Restricted accessResearch articleFirst published online 2016-09
Osteogenic Differentiation of Human Embryonic Stem Cell-Derived Mesenchymal Progenitor Cells as a Model for Assessing Developmental Bone Toxicity In Vitro
The assessment of adverse effects of substances on human embryonic bone formation is an integral part of regulatory in vivo studies that cover prenatal developmental toxicity. One of the most promising approaches to reduce such in vivo testing is the application of stem cell-based in vitro methods. In the present study, we aimed to establish an in vitro system for developmental bone toxicity testing using human embryonic stem cell-derived mesenchymal progenitors (hES-MPs). Cell-based and biochemical assays as well as cytochemical stainings and flow cytometry confirmed the presence of a differentiation pattern characteristic for osteogenesis. Nondifferentiating hES-MPs highly expressed the early protein markers Runx2 and Dlx5, while alkaline phosphatase expression and activity strongly increased during differentiation. Moreover, immunofluorescence staining revealed the formation of a compact collagen-rich extracellular matrix, which quickly mineralized. Based on these results, we established toxicological endpoints. Cells were differentiated in the presence of test chemicals from day 1 to 15 and analyzed for osteogenic differentiation and cytotoxicity. Differentiation was assessed by applying an easy-to-use fluorescence-based mineralization assay, and cell viability was determined by resazurin reduction. Treatments with the developmental toxicants sodium valproate, boric acid, and warfarin revealed a specific effect on mineralization (with mean EC50 values of 0.27, 3.2, and 0.0028 mM, respectively) at subtoxic concentrations (mean EC50 values for cell viability were 7.8, > 8.3, and > 0.3 mM, respectively). A significantly stronger effect on mineralization in comparison to cell viability was observed with p values of p = 0.013, 0.0049, and 0.015, respectively. In contrast, the antineoplastic agent 5-fluorouracil and the nondevelopmental toxicants diphenhydramine, metoclopramide, and penicillin G had no significantly stronger impact on mineralization in comparison to cell viability with p values of p ≥ 0.46. In summary, the teratogenic potential for six of the seven test chemicals was correctly predicted with this assay.
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References
1.
European Commission. Seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the European Union, 05.12.2013 Brussel, Belgium. http://ec.europa.eu/environment/chemicals/lab_animals/reports_en.htm (last accessed March19, 2015).
2.
Augustine-RauchK. Alternative experimental approaches for interpreting skeletal findings in safety studies. Birth Defects Res B Dev Reprod Toxicol. 2007:80; 497–504.
BaileyJ, KnightA, BalcombeJ. The future of teratology research is in vitro. Biog Amines, 2005:19; 97–145.
5.
KnightA. Systematic reviews of animal experiments demonstrate poor human clinical and toxicological utility. Altern Lab Anim, 2007:35; 641–659.
6.
European Parliament and Council. Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Off J Eur Union, 2006:L396/1-849.
National Research Council.. Toxicity Testing in the 21st century: A vision and a strategy. The National Academies Press. 2007. www.nap.edu/catalog.php?record_id=11970. (last accessed March19, 2015).
9.
ChoE, LiW-J. Human stem cells, chromatin, and tissue engineering: Boosting relevancy in developmental toxicity testing. Birth Defects Res C Embryo Today, 2007:81; 20–40.
10.
WobusAM, LöserP. Present state and future perspectives of using pluripotent stem cells in toxicology research. Arch Toxicol, 2011:85; 79–117.
11.
ParkerSE, MaiCT, CanfieldMA, et al.Updated National Birth Prevalence estimates for selected birth defects in the United States, 2004–2006. Birth Defects Res A Clin Mol Teratol, 2010:88; 1008–1016.
12.
International Clearinghouse for Birth Defects Surveillance and Research.. Annual report. 2012. www.icbdsr.org/page.asp?p=10065& (last accessed March19, 2015).
International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Harmonised tripartite guideline: Detection of toxicity to reproduction for medicinical products & toxicity to male fertility S5(R2), Step 4 version. 2005. www.ich.org/products/guidelines/safety/article/safety-guidelines.html (last accessed March19, 2015).
15.
