Expression of BMP4 mature peptide in eukaryotic cells and its differentiation-inhibiting effect in culturing induced pluripotent stem cells

Transcription

1 2012;32(10) J South Med Univ 1383 Original rticle Expression of MP4 mature peptide in eukaryotic cells and its differentiation-inhibiting effect in culturing induced pluripotent stem cells DING Daofang 1, WNG Zhen 2, XU Hao 1, XU Leqin 1, LING Qianqian 1, ZHO Yongjian 1, SHI Qi 1, WNG Yongjun 1 1 Institute of Spinal Disease, Longhua Hospital ffiliated to Shanghai University of Traditional hinese Medicine, Shanghai , hina; 2 entral Laboratory of ancer Hospital, Fudan University, Shanghai , hina bstract: Objective To investigate the role of bone morphogenetic protein 4 (MP4) in culturing induced pluripotent stem cells (ipss) and the related signal pathways. Methods We amplified the mature peptide of MP4 from the placenta through RT-PR, and IgK secretion peptide was ligated to the N-terminal of MP4 mature peptide. The recombinant plasmid ppyg-igk-mp4 was transfected into 293T cells and screened with puromycin, and the positive clones for expressing MP4 were verified by cell immunofluorescence and Western blotting. To test the bioactivity of MP4, ipss were cultured in the medium supplemented with leukemia inhibitory factor (LIF) plus the supernatant containing MP4, and the cell phenotype, cell differentiation capacity into lineages of the 3 germ layers and expression levels of pluripotency-associated genes were investigated. Results Smad1 was phosphorylated by MP4 from the culture medium. ipss cultured in the medium supplemented with LIF plus the supernatant containing MP4 for 3 passages maintained the phenotype of stem cells with the expression levels of pluripotency-associated genes not affected. These ipss also maintained the capacity to differentiate into cell lineages of the 3 germ layers. onclusion MP4 can be efficiently expressed in mammalian cells to maintain the multipotent differentiation capacity of the ipss in in vitro culture. Key words: bone morphogenetic protein 4; immunoglobulin kappa chains; over-expression; induced pluripotent stem cells; cell differentiation INTRODUTION Embryonic stem (ES) cells with self-renewal and multipotent differentiation capacities can be isolated from the inner cell mass (IM) of an embryo and cultured in vitro [1]. s a member of the transforming growth factor-β (TGF-β) family, bone morphogenetic protein 4 (MP4) plays pivotal roles in the modulation of the self-renewal and proliferative activities of stem cells [2], and can induce human ES cells to differentiate into the trophoblast lineage in the presence of fibroblast Received: ccepted: Supported by National asic Research Program of hina (973 Program, ), Key Project of National Natural Science Foundation of hina ( ), hangjiang Scholar hair Professor Project ([2009]17), Shanghai Education Innovation Project (08YZ56), Shu Guang Project supported by Shanghai Municipal Education ommission and Shanghai Education Development Foundation (10GG20), Shanghai University Innovation Team Programmer (Shanghai Education ommission, Division 6[2009]),and the hina Postdoctoral Science Foundation ( ). orresponding author: WNG Yongjun, MD, PhD, professor, Tel: , yjwang88@hotmail.com growth factor (FGF) [3] and the expression of trophoblastassociated genes in the ES cells without FGF [4]. MP4 is also capable of inducing the differentiation of mesendoderm in cooperation with activin [5]. ll these evidences suggest that MP4 importantly participates in the differentiation of human ES cells. MP4 assumes different roles in murine ES cell cultures. The differentiation of murine ES cells into neural lineage can be inhibited by treatment with MP4 [6]. MP4 in collaboration with leukemia inhibitory factor (LIF) sustains the self-renewal capacity of murine ES cells [7]. Further studies also confirmed the effect of MP4 on murine ES self-renewal [8]. oth ES cells and induced pluripotent stem cells (ipss) are pluripotent stem cells derived from different tissues by different methods. ipss are commonly obtained by combinatorial expression of Oct3/4, Sox2, c-myc and Klf4 transcription factors in embryonic fibroblast cells [9-11], and they share similarities to ES cells in gene expression profiles and differentiation potentials [12-14]. Genetically, ipss appear indistinguishable from ES cells, but they are not truly equivalent in comparative genomic analyses [15-16]. In this study, we aimed to express MP4 mature peptide and analyze the bioactivity of MP4 in ips culture and explore the pathways involved in the action of MP4.

