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Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells Hiroyuki Imai a, Ken Takeshi Kusakabe b, Yasuo Kiso b, Shosaku Hattori c, Chieko Kai c, Etsuro Ono a, Kiyoshi Kano d, * a
Department of Biomedicine, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan Laboratory of Veterinary Anatomy, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan Amami Laboratory of Injurious Animals, Institute of Medical Science, The University of Tokyo, Kagoshima, Japan d Laboratory of Veterinary Developmental Biology, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 September 2019 Accepted 1 October 2019 Available online xxx
Background: Cell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells. Methods: Fused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells. Results: Colonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells. Conclusions: Our results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species. © 2019 Published by Elsevier Inc.
Keywords: Pluripotency Cell fusion Embryonic stem cell Induced pluripotent stem cell Mammal
1. Introduction Physiological cell fusion is observed in various cells of the liver and the placenta in mammals. In the liver, hepatocytes could acquire differentiation potency by cell fusion with hematopoietic stem cells [1]. It could also have adaptive ability by obtaining genetic diversity of polyploidization, accompanying hepatocytes fusion [2]. In the placenta, cell fusion is involved in maintaining pregnancy through syncytiotrophoblasts [3]. In addition to physiological cell fusion, artificially fused cells have been used in various investigations of cellular functions. The establishment of pathological models of Huntington’s disease by producing hybrid cells with different genetic background has been reported [4]. Cell fusion with immortalized cell lines and fibroblasts revealed that loci associated with immortalization were identified
* Corresponding author. Yamaguchi University, 1677-1, Yoshida, Yamaguchi, 7538511, Japan. E-mail address: [email protected] (K. Kano).
[5,6]. Other study demonstrated that tumor malignancy grading was attenuated by fusion between tumor cells and normal somatic cells [7]. Furthermore, hybridoma cells were produced by fusion between splenocytes and myeloma cells, which have frequently been used to produce monoclonal antibodies as biotechnological method [8]. Application of cell fusion has been used to generate transgenic mice from pluripotent stem cells as a tetraploid complementation method development [9]. Thus, artificial cell fusion has been applied in various research fields such as genetics, tumor biology, antibody medicine, and experimental animals. In stem cell research area, artificial cell fusion has been applied to induce cellular reprogramming in somatic cells. Mouse fibroblasts that were fused with mouse embryonic stem cells (ESCs) have acquired pluripotency [10]. Other studies have reported interspecific cell fusion. For example, Li et al. have succeeded in establishing interspecific diploid pluripotent stem cells by fusion of mouse haploid ESCs and rat haploid ESCs [11]. Small murine molecules possessed the potential to maintain undifferentiated state in rat pluripotent stem cells [11]. Since the mechanism of maintaining
https://doi.org/10.1016/j.bbrc.2019.10.026 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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pluripotent embryonic stem cells is still unknown in most mammals, it has been unsuccessful to establish pluripotent cells in most non-experimental model animals. This experimental system involving fusion with mouse pluripotent cells could be applied to establish pluripotent stem cells and help identify key molecules that maintain pluripotency in non-model mammals, such as domestic animals, companion animals, or wild animals. In this present study, we succeeded in triggering pluripotent state in the nuclei of mammalian fibroblasts by cell fusion with mouse ESCs. The result indicated that cell fusion with pluripotent stem cells provides novel method of cellular reprogramming in somatic cells, and establishes pluripotent stem cells in nonexperimental animals. 2. Materials and methods 2.1. Mouse ESCs and animal fibroblasts Mouse embryonic stem cells (ESCs) expressing EGFP were used as established in previous report [12]. The stem cells were cultured on feeder cells in ESGRO complete plus clonal grade medium (Merck, NJ, USA). Rabbit and horse fibroblasts were isolated from the subcutaneous tissues autopsied at Veterinary Anatomy laboratory of Yamaguchi University. Bovine fibroblasts were isolated from the subcutaneous tissues received from the meat center (Hofu city). Owl monkey (Aotus lemurinus) and squirrel monkey (Saimiri boliviensis) fibroblasts from autopsied subcutaneous tissues obtained from Amami laboratory of injurious animals, the University of Tokyo. Large Japanese field mouse (Apodemus speciosis) and zebra (Equus quagga) fibroblasts from the subcutaneous tissues autopsied at Veterinary System Physiology laboratory of Yamaguchi University. Black rhinoceros (Diceros bicornis) fibroblasts from aborted placental vessels distributed from Kanazawa Zoological Gardens. These fibroblasts were cultured with DMEM supplemented with 10% fetal bovine serum. Karyotyping of each fibroblast was performed according to previous report [12]. Transduction of puromycin resistance gene into the fibroblasts was performed using the retroviral vector. Transduced cells were selected for 10 days with puromycin (25 mg/ml). 2.2. Cell fusion Cell fusion was performed using LF301 (BEX) and an electrofusion chamber (EG450-30BG; Bioresource Center). Mouse ESCs and fibroblasts were suspended with e-hybri buffer (0.3 M mannitol, 1 mM MgCl2, 1 mM CaCl2) and mixed together. The suspensions were electrified with 50 V AC for 20 s to form pearl chains and with 400 V DC for 30 msec, three times at 0.5 msec intervals for electrofusion. The fused cells were cultured in puromycin-added ESGRO medium for 1 week. EGFP expressing cells were fractionated using a cell sorter (SH800; Sony) to isolate only the fused cells. Thereafter, expansion culture of the fused cells was performed for at least 15 passages in ESGRO medium without puromycin. ALP staining was performed by using an Alkaline Phosphatase Detection Kit (Merck). 2.3. Genomic DNA extraction and PCR Extraction of genomic DNA was performed using Nucleospin TissueXS (Takara). For PCR reaction, iCycler (Biorad) and BIOTAQ (Bioline) were used. The primer sequences are listed in Supplementary Table 1. The amplification program was performed with an initial activation step (3 min at 94 C), followed by 35 cycles of denaturation (30 s at 94 C), annealing (45 s at 52 C), an initial
