Isolation of Mesenchymal Stem Cells (MSCs) from Green Fluorescent Protein Positive (GFP+) Transgenic Rodents: The Grass Is Not Always Green(er)

Traumatic brain injury is one of the leading causes of mortality and morbidity worldwide. At present there is no effective treatment. Previous studies have demonstrated that topical application of adipose tissue-derived mesenchymal stem cells can improve functional recovery in experimental traumatic brain injury. In this study, we evaluated whether hypoxic preconditioned mesenchymal stem cells could enhance the recovery from traumatic brain injury. Traumatic brain injury was induced with an electromagnetically controlled cortical impact device. Two million mesenchymal stem cells derived from the adipose tissue of transgenic green fluorescent protein Sprague-Dawley rats were cultured under either hypoxic (2.5% O2 for 18 hours) ( N = 30) or normoxic (18% O2) ( N = 30) conditions, then topically applied to the exposed cerebral cortex within 1 hour after traumatic brain injury. A thin layer of fibrin was used to fix the cells in position. No treatment was given to the animals with traumatic brain injury ( N = 30). Animals that underwent craniectomy without traumatic brain injury were treated as the sham group ( N = 15). Neurological functions were evaluated with water maze, Roto-rod and gait analysis. Animals were sacrificed at days 3, 7, and 14 for microscopic examinations and real-time polymerase chain reaction analysis. The rats treated with hypoxic mesenchymal stem cells showed the greatest improvement in neurological function recovery. More green fluorescent protein-positive cells were found in the injured brain parenchyma treated with hypoxic mesenchymal stem cells that co-expressed glial fibrillary acidic protein, Nestin, and NeuN. Moreover, there was early astrocytosis triggered by the infiltration of more glial fibrillary acidic protein-positive cells and microgliosis was suppressed with fewer ionized calcium binding adapter molecule 1-positive cells in the penumbra region of hypoxic mesenchymal stem cells group at day 3. Compared with normoxic mesenchymal stem cells and traumatic brain injury only groups, there was significantly ( p < 0.05) less neuronal death in both the hippocampus and penumbral regions in sections treated with hypoxic mesenchymal stem cells as determined by Cresyl violet and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining respectively. The expression of pro-inflammatory genes (interleukin 6, interleukin 1a, interleukin 1b, tumor necrosis factor α) was upregulated and apoptotic gene (Caspase-3) expression was suppressed at day 3. Anti-inflammatory (interleukin 10) and anti-apoptotic (BCL2 associated agonist of cell death) gene expression was upregulated at days 7 and 14. Our study showed that a hypoxic precondition enhanced the beneficial effects of mesenchymal stem cells on neurological recovery after traumatic brain injury.

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Enhanced green fluorescent protein-expressing human mesenchymal stem cells retain neural marker expression

Journal of Neuroimmunology ◽

10.1016/j.jneuroim.2007.10.019 ◽

2008 ◽

Vol 193 (1-2) ◽

pp. 59-67 ◽

Cited By ~ 11

Author(s):

David Gordon ◽

Colin P. Glover ◽

Andria M. Merrison ◽

James B. Uney ◽

Neil J. Scolding

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Green Fluorescent Protein ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Human Mesenchymal Stem Cells ◽

Neural Marker ◽

Marker Expression ◽

Green Fluorescent

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Survival and Immunogenicity of Mesenchymal Stem Cells From the Green Fluorescent Protein Transgenic Rat in the Adult Rat Brain

Neurorehabilitation and Neural Repair ◽

10.1177/1545968309357745 ◽

2010 ◽

Vol 24 (7) ◽

pp. 645-656 ◽

Cited By ~ 30

Author(s):

Teresa C. Moloney ◽

Peter Dockery ◽

Anthony J. Windebank ◽

Frank P. Barry ◽

Linda Howard ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Adult Brain ◽

Transgenic Rat ◽

Reporter Protein ◽

Technical Limitation ◽

Green Fluorescent ◽

The Brain

Background. A major technical limitation in preclinical cell replacement research is the ability to discriminate between donor and host tissue after transplantation. This problem has been lessened by the availability of transgenic animals that express “reporter” genes, such as green fluorescent protein (GFP). Objective. We determined the usefulness of one such transgenic reporter rat to assess the survival of bone marrow—derived rat mesenchymal stem cells (MSCs) following direct transplantation into the intact adult brain. We also sought to determine if the expression of GFP in the brain affected the survival of the MSCs or the host’s neuroimmune response to the cells. Methods. Rats received intrastriatal injections of sterile transplantation medium, 100 000 normal MSCs, or 100 000 GFP-MSCs and were killed humanely 1, 4, 7, 28, and 42 days posttransplantation for astrocyte and microglial immunohistochemical staining. Results. GFP-MSCs were evident at each examination, although their survival declined over time. Graft volume estimates comparing normal and GFP-MSCs revealed that GFP expression did not adversely affect the survival of the stem cells in the brain. Furthermore, immunostaining for astrocytes and microglia revealed that expression of the reporter protein did not affect the immunogenicity of the stem cells. Conclusions. These data indicate the usefulness of GFP for investigating the survival of MSCs following transplantation to the brain. However, the mechanisms responsible for the poor survival of the stem cells must be elucidated if these cells are to serve cell-based therapies for neurodegenerative disorders.

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Comparison of chloromethyl-dialkylcarbocyanine and green fluorescent protein for labeling human umbilical mesenchymal stem cells

Biotechnology Letters ◽

10.1007/s10529-014-1692-1 ◽

2014 ◽

Vol 37 (2) ◽

pp. 437-447 ◽

Cited By ~ 8

Author(s):

Fang Ji ◽

Hong-Gang Duan ◽

Chun-Quan Zheng ◽

Jing Li

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Green Fluorescent ◽

Umbilical Mesenchymal Stem Cells

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In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

Stem Cells International ◽

10.1155/2016/1659275 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Intekhab Islam ◽

Gopu Sriram ◽

Mingming Li ◽

Yu Zou ◽

Lulu Li ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Green Fluorescent Protein ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Fluorescent Protein ◽

Embryonic Stem ◽

Differentiation Potential ◽

Green Fluorescent

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

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