Methods to determine the effect of an agent on mammalian embryonic development

Abstract

The invention relates to the fields of developmental toxicity. In particular, it relates to novel reporter cell types that may be used in in vitro methods to determine the effect of an agent on mammalian embryonic development.

Claims

1. An in vitro method of determining an effect of an agent on mammalian embryonic development, the method comprising: a) providing at least seven types of reporter cells, wherein each type of reporter cell comprises a reporter sequence operatively linked to a different regulatory element of a gene; b) contacting the at least seven type of reporter cells with the agent; c) comparing the expression of the reporter sequences in the at least seven types of reporter cells contacted with the agent to a corresponding cell not contacted with the agent; and d) determining that the agent has an effect on mammalian embryonic development if in step c) a difference in expression of the reporter sequences is detected between the reporter cells contacted with the agent and the corresponding cells not contacted with the agent for at least one type of reporter cell; and, wherein the different regulatory elements for the at least seven types of reporter cells comprise: a regulatory element of the OCT4 gene comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 1 and SEQ ID NO: 21; a regulatory element of the BMP4 gene, comprising a polynucleotide sequence that has at least 60% sequence identity at least one of SEQ ID NO: 2 and SEQ ID NO: 22; a regulatory element of the MYH6 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 3 and SEQ ID NO:23; a regulatory element of the PAX6 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 4 and SEQ ID NO: 24; a regulatory element of the FOXA2 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 5 and SEQ ID NO: 25; a regulatory element of the AFP gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 8 and SEQ ID NO: 28; or a regulatory element of the SOX1 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 101 or SEQ ID NO: 103.

2. The method according to claim 1, wherein the method comprises contacting the agent in step b) with at least one additional type of reporter cells selected from the group consisting of: a regulatory element of the SOX17 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 6 and SEQ ID NO: 26; a regulatory element of the ALB gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 7 and SEQ ID NO: 27; a regulatory element of the Ck18 gene comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 9 and SEQ ID NO: 29; and a regulatory element of the Vegfr1 gene comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 10 and SEQ ID NO: 30.

3. The method according to claim 1, wherein the reporter sequence is a gene encoding a protein.

4. The method according to claim 1, wherein the agent is a pharmaceutical agent, a chemical agent, a dye, an agrochemical agent, a cosmetic, a plasticizer or a food-ingredient.

5. The method according to claim 1, wherein the agent is a polypeptide, a peptide, a nucleic acid, a small molecule, or a natural product.

6. A kit of parts comprising: at least seven types of reporter cell, wherein each type of reporter cell comprises a reporter sequence operatively linked to a regulatory element of a gene and wherein the at least seven type of reporter cells each comprise: a regulatory element of the OCT4 gene comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 1 and SEQ ID NO: 21; a regulatory element of the BMP4 gene, comprising a polynucleotide sequence that has at least 60% sequence identity at least one of SEQ ID NO: 2 and SEQ ID NO: 22; a regulatory element of the MYH6 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 3 and SEQ ID NO:23; a regulatory element of the PAX6 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 4 and SEQ ID NO: 24; a regulatory element of the FOXA2 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 5 and SEQ ID NO: 25; or a regulatory element of the AFP gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 8 and SEQ ID NO: 28. a regulatory element of the SOX1 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 101 or SEQ ID NO: 103.

7. The kit of parts according to claim 6, wherein the kit further comprises at least one additional type of reporter cell selected from the group consisting of: a regulatory element of the SOX17 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 6 and SEQ ID NO: 26; a regulatory element of the ALB gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 7 and SEQ ID NO: 27; a regulatory element of the Ck18 gene comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 9 and SEQ ID NO: 29; and a regulatory element of the Vegfr1 gene comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 10 and SEQ ID NO: 30.