extension (20 s at 72 C), and a final extension (1 min at 72 C). 2.4. RT-PCR and qRT-PCR After total RNA was isolated using ReliaPrep RNA Cell MiniPrep System (Promega), cDNA was synthesized with QuantiTect RT kit (Qiagen). RT-PCR was performed using PrimeSTAR HS (Takara) with the touchdown amplification program was performed with an initial activation step (30 s at 98 C), followed by 17 cycles of denaturation (10 s at 98 C), annealing (15 s at 72 C, decreasing by 1 C/cycle), and extension (30 s at 72 C). This step was followed by 18 cycles of denaturation, annealing (54 C), initial extension, and a final extension (1 min). qRT-PCR was performed using LightCycler Nano (Roche) and OneStep TB Green PrimeScript PLUS RT-PCR Kit (Takara) according to manufactures’ protocol. In Supplementary Table 1, the primer sequences are listed. Since the nucleic acid sequence information about zebra was not enough in database, primers for zebra analysis were designed with reference to the information about donkeys (Equus asinus), which is closely related to zebra [13]. DNA fragments were extracted using Monarch DNA gel extraction kit (NEB), and sequence analysis was performed. Primers were designed using primer3 software or referred to previous studies [14]. 2.5. Statistical analysis Student’s t-test was used to detect significant differences. 3. Results 3.1. Experiment scheme and cell lines The experimental scheme in this present study is shown in Fig. 1A. To selectively culture fused cells, fibroblasts were transfected with puromycin resistance gene by retroviral vector, and mouse ESCs were labeled with EGFP. After electrofusion, the cells were cultured with puromycin and sorted with fluorescence of EGFP. All established mammalian cells showed fibroblast-like shape after several passages (Fig. 1B). Details of fibroblast species used in this study were shown in materials and methods. The chromosome numbers were maintained their original state (Supplementary Fig. 1). After selective culture, puromycin resistance genes were detected in the genome of transfected fibroblasts (Supplementary Fig. 2). EFGP expressing mouse ESCs were established in our previous study [12]. The ESCs showed naive colony morphology and alkaline phosphatase activity (Fig. 1C). 3.2. Cell fusion process between somatic cells and pluripotent cells After mixing fibroblasts with mouse ESCs, the cells formed cell pearl chains under AC (Fig. 2A), and then cell fusion was performed by DC pulse. After electrostimulation, colonized cells like mouse ESCs appeared with EGFP signal in rabbit, large Japanese field mouse, squirrel monkey, zebra, and bovine fused cells (Fig. 2B). In owl monkey, horse, and black rhinoceros, the electrostimulated cells did not transform to colonies (Fig. 2B). 3.3. Effect of pluripotent stem cell fusion in animal somatic cells For subsequent analyses, selective culturing with puromycin and cell sorting were performed in the colonies of rabbit, large Japanese field mouse, squirrel monkey, zebra, and bovine of the
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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Fig. 1. Experiment scheme and cell lines. (A) Mammalian fibroblasts induced with puromycin resistance gene using retroviral vector, and EGFP expressing mouse ESCs were electrofused . Thereafter, only the fused cells were selectively separated using cell sorting and selective culture.(B) The migrating fibroblasts were isolated from tissue pieces. (C) Dome-like colonies in the bright field and fluorescence images of EGFP expressing mouse ESCs. ESCs stained positive for alkaline phosphatase activity. Bar; 50 mm.
Fig. 2. Production of fused cells (A) Pearl chains were formed by 50 V AC, and cell fusion was induced by 400 V DC. The details were described in materials and methods section.(B) The morphology of the fused cells was shown. Colonizing and non-colonizing cells expressions EGFP were observed. Bar; 50 mm.
fused cells. This revealed that the fused cells of rabbit, bovine, and zebra were well proliferated in the selection condition. After this purification process, the selected cells were defined as fused cell lines. Next, flow cytometric analysis was performed in the
puromycin selected fused cells, which revealed that EGFP was expressed in all fused cell line at the same levels with mouse ESCs (Fig. 3A). Genomic PCR analysis detected both EGFP of the mouse ESCs and puromycin resistance gene of the fibroblasts in the fused
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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Fig. 3. Cytogenetics of isolated fused cells. (A) Flow cytometric analysis of EGFP expression after cell sorting. Mammalian fibroblasts before electrofusion were used as negative control (Upper) and EGFP-ESCs were used as positive. Fused cells expressed EGFP similar to EGFP-ESCs.(B) PCR analysis of genetic biomarkers in the genome of each cell. EGFP derived from mouse ESCs and puromycin resistance gene from fibroblasts were detected in the fused cells.