2 1384 J South Med Univ Vol.32 MTERILS ND METHODS ell line and culture Human 293T17 cell line was obtained from merican Type ulture ollection (T) and maintained routinely in high-glucose DMEM supplemented with 10% fetal bovine serum (FS, iowest). Mouse ipss were produced in our lab previously in ES medium prepared with DMEM containing 10% FS (Gibco), 1 NE, 1 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, and 0.1 mmol/l 2-mercaptoethanol, supplemented with 10 ng/ml LIF. The ipss were cultured in prepared with DMEM containing N227 (Gibco), 1 NE, 1 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, and 0.1 mmol/l 2-mercaptoethanol, supplemented with 10 ng/ml LIF and with diluted supernatant of transfected 293T cells or not (MP4 + or MP4 - ). onstruction of recombinant plasmids and stable transfection The total RN was extracted from mouse placenta and reverse-transcribed into cdn with reverse transcriptase MV (D2620, Takara). MP4 mature peptide was amplified directly from cdn, and Igk signal peptide was ligated to MP4 mature peptide by multiplex PR. The restriction endonuclease sites such as Not I/Xho I were introduced into IgK-MP4, and Igk-MP4 was finally cloned to ppyg to obtain the recombinant plasmid ppyg-igk-mp4. The primers used are listed in Tab.1. ppyg-igk-mp4 and ppyg plasmids were separately transfected into 293T cells, and 72 h later, 2 mg puromycin/l was added to screen the positive clones for a week. oth the supernatant and cells were collected at 2, 4, 6 and 8 weeks after the transfection for analyzing MP4 expressions. Tab.1 Primers for amplifying IgK-MP4 Primer MP4-1F MP4-1R IgK-MP4-2F IgK-MP4-2R IgK-MP4-3F IgK-MP4-3R Primer sequence (5'-3') TGGTGGGTGG TGGGGTGGTTT TGTGTTGGGTTGGTTTGGTGTGGT TGGGGTGGTTT GGTGGTGGGGTTGTTGGGTGTGTGT TGGGTTGGTTTGGTG TGGGGTGGTTT First round Second round Third round Immunofluorescence assay The cells were fixed with 4% paraformaldehyde, washed in PS for 3 times, and permeabilized with 0.1% Triton X-100 for 30 min. fter washing with PS for 3 times, the cells were blocked with 1% bovine serum albumin (S) and then incubated with MP4 primary antibody (hemicon, M1049) for 1 h at room temperature. fter washing with PS for 3 times, the cells were incubated with the secondary antibody for 30 min at room temperature, followed by staining of the cell nuclei with 10 ng/ml DPI. Differentiation of ipss The ipss were cultured in MP4 + for 3 passages. Without LIF and MP4, the cells could form embryoid bodies in noncoated plastic dishes in a week. The embryoid bodies were attached to the dish bottom and began to differentiate. fter 4 days, the cells were examined by immunostaining for expressions of ol2ai (sc-52658, US), a-fetoprotein (Glostrup, Denmark), and vimentin (ab20346). Western blotting The ipss were lysed with the lysis buffer (eyotime, P0013) containing PMSF. To determine the phosphorylation level of Smad1, the cells were stimulated by the supernatants of transfected 293T cells for 15 min. nti-oct4 (S-5279, US), anti-sox2 (S-17320, US) and P-smad1/5/8 (#9511, ST) antibodies were used. Semi-quantitative PR of marker genes in ips cells Reverse transcription of the total RN (2 μg) was performed with RT Reagent Kit (Takara code DRR037). PR was performed with ExTaq (Takara code DRR041). fter amplification, 5 μl of the product from each reaction was loaded onto 2% agarose gel containing ethidium bromide (0.5 μg/ml). The sequences of the primers are listed in Tab.2. The experiments were repeated for 3 times. RESULTS loning MP4 mature peptide from the mouse placenta The signal peptide and leader peptide from MP4 were replaced by IgK signal peptide (Fig.1-). The length of MP4 was 348 bp (Fig.1). IgK-MP4 was inserted between XhoI and NotI restriction sites of ppyg plasmid (Fig.1D). Expression of ppyg-igk-mp4 plasmid and bioactivity of MP4 MP4 was expressed in the cytoplasm of 293T