8. A combination of at least seven transgenic non-human mammals, comprising at least one cell comprising a reporter sequence operatively linked to a different regulatory element and wherein the regulatory element is selected from: a regulatory element of the OCT4 gene comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 1 and SEQ ID NO: 21; a regulatory element of the BMP4 gene, comprising a polynucleotide sequence that has at least 60% sequence identity at least one of SEQ ID NO: 2 and SEQ ID NO: 22; a regulatory element of the MYH6 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 3 and SEQ ID NO: 23; a regulatory element of the PAX6 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 4 and SEQ ID NO: 24; a regulatory element of the FOXA2 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 5 and SEQ ID NO: 25; a regulatory element of the AFP gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 8 and SEQ ID NO: 28; and a regulatory element of the SOX1 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 101 or SEQ ID NO: 103.

9. The combination according to claim 8, wherein the combination comprises one or more additional transgenic non-human mammals selected from the group consisting of: a transgenic non-human mammal comprising a reporter sequence operatively linked to a different regulatory element and wherein the regulatory element is a regulatory element of the SOX17 gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 6 and SEQ ID NO: 26; a transgenic non-human mammal comprising a reporter sequence operatively linked to a different regulatory element and wherein the regulatory element is a regulatory element of the ALB gene, comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 7 and SEQ ID NO: 27; a transgenic non-human mammal comprising a reporter sequence operatively linked to a different regulatory element and wherein the regulatory element is a regulatory element of the Ck18 gene comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 9 and SEQ ID NO: 29; and a transgenic non-human mammal comprising a reporter sequence operatively linked to a different regulatory element and wherein the regulatory element is a regulatory element of the Vegfr1 gene comprising a polynucleotide sequence that has at least 60% sequence identity with at least one of SEQ ID NO: 10 and SEQ ID NO: 30.

10.-11. (canceled)

12. The method according to claim 3, wherein the reporter sequence encodes a protein that is readily detectable.

13. The combination according to claim 8, wherein the transgenic non-human mammals are rodents.

Description

DESCRIPTION OF THE FIGURES

[0209] FIG. 1: hiPSC cells were differentiated towards cardiomyocytes, hepatocyte and neural rosettes by addition of specific growth factors at specific times during differentiation (as described herein). RNA samples were collected at Day 0, Day 7 and Day 10, 14 or 21 and biomarker expression was analyzed using qPCR.

[0210] A) Cardiomyocyte differentiation. OCT4 was expressed in pluripotent stem cells and during differentiation the expression decreased. BMP4 marked the middle stage of the differentiation and expression peaks around day 7. MYH6 was expressed in mature cardiomyocytes and expression increased overtime.

[0211] B) Hepatocyte differentiation. OCT4 was expressed in pluripotent stem cells and during differentiation the expression decreased. SOX17 and FOXA2 were expressed around day 7 which marks the intermediate stage of differentiation. ALB and AFP were expressed during late stages of differentiation.

[0212] C) Neural rosette formation. OCT4 was expressed in pluripotent stem cells and during differentiation the expression decreases. SOX1 marked the middle stage of the differentiation and expression peaks around day 7. PAX6 is expressed in neural rosettes and expression increases over time.

[0213] FIG. 2: Differentiation in the presence of retinoic acid. To measure the effect of the teratogenic compound retinoic acid, hiPSC cells were differentiated towards hepatocytes (A), cardiomyocytes (B) and neural rosettes (C) in the presence of retinoic acid. During and at the end of differentiation, RNA samples were collected and biomarker expression was analyzed using qPCR. * p<0.05 in T-test compared to vehicle control.

[0214] FIG. 3: Differentiation in the presence of acrylamide. To measure the effect of the non-teratogenic compound acrylamide, hiPSC cells were differentiated towards hepatocytes (A) and cardiomyocytes (B) in the presence of acrylamide. During and at the end of differentiation, RNA samples were collected and biomarker expression was analyzed using qPCR. * p<0.05 in T-test compared to vehicle control (no changes were significant).

[0215] FIG. 4: OCT4-GFP reporter cell line. hiPSC were genetically modified to express OCT4-GFP from the endogenous locus. HiPSC express OCT4 in the pluripotent state (Day 0). (A) FACS plots show the GFP intensity of wild-type unmodified hiPSC and OCT4-GFP hiSPC cells. (B) percentage of GFP positive cells and GFP intensity of wild-type and OCT-GFP hiPSC (clone 1) during differentiation towards cardiomyocytes.