cells well proliferated (Fig. 3B). These results indicated that the fused cells inherited the genetic state of both mouse ESCs and the fibroblasts. Subsequently, the purified fused cells were cultured on feeder cells for expansion culture. This indicated that mouse ESC-like naive colonized cells expressing EGFP were observed in rabbit, bovine, and zebra fused cells (Fig. 4A). In addition, these fused cells showed ALP positive (Fig. 4B). To characterize molecular biological features as pluripotent cells, RT-PCR and qRT-PCR analyses were performed for these fused cell lines. These results revealed that expression level of Tert gene, subunit of telomerase complex, was expressed as mouse ESCs after cell fusion (Fig. 4C). In rabbit, intrinsic Oct3/4 gene was normally expressed in fibroblasts, but strongly induced in the fused cells with mouse ESCs. Similarly, bovine and zebra intrinsic Oct3/4 expression was strongly induced in bovine and zebra fused cells with mouse ESCs, respectively. Mouse Oct3/4 was expressed in all types of fused cells (Fig. 4D). RTPCR analysis revealed that the expression of other mouse pluripotent marker genes including Sox2 and Nanog, were detected in the fused cells (Supplementary Fig. 3A). In addition, intrinsic rabbit Klf4, bovine Nanog, Klf4 and zebra Nanog were also detected in the fused cells (Supplementary Fig. 3B). However, rabbit and zebra
Nanog were not detected in this present study (Data not shown, primer sequences in Supplementary Table 1). Gene expression was summarized in Supplementary Table 2. PCR fragments of zebra Oct3/4 and Gapdh genes were sequenced and registered in DDBJ database (Accession number: LC386206, LC386207). Hence, the expression of pluripotent marker genes that are unique to mammalian species was induced by cell fusion with mouse ESCs.
4. Discussion In this present study, we succeeded in producing fused cells by electrofusion between mouse embryonic stem cells (ESCs) and fibroblasts derived from domestic or wild animals. The fused cell lines formed domed colonies and expressed pluripotent marker genes unique to each species of the fused cells. These results indicated that pluripotent state might be inducible in mammalian somatic cells by cell fusion with mouse ESCs, and suggested that it could be applied to elucidate the mechanism of maintenance of pluripotency in various mammals for which induced pluripotent stem cells (iPSCs) has not yet been established. Cell fusion is generally accomplished by two methods. One is application of polyethylene glycol (PEG) and the other is
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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Fig. 4. Morphology and gene expression of fused cells. (A) Morphology of fused cells colonies in bright field and EGFP expression. Fused cells of rabbit, bovine, and zebra are shown above. Colonies of fused cells were positive for EGFP and showed the same morphology with mouse ESCs.(B) Fused cells of rabbit, bovine, and zebra stained positive for alkaline phosphatase activity. (C) The relative expression level of Tert. Data represent the mean ± SE. The different letters (aeb) indicate significant differences at P < 0.01. (D) RT-PCR analysis of rabbit fused cells. Expression of rabbit, bovine and zebra Oct3/4 was strongly induced after cell fusion.
electrofusion method. PEG-mediated cell fusion had long been used, but had been known as low efficiency with high cytotoxicity. In our study, we selected the electrofusion method because fusion efficiency and reproducibility is higher than that in PEG [15,16]. In consequence, EGFP signal derived from mouse ESCs was observed in the fused cells of all mammalian species used in this research, indicating that this electrofusion method has possibility of application for cell fusion of all mammalian species. Following stimulation of somatic cells of domestic or wild animals with mouse ESCs by cell fusion, two cell types emerged in fusion cells; fibroblast-like cells and naive pluripotent stem celllike colony. Based on appearance, only fused cells could be separated, collected, and culture, but un-fused cells were eliminated after cell fusion. Generally, hybridomas, one of fused cells, could be selectively cultured using a cell line deficient in Hprt gene. In the primary fibroblasts derived from domestic or wild animals, fused cells are efficiently purified using a combination of EGFP expression mouse ESCs [17] and puromycin resistant fibroblasts transduced with a retroviral vector; pMCs [18]. We first succeeded in establishing cell fusion between nonexperimental, domestic or wild animals, and mouse ESCs. The fused cells among experimental animals such as between mouse ESCs and mouse fibroblasts or between mouse haploid ESCs and rat
