OBJECTIVE: The discovery of stem cells in the adult human brain and developing stem cell technology open a possible future scenario of autotransplantation, where stem cells are harvested from the patient and propagated in vitro before they are used as transplants. The objectives of this study were: 1) to investigate the feasibility of harvesting tissue containing neural stem cells by endoscopy; 2) to study the possibility of propagating and multiplying stem cells from this tissue efficiently in vitro; and 3) to examine whether the stem cells differentiate into functional neurons. METHODS: In 13 patients with hydrocephalus undergoing routine neurosurgical procedures, we used an endoscope and a 3-mm biopsy forceps (Medtronic) to harvest the small piece of the ventricular wall that is detached by the introduction of the endoscope. Cells were cultured as neurospheres, and after induced differentiation, they were investigated with immunocytochemistry and whole-cell patch-clamp recordings. All cells characterized were propagated under strict clonal conditions. RESULTS: We found it uncomplicated to harvest the part of the lateral ventricular wall that compares with the inner lumen of the endoscope. Single cells, isolated and cultivated in vitro, multiplied to form neurospheres in a serum-free environment. A single stem cell had the potential to give rise to approximately 9 x 10(5) new cells after two passages. The total number of cells produced from a single biopsy was already, after the second passage, far beyond the number required in, for instance, Parkinson's disease. Within 1 week of induced differentiation, cells expressing markers for neurons (beta-III-tubulin or NeuN), oligodendrocytes (RIP or O4), and astrocytes (glial fibrillary acidic protein) appeared. After 3 weeks, cells with a neuronal phenotype showed a firing pattern distinctive of mature neurons, including repetitive, short-lasting, and overshooting action potentials that were blocked by inhibiting voltage-dependent Na+-channels with tetrodotoxin. CONCLUSION: These results indicate that it may be feasible to produce neural tissue for autotransplantation from endoscopically harvested stem cells, but further work is needed in refining culture protocols to control phenotype fate.
OBJECTIVE: The discovery of stem cells in the adult human brain and developing stem cell technology open a possible future scenario of autotransplantation, where stem cells are harvested from the patient and propagated in vitro before they are used as transplants. The objectives of this study were: 1) to investigate the feasibility of harvesting tissue containing neural stem cells by endoscopy; 2) to study the possibility of propagating and multiplying stem cells from this tissue efficiently in vitro; and 3) to examine whether the stem cells differentiate into functional neurons. METHODS: In 13 patients with hydrocephalus undergoing routine neurosurgical procedures, we used an endoscope and a 3-mm biopsy forceps (Medtronic) to harvest the small piece of the ventricular wall that is detached by the introduction of the endoscope. Cells were cultured as neurospheres, and after induced differentiation, they were investigated with immunocytochemistry and whole-cell patch-clamp recordings. All cells characterized were propagated under strict clonal conditions. RESULTS: We found it uncomplicated to harvest the part of the lateral ventricular wall that compares with the inner lumen of the endoscope. Single cells, isolated and cultivated in vitro, multiplied to form neurospheres in a serum-free environment. A single stem cell had the potential to give rise to approximately 9 x 10(5) new cells after two passages. The total number of cells produced from a single biopsy was already, after the second passage, far beyond the number required in, for instance, Parkinson's disease. Within 1 week of induced differentiation, cells expressing markers for neurons (beta-III-tubulin or NeuN), oligodendrocytes (RIP or O4), and astrocytes (glial fibrillary acidic protein) appeared. After 3 weeks, cells with a neuronal phenotype showed a firing pattern distinctive of mature neurons, including repetitive, short-lasting, and overshooting action potentials that were blocked by inhibiting voltage-dependent Na+-channels with tetrodotoxin. CONCLUSION: These results indicate that it may be feasible to produce neural tissue for autotransplantation from endoscopically harvested stem cells, but further work is needed in refining culture protocols to control phenotype fate.
Authors: Kaisorn L Chaichana; Hugo Guerrero-Cazares; Vivian Capilla-Gonzalez; Grettel Zamora-Berridi; Pragathi Achanta; Oscar Gonzalez-Perez; George I Jallo; Jose Manuel Garcia-Verdugo; Alfredo Quiñones-Hinojosa Journal: J Neurosci Methods Date: 2009-03-05 Impact factor: 2.390