DastonGP, SeedJ. Skeletal malformations and variations in developmental toxicity studies: Interpretation issues for human risk assessment. Birth Defects Res B Dev Reprod Toxicol, 2007:80; 421–424.
16.
CarneyEW, KimmelCA. Interpretation of skeletal variations for human risk assessment: Delayed ossification and wavy ribs. Birth Defects Res B Dev Reprod Toxicol, 2007:80; 473–496.
17.
SpielmannH, PohlI, DöringB, et al.The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In Vitro Toxicol, 1997:10; 119–127.
18.
SeilerAEM, SpielmannH. The validated embryonic stem cell test to predict embryotoxicity in vitro. Nat Protoc, 2011:6; 961–978.
19.
GenschowE, SpielmannH, ScholzG, et al.The ECVAM international validation study on in vitro embryotoxicity tests: Results of the definitive phase and evaluation of prediction models. Altern Lab Anim, 2002:30; 151–176.
20.
GenschowE, SpielmannH, ScholzG, et al.Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern Lab Anim, 2004:32; 209–244.
21.
PaquetteJA, KumpfSW, StreckRD, et al.Assessment of the embryonic stem cell test and application and use in the pharmaceutical industry. Birth Defects Res B Dev Reprod Toxicol, 2008:83; 104–111.
22.
De JongE, LouisseJ, VerweiM, et al.Relative developmental toxicity of glycol ether alkoxy acid metabolites in the embryonic stem cell test as compared with the in vivo potency of their parent compounds. Toxicol Sci, 2009:110; 117–124.
23.
SchenkB, WeimerM, BremerS, et al.The ReProTect feasibility study, a novel comprehensive in vitro approach to detect reproductive toxicants. Reprod Toxicol, 2010:30; 200–218.
24.
RiebelingC, PirowR, BeckerK, et al.The embryonic stem cell test as tool to assess structure-dependent teratogenicity: The case of valproic acid. Toxicol Sci, 2011:120; 360–370.
25.
Marx-StoeltingP, AdriaensE, BremerS, et al.A review of the implementation of the embryonic stem cell test (EST). Altern Lab Anim, 2009:37; 313–328.
26.
StrikwoldM, WoutersenRA, SpenkelinkB, et al.Relative embryotoxic potency of p-substituted phenols in the embryonic stem cell test (EST) and comparison to their toxic potency in vivo and in the whole embryo culture (WEC) assay. Toxicol Lett, 2012:213; 235–242.
27.
SpielmannH. The way forward in reproductive/developmental toxicity testing. Altern Lab Anim, 2009:37; 641–656.
28.
RiebelingC, HayessK, PetersAK, et al.Assaying embryotoxicity in the test tube: Current limitations of the embryonic stem cell test (EST) challenging its applicability domain. Crit Rev Toxicol, 2012:42; 443–464.
29.
Zur NiedenNI, KempkaG, AhrHJ. Molecular multiple endpoint embryonic stem cell test—A possible approach to test for the teratogenic potential of compounds. Toxicol Appl Pharmacol, 2004:194; 257–269.
30.
Zur NiedenNI, DavisLA, RancourtDE. Comparing three novel endpoints for developmental osteotoxicity in the embryonic stem cell test. Toxicol Appl Pharmacol, 2010:247; 91–97.
31.
De JongE, van BeekL, PiersmaAH. Osteoblast differentiation of murine embryonic stem cells as a model to study the embryotoxic effect of compounds. Toxicol In Vitro, 2012:26; 970–978.
32.
De JongE, van BeekL, PiersmaAH. Comparison of osteoblast and cardiomyocyte differentiation in the embryonic stem cell test for predicting embryotoxicity in vivo. Reprod Toxicol, 2014:48; 62–71.
33.
BarberiT, WillisLM, SocciND, et al.Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med, 2005:2; e161.
34.
OlivierEN, RybickiAC, BouhassiraEE. Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells. Stem Cells, 2006:24; 1914–1922.
35.
LianQ, LyeE, Suan YeoK, et al.Derivation of clinically compliant MSCs from CD105+, CD24- differentiated human ESCs. Stem Cells, 2007:25; 425–436.
36.
LiuY, GoldbergAJ, DennisJE, et al.One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating. PLoS One, 2012:7; e33225.