3 No.10 DING Daofang, et al. Expression of MP4 mature peptide in eukaryotic cells and its differentiation-inhibiting effect in culturing induced pluripotent stem cells 1385 Tab.2 Primers for marker genes expressed in ipss Gene actin-f actin-r ET1-F ET1-R Fgf4-F Fgf4-R Foxd3-F Foxd3-R Gdf3-F Gdf3-R Nanog-F Nanog-R Genank accession no. NM_ NM_ NM_ NM_ '-3' aaggccaaccgtgaaaagat gtggtacgaccagaggcatac gaaaggcgagctgagatttg ccagcctccagagcctctat gcaagctcttcggtgtgc cgtaggattcgtaggcgttg ccccaacactgaccaacag gtttgctccgccagctta tgttcgtgggaacctgct gccatcttggaaaggtttctg ttcttgcttacaagggtctgc agaggaagggcgaggaga cells transfected with ppyg-igk-mp4, but not detected in cells transfected with ppyg (Fig.2). Quantitative analysis of the supernatant by Western blotting demonstrated MP4 release in the medium by ppyg-igk-mp4-transfected cells (Fig.2). No significant variations were observed in the expression levels of MP4 in the cytoplasm at different time points, nor in MP4 levels in the supernatant (Fig.2). Smad1 was phosphorylated by MP4 in the supernatant of ppyg-igk-mp4-transfected cells within 15 min (Fig.2D). Self-renewal of ipss ipss were maintained in MP4 + or MP4 - and passaged for three times. LIF combined with MP4 in the medium efficiently prevented ips differentiation Signal peptide leader peptide Mature peptide MP4 xhoi lgk Mature peptide NotI IgK sequence: TGGGGTTGTTGGGTGTGTGTTGGGTTGGTTTGGTG D ppyg 500 bp 300 bp MP4 500 bp 300 bp IgK-MP4 Fig.1 Plasmid construction of ppyg-igk-mp4. :Map of ppyg-igk-mp4 construction; : IgK signal peptide sequence; : RT-PR analysis of MP4 mature peptide from mouse placenta tissue (the size of MP4 is 348 bp); D: Restriction enzyme analysis of ppyg- Igk-MP4 with NotI and XhoI. The size of Igk-MP4 is 411 bp. (Fig.3 and ), but in the absence of MP4, LIF alone failed to maintain the phenotype of stem cells (Fig.3). The cells cultured in MP4 + did not exhibit significant changes in Sox2/Oct4 protein expressions compared to the cells cultured in ESM. In MP4 -, the cells showed down-regulated Sox2/Oct4 expression compared to the cells cultured in ESM or in MP4 + (Fig.3D). The mrn expression levels of other pluripotency-associated genes also showed changes similar to those of Sox2/Oct4 protein expression (Fig.3E). ips differentiation into three germ layers ipss still maintained the differentiation potential after culturing for 3 passages in MP4 + and expressed the marker genes (FP, ol2a1 and vimentin) of the three germ layers (Fig.4). DISUSSION Numerous evidences have been obtained concerning the transcriptional networks that regulate ES cells, but currently little is known of the signaling pathways that control the reprogramming and maintain the pluripotent state of the cells. recent study showed that MP signaling promotes reprogramming during the initiation stage, and the effect of MPs on reprogramming depends on the presence of Oct3/4, Sox2, c-myc and Klf4 [17]. MP4 also greatly improves the efficiency of Oct4/Sox2 or Oct4 reprogramming [18]. The role of MP4

4 1386 J South Med Univ Vol.32 MP4 DPI Merged ppyg-igk-mp4 ppyg ppyg-igk-mp4 ppyg supernatant cytoplasm actin D ppyg-igk-mp4 ppyg 2 weeks 4 weeks 6 weeks 8 weeks 15 min P-smad1 actin Fig.2 Expression of MP4 in 293T17 cells after stable transfection with the episomal vector. : Immunofluorescence analysis of MP4 expression in 293T cells ( 200); : MP4 (protein molecular weight ) from the supernatant 14 days post-transfection detected by Western blotting: : MP4 expression in the cytoplasm and supernatant at different time points post-transfection; D: MP4 from the supernatant activates Smad1 in comparison with the control supernatant. in culturing ipss is still unknown. In the present study, the effects of the supernatant containing MP4 on ipss were analyzed. Several measures were taken to obtain higher