haploid ESCs has been reported which indicate that the fused cells show naive type cell colonies and maintained pluripotent state [10,11,19]. In our study, naive-type pluripotent stem cell-like colonies [20] were also emerged shortly after cell fusion between all fibroblasts of rabbit, large Japanese field mouse (Apodemus speciosis), squirrel monkey (Saimiri boliviensis), bovine, and zebra (Equus quagga) and mouse ESCs. In mouse iPSCs, four factors are essential for its establishment [21]. However, in non-experimental animals, it is still difficult to establish of pluripotent cells because essential factors converting cellular state from somatic state to pluripotent state are not well understood. In our study, we employed mouse ESCs as a whole cocktail of reprogramming factors, resulting in some mammalian fibroblasts being finally reprogrammed as establishment of iPSCs. However, only fibroblastlike cells were observed after cell fusion with mouse ESCs in some species of these animals, which indicates that the mouse reprogramming factors could be insufficient to convert cell state from somatic to pluripotent for specific animal species. It had been known that reprogramming highly depend on metabolic dynamics of original cells [22,23]. Furthermore, after cell fusion, DNA replication is required for the acquisition of pluripotency in the fused cells between mouse ESCs and mouse embryonic fibroblasts [24]. Both metabolic status and DNA replication might be important to
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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impart pluripotency to somatic cells through cell fusion with pluripotent cells. Among domestic or wild animal cells, cell fusion with mouse ESCs was successfully purified and cultured for further analysis in rabbit, bovine, and zebra. It is unclear why the fused cells using large Japanese field mouse and squirrel monkey failed to proceed cell culture after purification. In previous reports, cell fusion affect cell cycle and epigenome status in fusion cells among embryonic cells [25,26], cell division and cell proliferation in fusion cells between normal fibroblasts and immortalized cells [27]. Abnormal genome modifications or cell cycles might be provoked in large Japanese field mouse and squirrel monkey, resulting in unavailability for expansion culture and furthermore analysis. It would be necessary for detailed analysis after cell fusion to trace the fusion process at the single cell level by time-lapse observation with multi-colored fluorescence labeled cells. Even after cell fusion, expression of mouse pluripotent marker genes, Oct3/4, Nanog, Sox2 were detected and expression level of Tert in fusion cells was similar to that in mouse ESCs. These results suggested that the nuclei of the fused cells derived from mouse ESCs have been maintained stem cell state after fusion with domestic or animal fibroblast cells. Although rabbit and zebra Nanog were not detected in this present study, we could not determine whether these genes were not expressed or the sequence data used for primer design was inconsistent to detect mRNA of these fused cells. Pluripotent marker genes, Oct3/4, Klf4 and Nanog that are unique to each domestic and wild animal were expressed in the purified fused cells, indicating that pluripotent factors of mouse ESCs induced somatic cell nuclear reprogramming in each animal fibroblasts. Establishment of iPSCs has been reported in domestic and companion animals such as dog, bovine, horse, and wild animals such as white rhinoceros and Amami spiny rat (Tokudaia osimensis) [28e32]. Somatic cell reprogramming is partially observed in these non-model organisms [33]; however, these cells are unstable to maintain pluripotent state and show different gene expression pattern from well-established mouse and human pluripotent stem cells [34]. In this study, the fused cells using several animals formed naive type of colonies and expressed pluripotent marker genes, suggesting that the fused cells might successfully mimic original embryonic stem cells of individual animals. As pluripotency maintenance mechanisms is different between mouse and human [35,36], clarification of the mechanisms in each mammalian species would be essential for establishing and maintaining pluripotent stem cells. Comprehensive analysis of transcriptome or genome modification of fused cells might clarify pluripotency maintenance mechanism that is unique to each mammalian species. However, for this purpose, highly accurate reference to sequences of nucleic acid of each species would be necessary. In the future, highly reprogrammed pluripotent stem cells could be established without cell fusion method by elucidating pluripotency maintenance mechanisms in various mammalian species based on the fused cells in this study. Furthermore, production of germ cells could be expected from high-quality pluripotent stem cells [37,38]. The progress of our present study could establish pluripotent stem cells that maintain undifferentiated state in various animal species. Furthermore, our study might contribute to promote researches on genetic resources of endangered mammals through establishment of pluripotent stem cells from these animals [39]. In this study, we succeeded to convert cellular status somatic to pluripotent in various wild or domestic mammalian fibroblasts through cell fusion with mouse ESCs. Further study using cell fusion method might help establishment of highly reprogrammed stem
