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Efficient Production of Trophoblast Lineage Cells from Human Induced Laboratory Investigation (2017) 97, 1188–1200 © 2017 USCAP, Inc All rights reserved 0023-6837/17 Efficient production of trophoblast lineage cells from human induced pluripotent stem cells Junya Kojima1,2, Atsushi Fukuda1, Hayato Taira1, Tomoyuki Kawasaki1, Hiroe Ito2, Naoaki Kuji2, Keiichi Isaka2, Akihiro Umezawa1 and Hidenori Akutsu1,3 Human induced pluripotent stem cells (hiPSCs) are potentially useful in both clinical applications and basic biological research. hiPSCs can differentiate into extra-embryonic cells in the presence of BMP4. However, the differentiation potential of hiPSCs can be affected by culture conditions or genetic variation. In this study, we investigated the effect of various BMP4 concentrations on the expression states of trophoblast markers and the optimal conditions for trophoblast induction. A high-fidelity gene expression assay using hiPSC lines showed that the expression levels of various trophoblast marker genes, such as KRT7, GCM1, CGB, and HLA-G, were upregulated by BMP4 in a dose-dependent manner in all types of hiPSCs used in this study. Treatment with high doses of BMP4 for prolonged periods increased the ratio of cells with trophoblast markers irrespective of the presence of bFGF. We found that the expression states of major pluripotency- and differentiation-related protein-coding genes in BMP4-treated cells depended on culture conditions rather than donor cell types. However, miRNA expression states were affected by donor cell types rather than BMP4 dose. Furthermore, the effect of the presence of bFGF on differentiation potential of KRT7-positive cells differed among iPSC types. Mechanistically, chromatin states around KRT7 promoter regions were comparable among the iPSC types used in this study, indicating that hiPSC chromatin state at these regions is not a parameter for cytotrophoblast differentiation potential. In conclusion, the optimal conditions for trophoblast differentiation from hiPSCs differ according to parental cell line. Laboratory Investigation (2017) 97, 1188–1200; doi:10.1038/labinvest.2016.159; published online 13 March 2017 Human embryonic stem cells and induced pluripotent stem The most common method for derivation of trophoblasts cells are useful as research tools for resolving questions about from hPSCs involves exposing a culture of pluripotent stem basic biological phenomena and for cell transplantation and cells to bone morphogenetic protein 4 (BMP4) in the absence drug screening in regenerative medicine.1 Although most of basic fibroblast growth factor (bFGF).6,7 BMP4 is a key studies demonstrated that human pluripotent stem cells protein of the TGF-β super family that is essential for various (hPSCs) can differentiate into embryonic tissue,2 they can cellular activities.8 In myeloma cell lines, BMP4 inhibits DNA also differentiate into extra-embryonic tissues.3 As it is synthesis in a dose-dependent manner.9 An initial study of the difficult to study differentiating trophoblasts in vivo, hPSCs generation of trophoblast cells from human embryonic stem offer an attractive ex vivo model for human placentation. cells revealed dose-dependent morphological changes of Moreover, the placenta is an essential organ for proper BMP4.3 However, a study reported that BMP4-treated hPSCs embryonic development and pregnancy maintenance in are not able to differentiate into trophoblast cells, instead humans and its dysfunction is thought to be related with differentiating into mesoderm.10 Different differentiation pregnancy-specific diseases, such as fetal growth restriction protocols are often used in different studies, and varying and pregnancy-induced hypertension.4,5 Therefore, the culture conditions may have resulted in conflicting determination of optimal conditions for trophoblast differ- observations.11,12 Furthermore, given that the genetic back- entiation from hiPSCs is useful for understanding pregnancy- ground of human induced pluripotent stem cells (hiPSCs) is associated diseases. predominantly responsible for transcriptional and epigenetic 1Center for Regenerative Medicine, National Research Institute for Child Health and Development, Tokyo, Japan; 2Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan and 3Department of Stem Cell Research, Fukushima Medical University, Fukushima City, Fukushima, Japan Correspondence: Dr H Akutsu, MD, PhD, Center for Regenerative Medicine, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan. E-mail: [email protected] Received 12 November 2016; revised 14 December 2016; accepted 19 December 2016 1188 Laboratory Investigation | Volume 97 October 2017 | www.laboratoryinvestigation.org BMP4 dose effect on human iPSCs J Kojima et al Figure 1 