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
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[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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