cells in non-experimental animals. Acknowledgments The authors thank Dr. Nobuhide Kido (Kanazawa Zoological Gardens) for the gift of an aborted placenta of black rhinoceros, Taiki Matsuo and Prof. Naomi Wada (Veterinary System Physiology, Yamaguchi University) for autopsied tissues, and Prof. Toshio Kitamura (Institute of Medical Science, the University of Tokyo) for pMCs-puro. This work was supported by JSPS KAKENHI; Grant Numbers JP25660254, JP15K14880, JP17J07902, grants-in-aid from the Foundation for Growth Science and grant for Joint Research Project of Institute of Medical Science, the University of Tokyo; Number 3015. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.026. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.026. References [1] G. Vassilopoulos, P.R. Wang, D.W. Russell, Transplanted bone marrow regenerates liver by cell fusion, Nature 422 (6934) (2003) 901e904. [2] A.W. Duncan, M.H. Taylor, R.D. Hickey, A.E. Hanlon Newell, M.L. Lenzi, S.B. Olson, et al., The ploidy conveyor of mature hepatocytes as a source of genetic variation [Internet], Nature 467 (7316) (2010) 707e710, https:// doi.org/10.1038/nature09414. Available from:. [3] A. Moffett, C. Loke, Immunology of placentation in eutherian mammals, Nat. Rev. Immunol. 6 (8) (2006) 584e594. [4] C. Laowtammathron, E.C. Cheng, P.-H. Cheng, B.R. Snyder, S.-H. Yang, Z. Johnson, et al., Monkey hybrid stem cells develop cellular features of Huntington’s disease, BMC Cell Biol. 11 (2010) 12. [5] O. Sugawara, M. Oshimura, M. Koi, L.A. Annab, J.C. Barrett, Induction of cellular senescence in immortalized cells by human chromosome 1, Science 247 (4943) (1990) 707e710, 80-. [6] D. Peter, M. van Vliet, A. Bardoel, T. Kievits, N. Kuipers-Dijkshoorn, L.P. Pearson, et al., Frequent somatic imbalance of marker alleles for chromosome 1 in human primary breast carcinoma, Cancer Res. 51 (3) (1991) 1020e1025. [7] H. Harris, O.J. Miller, G. Klein, P. Worst, T. Tachibana, Suppression of malignancy by cell fusion [Internet], Nature 223 (1969 Jul 26) 363, https://doi.org/ 10.1038/223363a0. Available from:. €hler, C. Milstein, Continuous cultures of fused cells secreting antibody of [8] G. Ko predefined specificity [Internet], Nature 256 (1975 Aug 7) 495, https://doi.org/ 10.1038/256495a0. Available from:. nek, E.F. Wagner, Generation of completely em[9] Z.Q. Wang, F. Kiefer, P. Urba bryonic stem cell-derived mutant mice using tetraploid blastocyst injection, Mech. Dev. 62 (2) (1997) 137e145. [10] A.A. Kruglova, E.A. Kizilova, A.I. Zhelezova, M.M. Gridina, A.N. Golubitsa, O.L. Serov, Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras, Cell Tissue Res. 334 (3) (2008) 371e380. [11] X. Li, X.L. Cui, J.Q. Wang, Y.K. Wang, Y.F. Li, L.Y. Wang, et al., Generation and application of mouse-rat allodiploid embryonic stem cells, Cell 164 (1e2) (2016) 279e292. [12] H. Imai, K. Kano, W. Fujii, K. Takasawa, S. Wakitani, M. Hiyama, et al., Tetraploid embryonic stem cells maintain pluripotency and differentiation potency into three germ layers [Internet], PLoS One 10 (6) (2015), e0130585. Available from: http://dx.plos.org/10.1371/journal.pone.0130585. [13] C.C. Steiner, A. Mitelberg, R. Tursi, O.A. Ryder, Molecular phylogeny of extant equids and effects of ancestral polymorphism in resolving species-level phylogenies [Internet], Mol. Phylogenet. Evol. 65 (2) (2012) 573e581, https://doi.org/10.1016/j.ympev.2012.07.010. Available from:. [14] M.H. Hsieh, Y.T. Chen, Y.T. Chen, Y.H. Lee, J. Lu, C.L. Chien, et al., PARP1 controls KLF4-mediated telomerase expression in stem cells and cancer cells, Nucleic Acids Res. 45 (18) (2017) 10492e10503. [15] X. Yu, P.A. McGraw, F.S. House, J.E. Crowe, An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies, J. Immunol. Methods 336 (2) (2008) 142e151. [16] J. Vienken, U. Zimmermann, An improved electrofusion technique for
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
H. Imai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx production of mouse hybridoma cells, FEBS Lett. 182 (2) (1985) 278e280. [17] T. Abe, H. Kiyonari, G. Shioi, K.I. Inoue, K. Nakao, S. Aizawa, et al., Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging, Genesis 49 (7) (2011) 579e590. [18] T. Kitamura, M. Onishi, S. Kinoshita, a Shibuya, a Miyajima, G.P. Nolan, Efficient screening of retroviral cDNA expression libraries, Proc. Natl. Acad. Sci. U. S. A. 92 (20) (1995) 9146e9150. [19] N.R. Battulin, I.E. Pristyazhnyuk, N.M. Matveeva, V.S. Fishman, A.A. Vasilkova, O.L. Serov, Allelic expression and DNA methylation profiles of promoters at the parental Oct4 and Nanog genes in Mus musculus ES cell/Mus caroli splenocyte hybrid cells, Cell Tissue Res. 337 (3) (2009) 439e448. [20] J. Nichols, A. Smith, Naive and primed pluripotent states [Internet], Cell Stem Cell 4 (6) (2009) 487e492, https://doi.org/10.1016/j.stem.2009.05.015. Available from:. [21] K. Takahashi, S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell 126 (4) (2006) 663e676. [22] N.M. Vacanti, C.M. Metallo, Exploring metabolic pathways that contribute to the stem cell phenotype [Internet], Biochim. Biophys. Acta Gen. Subj. 1830 (2) (2013) 2361e2369, https://doi.org/10.1016/j.bbagen.2012.08.007. Available from:. [23] M. Sone, N. Morone, T. Nakamura, A. Tanaka, K. Okita, K. Woltjen, et al., Hybrid cellular metabolism coordinated by Zic3 and Esrrb synergistically Enhances induction of naive pluripotency [Internet], Cell Metab. 25 (5) (2017) 1103e1117, https://doi.org/10.1016/j.cmet.2017.04.017. e6. Available from:. [24] T. Tsubouchi, J. Soza-Ried, K. Brown, F.M. Piccolo, I. Cantone, D. Landeira, et al., DNA synthesis is required for reprogramming mediated by stem cell fusion [Internet], Cell 152 (4) (2013) 873e883, https://doi.org/10.1016/ j.cell.2013.01.012. Available from:. [25] J. Kawaguchi, K. Kano, K. Naito, Expression profiling of tetraploid mouse embryos in the developmental stages using a cDNA microarray analysis [Internet], J. Reprod. Dev. 55 (6) (2009) 670e675. Available from: http://www. ncbi.nlm.nih.gov/pubmed/19789425. [26] B.J. Wu, L.X. Zhao, C.C. Zhu, Y.L. Chen, M.Y. Wei, S.Q. Bao, et al., Altered apoptosis/autophagy and epigenetic modifications cause the impaired postimplantation octaploid embryonic development in mice, Cell Cycle 16 (1) (2017) 82e90. [27] S. Ohshima, A. Seyama, Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts, Genes Chromosomes