[0216] FIG. 5: Defective cardiomyocyte differentiation upon teratogenic compound exposure. mES cells were exposed to teratogenic compounds during mES cell differentiation towards cardiomyocytes. The percentage of beating 3D embryoid bodies (examples in A), representing functional cardiomyocytes, was counted. (B) Quantification of beating bodies. Presented values are expressed as percentages of control. Students t-test was used to determine significance. *p<0.05, **p<0.01, ***p<0.001.

[0217] FIG. 6: mES cells were differentiated towards cardiomyocytes or hepatocytes by the addition of specific growth factors at specific times during differentiation (as described herein). RNA samples were collected at Day 0, Day 5, Day 12 and Day 18 and biomarker expression was analyzed using qPCR.

[0218] A) Cardiomyocyte differentiation. OCT4 was expressed in pluripotent mES cells and expression decreased as differentiation progressed. BMP4 was expressed at the intermediate stage of differentiation and expression peaked around day 5. Vegfr1 marked maturing cardiomyocytes and expression peaked around day 12.

[0219] B) Hepatocyte differentiation. OCT4 was expressed in pluripotent mES cells and expression decreased as differentiation progressed. FOXA2 and SOX17 were expressed at the intermediate stage of differentiation and expression peaks around day 10.

[0220] FIG. 7: GFP-Ck18 reporter cells were differentiated towards hepatocytes. During differentiation, the expression of GFP-CK18, a marker for mature hepatocytes, increased overtime, which can be observed using microscopy (A). (B) Expression of GFP-CK18 was measured by quantifying the GFP expression. GFP-intensity calculated with Fiji image processing package.

[0221] FIG. 8: Defective hepatocyte differentiation upon teratogenic compound exposure. mES GFP-Ck18 reporter cells were exposed to different teratogenic compounds during mES cell differentiation towards hepatocytes. After 21 days, fluorescence intensity of the hepatocyte specific GFP-Ck18 reporter was measured using microscopy and quantified using Fiji image processing software. GFP intensity was normalized to nuclear intensity measured using DAPI. Presented values are expressed as percentages of control. Student t-test was used for determination of significance. *p<0.05 **p<0.01.*** p<0.001.

[0222] FIG. 9: Table summarizing the correlation of the in vivo classification of 5 well-established teratogenic and 1 non-teratogenic compounds with their respective in vitro prediction based method as described herein which utilizes seven different types of reporter cells as indicated in the table. Reduction (+) of the biomarker expression means disruption of the developmental processes, which flags the teratogenic potential of the respective compound under assessment. No effect on the biomarker expression (−) indicates the non-teratogenic compounds. Inconclusive results have been additionally highlighted (i).

DESCRIPTION OF THE SEQUENCES

[0223]

