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Efficient Generation of Induced Pluripotent Stem and Neural Progenitor Cells From Acutely Harvested Dura Mater Obtained During Ventriculoperitoneal Shunt Surgery.

ABSTRACT: BACKGROUND:The dura mater can be easily biopsied during most cranial neurosurgical operations. We describe a protocol that allows for robust generation of induced pluripotent stem cells (iPSCs) and neural progenitors from acutely harvested dura mater. OBJECTIVE:To generate iPSCs and neural progenitor cells from dura mater obtained during ventriculoperitoneal shunt surgery. METHODS:Dura was obtained during ventriculoperitoneal shunt surgery for normal pressure hydrocephalus from a 60-year-old patient with severe cognitive impairment. Fibroblasts were isolated from the dural matrix and transduced with nonintegrating Sendai virus for iPSC induction. A subset of successfully generated iPSC clones underwent immunocytochemical analysis, teratoma assay, karyotyping, and targeted neural differentiation. RESULTS:Eleven iPSC clones were obtained from the transduction of an estimated 600,000 dural fibroblasts after 3 passages. Three clones underwent immunocytochemical analysis and were shown to express the transcription factors OCT-4, SOX2, and the embryonic cell markers SSEA-4, TRA-1-60, and Nanog. Two clones were tested for pluripotency and formed teratomas at the injection site in immunodeficient mice. Three clones underwent chromosomal analysis and were found to have a normal metaphase spread and karyotype. One clone underwent targeted neural differentiation and formed neural rosettes as well as TuJ1/SOX1-positive neural progenitor cells. CONCLUSIONS:IPSCs and neural progenitor cells can be efficiently derived from the dura of patients who need to undergo cranial neurosurgical operations. IPSCs were obtained with a nonintegrating virus and exhibited a normal karyotype, making them candidates for future autotransplantation after targeted differentiation to treat functional deficits.

SUBMITTER: Cary WA

PROVIDER: S-EPMC7602677 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Efficient Generation of Induced Pluripotent Stem and Neural Progenitor Cells From Acutely Harvested Dura Mater Obtained During Ventriculoperitoneal Shunt Surgery.

Cary Whitney Ann WA Hori Courtney Namiko CN Pham Missy Trananh MT Nacey Catherine Ann CA McGee Jeannine Logan JL Hamou Mattan M Berman Robert F RF Bauer Gerhard G Nolta Jan A JA Waldau Ben B

World neurosurgery 20150611 5

<h4>Background</h4>The dura mater can be easily biopsied during most cranial neurosurgical operations. We describe a protocol that allows for robust generation of induced pluripotent stem cells (iPSCs) and neural progenitors from acutely harvested dura mater.<h4>Objective</h4>To generate iPSCs and neural progenitor cells from dura mater obtained during ventriculoperitoneal shunt surgery.<h4>Methods</h4>Dura was obtained during ventriculoperitoneal shunt surgery for normal pressure hydrocephalus ...[more]

PMID: 26074438

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Project description:BackgroundDespite greatly renewed interest concerning meningeal lymphatic function over recent years, the lymphatic structures of human dura mater have been less characterized. The available information derives exclusively from autopsy specimens. This study addressed methodological aspects of immunohistochemistry for visualization and characterization of lymphatic vessels in the dura of patients.MethodsDura biopsies were obtained from the right frontal region of the patients with idiopathic normal pressure hydrocephalus (iNPH) who underwent shunt surgery as part of treatment. The dura specimens were prepared using three different methods: Paraformaldehyde (PFA) 4% (Method #1), paraformaldehyde (PFA) 0.5% (Method #2), and freeze-fixation (Method #3). They were further examined with immunohistochemistry using the lymphatic cell marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and as validation marker we used podoplanin (PDPN).ResultsThe study included 30 iNPH patients who underwent shunt surgery. The dura specimens were obtained average 16.1 ± 4.5 mm lateral to the superior sagittal sinus in the right frontal region (about 12 cm posterior to glabella). While lymphatic structures were seen in 0/7 patients using Method #1, it was found in 4/6 subjects (67%) with Method #2, while in 16/17 subjects (94%) using Method #3. To this end, we characterized three types of meningeal lymphatic vessels: (1) Lymphatic vessels in intimate contact with blood vessels. (2) Lymphatic vessels without nearby blood vessels. (3) Clusters of LYVE-1-expressing cells interspersed with blood vessels. In general, highest density of lymphatic vessels were observed towards the arachnoid membrane rather than towards the skull.ConclusionsThe visualization of meningeal lymphatic vessels in humans seems to be highly sensitive to the tissue processing method. Our observations disclosed most abundant lymphatic vessels towards the arachnoid membrane, and were seen either in close association with blood vessels or remote from blood vessels.

Project description:PurposeSpinal surgery in a previously irradiated field carries increased risk of perioperative complications, such as delayed wound healing or wound infection. In addition, adhesion around the dura mater is often observed clinically. Therefore, similar to radiation-induced fibrosis--a major late-stage radiation injury in other tissue--epidural fibrosis is anticipated to occur after spinal radiation. In this study, we performed histopathologic assessment of postirradiation changes in the spinal dura mater and peridural tissue in mice.Materials and methodsThe thoracolumbar transition of ddY mice was irradiated with a single dose of 10 or 20 Gy. After resection of the irradiated spine, occurrence of epidural fibrosis and expression of transforming growth factor beta 1 in the spinal dura mater were evaluated. In addition, microstructures in the spinal dura mater and peridural tissue were assessed using an electron microscope.ResultsIn the 20-Gy irradiated mice, epidural fibrosis first occurred around 12 weeks postirradiation, and was observed in all cases from 16 weeks postirradiation. In contrast, epidural fibrosis was not observed in the nonirradiated mice. Compared with the nonirradiated mice, the 10- and 20-Gy irradiated mice had significantly more overexpression of transforming growth factor beta 1 at 1 week postirradiation and in the late stages after irradiation. In microstructural assessment, the arachnoid barrier cell layer was thinned at 12 and 24 weeks postirradiation compared with that in the nonirradiated mice.ConclusionIn mice, spinal epidural fibrosis develops in the late stages after high-dose irradiation, and overexpression of transforming growth factor beta 1 occurs in a manner similar to that seen in radiation-induced fibrosis in other tissue. Additionally, thinning of the arachnoid barrier cell layer was observed in the late stages after irradiation. Thus, consideration should be given to the possibility that these phenomena can occur as radiation-induced injuries of the spine.

Dataset Information

Efficient Generation of Induced Pluripotent Stem and Neural Progenitor Cells From Acutely Harvested Dura Mater Obtained During Ventriculoperitoneal Shunt Surgery.

Publications

Efficient Generation of Induced Pluripotent Stem and Neural Progenitor Cells From Acutely Harvested Dura Mater Obtained During Ventriculoperitoneal Shunt Surgery.

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