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Cancer 47 (April) (2008) 238e246. [28] A.E.G. Baird, T. Barsby, D.J. Guest, Derivation of canine induced pluripotent stem cells, Reprod. Domest. Anim. 50 (4) (2015) 669e676. [29] T. Kawaguchi, T. Tsukiyama, K. Kimura, S. Matsuyama, N. Minami, M. Yamada, et al., Generation of naïve bovine induced pluripotent stem cells using piggybac transposition of doxycycline-inducible transcription factors, PLoS One 10 (8) (2015) 1e18. [30] T. Nishimura, S. Hatoya, R. Kanegi, D.P.H. Wijesekera, K. Sanno, E. Tanaka, et al., Feeder-independent canine induced pluripotent stem cells maintained under serum-free conditions, Mol. Reprod. Dev. 84 (4) (2017) 329e339. [31] I. Friedrich Ben-Nun, S.C. Montague, M.L. Houck, H.T. Tran, I. Garitaonandia, T.R. Leonardo, et al., Induced pluripotent stem cells from highly endangered species, Nat. Methods 8 (10) (2011) 829e831. [32] A. Honda, N. Choijookhuu, H. Izu, Y. Kawano, M. Inokuchi, K. Honsho, et al., Flexible adaptation of male germ cells from female iPSCs of endangered Tokudaia osimensis, Sci. Adv. 3 (5) (2017) 1e10. , C.C. Chen, R. Dai, J.T. Howard, U. Hochgeschwender, E.D. Jarvis, [33] R.A. Rossello Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species, Elife 2013 (2) (2013) 1e24. [34] Y.Z. Paterson, C. Kafarnik, D.J. Guest, Characterization of companion animal pluripotent stem cells, Cytometry A 93 (1) (2018) 137e148. [35] J. Deng, R. Shoemaker, B. Xie, A. Gore, E.M. Leproust, J. Antosiewicz-Bourget, et al., Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming, Nat. Biotechnol. 27 (4) (2009) 353e360. [36] B. Papp, K. Plath, Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape [Internet], Cell. Res. 21 (3) (2011) 486e501, https:// doi.org/10.1038/cr.2011.28. Available from:. [37] K. Hayashi, S. Ogushi, K. Kurimoto, S. Shimamoto, H. Ohta, M. Saitou, Offspring from oocytes derived from in vitro primordial germ cellelike cells in mice Katsuhiko [Internet], Science 80 (2012) 971 (2012):10e5. Available from: http://science.sciencemag.org/content/338/6109/971.full. [38] O. Hikabe, N. Hamazaki, G. Nagamatsu, Y. Obata, Y. Hirao, N. Hamada, et al., Reconstitution in vitro of the entire cycle of the mouse female germ line [Internet], Nature 539 (7628) (2016) 299e303, https://doi.org/10.1038/nature20104. Available from:. [39] J. Saragusty, S. Diecke, M. Drukker, B. Durrant, I. Friedrich Ben-Nun, C. Galli, et al., Rewinding the process of mammalian extinction, Zoo Biol. 35 (4) (2016) 280e292.
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Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells Hiroyuki Imai a, Ken Takeshi Kusakabe b, Yasuo Kiso b, Shosaku Hattori c, Chieko Kai c, Etsuro Ono a, Kiyoshi Kano d, * a
Department of Biomedicine, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan Laboratory of Veterinary Anatomy, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan Amami Laboratory of Injurious Animals, Institute of Medical Science, The University of Tokyo, Kagoshima, Japan d Laboratory of Veterinary Developmental Biology, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 September 2019 Accepted 1 October 2019 Available online xxx
Background: Cell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells. Methods: Fused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells. Results: Colonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells. Conclusions: Our results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species. © 2019 Published by Elsevier Inc.
Keywords: Pluripotency Cell fusion Embryonic stem cell Induced pluripotent stem cell Mammal
1. Introduction Physiological cell fusion is observed in various cells of the liver and the placenta in mammals. In the liver, hepatocytes could acquire differentiation potency by cell fusion with hematopoietic stem cells [1]. It could also have adaptive ability by obtaining genetic diversity of polyploidization, accompanying hepatocytes fusion [2]. In the placenta, cell fusion is involved in maintaining pregnancy through syncytiotrophoblasts [3]. In addition to physiological cell fusion, artificially fused cells have been used in various investigations of cellular functions. The establishment of pathological models of Huntington’s disease by producing hybrid cells with different genetic background has been reported [4]. Cell fusion with immortalized cell lines and fibroblasts revealed that loci associated with immortalization were identified
* Corresponding author. Yamaguchi University, 1677-1, Yoshida, Yamaguchi, 7538511, Japan. E-mail address: [email protected] (K. Kano).
