| 1. |
- Guloglu, Oktar
(författare)
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A long and winding road: Differentiating human embryonic stem cells into dopaminergic neurons
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Parkinson’s disease is a progressive neurodegenerative disorder. Since the motor manifestations of the disease is associated with the loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta, cell replacement therapy is a promising approach for treatment. Although several different cell types have been used in clinical trials, with the recent findings of somatic cell nuclear transfer in humans, human embryonic stem cells (hESCs) offer a promising “personalized” source of cells in this quest. In the present study, we established a simple protocol for large-scale generation of dopaminergic neurons from hESCs using PA6 cells as feeders. We showed that the intermediate cell type, DAergic neural progenitors (NP), generated during the differentiation could be expanded for long time and cryopreserved without loosing their full capacity to differentiate into DAergic neurons. We further revealed that within the heterogenous PA6 population, adipocytes robustly express all the factors previously identified to be associated with SDIA. Coculturing with PA6-derived adipocytes reliably promoted the dopaminergic differentiation of the hESCs. Moreover, we identified three candidate molecules that might be involved in the SDIA of PA6 and PA6-derived adipocytes. We also demonstrated that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) efficiently induced apoptosis in proliferating neural progenitors through the TRAIL pathway, and promoted the DAergic differentiation of the hESCs. Additionally, we also identified twelve candidate surface markers that can be used in MACS for purifying DAergic neural progenitors within the heterogeneous population.
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| 2. |
- Guloglu, Oktar, et al.
(författare)
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Adipocytes derived from PA6 cells reliably promote the differentiation of dopaminergic neurons from human embryonic stem cells.
- 2014
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Ingår i: Journal of Neuroscience Research. - : Wiley. - 1097-4547 .- 0360-4012. ; 92:5, s. 564-573
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Tidskriftsartikel (refereegranskat)abstract
- The PA6 stromal cell line comprises a heterogeneous population of cells that can induce both mouse and human embryonic stem cells to differentiate into dopaminergic neurons. This ability of PA6 cells has been termed stromal cell-derived inducing activity (SDIA). The level of SDIA has been found to vary considerably between and within batches of PA6 cells. Not only are the molecular mechanisms that underlie SDIA unknown but also the cell type(s) within the heterogeneous PA6 cultures that underlie SDIA remain poorly defined. In this study, we reveal that adipocytes, which are present within the heterogeneous PA6 cell population, robustly release the factors mediating SDIA. Furthermore, we report that the coculture of human embryonic stem cells with PA6-derived adipocytes reliably induces their differentiation into midbrain dopaminergic neurons. © 2014 Wiley Periodicals, Inc.
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| 3. |
- Guloglu, Oktar, et al.
(författare)
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Dopaminergic Differentiation of Human Embryonic Stem Cells on PA6-Derived Adipocytes.
- 2016
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029. ; 1341, s. 235-244
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESCs) are a promising source for cell replacement therapies. Parkinson's disease is one of the candidate diseases for the cell replacement therapy since the motor manifestations of the disease are associated with the loss of dopaminergic neurons in the substantia nigra pars compacta. Stromal cell-derived inducing activity (SDIA) is the most commonly used method for the dopaminergic differentiation of hESCs. This chapter describes a simple, reliable, and scalable dopaminergic induction method of hESCs using PA6-derived adipocytes. Coculturing hESCs with PA6-derived adipocytes markedly reduces the variable outcomes among experiments. Moreover, the colony differentiation step of this method can also be used for the dopaminergic induction of mouse embryonic stem cells and NTERA2 cells as well.
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| 4. |
- Morizane, Asuka, et al.
(författare)
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A simple method for large-scale generation of dopamine neurons from human embryonic stem cells.
- 2010
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Ingår i: Journal of Neuroscience Research. - : Wiley. - 1097-4547 .- 0360-4012. ; 88:16, s. 3467-3478
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Tidskriftsartikel (refereegranskat)abstract
- Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinson's disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC-derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT-PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation. © 2010 Wiley-Liss, Inc.
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