Human pluripotent stem cell-based models for predictive developmental neural toxicity

Patent metadata

Field	Value
Publication number	US-11060066-B2
Application number	US-201514986363-A
Country	US
Kind code	B2
Filing date	Dec 31, 2015
Priority date	Dec 31, 2014
Publication date	Jul 13, 2021
Grant date	Jul 13, 2021

Abstract

The present invention relates to three-dimensional (3D) tissue constructs and methods of using such 3D tissue constructs to screen for neurotoxic agents. In particular, provided herein are methods of producing and using complex, highly uniform human tissue models comprising physiologically relevant human cells, where the tissue models have the degree of sample uniformity and reproducibility required for use in quantitative high-throughput screening applications.

First claim

We claim:

1. A method of producing a chemically defined, xenogen-free vascularized neural tissue construct, comprising:
(a) seeding a xenogen-free, three-dimensional (3D) RGD-containing peptide-functionalized, matrix metalloproteinase (MMP)-degradable, porous hydrogel with human neural progenitor cells, wherein the hydrogel is formed by thiol-ene photopolymerization;
(b) culturing the seeded hydrogel under chemically defined, xenogen-free conditions for a length of time sufficient to detect differentiation of at least a portion of the neural progenitor cells;
(c) dispersing on or within the cultured seeded hydrogel human endothelial cells and human mesenchymal cells and, optionally, one or more of primitive macrophages and pericytes; and
(d) culturing the seeded hydrogel comprising the dispersed human endothelial cells and human mesenchymal stem cells under chemically defined, xenogen-free culture conditions that promote cell differentiation, whereby a 3D chemically defined, xenogen-free, vascularized neural tissue construct comprising stratified layers of human neurons and glial cells is produced within about 14 days from seeding step (a).

2. The method of claim 1 , wherein the hydrogel comprises polymerized poly(ethylene glycol) (PEG) or polymerized polysaccharide.

3. The method of claim 1 , wherein the dispersed human endothelial cells are derived under chemically defined, xenogen-free conditions from a human pluripotent stem cell.

4. The method of claim 3 , wherein the human pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.

5. The method of claim 1 , wherein cells dispersed on or within the seeded hydrogel further comprise human pluripotent stem cell-derived primitive macrophages, whereby the 3D vascularized neural tissue construct comprises human neurons, glial cells, and mature microglia.

6. The method of claim 1 , wherein seeding the porous hydrogel comprises contacting the porous hydrogel with at least one human neural progenitor cell.

7. The method of claim 1 , further comprising dispersing within or on the porous hydrogel a bioactive agent that modulates a morphological feature, function, or differentiation status of a cell seeded or dispersed therein.

8. The method of claim 7 , wherein the bioactive agent is selected from the group consisting of a growth factor, a cytokine, and a bioactive peptide, or a combination thereof.

9. The method of claim 1 , wherein the vascularized neural tissue construct exhibits one or more properties selected from the group consisting of: (i) an interconnected vasculature; (ii) differentiated cells within the neural tissue construct mutually contact each other in three dimensions; (iii) more than one layer of cells; and (iv) a function or property characteristic of human neural tissue in vivo or in situ.

10. The method of claim 1 , wherein the neurons and glial cells are selected from the group consisting of GABAergic neurons, glutamatergic neurons, astrocytes, and oligodendrocytes.

11. A three-dimensional (3D), xenogen-free, vascularized neural tissue construct obtained according to the method of claim 1 .

12. A three-dimensional (3D), xenogen-free, vascularized neural tissue construct comprising mature microglia obtained according to the method of claim 5 .

13. The neural tissue construct of claim 11 , comprising stratified layers of neurons and glia.

14. The method of claim 1 , wherein the hydrogel is a CRGDS peptide-functionalized hydrogel.

Assignees

• Wisconsin Alumni Res Found

Inventors

• Thomson James A
• Murphy William L
• Page Charles D
• Schwartz Michael P
• Hou Zhonggang

Classifications

• G01N33/5058

  Neurological cells · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/142

  Toxicological screening, e.g. expression profiles which identify toxicity · CPC title

• C12Q1/6883

  for diseases caused by alterations of genetic material · CPC title

• C12N2533/90

  Substrates of biological origin, e.g. extracellular matrix, decellularised tissue · CPC title