TABLE-US-00001 TABLE 1 Sequences SEQ ID NO: Name 1 OCT4 promotor region (human) 2 BMP4 promotor region (human) 3 MYH6 promotor region (human) 4 PAX6 promotor region (human) 5 FOXA2 promotor region (human) 6 SOX17 promotor region (human) 7 ALB promotor region (human) 8 AFP promotor region (human) 9 Ck18 promotor region (human) 10 Vegfr1 promotor region (human) 11 OCT4 regulatory element (human) 12 BMP4 regulatory element (human) 13 MYH6 regulatory element (human) 14 PAX6 regulatory element (human) 15 FOXA2 regulatory element (human) 16 SOX17 regulatory element (human) 17 ALB regulatory element (human) 18 AFP regulatory element (human) 19 Ck18 regulatory element (human) 20 Vegfr1 regulatory element (human) 21 OCT4 promotor region (mouse) 22 BMP4 promotor region (mouse) 23 MYH6 promotor region (mouse) 24 PAX6 promotor region (mouse) 25 FOXA2 promotor region (mouse) 26 SOX17 promotor region (mouse) 27 ALB promotor region (mouse) 28 AFP promotor region (mouse) 29 Ck18 promotor region (mouse) 30 Vegfr1 promotor element (mouse) 31 OCT4 regulatory element (mouse) 32 BMP4 regulatory element (mouse) 33 MYH6 regulatory element (mouse) 34 PAX6 regulatory element (mouse) 35 FOXA2 regulatory element (mouse) 36 SOX17 regulatory element (mouse) 37 ALB regulatory element (mouse) 38 AFP regulatory element (mouse) 39 Ck18 regulatory element (mouse) 40 Vegfr1 regulatory element (mouse) 41 GFP with PGK-NEO selection (N-terminal) 42 hOCT4_BAC_GFP_FW 43 hOCT4_BAC_GFP_REV 44 hALB_BAC_GFP-N-F 45 hALB_BAC_GFP-N-R 46 hAFP_BAC_GFP-N_F 47 hAFP_BAC_GFP-N_R 48 hMYH6_BAC_GFP-N_F 49 hMYH6_BAC_GFP-N_R 50 mCK18_BAC_GFP_NF 51 mCK18_BAC_GFP_NR 52 hOCT4_homDonor_F 53 hOCT4_homDonor_R 54 hALB_Hom_N_FW 55 hALB_Hom_N_REV 56 hAFP_Hom_N_FW 57 hAFP_Hom_N_REV 58 hMYH6_Hom_N_FW 59 hMYH6_Hom_N_REV 60 hOct4-Crispr_F 61 hOct4-Crispr-R 62 ALB-Cripsr-N1 63 ALB-Cripsr-N1 64 AFP-Crispr-N1 65 AFP-Crispr-N1 66 MYH6-Crispr-N1 67 MYH6-Crispr-N1 68 hBMP4-FW2 69 hBMP4-Rev2 70 hSOX17-FW2 71 hSOX17 Rev2 72 hAFP-FW3 73 hAFP-Rev3 74 hALB-FW3 75 hALB-Rev3 76 GAPDH-human-F 77 GAPDH-human-R 78 Hprt-human-F 79 Hprt-human-R 80 MYH6-FW 81 MYH6-Rev 82 FoxA2-human-F 83 FoxA2-human-R 84 Oct-4-human-F 85 Oct4-human-R 86 Hprt-F 87 Hprt-R 88 GAPDH-F 89 GAPDH-R 90 Oct4 91 Oct4 92 BMP-4 93 BMP-4 94 SOX17 95 SOX17 96 FOXA2 97 FOXA2 98 VEGFR 99 VEGFR 100 GFP with PGK-NEO selection (C-terminal) 101 SOX1 promotor region (human) 102 SOX1 regulatory region (human) 103 SOX1 promotor region (mouse) 104 SOX1 regulatory region (mouse) 105 SOX1_FW 106 SOX1_Rev

EXAMPLES

[0224] Materials and Methods

[0225] HiPSC Cell Culture

[0226] Human Episomal iPSC Line (hiPSC) obtained from Thermo Scientific (A18945) were passaged as clumps using ReleasR and maintained in mTESR medium (StemCell Technologies) with 0.5% PS on Matrigel (Corning) according to established protocols.

[0227] mES Cell Culture

[0228] C57/Bl6 B4418 wild type mouse ES (mES) cells were cultured in ES knockout medium (Gibco) containing 10% FCS, 2 mM glutamax, 1 mM sodium pyruvate, 1% non-essential amino acids (NEAA), 1% pencillin/streptomycin (PS), 0.1 mM 2-mercaptoethanol and leukemia inhibitory factor (LIF) as previously described (Hendriks et al., 2016). Mouse ES cells were propagated on irradiated primary mouse embryonic fibroblasts as feeders according to established protocols.

Example 1: Human iPSC Differentiation

[0229] For differentiation, single cell suspensions were created using Tryple select (Thermo) and single cell suspensions were counted using a flow cytometer before seeding.