37.
KarlssonC, EmanuelssonK, WessbergF, et al.Human embryonic stem cell-derived mesenchymal progenitors-potential in regenerative medicine. Stem Cell Res, 2009:3; 39–50.
38.
De PeppoGM, SvenssonS, LenneråsM, et al.Human embryonic mesodermal progenitors highly resemble human mesenchymal stem cells and display high potential for tissue engineering applications. Tissue Eng Part A, 2010:16; 2161–2182.
39.
De PeppoGM, SjovallP, LenneråsM, et al.Osteogenic potential of human mesenchymal stem cells and human embryonic stem cell-derived mesodermal progenitors: A tissue engineering perspective. Tissue Eng Part A, 2010:16; 3413–3426.
40.
KärnerE, UngerC, SloanAJ, et al.Bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro. Stem Cells Dev, 2007:16; 39–52.
41.
LiH, MarijanovicI, KronenbergMS, et al.Expression and function of Dlx genes in the osteoblast lineage. Dev Biol, 2008:316; 458–470.
42.
McGee-RussellSM. Histochemical methods for calcium. J Histochem Cytochem, 1958:6; 22–42.
43.
DieneltA, zur NiedenNI. Hyperglycemia impairs skeletogenesis from embryonic stem cells by affecting osteoblast and osteoclast differentiation. Stem Cells Dev, 2011:20; 465–474.
44.
RitzC, StreibigJ. Bioassay analysis using R. J Stat Softw, 2005:12; 1–22.
45.
R Core Team.. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2003. www.R-project.org/ (last accessed March19, 2015).
46.
AkaikeH. Information theory and an extension of the maximum likelihood principle. In: Breakthroughs in statistics, Vol. 1 foundations and basic theory. ParzenE, TanabeK, KitagawaG (eds); pp. 199–213. New York: Springer-Verlag; 1998.
47.
Zur NiedenNI, KempkaG, AhrHJ. In vitro differentiation of embryonic stem cells into mineralized osteoblasts. Differentiation, 2003:71; 18–27.
48.
HansenDK, GraftonTF. Attenuation of 5-fluorouracil-induced embryotoxicity by exogenous thymidine in vitro. Toxicol In Vitro, 1988:2; 249–258.
49.
KomoriT. Regulation of osteoblast differentiation by Runx2. ChoiY, ed. Adv Exp Med Biol, 2010:658; 43–49.
50.
KrausP, LufkinT. Dlx homeobox gene control of mammalian limb and craniofacial development. Am J Med Genet Part A, 2006:140; 1366–1374.
51.
LeeHL, WooKM, RyooHM, et al.Distal-less homeobox 5 inhibits adipogenic differentiation through the down-regulation of peroxisome proliferator-activated receptor gamma expression. J Cell Physiol, 2013:228; 87–98.
52.
FrithJ, GeneverP. Transcriptional control of mesenchymal stem cell differentiation. Transfus Med Hemotherapy, 2008:35; 216–227.
53.
SoltanoffCS, ChenW, YangS, et al.Signaling networks that control the lineage commitment and differentiation of bone cells. Crit Rev Eukaryot Gene Expr, 2009:19; 1–46.
54.
WhyteMP. Physiological role of alkaline phosphatase explored in hypophosphatasia. Ann N Y Acad Sci, 2010:1192; 190–200.
55.
CohenMM. The new bone biology: Pathologic, molecular, and clinical correlates. Am J Med Genet A, 2006:140A; 2646–2706.
56.
NudelmanF, PieterseK, GeorgeA, et al.The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nat Mater, 2010:9; 1004–1009.
BonucciE. Bone mineralization. Front Biosci, 2012:17; 100–128.
59.
ChangYL, StanfordCM, KellerJC. Calcium and phosphate supplementation promotes bone cell mineralization: Implications for hydroxyapatite (HA)-enhanced bone formation. J Biomed Mater Res, 2000:52; 270–278.
SteinGS, LianJB, van WijnenAJ, et al.Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression. Oncogene, 2004:23; 4315–4329.
62.
OrnoyA. Valproic acid in pregnancy: How much are we endangering the embryo and fetus?. Reprod Toxicol, 2009:28; 1–10.
63.
