronal behavior in rats with TBI. Mechanisms of neuroprotection and regeneration were involved, which could be associated with the GDNF regulating the apoptosis signals through BAX and BAD.

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Green Fluorescent Protein: A Tool for Gene Expression and Cell Biology in Mycobacteria

Mycobacteria Protocols ◽

10.1385/0-89603-471-2:245 ◽

2003 ◽

pp. 245-260

Author(s):

Laura E. Via ◽

Subramanian Dhandayuthapani ◽

Dusanka Deretic ◽

V. Deretic

Keyword(s):

Gene Expression ◽

Green Fluorescent Protein ◽

Cell Biology ◽

Fluorescent Protein ◽

Protein A ◽

Green Fluorescent

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Stem Cell Defect in Ubiquitin-Green Fluorescent Protein Mice Facilitates Engraftment of Lymphoid-Primed Hematopoietic Stem Cells

Stem Cells ◽

10.1002/stem.2828 ◽

2018 ◽

Vol 36 (8) ◽

pp. 1237-1248

Author(s):

Kateřina Faltusová ◽

Katarína Szikszai ◽

Martin Molík ◽

Jana Linhartová ◽

Petr Páral ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Green Fluorescent Protein ◽

Hematopoietic Stem Cells ◽

Fluorescent Protein ◽

Hematopoietic Stem ◽

Green Fluorescent ◽

Cell Defect

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Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells

Blood ◽

10.1182/blood.v90.9.3304 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3304-3315 ◽

Cited By ~ 40

Author(s):

Marti F.A. Bierhuizen ◽

Yvonne Westerman ◽

Trudi P. Visser ◽

Wati Dimjati ◽

Albertus W. Wognum ◽

...

Keyword(s):

Bone Marrow ◽

Green Fluorescent Protein ◽

Gene Transfer ◽

Fluorescent Protein ◽

Bone Marrow Cells ◽

Retroviral Vector ◽

Egfp Expression ◽

Green Fluorescent ◽

Marrow Cells

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

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Interleukin-1 (IL-1) Inhibits Growth of Cytomegalovirus in Human Marrow Stromal Cells: Inhibition Is Reversed Upon Removal of IL-1

Blood ◽

10.1182/blood.v94.2.572 ◽

1999 ◽

Vol 94 (2) ◽

pp. 572-578 ◽

Cited By ~ 16

Author(s):

Mineo Iwata ◽

Jeff Vieira ◽

Michael Byrne ◽

Heidi Horton ◽

Beverly Torok-Storb

Keyword(s):

Stromal Cells ◽

Neutralizing Antibodies ◽

Fluorescent Protein ◽

Interleukin 1 ◽

Elongation Factor ◽

Marrow Stromal Cells ◽

Type I ◽

Untreated Culture ◽

Green Fluorescent ◽

Induced Changes

Abstract A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1 (IL-1) and IL-1β completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1β was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1–treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 ± 3 v 151 ± 12/well, respectively; GFP intensity was 535 ± 165 v 6,516 ± 652/well, respectively, in 4 separate experiments). At day 21, when IL-1β–treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1β, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1β effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1–induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1β–treated and nontreated HS-27a cells has identified some candidate molecules.

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