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New method for inducing dopamine-producing neural precursor cells

A technology of dopamine nerve and precursor cells, applied in the direction of nervous system cells, artificially induced pluripotent cells, non-embryonic pluripotent stem cells, etc., can solve the problems of non-administration and achieve high implantation rate

Pending Publication Date: 2016-08-10
KYOTO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Therefore, it has been proposed to use marker genes for dopamine-producing neuronal cells or dopamine-producing neural precursor cells to screen suitable cells for transplantation (Patent Documents 1 to 4), but there is still room for improvement in the selection of markers
In addition, in these documents, there is no study on whether the selected cells are suitable for drug administration, or whether the cells induced from the intermediate cells can be used for drug administration.
[0005] Furthermore, it is believed that there is room for improvement in the production method of dopamine-producing nerve cells in order to suppress the influence of individual differences caused by the inclusion of biologically derived components and the sharp increase in prices

Method used

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  • New method for inducing dopamine-producing neural precursor cells
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  • New method for inducing dopamine-producing neural precursor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0175] cells and culture

[0176] Human ES cells (KhES-1) were kindly provided by the Institute of Regenerative Medicine, Kyoto University (Suemori H, et al. Biochem Biophys Res Commun. 345:926-32, 2006). 404C2 and 836B3, which are human iPS cells, are obtained by introducing Oct3 / 4, Sox2, Klf4, L-MYC, LIN28, and p53shRNA into human fibroblasts using episomal vectors. Professor Yamanaka from Kyoto University et al. (Okita, et al, Nat Methods. 8:409-412, 2011).

[0177] ES cells and iPS cells were cultured based on the method described in Miyazaki T et al., Nat Commun. 3:1236, 2012. Briefly, 6-well plates coated with laminin 511E8 were used for culture.

[0178] The ES cells and iPS cells obtained in this way were dissociated with TrypLE CTS (Life Technologies), and each well was 4×10 4 Transfer each into an additionally prepared 6-well plate coated with laminin 511E8 (iMatrix-511, Nippi). After culturing for 4 days with the above-mentioned culture method, it was confirmed...

Embodiment 2

[0200] cell culture

[0201] ES cells (Kh-ES1) were dissociated using TrypLE CTS (Life Technologies), and all were transferred into an additionally prepared 6-well plate coated with laminin 511E8. 01 in minimal medium A (GMEM containing 8% KSR, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids and 0.1 mM 2-mercaptoethanol). One day after the initiation of culture (Day 1), the medium was replaced with minimal medium A containing 0.1 μM LDN193189, 0.5 μM A83-01, 2 μM purmorphamine, and 100 ng / mL FGF8. Three days after the initiation of culture (Day 3), the medium was replaced with minimal medium A containing 0.1 μM LDN193189, 0.5 μM A83-01, 2 μM purmorphamine, 100 ng / mL FGF8, and 3 μM CHIR99021. Seven days after the initiation of culture (day 7), the medium was replaced with minimal medium A containing 0.1 μM LDN193189 and 3 μM CHIR99021. On the 12th day (12 days after the start of culture), the medium was replaced with B27 supplement without vitamin A, 2 mM L-gluta...

Embodiment 3

[0209] cell culture

[0210] ES cells (Kh-ES1) were dissociated using TrypLE CTS (Life Technologies), and 4 × 10 5Each was transferred into a separately prepared 6-well plate coated with laminin 511E8, and cultured with StemFit medium (Ajinomoto) containing 10 μM Y-27632. After 4 days, the medium was replaced with the above minimal medium A containing 0.1 μM LDN193189, 0.5 μM A83-01 (day 0). One day after the initiation of culture (Day 1), the medium was replaced with minimal medium A containing 0.1 μM LDN193189, 0.5 μM A83-01, 2 μM purmorphamine, and 100 ng / mL FGF8. Three days after the initiation of culture (Day 3), the medium was replaced with minimal medium A containing 0.1 μM LDN193189, 0.5 μM A83-01, 2 μM purmorphamine, 100 ng / mL FGF8, and 3 μM CHIR99021. Seven days after the initiation of culture (day 7), the medium was replaced with minimal medium A containing 0.1 μM LDN193189 and 3 μM CHIR99021. On the 14th day (14 days after the start of culture), sort or replac...

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Abstract

Provided is a method for manufacturing dopamine-producing neural precursor cells from pluripotent stem cells, the method comprising: (i) a step in which pluripotent stem cells are cultured adherently on an extracellular matrix in a culture solution containing a reagent selected from the group consisting of BMP inhibitors, TGF beta inhibitors, SHH signal stimulants, and FGF8 and GSK3 beta inhibitors; (ii) a step in which a substance that binds to Corin and / or a substance that binds to Lrtm1 is used to collect Corin and / or Lrtm1 positive cells from the cells obtained in step (i); and (iii) a step in which the cells obtained in step (ii) are cultured in suspension in a culture solution containing a neurotrophic factor.

Description

technical field [0001] The present invention relates to a method for producing dopamine-producing neural precursor cells. Background technique [0002] Parkinson's disease is a neurodegenerative disease caused by the shedding of dopamine-producing neurons in the substantia nigra of the midbrain. At present, there are about 4 million patients in the world. For the treatment of Parkinson's disease, drug therapy with L-DOPA or a dopamine agonist, coagulation surgery with localized brain surgery, deep electrical stimulation therapy, and fetal midbrain transplantation are performed. [0003] Fetal midbrain transplantation has a high risk of infection as well as ethical problems in the source tissue. Therefore, therapeutic methods using neural cells induced to differentiate from pluripotent stem cells such as embryonic stem cells (ES cells) and artificial pluripotent stem cells (iPS cells) have been proposed (Non-Patent Document 1). However, the possibility of benign tumor forma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797A61K35/30A61P25/16
CPCA61K35/30C12N5/0619C12N2501/119C12N2501/13C12N2501/15C12N2501/155C12N2501/41C12N2501/415C12N2501/727C12N2506/02C12N2506/45C12N2533/52A61P25/16C12N5/0623C12N2506/03
Inventor 高桥淳土井大辅佐俣文平关口清俊尾野雄一
Owner KYOTO UNIV
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