[0230] Cardiomyocyte

[0231] The protocol for cardiomyocyte differentiation is based on (Den Hartogh et al., 2015, which is incorporated herein by reference). Briefly, on day −4, hiPSC cells were seeded in 24-well plates in mTESR medium on Matrigel. On day 0, the medium was replaced with BEL medium (IMDM, HF12 medium, PFHMII medium, BSA, ITS-X, CD lipids, α-Monothioglycerol, Glutamax, PS and ascorbic acid) containing BMP4, Activin A and Chir99021. At day 3 of differentiation, the medium was refreshed with BEL containing Xav939. On day 7 and 10 of differentiation, the medium was replaced with BEL without growth factors. RNA samples were collected on day 0, day 7 and day 14 of differentiation.

[0232] The results can be seen in FIG. 1A: OCT4, a marker of pluripotency was expressed at day 0 and, as expected, the expression decreased during differentiation. It was further found that BMP4 is a good marker for the middle stage of differentiation as the expression peaks around day 7, while MYH6 was found to be a good marker for mature cardiomyocytes, its expression increasing over time.

[0233] Hepatocyte

[0234] The hepatocyte differentiation protocol is based on (Chen et al., 2012, which is incorporated herein by reference). In brief, hiPSC cells were seeded on day −4 in mTESR medium on Matrigel. On day 0 the mTESR medium was replaced with RPMI medium containing B27, Activin A, Chir99021, HGF and PS. On day 3, the cells were refreshed with DMEM medium containing KO serum replacement, Glutamax, NEAA, 2-mercaptoethanol, DMSO and PS. On day 7, the medium was replaced with IMDM medium containing oncostatin M, dexamethasone, ITS, Glutamax, NEAA and PS. On day 10, 14 and 17, cells are refreshed with IMDM medium containing ITS, Glutamax, NEAA and PS. RNA samples were collected on day 0, day 7 and day 21 of differentiation.

[0235] The results can be seen in FIG. 1B: OCT4, a marker of pluripotency was expressed at day 0 and, as expected, the expression decreased during differentiation. SOX17 and FOXA2 were expressed around day 7 and can therefore be used to test whether differentiation is affected at the intermediate stage. ALB and AFP were expressed during late stages of differentiation and these markers were used to see whether differentiation is affected at the late stage.

[0236] Neural Rosette

[0237] The protocol for neural differentiation is based on (Lippmann, Estevez-Silva, & Ashton, 2014, which is incorporated herein by reference). In short, hiPSC were seeded in mTESR medium on Matrigel on day −1. On day 0, medium was replaced with E6 medium with PS to start differentiation. Medium was refreshed with E6 medium on Day 3 and 7. RNA samples were collected on day 0, day 7 and day 10.

[0238] The results can be seen in FIG. 1C: OCT4, a marker of pluripotency, is expressed at Day 0 and, as expected, the expression decreases during differentiation. SOX1 is expressed around day 7 and can therefore be used to test whether differentiation is affected at the intermediate stage. PAX6 is expressed in maturing neural rosettes and thus expression increased overtime.

Example 2: Effect of Teratogenic Compounds on hiPSCs

[0239] Compound Exposure During Differentiation

[0240] To assess the effect of potential teratogenic agents, hiPSC were treated with the test material from day 0 until the end of differentiation. When differentiation medium was refreshed, fresh compound was added. The maximum concentration of the vehicle was 0.1% for DMSO and All-trans retinoic acid, acrylamide, 5-fluorouracil, diphenylhydantoin and thalidomide were dissolved in DMSO.

[0241] RNA Isolation, cDNA Synthesis and qPCR

[0242] Induction of the biomarker expression was compared with the expression of the gene in undifferentiated cells using quantitative real-time PCR (qRT-PCR). At several time points during differentiation, total RNA was isolated using Trizol (Qiagen). Complementary DNA was synthesized using oligo(dT) primers and SuperScript VI ReverseTranscriptase (Invitrogen) according to the manufacturer's protocol. Expression of biomarker genes was determined using specific primers (SEQ ID NOs: 68-99, 105 and 106) spanning the exon-exon boundaries of the genes with the PowerUP SYBR Green Master Mix (Applied Biosystems) on a Quantstudio 5 Real-Time PCR System (Applied Biosystems) using ROX as a passive reference. Relative expression was normalized using expression of the GAPDH and HPRT genes.