expressions of MP4. highly efficient eukaryotic expression system exploiting the episomal vector ppyg and 293T cell line was used. ppyg expression vector contains the hybrid promoter G which is a combination of the cytomegalovirus (MV), early enhancer element and chicken beta-actin promoter. Episomal vector can be replicated continuously after transfection into cells that contain a large T antigen such as 293T cells [19-20]. Such replication allows for maintenance of stable expression of the recombinant protein. The expression of a recombinant protein is also associated with its gene length. MP4 gene is constituted by 3 elements, namely the signal peptide, leader peptide, and mature peptide, and the mature peptide executes the major function of MP4 [21]. We amplified the mature peptide of MP4 by RT-PR from mouse placenta. To express MP4 in the form of secretion in the supernatant, the coding sequences for a mouse immunoglobulin k chain (Igk) signal peptide was fused to the N-terminal of MP4 mature peptide. This signal peptide also increased the expression level of MP4 in the supernatant. ell immunofluorescence assay was employed to verify the expression of MP4 in 293T cells. ompare to the control cells, the transfected cells showed obvious

5 DING Daofang, et al. Expression of MP4 mature peptide in eukaryotic cells and its differentiation-inhibiting effect in culturing induced pluripotent stem cells No.10 (LIF+,MP4+) ESM ESM 1387 (LIF+MP4+) (LIF+,MP4-) (LIF+MP4-) ESM (LIF+,MP4+) (LIF+,MP4-) ecat1 Sox2 nanog fgf4 Oct4 gdf3 foxd3 actin E actin D Fig.3 Phenotype of ipss and expression of pluripotency-associated genes after cell culture in ESM (), MP4+ (), and MP4 () for 3 passages ( 100). D: omparison of Sox2/Oct4 protein expression levels in different media; E: Semi-quantitative PR analysis of mrn expression of Ecat1, Nanog, FGF4, Gdf3 and Foxd3 in different media. endoderm marker FP mesoderm marker ol2a1 ectoderm marker Vimentin Fig.4 Differentiation of ipss in vitro cultured in MP4 + for 3 passages. The differentiated ipss expressed endoderm marker FP, mesoderm marker ol2a1 and ectoderm marker vimentin (Immunostaining, original magnification: 200). MP4 expression in the cytoplasm. To eliminate the trace of MP4 in FS, 293T cells screened by puromycin were cultured in the medium in the absence of LIF and MP4 before the supernatants were collected. Thus, the proteins present in medium were totally cell-secreted proteins. Western blotting showed that MP4 was released in the medium 14 days after the transfection. We also found that the expression levels of MP4 were stable in both the cytoplasm and supernatant 2, 4, 6, and 8 weeks after the transfection, which further confirmed the correct construction of ppyg-igk-mp4 vector and the stable expression of MP4 in 293T cells. ipss, like ES cells, also have self-renewal and differentiation potentials. ipss exhibits the phenotype of ES cells when cultured in ESM. Obvious differentiated cells were observed around the clone when ESM was replaced by MP4-. The cell differentiation was inhibited efficiently by MP4 +. The expression levels of pluripotency-associated genes in ipss cultured in different medium were consistent with the cell phenotypes. Differentiation of ipss into the three germ layers in vitro indicates that MP4 supports the selfrenewal of ipss in the presence of LIF. MPs including MP4 had proved to be essential for the reprogramming of somatic cells. Our findings confirmed that MP4 was indispensable for culturing ipss. However, Smad1 was activated by the supernatant containing MP4. This is a new finding for ipss, suggesting that ipss may share the same pathway of self-renewal that ES cells exploit. cknowledgments We are grateful to Dr. Yang Jian (Wellcome Trust Sanger Institute) for kindly providing us ppyg vector. References [1] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos J. Nature, 1981, 292: [2] Watabe T, Miyazono K. Roles of TGF-beta family signaling in stem cell renewal and differentiation J. ell Res, 2009, 19(1): [3] Xu RH, Peck RM, Li DS, et al. asic FGF and suppression of MP

