Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Summary

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Abstract

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

Introduction

The ability of human embryonic stem cells (hESC) to differentiate into various types of cells opened up a new era of in vitro toxicity testing¹, disease modelling and regenerative medicine². The stem cells are endowed with the capacity to self-replicate, to keep their pluripotent state, and to differentiate into specialized cells^3,4. The properties of hESC (capacity to differentiate to all major cell types) are also found in other human pluripotent stem cells, such as human induced pluripotent stem cells (hiPSC) or cells generated by nuclear transfer⁵. For instance, many different hESC lines have been differentiated....

Protocol

The following protocol was performed using human Embryonic Stem Cell line (hESC) H9. This cell line was routinely cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) in hESC culture media supplemented with bFGF and then cultured in stem cell media on 6 cm Petri plates coated with basement membrane matrix such as matrigel, to get rid of MEFs. The H9 cells from >80% confluent plates were used for further passage. H9 cells cultured on basement membrane matrix plates were used for EBs formation. All procedures mentioned in the following protocol have been performed using standard methods for aseptic and good cell culture....

Results

Methyl mercury exposure in UKK test system

The cytotoxicity assay was performed with H9 EBs to obtain an IC₁₀ value (reduction of viability by 10%) for the cytotoxicity of methyl mercury (Figure 1). We also performed a microarray based (affymetrix platform) biomarker study. The H9 EBs have been exposed to methyl mercury (0.25 and 1 µM) for 14 days. On day 14, samples have been collected using TRIzol and RNA was isolated. Transcriptional profiling was performed using.......

Discussion

Traditional approaches to toxicological testing involve extensive animal studies thus making testing costly and time-consuming. Moreover, due to the interspecies differences the preclinical animal safety studies are not always valid to predict toxicity effects of potential drugs relevant for humans. Although non-human primates are most predictable, still strong ethical, and socioeconomical demands are rapidly raising by modern societies for developing sensitive and robust in vitro test system relevant t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/F-12	Life Technologies	11320082	Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12
KOSR	Life Technologies	10828028	Knockout Serum Replacement
GlutaMAX	Life Technologies	35050061	GlutaMAX supplement
NEAA	Life Technologies	11140050	MEM Nonessential Amino Acids Solution
DPBS	Life Technologies	14190-0144	Dulbecco's Phosphate-Buffered Saline, without calcium, without magnesium
mTeSR medium	Stemcell Technologies	5850
Pluronic F-127	Sigma	P2443-250G
V bottom plate	VWR	734-0483	Plate,Microwell,V BTTM,96 Well,Sterile 1 * 50 ST
V bottom plate lid	VWR	634-0011	Lid, Microtitre plates, Cond. Ring 1 * 50 ST
Pen/Strep	Life Technologies	15140-122	Penicillin-Streptomycin, Liquid
Distilled Water	Life Technologies	15230-089.	Sterile Distilled Water
Human FGF-2 (bFGF)	Millipore	GF003AF-100UG	Fibroblast Growth Factor basic, human recombinant, animal-free
Filter 0.22 μm	Millipore	SCGPU02RE	Stericup-GP, 0.22 μm, polyethersulfone, 250 ml, radio-sterilized
StemPro EZPassageTM Disposablte	Invitrogen	23181010
BD MatrigelTM, hESC qualified Matrix	Stemcell Technologies	354277	5 ml vial
DMSO	Sigma	D-2650
RNAlater Stabilization Solution	Life Technologies	AM7020	It stabilizes and protect the RNA integrity in unfrozen samples.
70 μm Cell Strainer	Becton Dickinson	352350	Cell strainer with 70 μm Nylon mesh
35 μm Lid cell strainer, 5 ml tube	Becton Dickinson	352235	5 ml polystyrene round bottom test tube, with a cell strainer cap (35 μm)
50 ml sterile Polypropylene tube	Greiner Bio-One	227261	50 ml Polypropylene tube with conical bottom, Sterile
T75 flask	Greiner Bio-One	658175	CELLSTAR Filter Cap Cell Culture 75 cm2 Flasks
TRIzol	Life Technologies	10296010
96 well optical bottom plates	Thermo Scientific	165305
CellTiter-Blue	Promega	G8081
Accutase	PAA	L11-007
Apotransferin	Sigma-Aldrich	T-2036
Dispase	Worthington Biochemicals	LS002104
Dorsomorphin	Tocris Bioscience	3093
EDTA	Roth	8043.2
FBS	PAA	A15-101
FGF-2	R&D Systems	233-FB
Gelatine	Sigma-Aldrich	G1890-100G
Glucose	Sigma-Aldrich	G7021-100G
GlutaMAX	Gibco Invitrogen	35050-038
HEPES	Gibco Invitrogen	15630-056
Insulin	Sigma-Aldrich	I-6634
Knockout DMEM	Gibco Invitrogen	10829-018
Matrigel	BD Biosciences	354234
Noggin	R&D Systems	719-NG
PBS	Biochrom AG	L1825
Progesteron	Sigma-Aldrich	P7556
Putrescine	Sigma-Aldrich	P-5780
ROCK inhibitor Y-27632	Tocris Biosciences	1254
SB431542	Tocris Biosciences	1614
SDS	Bio-Rad	161-0416
Selenium	Sigma-Aldrich	S-5261
β-Mercaptoethanol	Gibco Invitrogen	31350-010
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List of Kits
RNeasy Mini Kit (250)	QIAGEN	74106
GeneChip Hybridization, Wash, and Stain Kit	Affymetrix	900721, 22, 23	This kit provides all reagents required for hybridization wash and staining of microarrays.
Rnase-Free DNase Set	QIAGEN	79254
[header]
List of equipment
Inverted microscope	Olympus		IX71
Genechip Hybridisation Oven - 645	Affymetrix
Genechip Fluidics Station-450	Affymetrix
Affymetrix Gene-Chip Scanner-3000-7 G	Affymetrix
Spectramax M5	Molecular Devices
[header]
List of softwares
Prism 4
Affymetrix GCOS
Partek Genomic Suite 6.25
Online tools for Functional annotation DAVID Onto-tools Intelligent Systems and Bioinformatics Laboratory