Effect of BMP4 concentration on cellular viability. (a) Expression of NANOG and OCT4 in hiPSC lines. Blue (DAPI), red (NANOG), and green (OCT4). Scale bar = 50 μm. (b) Morphology of cells treated with various concentrations of BMP4. Scale bar = 500 μm. (c) Population doublings (PDs) of BMP4-treated cells. The average PDs of three independent experiments are shown. Error bars are s.e. (d) Apoptosis assay of cells treated with 10 and 100 ng/ml BMP4 for 10 days. Values shown are averages of three independent experiments. differences,13 there may be different optimal conditions for treatment of differentiated cells with various concentrations trophoblast differentiation of hiPSCs. of BMP4. The results indicated that BMP4 treatment robustly In this study, in order to define molecular features of induced trophoblast differentiation. The expression levels of BMP4-mediated differentiated cells and optimal conditions protein-coding genes associated with pluripotency and for efficient production of trophoblast cells, we conducted a differentiation in BMP4-treated cells were dependent on large-scale, high-fidelity gene expression assay following culture conditions, while those of miRNAs were affected by www.laboratoryinvestigation.org | Laboratory Investigation | Volume 97 October 2017 1189 BMP4 dose effect on human iPSCs J Kojima et al Figure 1 Continued. cell type. We also found that the presence or absence of bFGF MATERIALS AND METHODS had an impact on trophoblast differentiation. Thus our Ethics Statement findings demonstrate that there are optimal conditions for Human placental cells were collected from surgical specimens BMP4-induced trophoblast differentiation from hiPSCs. with signed informed consent and ethical approval of the 1190 Laboratory Investigation | Volume 97 October 2017 | www.laboratoryinvestigation.org BMP4 dose effect on human iPSCs J Kojima et al Figure 1 Continued. Institutional Review Board of the National Institute for Child hiPSC Culture and Differentiation into Trophoblast Health and Development, Japan (permit numbers EDOM no. Lineages 146, PL no. 55, and UTE no. 89). All experiments conducted We used three types of hiPSCs that had been previously in this study using human cells and tissues were performed in established: cells derived from menstrual blood (EDOM), accordance with the tenets of the Declaration of Helsinki. placental artery endothelium (PL), and uterine endometrium (UTE). These lines were established by retroviral infection of four reprogramming factors (OCT-3/4, SOX2, c-MYC, and Mouse Experiments KLF4), and transgene silencing was confirmed.15 All mouse experiments in this study were conducted The hiPSCs were maintained on irradiated mouse embryo- according to protocols approved by the Institutional Animal nic fibroblasts in medium consisting of 80% KnockOut Care and Use Committee of the NRICHD (Permit Number: DMEM (ThermoFisher Scientific, Waltham, MA, USA), A2016-003). Adult female B6D2F1 mice were purchased from 20% KnockOut Serum Replacement (ThermoFisher Clea Japan (Tokyo, Japan) and oocytes were collected Scientific), 2 mM L-glutamine (ThermoFisher Scientific), following standard methods.14 For functional assay of 100 U/ml penicillin (ThermoFisher Scientific), 100 μg/ml in vitro prepared human chorionic gonadotropin (HCG), streptomycin (ThermoFisher Scientific), 0.1 mM nonessential pregnant mare serum gonadotropin (PMSG; 7.5 IU; Merck amino acids (ThermoFisher Scientific), 0.2 mM 2-mer- Millipore, Germany) was injected into the abdominal cavity captoethanol (Sigma, St Louis, MO, USA), and 10 ng/ml followed by injection of the supernatant of BMP4-treated recombinant human bFGF (Wako Pure Chemical Industries, culture medium (1.5 ml) within 48 h. At 16 h after super- Osaka, Japan). natant injection, the oviducts were retrieved and the presence For trophoblast differentiation, CTK solution (Reprocell, of superovulation was confirmed by microscopy. Yokohama, Japan) was added for 1–2 min at room www.laboratoryinvestigation.org | Laboratory Investigation | Volume 97 October 2017 1191 BMP4 dose effect on human iPSCs J Kojima et al PAX6 SOX2 UTF1 FOXD3 SYP HLXB9 T GFAP OLIG2 GBX2 DES FOXA2 EBAF IFITM1 NRBA1 NODAL LIN28 DNMT3B ZFP42 EEF1A1 SEMA3A KIT GABRB3 GDF3 NR5A2 FGF4 GRB7 LEFTB TRGF1 TERT Nanog POU5F1 SFRP2 IFITM2 PODXL AFP TFCP2L1 RAF1 EOMES CDX2 GATA4 ISL1 IMP2 CRABP2 COL2A1 REST 18S NES CTNNB1 PTEN LAMC1 CD9 COMMD3 LAMB1 RUNX2 COL1A1 IL6ST GAL GATA6 SOX17 SST BRIX LAMA1 CD34 ACTB FLT1 PECAM1 WT1 ACTC FN1 LIFR NOG CDH5 CGB GCM1 Day5 <-5 (Low) 0 (High) >5 PL iPSC PL iPSC Day10 UTE iPSC Day5 UTE iPSC Day10 EDOM iPSC Day5 EDOM iPSC Day10 (log2) PL BMP4 5ng/mL Day5 PL BMP4 5ng/mL PL BMP4 10ng/mL Day5 PL BMP4 5ng/mL Day10 PL BMP4 5ng/mL PL BMP4 50ng/mL PL BMP4 50ng/mL Day5 UTE
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