6 1388 J South Med Univ signaling sustain undifferentiated proliferation of human ES cells J. Nat Methods, 2005, 2(3): [4] Yu P, Pan G, Yu J, et al. FGF2 sustains NNOG and switches the outcome of MP4-induced human embryonic stem cell differentiation J. ell Stem ell, 2011, 8(3): [5] Vallier L, Touboul T, hng Z, et al. Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways J. PLoS ONE, 2009, 4(6): e6082. [6] Finley MF, Devata S, Huettner JE. MP-4 inhibits neural differentiation of murine embryonic stem cells J. J Neurobiol, 1999, 40(3): [7] Ying QL, Nichols J, hambers I, et al. MP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STT3 J. ell, 2003, 115(3): [8] Qi X, Li TG, Hao J, et al. MP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways J. Proc Natl cad Sci US, 2004, 101(16): [9] Takahashi K, Yamanaka S. Induction of Pluripotent Stem ells from Mouse Embryonic and dult Fibroblast ultures by Defined Factors J. ell, 2006, 126(4): [10] Takahashi K, Okita K, Nakagawa M, et al. Induction of pluripotent stem cells from fibroblast cultures J. Nat Protoc, 2007, 2(12): [11] Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells J. Nature, 2007, 448(7151): [12] Kang L, Wang J, Zhang Y, et al. ips cells can support full-term development of tetraploid blastocystcomplemented embryos J. ell Stem ell, 2009, 5(2): Vol.32 [13] Zhao XY, Li W, Lv Z, et al. ips cells produce viable mice through tetraploid complementation J. Nature, 2009, 461(7260): [14] hin MH, Mason MJ, Xie W, et al. Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures J. ell Stem ell, 2009, 5(1): [15] Deng J, Shoemaker R, Xie, et al. Targeted bisulfite sequencing reveals changes in DN methylation associated with nuclear reprogramming J. Nat iotechnol, 2009, 27(4): [16] Doi, Park IH, Wen, et al. Differential methylation of tissue-and cancer-specific pg island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts J. Nat Genet, 2009, 41(12): [17] Samavarchi-Tehrani P, Golipour, David L, et al. Functional genomics reveals a MP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming J. ell Stem ell, 2010, 7(1): [18] hen J, Liu J, Yang J, et al. MPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone J. ell Res, 2011, 21(1): [19] Gassmann M, Donoho G, erg P. Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells J. Proc Natl cad Sci US, 1995, 92(5): [20] amenisch G, Gruber M, Donoho G, et al. polyoma-based episomal vector efficiently expresses exogenous genes in mouse embryonic stem cells J. Nucleic cids Res, 1996, 24(19): [21] ui Y, Hackenmiller R, erg L, et al. The activity and signaling range of mature MP-4 is regulated by sequential cleavage at two sites within the prodomain of the precursor J. Genes Dev, 2001, 15 (21): 真核表达 MP4 成熟肽及其对 ips 细胞的分化抑制作用 丁道芳 1 王 臻 2 徐 浩 1 徐乐勤 1 梁倩倩 1 赵永见 1 施 杞 1 王拥军 1 1 上海中医药大学龙华医院脊柱病研究所 上海 复旦大学肿瘤医院中心实验室 上海 摘要 目的 研究 MP4 因子在诱导型干细胞 ips 细胞 培养过程中的作用和起作用的相关通路 方法 通过 RT-PR 的方法从 小鼠的胎盘组织中扩增 MP4 成熟肽 并且在其 N 末端连接 IgK 分泌肽 此融合的片段克隆至 ppyg 载体上 重组质粒 ppyg-igk-mp4 转染至 293T17 细胞中并进行嘌呤霉素的筛选 通过细胞免疫荧光和 Western blot 方法鉴定出表达 MP4 的阳性克隆 为进一步检测上清中 MP4 的生物活性 ips 细胞培养于含 MP4 因子的细胞培养上清和 LIF 因子中 连续培养 3 代 并进一步观察细胞表型 三胚层细胞的分化潜能和多潜能相关基因的表达水平 结果 Smad1被分泌至上清中的MP4磷酸 化 当培养基中含 MP4 同时加入 LIF 因子后 可以有效抑制 ips 细胞分化 在此种方法培养 3 代后 不仅 ips 细胞的表型可以 有效维持 ips细胞中多潜能相关的基因表达水平也未受到影响 同时这些iPS细胞仍具有向三个胚层分化的能力 结论 MP4 可高效表达在真核细胞中 有效地使培养iPS细胞保持其多向分化潜能 关键词 Igk-MP4 免疫球蛋白κ链 过表达 诱导型干细胞 细胞分化 中图分类号 R34 文献标志码 文章编号 doi: /j.issn 收稿日期 基金项目 国家基础研究973项目 国家自然科学基金重点项目 长江学者计划 Teach people 上海创新团队项目 08YZ56 曙光计划 10GG20 上海高校创新团队项目 Shanghai Education ommission, Division 中国博士后基金 作者简介 丁道芳 博士 @fudan.edu.cn 通讯作者 王拥军 博士 教授 yjwang88@hotmail.com