ABSTRACT
PURPOSE: Reelin is important in the guidance of neuronal stem cells in the central nervous system during normal development. We wished to determine whether reelin is expressed in the retina and cornea after injury. METHODS: Mice underwent laceration of their retina as well as corneal epithelial debridement. The mice were sacrificed at 3 days, and eyes were fixed and stained for reelin expression and reelin messenger ribonucleic acid (mRNA). RESULTS: In normal eyes, reelin was expressed only at very low levels in the ganglion cell layer of the retina and the endothelial cell layer of the cornea. In injured eyes, there was marked expression in reelin immunoreactivity in the retina and cornea. Reelin gene expression was seen in the retina and cornea. CONCLUSIONS: Reelin is expressed during normal retinogenesis. This study shows that reelin is also upregulated following injury to the retina and cornea. The expression of reelin following injury suggests that reelin may play an important role in regulating stem cell trafficking in neuronal and nonneuronal tissues following injury similar to its role in normal organogenesis.
Subject(s)
Cell Adhesion Molecules, Neuronal/genetics , Corneal Injuries , Extracellular Matrix Proteins/genetics , Eye Injuries, Penetrating/genetics , Nerve Tissue Proteins/genetics , RNA, Messenger/metabolism , Retina/injuries , Serine Endopeptidases/genetics , Up-Regulation/physiology , Animals , Blotting, Western , Cell Adhesion Molecules, Neuronal/metabolism , Cornea/metabolism , Extracellular Matrix Proteins/metabolism , Eye Injuries, Penetrating/metabolism , In Situ Hybridization , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Reelin Protein , Retina/metabolism , Serine Endopeptidases/metabolismABSTRACT
We have reported that transplantation of adrenal medullary chromaffin cells that release endogenous opioid peptides into pain modulatory regions in the CNS produce significant antinociceptive effects in patients with terminal cancer pain. However, the usefulness of this procedure is minimal because the availability of human adrenal tissue is very limited. Alternative xenogeneic materials, such as porcine and bovine adrenal chromaffin cells present problems of immune rejection and possible pathogenic contamination. In an attempt to develop opioid peptide-producing cells of autologous origin, we have transfected human mesenchymal stem cells (hMeSCs) with a mammalian expression vector containing a fusion gene of green fluorescent protein (GFP) and human preproenkephalin (hPPE), a precursor protein for enkephalin opioid peptides. Enkephalins are major neurotransmitters that play an important role in analgesia by activating peripheral opioid receptors. Following the establishment of stable transfection of hMeSCs, the expressions of hPPE and GFP were confirmed and the production of methionine enkephalin (Met-enkephalin) was significantly increased compared to control naive hMeSCs (p < 0.05). Our in vitro data demonstrated that genetically engineered hMeSCs with transfected hPPE gene can constitutively produce opioid peptide Met-enkephalin at an augmented high level. hMeSCs are relatively easy to isolate from a patient's bone marrow aspirates and expand in culture by repeated passages. Autologous hMeSCs would not require immunosuppression when transplanted back into the same patient. Through targeted gene manipulation such as hPPE gene transfection, this may offer a virtually unlimited safe cell supply for the treatment of opioid-sensitive pain in humans.
Subject(s)
Analgesics/metabolism , Enkephalin, Methionine/genetics , Enkephalin, Methionine/metabolism , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/metabolism , Analgesics/therapeutic use , Analgesics, Opioid/therapeutic use , Cell Proliferation , Cells, Cultured , DNA/genetics , Enkephalins/genetics , Gene Expression Regulation/genetics , Gene Fusion , Genetic Engineering , Genetic Vectors , Green Fluorescent Proteins/genetics , Humans , Mesenchymal Stem Cells/cytology , Pain/drug therapy , Protein Precursors/genetics , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
PURPOSE: Recent reports show that marrow derived mesenchymal stem cells (MeSCs) may have the ability to differentiate into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. While demonstrated "in vitro" and neonatally, efforts to demonstrate this ability in adult animal brains have had limited success. If it can be shown that human MeSC (HMeSC) can differentiate into neural cells in adult brain, it would open up the possibility that HMeSCs may be of potential therapeutic use in cell replacement therapies for neurological diseases. Here, we demonstrate that adult HMeSCs treated with 5-bromo-2-deoxyuridine (BrdU) for 3 weeks develop the capability to differentiate into neural and retinal cells when provided the appropriate lineage specific differentiation signals in vitro and in adult animals. HMeSC without BrdU treatment did not differentiate into neurons in vitro or adult animal or retinal cells in adult animal. METHODS: MeSCs isolated from adult human bone marrow were treated with BrdU (3 muM) for 3 weeks and then subjected to differentiation conditions both in vitro and in vivo. RESULTS: BrdU pretreated HMeSCs express neuronal and glial markers after co-culture with differentiated human neural stem cells and after transplantation into the adult rat brain. HMeSCs pretreated with BrdU and transforming growth factor-beta3 express a photoreceptor marker after transplantation into the adult rat vitreous. CONCLUSIONS: These results suggest that BrdU treatment may increase the multipotency of HMeSCs for possible use in autologous cell therapies for neurological and opthamological diseases.