[0243] Results

[0244] To measure the effect of the teratogenic compound retinoic acid, hiPSC cells were differentiated towards hepatocytes, cardiomyocytes and neural rosettes in the presence of retinoic acid as described here above. Retinoic acid is a known teratogenic compound and as such, this allowed us to measure whether or our selected biomarkers would reflect the teratogenic nature of retinoic acid. Expression of AFP and ALB in hepatocytes, MYH6 in cardiomyocytes and PAX6 in neural rosettes was reduced when retinoic acid was added during differentiation (see FIG. 2).

[0245] In contrast, when hiPSC cells were differentiated to hepatocytes and cardiomyocytes in the presence of the non-teratogenic compound, acrylamide, no significant reduction of biomarker expression was observed during hepatocyte or cardiomyocyte differentiation (see FIG. 3).

Example 3: OCT4-GFP Reporter Cell Line

[0246] Generation of GFP Reporter Cell Lines

[0247] The constructs for the GFP reporters were generated by BAC recombineering as described (Poser et al., 2008). Bacterial strains with a BAC containing the biomarker gene were selected using the mouse or human BAC finder and ordered from Thermo Scientific. The putative biomarker genes on the BAC were modified with a N-terminal or C-terminal GFP green fluorescent marker (Poser et al., 2008, supra) using BAC recombineering (SEQ ID NOs: 41,100). Electrocompetent BAC strains were first transformed with the pRed/ET plasmid that contains the RecE and RecT recombination enzymes. In the N-terminal GFP cassette, GFP consists of two exons, which are separated by the PGK promotor and neomycin selection cassette in the intron. The C-terminal GFP cassette consists of a GFP-tag linked to an IRES and a Neomycin/Kanamycin selection cassette. PCR fragments encoding the C-terminal or N-terminal GFP reporter cassette were generated using primers that each contain 50 nucleotide additional sequence homologous to the 5′ or 3′ sequence of the biomarker gene on the BAC (SEQ ID NOs: 42-51). These homologous sequences on both the 5′- and the 3′-ends of the PCR fragment allow Red/ET mediated site-specific recombination of the N-terminal or C-terminal GFP selection cassette at the 5′-end or 3′-end of the biomarker gene on the BAC. BAC strains that contain pRed/ET were grown at 37° C. for 45 min in the presence of L-arabinose to induce expression of the recombination enzymes. Subsequently, BAC strains were transformed with the GFP selection cassette PCR fragment by electroporation, incubated at 37° C. for 2 h to allow recombination of the PCR fragment with the BAC, and plated on kanamycin selection plates. Individual clones were analyzed for proper integration of the GFP cassette by PCR. Modified BACs were isolated using the Nucleobond PC100 DNA isolation kit (Macherey Nagel).

[0248] Creation of hiPSC Reporter Cell Lines

[0249] For hiPSC reporter cell lines, donor constructs were created from the BAC constructs containing the GFP selection cassette and homology arms matching the target sites within the reporter genes. PCR fragments were created using primers represented by SEQ ID NOs: 52-59. Furthermore, constructs containing Cas9 as well as a gRNA cutting near the START op STOP codon of the biomarker gene were created for gene targeting, as described in (Hsu et al., 2013), using the primers as represented by SEQ ID NOs:60-67.

[0250] hiPSC cells were seeded on Matrigel coated dishes 24 h prior to transfection. Donor constructs and gRNAs were transfected into hiPSC using lipofectamine 3000. Monoclonal hiPSC lines were selecting using neomycin and screened for integration of the construct by PCR.

[0251] Flow Cytometry

[0252] GPF reporter expression was generally determined by flow cytometry (Guava easyCyte 6HT, EMD Millipore). For this, differentiated cells were harvested as a single cell suspension using Tryple Select (Thermo) and resuspended in medium. Cell harvest was immediately followed by flow cytometry analysis.