[5,6]. Other study demonstrated that tumor malignancy grading was attenuated by fusion between tumor cells and normal somatic cells [7]. Furthermore, hybridoma cells were produced by fusion between splenocytes and myeloma cells, which have frequently been used to produce monoclonal antibodies as biotechnological method [8]. Application of cell fusion has been used to generate transgenic mice from pluripotent stem cells as a tetraploid complementation method development [9]. Thus, artificial cell fusion has been applied in various research fields such as genetics, tumor biology, antibody medicine, and experimental animals. In stem cell research area, artificial cell fusion has been applied to induce cellular reprogramming in somatic cells. Mouse fibroblasts that were fused with mouse embryonic stem cells (ESCs) have acquired pluripotency [10]. Other studies have reported interspecific cell fusion. For example, Li et al. have succeeded in establishing interspecific diploid pluripotent stem cells by fusion of mouse haploid ESCs and rat haploid ESCs [11]. Small murine molecules possessed the potential to maintain undifferentiated state in rat pluripotent stem cells [11]. Since the mechanism of maintaining
https://doi.org/10.1016/j.bbrc.2019.10.026 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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pluripotent embryonic stem cells is still unknown in most mammals, it has been unsuccessful to establish pluripotent cells in most non-experimental model animals. This experimental system involving fusion with mouse pluripotent cells could be applied to establish pluripotent stem cells and help identify key molecules that maintain pluripotency in non-model mammals, such as domestic animals, companion animals, or wild animals. In this present study, we succeeded in triggering pluripotent state in the nuclei of mammalian fibroblasts by cell fusion with mouse ESCs. The result indicated that cell fusion with pluripotent stem cells provides novel method of cellular reprogramming in somatic cells, and establishes pluripotent stem cells in nonexperimental animals. 2. Materials and methods 2.1. Mouse ESCs and animal fibroblasts Mouse embryonic stem cells (ESCs) expressing EGFP were used as established in previous report [12]. The stem cells were cultured on feeder cells in ESGRO complete plus clonal grade medium (Merck, NJ, USA). Rabbit and horse fibroblasts were isolated from the subcutaneous tissues autopsied at Veterinary Anatomy laboratory of Yamaguchi University. Bovine fibroblasts were isolated from the subcutaneous tissues received from the meat center (Hofu city). Owl monkey (Aotus lemurinus) and squirrel monkey (Saimiri boliviensis) fibroblasts from autopsied subcutaneous tissues obtained from Amami laboratory of injurious animals, the University of Tokyo. Large Japanese field mouse (Apodemus speciosis) and zebra (Equus quagga) fibroblasts from the subcutaneous tissues autopsied at Veterinary System Physiology laboratory of Yamaguchi University. Black rhinoceros (Diceros bicornis) fibroblasts from aborted placental vessels distributed from Kanazawa Zoological Gardens. These fibroblasts were cultured with DMEM supplemented with 10% fetal bovine serum. Karyotyping of each fibroblast was performed according to previous report [12]. Transduction of puromycin resistance gene into the fibroblasts was performed using the retroviral vector. Transduced cells were selected for 10 days with puromycin (25 mg/ml). 2.2. Cell fusion Cell fusion was performed using LF301 (BEX) and an electrofusion chamber (EG450-30BG; Bioresource Center). Mouse ESCs and fibroblasts were suspended with e-hybri buffer (0.3 M mannitol, 1 mM MgCl2, 1 mM CaCl2) and mixed together. The suspensions were electrified with 50 V AC for 20 s to form pearl chains and with 400 V DC for 30 msec, three times at 0.5 msec intervals for electrofusion. The fused cells were cultured in puromycin-added ESGRO medium for 1 week. EGFP expressing cells were fractionated using a cell sorter (SH800; Sony) to isolate only the fused cells. Thereafter, expansion culture of the fused cells was performed for at least 15 passages in ESGRO medium without puromycin. ALP staining was performed by using an Alkaline Phosphatase Detection Kit (Merck). 2.3. Genomic DNA extraction and PCR Extraction of genomic DNA was performed using Nucleospin TissueXS (Takara). For PCR reaction, iCycler (Biorad) and BIOTAQ (Bioline) were used. The primer sequences are listed in Supplementary Table 1. The amplification program was performed with an initial activation step (3 min at 94 C), followed by 35 cycles of denaturation (30 s at 94 C), annealing (45 s at 52 C), an initial
extension (20 s at 72 C), and a final extension (1 min at 72 C). 2.4. RT-PCR and qRT-PCR After total RNA was isolated using ReliaPrep RNA Cell MiniPrep System (Promega), cDNA was synthesized with QuantiTect RT kit (Qiagen). RT-PCR was performed using PrimeSTAR HS (Takara) with the touchdown amplification program was performed with an initial activation step (30 s at 98 C), followed by 17 cycles of denaturation (10 s at 98 C), annealing (15 s at 72 C, decreasing by 1 C/cycle), and extension (30 s at 72 C). This step was followed by 18 cycles of denaturation, annealing (54 C), initial extension, and a final extension (1 min). qRT-PCR was performed using LightCycler Nano (Roche) and OneStep TB Green PrimeScript PLUS RT-PCR Kit (Takara) according to manufactures’ protocol. In Supplementary Table 1, the primer sequences are listed. Since the nucleic acid sequence information about zebra was not enough in database, primers for zebra analysis were designed with reference to the information about donkeys (Equus asinus), which is closely related to zebra [13]. DNA fragments were extracted using Monarch DNA gel extraction kit (NEB), and sequence analysis was performed. Primers were designed using primer3 software or referred to previous studies [14]. 2.5. Statistical analysis Student’s t-test was used to detect significant differences. 