PalmerJA, SmithAM, EgnashLA, et al.Establishment and assessment of a new human embryonic stem cell-based biomarker assay for developmental toxicity screening. Birth Defects Res B Dev Reprod Toxicol, 2013:98; 343–363.
64.
WaldmannT, RempelE, BalmerNV, et al.Design principles of concentration-dependent transcriptome deviations in drug-exposed differentiating stem cells. Chem Res Toxicol, 2014:27; 408–420.
65.
BaroneLM, AronowMA, TassinariMS, et al.Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins, osteocalcin, and matrix GLA protein in vitro. J Cell Physiol, 1994:160; 255–264.
66.
AtkinsGJ, WelldonKJ, WijenayakaAR, et al.Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by {gamma}-carboxylation-dependent and -independent mechanisms. Am J Physiol Cell Physiol, 2009:297; C1358–C1367.
67.
Van DrielD, WesselingJ, SauerPJJ, et al.Teratogen update: Fetal effects after in utero exposure to coumarins overview of cases, follow-up findings, and pathogenesis. Teratology, 2002:66:127–140.
68.
PiersmaAH, GenschowE, VerhoefA, et al.Validation of the postimplantation rat Whole-embryo Culture Test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern Lab Anim, 2004:32; 275–307.
69.
WalmodPS, GravemannU, NauH, et al.Discriminative power of an assay for automated in vitro screening of teratogens. Toxicol In Vitro, 2004:18; 511–525.
70.
SelderslaghsIWT, BlustR, WittersHE. Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds. Reprod Toxicol, 2012:33; 142–154.
71.
ZimmermannB, WachtelC, NoppeC. Patterns of mineralization in vitro. Cell Tissue Res, 1991:263; 483–493.
72.
ProudfootD, SkepperJN, HegyiL, et al.Apoptosis regulates human vascular calcification in vitro: Evidence for initiation of vascular calcification by apoptotic bodies. Circ Res, 2000:87; 1055–1062.
73.
McCartyMF, DinicolantonioJJ. The molecular biology and pathophysiology of vascular calcification. Postgrad Med, 2014:126; 54–64.
74.
KirschT, WangW, PfanderD. Functional differences between growth plate apoptotic bodies and matrix vesicles. J Bone Miner Res, 2003:18; 1872–1881.
75.
KimKM. Apoptosis and calcification. Scanning Microsc, 1995:9; 1137–1178.
76.
SpeerMY, GiachelliCM. Regulation of cardiovascular calcification. Cardiovasc Pathol, 2004:13; 63–70.
77.
StummannTC, HarengL, BremerS. Hazard assessment of methylmercury toxicity to neuronal induction in embryogenesis using human embryonic stem cells. Toxicology, 2009:257; 117–126.
78.
GroebeK, HayessK, Klemm-MannsM, et al.Unexpected common mechanistic pathways for embryotoxicity of warfarin and lovastatin. Reprod Toxicol, 2010:30; 121–130.
79.
KleinstreuerNC, SmithAM, WestPR, et al.Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics. Toxicol Appl Pharmacol, 2011:257; 111–121.
80.
SmirnovaL, BlockK, SittkaA, et al.MicroRNA profiling as tool for in vitro developmental neurotoxicity testing: The case of sodium valproate. PLoS One, 2014:9; e98892.
81.
FullerHR, ManNT, LamLT, et al.Valproate and bone loss: iTRAQ proteomics show that valproate reduces collagens and osteonectin in SMA cells. J Proteome Res, 2010:9; 4228–4233.
82.
WatanabeT, TajimaH, HironoriH, et al.Sodium valproate blocks the transforming growth factor (TGF)-β1 autocrine loop and attenuates the TGF-β1-induced collagen synthesis in a human hepatic stellate cell line. Int J Mol Med, 2011:28; 919–925.
83.
GürgenSG, ErdoğanD, CoşkunZK, et al.The effect of valproic acid and oxcarbazepine on the distribution of adhesion molecules in embryo implantation. Toxicology, 2012:292; 71–77.
84.
HumphreyEL, MorrisGE, FullerHR. Valproate reduces collagen and osteonectin in cultured bone cells. Epilepsy Res, 2013:106; 446–450.
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