[0253] Results

[0254] hiPSC were genetically modified to express OCT4-GFP from the endogenous locus and GFP expression was assed using flow cytometry. The cells were then differentiated as described above. At Day 0, OCT4 was highly expressed as expected in pluripotent cells (see FIG. 4A). During differentiation towards cardiomyocytes, both the average GFP intensity and the number of GFP positive cells reduced in the OCT4-GFP expressing clone (FIG. 4B). The OCT4-GFP cells were able to form beating cardiomyocytes, indicating that the gene targeting did not affect the differentiation potential of the hiPSC (results not shown).

Example 4: Mouse ES Differentiation

[0255] Cardiomyocyte

[0256] For cardiomyocyte differentiation embryonic bodies were used, formed by hanging drops containing 750 cells in Iscoves's Modified Dulbecco's Medium (IMDM). Prior to hanging drop formation, cells were cultured on gelatine-coated dishes in IMDM supplemented with 20% FBS. Cells were detached with cell dissociation buffer (Gibco). After 3 days, embryonic bodies were transferred to a bacterial plate in differentiation medium (IMDM, 10% serum (unless specified otherwise), PS, NEAA and 2-mercapto ethanol). On day 5, the bodies were transferred to a 48-well plate in differentiation medium containing 2% serum, BMP, Activin A, Chir99021 and Xav939. On day 7 embryonic bodies were exposed to only Xav939 in differentiation medium and on day 11 all growth factors were removed and replaced with differentiation medium. Day 13 was used as endpoint for cardiomyocyte differentiation. On this day, beating bodies were quantified and RNA samples collected.

[0257] The results can be seen in FIG. 6A: OCT4, a marker of pluripotency was expressed at day 0 and, as expected, the expression decreased during differentiation. It was further found that BMP4 was a good marker for the middle stage of differentiation as the expression peaked around day 7, while Vegfr1 marked maturing cardiomyocytes and expression peaked around day 12. Exposure to the teratogenic agents 5′fluoracil, retinoic acid and diphenylhydantoin resulted in a decrease in beating cardiomyocytes after 13 days of differentiation, as can be seen in FIG. 5B.

[0258] Hepatocyte

[0259] Hepatocyte differentiation was started from a monolayer of mES cells on day −1. On day 0, differentiation medium supplemented with Activin A was added. On day 4, the medium was replaced with liver differentiation medium (DMEM containing 10% serum, PS, NEAA and 2-mercaptoethanol) with aFGF and sodium butyrate. On day 9, the medium was replaced with liver differentiation medium containing HGF. On day 14, cells were exposed to liver differentiation medium containing Dexamethasone and Oncostatin M. From day 17 onwards, liver differentiation medium was refreshed every 3-4 days. Endpoint of differentiation was set on day 21.

[0260] Proper differentiation of the mES cells was confirmed by a decrease in expression of the pluripotency marker gene OCT4, and expression of the FOXA2 and SOX17 genes during the intermediate stage of differentiation and expression peaks around day 10 (FIG. 6B).

Example 5: mES Reporter Cell Lines

[0261] Creation of mES Reporter Cell Lines

[0262] mES cells were seeded on gelatin-coated culture dishes 24 h prior to transfection. Modified BACs were transfected into the mES cells using Lipofectamine 2000 (Invitrogen) as described previously (Poser et al., 2008, supra). Monoclonal mES cell lines were selected based on the level of induction of the GFP reporter after differentiation. GFP expression in differentiated cells was determined by flow cytometry.

[0263] Results

[0264] GFP-Ck18 mES reporter cells were created as described above; Ck18 is a known marker for mature hepatocytes. The reporter cells were differentiated towards hepatocytes as described above. During differentiation, the expression of GFP-Ck18 increased overtime (FIG. 7) indicating that the reporter cells behave like unmodified mES cells. To test the effect of teratogenic compounds on GFP-CK18 mES reporter cell line differentiation, the cells were exposed to the teratogenic compounds 5′Fluoracil and retinoic acid. After 21 days, fluorescence intensity of the hepatocyte specific CK-18-GFP reporter was measured and quantified (FIG. 8). GFP-Ck18 intensity was significantly reduced upon exposure to 5′Fluoracil and retinoic acid, indicating that the reporter cell line is a reliable way to measure teratogenic effects.

REFERENCES

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