One-Step Induction of Neurons from Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B and Heparin
We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B 12 and heparin, but without any exp...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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SAGE Publishing
2006-02-01
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| Series: | Cell Transplantation |
| Online Access: | https://doi.org/10.3727/000000006783982061 |
| _version_ | 1818961399032840192 |
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| author | Hironori Yamazoe Masato Kobori Yoshinobu Murakami Keiichi Yano Mitsuo Satoh Kenji Mizuseki Yoshiki Sasai Hiroo Iwata |
| author_facet | Hironori Yamazoe Masato Kobori Yoshinobu Murakami Keiichi Yano Mitsuo Satoh Kenji Mizuseki Yoshiki Sasai Hiroo Iwata |
| author_sort | Hironori Yamazoe |
| collection | DOAJ |
| description | We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B 12 and heparin, but without any expensive cytokines. After 14 days in culture, β-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B 12 /heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B 12 /heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B 12 /heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinson's disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate. |
| first_indexed | 2024-12-20T12:12:48Z |
| format | Article |
| id | doaj.art-c29426db2d6341d29fba57ef9f3a5d53 |
| institution | Directory Open Access Journal |
| issn | 0963-6897 1555-3892 |
| language | English |
| last_indexed | 2024-12-20T12:12:48Z |
| publishDate | 2006-02-01 |
| publisher | SAGE Publishing |
| record_format | Article |
| series | Cell Transplantation |
| spelling | doaj.art-c29426db2d6341d29fba57ef9f3a5d532022-12-21T19:41:12ZengSAGE PublishingCell Transplantation0963-68971555-38922006-02-011510.3727/000000006783982061One-Step Induction of Neurons from Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B and HeparinHironori Yamazoe0Masato Kobori1Yoshinobu Murakami2Keiichi Yano3Mitsuo Satoh4Kenji Mizuseki5Yoshiki Sasai6Hiroo Iwata7 Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo, Kyoto 606-8507, Japan Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahimachi, Machida, Tokyo 194-8533, Japan Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo, Kyoto 606-8507, Japan Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahimachi, Machida, Tokyo 194-8533, Japan Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahimachi, Machida, Tokyo 194-8533, Japan Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo, Kyoto 606-8507, JapanWe present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B 12 and heparin, but without any expensive cytokines. After 14 days in culture, β-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B 12 /heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B 12 /heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B 12 /heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinson's disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.https://doi.org/10.3727/000000006783982061 |
| spellingShingle | Hironori Yamazoe Masato Kobori Yoshinobu Murakami Keiichi Yano Mitsuo Satoh Kenji Mizuseki Yoshiki Sasai Hiroo Iwata One-Step Induction of Neurons from Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B and Heparin Cell Transplantation |
| title | One-Step Induction of Neurons from Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B and Heparin |
| title_full | One-Step Induction of Neurons from Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B and Heparin |
| title_fullStr | One-Step Induction of Neurons from Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B and Heparin |
| title_full_unstemmed | One-Step Induction of Neurons from Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B and Heparin |
| title_short | One-Step Induction of Neurons from Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B and Heparin |
| title_sort | one step induction of neurons from mouse embryonic stem cells in serum free media containing vitamin b and heparin |
| url | https://doi.org/10.3727/000000006783982061 |
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