Cells Containing a Dopaminergic Fluorescent Reporter System and an Asynchronous Co-Culture Method

Abstract

The present invention relates to a genetically modified induced neural stem cell and an asynchronous co-culture method. By providing a CRISPR / Cas9-engineered Nurr1 dopaminergic reporter-induced neural stem cell model, the differentiation of dopaminergic neural progenitor cells and neurons can be monitored in real time, and key pathways related to differentiation heterogeneity can be revealed by RNA sequencing. An asynchronous co-culture method is established, which can improve the yield and purity of the dopaminergic neural progenitor cells, and its therapeutic potential for Parkinson's disease is confirmed by successful transplantation and differentiation into dopaminergic neurons in the mouse striatum.

Claims

1. An asynchronous co-culture method, which is used to culture dopaminergic neural precursor cells and improve the yield and purity of the dopaminergic neural precursor cells, and the dopaminergic neural precursor cells differentiate into dopaminergic neurons after being transplanted into the body and are used to treat Parkinson's disease, wherein: The asynchronous co-cultivation method comprises the following steps: Step 1: starting from induced neural stem cells, the early induced neural stem cell differentiation population and the mid-induced neural stem cell differentiation population are differentiated independently in the first stage differentiation medium; Step 2: co-culturing the early induced neural stem cell differentiation population and the mid-stage induced neural stem cell differentiation population obtained in step 1 in the first stage differentiation medium, wherein the early induced neural stem cell differentiation population and the mid-stage induced neural stem cell differentiation population are mixed at a ratio of 1:6-8 in terms of cell number; Step 3: replacing the first stage differentiation medium with the second stage differentiation medium, and continuing the co-culture to obtain dopaminergic neural precursor cells; Wherein, in step 1, the early induced neural stem cell differentiation population is a differentiation population differentiated for 4-6 days, and the mid-induced neural stem cell differentiation population is a differentiation population differentiated for 7-9 days; The co-cultivation time in step 2 is 1-3 days; The continued co-cultivation time in step 3 is 2-4 days; The composition of the first stage differentiation medium is 96% DMEM / F12, 1% B27, 1% N2, 1% NEAA, 1% GlutaMAX, 1 µM SAG1 and 100 ng / mL FGF8; the composition of the second stage differentiation medium is the first stage differentiation medium supplemented with 10 ng / mL BDNF, 10 ng / mL GDNF, 1 ng / mL TGF-β III, 10 µM DAPT, 0.2 mM ascorbic acid and 0.5 mM cAMP. 2 . The asynchronous co-culture method according to claim 1 , wherein in step 2, the early induced neural stem cell differentiation population and the mid-induced neural stem cell differentiation population are mixed at a ratio of 1:7 in terms of cell number.

3. The asynchronous co-culture method according to claim 1, wherein the differentiation time of the early induced neural stem cell differentiation population is 5 days, and the differentiation time of the mid-term induced neural stem cell differentiation population is 8 days; the co-culture time in step 2 is 2 days, and the continued co-culture time in step 3 is 3 days.

4. Use of dopaminergic neural precursor cells for preparing a drug for treating Parkinson's disease, wherein the dopaminergic neural precursor cells are dopaminergic neural precursor cells obtained by the asynchronous co-culture method according to any one of claims 1 to 3.

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