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

Patent metadata

Field	Value
Publication number	US-2016186146-A1
Application number	US-201514986363-A
Country	US
Kind code	A1
Filing date	Dec 31, 2015
Priority date	Dec 31, 2014
Publication date	Jun 30, 2016
Grant date	—

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 vascularized neural tissue construct, comprising
(a) seeding a three-dimensional porous biomaterial with human neural progenitor cells; (b) culturing the seeded biomaterial 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 biomaterial human endothelial cells and, optionally, one or more of human mesenchymal cells, primitive macrophages, and pericytes; and (d) culturing the seeded biomaterial comprising the dispersed human endothelial cells under culture conditions that promote cell differentiation, whereby a vascularized neural tissue construct comprising human neurons and glial cells is produced.

2 . The method of claim 1 , wherein the three-dimensional porous biomaterial is a hydrogel.

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

4 . The method of claim 1 , wherein the dispersed human endothelial cells are derived from a human pluripotent stem cell.

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

6 . The method of claim 1 , wherein the seeded biomaterial comprising the dispersed human endothelial cells further comprises human pluripotent stem cell-derived primitive macrophages and wherein the 3D vascularized neural tissue construct comprises mature microglia.

7 . The method of claim 1 , wherein seeding the porous biomaterial comprises contacting to the porous biomaterial at least one human neural progenitor cell.

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

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

10 . 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.

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

12 . The method of claim 1 , wherein the porous biomaterial is degradable.

13 . The method of claim 12 , wherein the degradable porous biomaterial is selected from the group consisting of an enzymatically degradable hydrogel, a hydrolytically degradable hydrogel, or a photodegradable hydrogel.

14 . The method of claim 13 , wherein the enzymatically degradable hydrogel is matrix metalloproteinase (MMP)-degradable.

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

16 . The neural tissue construct of claim 15 , comprising mature microglia.

17 . The neural tissue construct of claim 15 , comprising stratified layers of neurons and glia.

18 . A method of in vitro screening of an agent, comprising
(a) contacting a test agent to a vascularized neural tissue construct obtained according to the method of claim 1 ; and (b) detecting an effect of the agent on one or more cell types within the contacted neural tissue construct.

19 . The method of claim 18 , wherein the agent is screened for toxicity to human neural tissue.

20 . The method of claim 18 , wherein detecting comprises detecting at least one effect of the agent on morphology or life span of cells or tissues within the contacted tissue construct, whereby an agent that reduces the life span of the cells or tissues or has a negative impact on the morphology of the cells or tissues is identified as toxic to human neural tissue.

21 . The method of claim 18 , wherein detecting comprises performing a method selected from the group consisting of RNA sequencing, gene expression profiling, transcriptome analysis, metabolome analysis, detecting reporter or sensor, protein expression profiling, Førster resonance energy transfer (FRET), metabolic profiling, and microdialysis.

22 . The method of claim 18 , wherein the agent is screened for an effect on gene expression and wherein detecting comprises assaying for differential gene expression relative to an uncontacted tissue construct.

23 . The method of claim 18 , further comprising using a predictive model to determine the relationship of gene expression levels of a panel of markers for the test compound-contacted tissue construct to gene expression levels of markers that are characteristic of exposure to a neurotoxic agent, wherein the predictive model is constructed using transcription and metabolic profiles obtained for each component of a panel of agents having known neurotoxic effects as markers of toxicity to human neural tissue.

24 . A tissue construct screening system, comprising an analytical device configured to obtain data comprising measurements from a human vascularized neural tissue construct; a computer controller configured to receive the data from the analytical device; and a machine-based adaptive learning system trained using known gene expression data and configured to select a subset of features from the data using a feature selection algorithm, wherein the subset of features correspond to a change in a level of expression of at least one gene following exposure to a known or unknown compound.

25 . The system of claim 24 , wherein the human vascularized neural tissue construct is obtained according to the method of claim 1 .

26 . The system of claim 24 , wherein the measurements comprise gene expression data obtained from microarray analysis.

Assignees

• Wisconsin Alumni Res Found

Inventors

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

Classifications

• C12N5/0697 Primary

  Artificial constructs associating cells of different lineages, e.g. tissue equivalents (blood vessels C12N5/0691) · CPC title

• C12N2533/30

  Synthetic polymers · CPC title

• C12N2503/02

  Drug screening · CPC title

• G01N33/5058

  Neurological cells · CPC title

• C12N5/0622

  Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells · CPC title