Methods of producing bioengineered neuronal organoids (BENOS) and uses thereof

A bioengineering and neuron technology, applied in biochemical equipment and methods, microorganisms, biological tests, etc., can solve the problem of lack of neuron network function in organoids, limit disease modeling and drug development, limit neuron function and plasticity, etc. question

Pending Publication Date: 2020-01-31
乔治-奥古斯特-哥廷根大学公共利益基金会
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Another disadvantage associated with known methods is that the resulting organoids lack neuronal network functionality, thus significantly limiting any study of neuronal function and plasticity
Disease modeling and drug development are also limited by the known long-range phenotypic similarity of the resulting neuronal organoid structures compared to normal brain tissue

Method used

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  • Methods of producing bioengineered neuronal organoids (BENOS) and uses thereof
  • Methods of producing bioengineered neuronal organoids (BENOS) and uses thereof
  • Methods of producing bioengineered neuronal organoids (BENOS) and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0233] Embodiment 1: Optimizing the scheme of producing BENO

[0234] To optimize the disclosed protocol for neural induction, NPC amount and neuronal differentiation, the following studies were performed. The data obtained allowed to establish an optimal protocol for the production of BENO (Example 2).

[0235] neural induction optimization

[0236] In initial attempts, a single SMAD pathway inhibition (using Noggin) was used to elicit neural induction, with the addition of retinoic acid (RA). In the present study, it was tested whether dual SMAD signaling pathway inhibition using Noggin and SB431542 during the neural induction phase (days 0–8) might further enhance neurogenesis, for example at day 28. Therefore, NCMs containing only Noggin or NCMs containing Noggin and SB431542 were investigated, respectively. As analyzed by immunofluorescence ( Figure 2A ) and transcript analysis of PAX6 and MAP2 ( Figure 2B ), it was clearly observed that dual SMAD inhibition is ben...

Embodiment 2

[0248] Example 2: Optimization Protocol for Generation of Human Bioengineered Neuronal Organoids (BENO)

[0249] The following studies established optimal protocols for producing human bioengineered neuronal organoids (BENOs).

[0250] Material

[0251] The materials used in these experiments were the same as those used in Example 1.

[0252] Stock solution (-20℃)

[0253] The stock solutions used in these experiments were the same as those used in Example 1.

[0254] Working solution (4°C)

[0255] EDTA 0.5mM solution, 1:30 Matrigel solution, TeSR TM -E8 TM The medium and base medium are the same as those used in Example 1.

[0256] neural presence medium (NCM)

[0257] Basal medium (50 ml) was supplemented with 10 μM SB 431542 (50 μl), 50 ng / ml Noggin (10 μl) and 1 μM RA (5 μl).

[0258] neural progenitor cell expansion medium (NPEM)

[0259] Basal medium (50 ml) supplemented with 10 ng / ml FGF-2 (50 μl) and 5 ng / ml TGFB1.

[0260] neural differentiation...

Embodiment 3

[0277]Example 3: High-throughput transcriptome analysis of BENO

[0278] To characterize the cell types that emerged at different time points (e.g., days -1, 0, 3, 8, 15, 28, 40, 50, and 60) during BENO tissue production, cells were subjected to RNAseq analysis; each time point 3-6 tissues).

[0279] First, the differentiation status of the cells over the time course of cell culture was tested. As expected, the stem cell markers NANOG and POUF5A1 were highly expressed on day −1 and day 0, but by day 3 after neural induction, the expression of these markers was significantly reduced. In contrast, SOX2, which is both a marker of stem cells and a NPC marker, did not show any decrease at these time points, but reached maximal expression at day 15 along with other neuroectoderm and NPC markers. These data are consistent with the known proliferative role of FGF-2. Most neuronal structural markers (MAP2, MAPT, NFASC, TUBA1A) were significantly increased from day 28 to 40. Once ne...

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Abstract

The present invention relates to the field of in vitro 3D modeling of neural tissues, particularly of the brain. There is the need of developing cell culture models of neural tissue that reflect physiological aspects of neural tissue. The present invention provides methods of producing bioengineered neuronal organoids (BENOs) which form functional neuronal networks. The present invention also relates to uses and applications of the produced BENOs, e.g., in the fields of drug screening and personalized medicine.

Description

Background technique [0001] In vitro 3D modeling of neural tissue, especially brain tissue derived from mammals such as the human brain, represents a powerful tissue bioengineering tool that can be used to study complex neuronal cell systems. In general, 3D cell culture systems provide faster cell differentiation, higher cell complexity, and longevity than corresponding 2D culture systems. A promising approach for in vitro 3D modeling of neural tissue involves the use of pluripotent stem cells (PSCs), which have been used in the modeling of various human tissues and organs. These advantages of 3D modeling approaches, combined with recently used techniques such as the reprogramming of patient fibroblasts into induced PSCs, together provide new insights into the elucidation of the underlying molecular mechanisms responsible for a variety of human-based neuronal diseases. . [0002] Numerous protocols for inducing neural differentiation of human stem cells exist in the literatu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/071C12N5/00G01N33/50C12N5/0793
CPCC12N5/0031C12N5/0619C12N5/0622C12N5/0697G01N33/50G01N33/5005C12N2501/115C12N2501/15C12N2501/42C12N2501/727C12N2502/081C12N2502/086C12N2506/45C12N2533/54C12N2500/30C12N2500/90C12N2503/02C12N2506/02C12N2513/00G01N33/5058
Inventor W-H·齐默尔曼M·P·扎菲里奥
Owner 乔治-奥古斯特-哥廷根大学公共利益基金会
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