Method for producing dopamine-producing neural precursor cells

A technology of pluripotent stem cells and cells, which is applied in the field of preparation of dopamine-producing neural progenitor cells, and can solve problems such as the formation of benign tumors

Active Publication Date: 2018-12-21
KYOTO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it has been pointed out that induced differentiated nerve cells may form benign tumors during transplantation, and it is believed that cells other than dopamine-producing nerve cells may cause movement disorders. Therefore, people seek to select viable and safe cells for transplantation

Method used

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  • Method for producing dopamine-producing neural precursor cells
  • Method for producing dopamine-producing neural precursor cells
  • Method for producing dopamine-producing neural precursor cells

Examples

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preparation example Construction

[0204] III. Preparation method of dopamine-producing neural progenitor cells

[0205]

[0206] The step of collecting Corin and / or Lrtm1 positive cells in step (3) can be performed based on the above-mentioned .

[0207]

[0208] The medium used in step (4) can be prepared by using a medium used for culturing animal cells as a basal medium. As the base medium, include, for example: Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium , Ham's F12 medium, RPMI1640 medium, Fischer's medium, Neurobasal Medium (manufactured by Life Technologies) and mixed media thereof. Neurobasal Medium is preferred. The medium may contain serum, or may be serum-free. The medium may contain, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS in ES cell culture), N2 supplement (Invitrogen), B27 suppleme...

Embodiment 1

[0223] Cells and Culture

[0224] The protocol for preparing dopamine-producing cells is shown in figure 1 .

[0225] Human iPS cell QHJ-I01 was obtained from Professor Yamanaka of Kyoto University, etc., which is a dominant negative form of Oct3 / 4, Sox2, Klf4, L-MYC, LIN28 and p53 using episomal vectors (Okita, K., et al Stem Cells 31, 458-66 (2013); WO2013 / 176233) were obtained by introducing human PBMC.

[0226] iPS cells were cultured by the method described in Miyazaki T et al., Nat Commun. 3:1236, 2012. Briefly, cultures were maintained on 6-well plates coated with Laminin511E8 using undifferentiated maintenance medium containing FGF2 (bFGF).

[0227] 24 hours before the initiation of induction of differentiation (i.e. change to differentiation medium), change to undifferentiated maintenance medium containing SHH signaling stimulator SAG (Enzo Life Sciences, Inc, 300nM) - StemFit: AK-03N (Ajinomoto) , a cell population containing iPS cells was obtained...

Embodiment 2

[0234] In the same manner as in Example 1, experiments were performed in which the concentration of SAG to be added was changed, and in which A83-01 or LDN193189 was added simultaneously with SAG, and the positive rate of Corin was measured. Table 1 shows the study conditions and the Corin positive rates under the respective conditions.

[0235] [Table 1]

[0236] condition

[0237] From the above results, it was found that when SAG was added, or when A83-1 or LDN193189 was added in addition to SAG, the Corin positive rate increased in a manner dependent on the concentration of SAG added compared to the control. On the other hand, when only A83-1 and / or LDN193189 was added, no effect was shown.

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Abstract

Dopamine-producing neural precursor cells are produced by: producing a cell population containing Corin and / or Lrtm1 positive cells via steps (1) and (2) as will be shown below; collecting the Corin and / or Lrtm1 positive cells from the cell population thus obtained by using a substance that binds to Corin and / or a substance that binds to Lrtm1; and suspension-culturing the Corin and / or Lrtm1 positive cells in a liquid culture medium that contains one or more neurotrophic factors: (1) a step for adhesion-culturing pluripotent stem cells in the absence of any feeder cells in an undifferentiatedstate-maintaining culture medium that contains a sonic hedgehog (SHH) signal stimulant and an undifferentiated state-maintaining factor in the presence of an extracellular matrix; and (2) a step for culturing the cell population obtained in step (1) in a liquid culture medium that contains one or more differentiation inducing factors.

Description

technical field [0001] The present invention relates to a preparation method of dopamine-producing neural progenitor cells and a preparation method of a cell population containing Corin and / or Lrtm1 positive cells that can differentiate into dopamine-producing neural progenitor cells. Background technique [0002] Parkinson's disease is a neurodegenerative disease caused by the shedding of dopamine-producing nerve cells in the substantia nigra of the midbrain. Currently, there are about 4 million patients worldwide. As a treatment for Parkinson's disease, drug therapy based on L-DOPA or a dopamine agonist, coagulation or deep electrical stimulation therapy based on localized brain surgery, fetal midbrain transplantation, and the like have been performed. [0003] Fetal midbrain transplantation has ethical issues regarding the source of its supply, and the risk of infection is also high. Therefore, therapeutic methods using neural cells induced to differentiate from pluripot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797A61K35/30A61P25/16
CPCA61K35/30C12N5/0619C12N2506/45C12N2501/119C12N2501/13C12N2501/15C12N2501/155C12N2501/41C12N2501/727A61P25/16C12N2501/01C12N2501/999C12N2533/90A61K31/137C12N2501/115C12N2506/02C12N2500/38C12N2533/52C12N5/0696
Inventor 高桥淳土井大辅吉田贤司桑原笃高桥政代
Owner KYOTO UNIV
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