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Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

ABSTRACT: Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts - MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3?±?2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed.

SUBMITTER: Molcanyi M

PROVIDER: S-EPMC4155790 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

Molcanyi Marek M Mehrjardi Narges Zare NZ Schäfer Ute U Haj-Yasein Nadia Nabil NN Brockmann Michael M Penner Marina M Riess Peter P Reinshagen Clemens C Rieger Bernhard B Hannes Tobias T Hescheler Jürgen J Bosche Bert B

Frontiers in cellular neuroscience 20140905

Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells i ...[more]

PMID: 25249934

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Project description:In this study two regions of embryonic (E11) mouse central nervous system (CNS) have been profiled for their unesterified sterol content. Using high-performance liquid chromatography (HPLC)-mass spectrometry (MS) and tandem mass spectrometry (MS(n)) low levels of oxysterols (estimated 2-165 ng g(-1) wet weight) were identified in cortex (Ctx) and spinal cord (Sc). The identified oxysterols include 7 alpha-, 7 beta-, 22R-, 24S-, 25- and 27-hydroxycholesterol; 24,25- and 24,27-dihydroxycholesterol; and 24S,25-epoxycholesterol. Of these, 24S-hydroxycholesterol is biosynthesised exclusively in brain. In comparison to adult mouse where the 24S-hydroxycholesterol level is about 40 microg g(-1) in brain the level of 24S-hydroxycholesterol reported here (estimated 26 ng g(-1) in Ctx and 13 ng g(-1) in Sc) is extremely low. Interestingly, the level of 24S,25-epoxycholesterol in both CNS regions (estimated 165 ng g(-1) in Ctx and 91 ng g(-1) in Sc) is somewhat higher than the levels of the hydroxycholesterols. This oxysterol is formed in parallel to cholesterol via a shunt of the mevalonate pathway and its comparatively high abundance may be a reflection of a high rate of cholesterol synthesis at this stage of development. Levels of cholesterol (estimated 1.25 mg g(-1) in Ctx and 1.15 mg g(-1) in Sc) and its precursors were determined by gas chromatography-mass spectrometry (GC-MS). In both CNS regions cholesterol levels were found to be lower than those reported in the adult, but in relation to cholesterol the levels of cholesterol precursors were higher than found in adult indicating a high rate of cholesterol synthesis. In summary, our data provide evidence for the presence of endogenous oxysterols in two brain regions of the developing CNS. Moreover, while most of the enzymes involved in hydroxysterol synthesis are minimally active at E11, our results suggest that the mevalonate pathway is significantly active, opening up the possibility for a function of 24S,25-epoxycholesterol during brain development.

Project description:BackgroundFolates are a family of B9 vitamins that serve as one-carbon donors critical to biosynthetic processes required for the development and function of the central nervous system (CNS) in mammals. Folate transport is mediated by three highly specific systems: (1) folate receptor alpha (FRα; FOLR1/Folr1), (2) the reduced folate-carrier (RFC; SLC19A1/Slc19a1) and (3) the proton-coupled folate transporter (PCFT; SLC46A1/Slc46a1). Folate transport into and out of the CNS occurs at the blood-cerebrospinal fluid barrier (BCSFB), mediated by FRα and PCFT. Impairment of folate transport at the BCSFB results in cerebral folate deficiency in infants characterized by severe neurological deficiencies and seizures. In contrast to the BCSFB, CNS folate transport at other brain barriers and brain parenchymal cells has not been extensively investigated. The aim of this study is to characterize folate transport systems in the murine CNS at several known barriers encompassing the BCSFB, arachnoid barrier (AB), blood-brain barrier (BBB) and parenchymal cells (astrocytes, microglia, neurons).MethodsApplying immunohistochemistry, localization of folate transport systems (RFC, PCFT, FRα) was examined at CNS barriers and parenchymal sites in wildtype (C57BL6/N) mice. Subcellular localization of the folate transport systems was further assessed in an in vitro model of the mouse AB. Gene and protein expression was analyzed in several in vitro models of brain barriers and parenchyma by qPCR and western blot analysis.ResultsRFC, PCFT, and FRα expression was localized within the BCSFB and BBB consistent with previous reports. Only RFC and PCFT expression was detected at the AB. Varied levels of RFC and PCFT expression were detected in neuronal and glial cells.ConclusionsLocalization of RFC and PCFT within the AB, described here for the first time, suggest that AB may contribute to folate transport between the peripheral circulation and the CSF. RFC and PCFT expression observed in astrocytes and microglia is consistent with the role that one or both of these transporters may play in delivering folates into cells within brain parenchyma. These studies provide insights into mechanisms of folate transport in the CNS and may enhance our understanding of the critical role folates play in neurodevelopment and in the development of novel treatment strategies for disorders of brain folate deficiency due to impaired transporter function.

Dataset Information

Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

Publications

Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

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