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Method for differentiating pluripotent stem cells into midbrain black matter dopaminergic nerve cells

A technology of dopaminergic and neural cells, applied in the field of neural stem cell science, can solve the problem of not clearly distinguishing subtypes of brain dopaminergic neurons

Pending Publication Date: 2022-04-08
UNIXELL BIOTECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, subtypes of midbrain dopaminergic neurons (A9 / A10) in transplanted cells were not clearly distinguished in any of these studies

Method used

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  • Method for differentiating pluripotent stem cells into midbrain black matter dopaminergic nerve cells
  • Method for differentiating pluripotent stem cells into midbrain black matter dopaminergic nerve cells
  • Method for differentiating pluripotent stem cells into midbrain black matter dopaminergic nerve cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0229] Example 1. Transplanted human dopaminergic neurons and glutamatergic neurons project to different brain regions

[0230] During development, the directional projection of axons often depends on intrinsic properties of the cell. In order to understand whether the intrinsic properties of cells can still determine the projection targets of cells in the adult brain, the inventors transplanted mDA or forebrain glutamatergic neuron precursor cells differentiated from hESC into the midbrain of PD model mice . The inventors' method enables the differentiation of hESCs into neural precursors of mDA or Glu. At day 32 of mDA neuron differentiation (the day of transplantation), most precursor cells expressed the floor plate and midbrain markers CORIN, FOXA2, LMX1A, and EN1 ( Figure 8 A and 8C). By day 42, 69% of total cells or 84% of TUJ1+ neurons expressed tyrosine hydroxylase (TH) as well as EN1, FOXA2, LMX1A and NURR1 ( Figure 8 D and 8F), indicating mDA neuronal propertie...

Embodiment 2

[0234] Example 2, Transplanted human mDA neurons project through homologous pathways

[0235] From serial sagittal sections ( figure 2 A) shows that transplanted mDA neurons extend rostral axons along the well-defined medial forebrain bundle (MFB) ( figure 2 B, L1.08 and the red arrow at figure 2 C). Viewed from the side, they extend across SI ( figure 2 B, L1.44) and amygdala ( figure 2 B, L2.04). Viewed from the rostral side of the beak, hNCAM+ fibers passed through the Acb( figure 2 B-2E), where most of the hNCAM+ fibers enter the CPu along the border between cortex and striatum ( figure 2 A and figure 2 red arrow in D). Few hNCAM+ fibers were detected in the cortex ( figure 2 Blue arrows in D and 2F). These results indicate that most of the axonal projections of transplanted mDA neurons follow the endogenous substantia nigra-striatum pathway.

[0236] In the dorsal / lateral striatum, the dense hNCAM+ fibers were branched, forming a dense branched network...

Embodiment 3

[0238] Example 3. Genetic markers reveal specific axonal innervation of human mDA neurons

[0239] To elucidate the specific axonal innervation elicited by transplanted mDA neurons, the inventors established a TH reporter hESC line with tdTomato expression recapitulating the expression of the endogenous TH gene (Methods Details). The inventors further knocked the ChR2-EYFP fusion protein expression cassette into the AAVS1 gene locus to realize specific markers and manipulation of transplanted human cells ( image 3 A, 3B, 10A and 10B). The final hESC, termed TH-tdTomato / AAVS1-ChR2-EYFPhESC, constitutively expressed ChR2-EYFP throughout mDA neuronal differentiation, whereas tdTomato was expressed only at later stages of mDA neuronal differentiation ( Figure 10 C), and is expressed only in TH+ mDA neurons (Figure 3C and 10D), emphasizing TH+ cell-specific expression.

[0240] Then, the inventors transplanted mDA precursor cells derived from TH-tdTomato / AAVS1-ChR2-EYFP hESCs i...

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Abstract

The invention provides a specific method for differentiating pluripotent stem cells into midbrain nigra dopaminergic (A9mDA) nerve cells. According to the method, mature A9 mDA neurons are differentiated to form mature A9 mDA neurons, surface molecular markers capable of expressing dopaminergic neurons of midbrain nigra, including TH, FOXA2, EN1, LMX1A, NURR1 and GIRK2, and little expression of a dopaminergic neuron marker CB of a covered region on the ventral side. The A9mDA nerve cells are transplanted to the nigra, and the axons of the A9mDA nerve cells can be specifically projected to a target brain region-dorsal striatum dominated by endogenous nigra dopaminergic neurons; the transplanted A9mDA neurons show classical electrophysiological characteristics of endogenous nigra dopaminergic neurons, including low-frequency spontaneous discharge frequency, and sag can be induced by hyperpolarization current stimulation; a9mDA nerve cells are transplanted to the nigra or striatum of a neurodegenerative disease individual, so that dyskinesia can be improved.

Description

technical field [0001] The invention belongs to the field of neural stem cell science, and more specifically, the invention relates to a method for differentiating pluripotent stem cells into dopaminergic neurons in the substantia nigra of the midbrain. Background technique [0002] Parkinson's disease (PD) is a long-term degenerative disease of the central nervous system, with an average age of onset of 60 years and an incidence rate of 1.7% among the elderly over 65 years old. According to the international epidemiological survey, there were about 2 million PD patients in China in 2005, accounting for half of the global incidence. It is expected that by 2030, there will be nearly 5 million PD patients in China. Patients with Parkinson's disease often present with movement disorders, such as resting tremor, increased muscle tone, slowness of movement, and postural instability. The pathological basis of Parkinson's disease is the degeneration and loss of dopaminergic neuro...

Claims

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Application Information

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IPC IPC(8): C12N5/0793C12Q1/02A61K35/30A61P25/14A61P25/16A61P25/28A61P25/00
CPCA61K35/30A61P25/00C12N5/0619C12N2501/119C12N2506/08
Inventor 陈跃军周文浩熊曼
Owner UNIXELL BIOTECHNOLOGY
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