3. Results 3.1. Experiment scheme and cell lines The experimental scheme in this present study is shown in Fig. 1A. To selectively culture fused cells, fibroblasts were transfected with puromycin resistance gene by retroviral vector, and mouse ESCs were labeled with EGFP. After electrofusion, the cells were cultured with puromycin and sorted with fluorescence of EGFP. All established mammalian cells showed fibroblast-like shape after several passages (Fig. 1B). Details of fibroblast species used in this study were shown in materials and methods. The chromosome numbers were maintained their original state (Supplementary Fig. 1). After selective culture, puromycin resistance genes were detected in the genome of transfected fibroblasts (Supplementary Fig. 2). EFGP expressing mouse ESCs were established in our previous study [12]. The ESCs showed naive colony morphology and alkaline phosphatase activity (Fig. 1C). 3.2. Cell fusion process between somatic cells and pluripotent cells After mixing fibroblasts with mouse ESCs, the cells formed cell pearl chains under AC (Fig. 2A), and then cell fusion was performed by DC pulse. After electrostimulation, colonized cells like mouse ESCs appeared with EGFP signal in rabbit, large Japanese field mouse, squirrel monkey, zebra, and bovine fused cells (Fig. 2B). In owl monkey, horse, and black rhinoceros, the electrostimulated cells did not transform to colonies (Fig. 2B). 3.3. Effect of pluripotent stem cell fusion in animal somatic cells For subsequent analyses, selective culturing with puromycin and cell sorting were performed in the colonies of rabbit, large Japanese field mouse, squirrel monkey, zebra, and bovine of the
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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Fig. 1. Experiment scheme and cell lines. (A) Mammalian fibroblasts induced with puromycin resistance gene using retroviral vector, and EGFP expressing mouse ESCs were electrofused . Thereafter, only the fused cells were selectively separated using cell sorting and selective culture.(B) The migrating fibroblasts were isolated from tissue pieces. (C) Dome-like colonies in the bright field and fluorescence images of EGFP expressing mouse ESCs. ESCs stained positive for alkaline phosphatase activity. Bar; 50 mm.
Fig. 2. Production of fused cells (A) Pearl chains were formed by 50 V AC, and cell fusion was induced by 400 V DC. The details were described in materials and methods section.(B) The morphology of the fused cells was shown. Colonizing and non-colonizing cells expressions EGFP were observed. Bar; 50 mm.
fused cells. This revealed that the fused cells of rabbit, bovine, and zebra were well proliferated in the selection condition. After this purification process, the selected cells were defined as fused cell lines. Next, flow cytometric analysis was performed in the
puromycin selected fused cells, which revealed that EGFP was expressed in all fused cell line at the same levels with mouse ESCs (Fig. 3A). Genomic PCR analysis detected both EGFP of the mouse ESCs and puromycin resistance gene of the fibroblasts in the fused
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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Fig. 3. Cytogenetics of isolated fused cells. (A) Flow cytometric analysis of EGFP expression after cell sorting. Mammalian fibroblasts before electrofusion were used as negative control (Upper) and EGFP-ESCs were used as positive. Fused cells expressed EGFP similar to EGFP-ESCs.(B) PCR analysis of genetic biomarkers in the genome of each cell. EGFP derived from mouse ESCs and puromycin resistance gene from fibroblasts were detected in the fused cells.
cells well proliferated (Fig. 3B). These results indicated that the fused cells inherited the genetic state of both mouse ESCs and the fibroblasts. Subsequently, the purified fused cells were cultured on feeder cells for expansion culture. This indicated that mouse ESC-like naive colonized cells expressing EGFP were observed in rabbit, bovine, and zebra fused cells (Fig. 4A). In addition, these fused cells showed ALP positive (Fig. 4B). To characterize molecular biological features as pluripotent cells, RT-PCR and qRT-PCR analyses were performed for these fused cell lines. These results revealed that expression level of Tert gene, subunit of telomerase complex, was expressed as mouse ESCs after cell fusion (Fig. 4C). In rabbit, intrinsic Oct3/4 gene was normally expressed in fibroblasts, but strongly induced in the fused cells with mouse ESCs. Similarly, bovine and zebra intrinsic Oct3/4 expression was strongly induced in bovine and zebra fused cells with mouse ESCs, respectively. Mouse Oct3/4 was expressed in all types of fused cells (Fig. 4D). RTPCR analysis revealed that the expression of other mouse pluripotent marker genes including Sox2 and Nanog, were detected in the fused cells (Supplementary Fig. 3A). In addition, intrinsic rabbit Klf4, bovine Nanog, Klf4 and zebra Nanog were also detected in the fused cells (Supplementary Fig. 3B). However, rabbit and zebra
Nanog were not detected in this present study (Data not shown, primer sequences in Supplementary Table 1). Gene expression was summarized in Supplementary Table 2. PCR fragments of zebra Oct3/4 and Gapdh genes were sequenced and registered in DDBJ database (Accession number: LC386206, LC386207). Hence, the expression of pluripotent marker genes that are unique to mammalian species was induced by cell fusion with mouse ESCs.
4. Discussion In this present study, we succeeded in producing fused cells by electrofusion between mouse embryonic stem cells (ESCs) and fibroblasts derived from domestic or wild animals. The fused cell lines formed domed colonies and expressed pluripotent marker genes unique to each species of the fused cells. These results indicated that pluripotent state might be inducible in mammalian somatic cells by cell fusion with mouse ESCs, and suggested that it could be applied to elucidate the mechanism of maintenance of pluripotency in various mammals for which induced pluripotent stem cells (iPSCs) has not yet been established. Cell fusion is generally accomplished by two methods. One is application of polyethylene glycol (PEG) and the other is
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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Fig. 4. Morphology and gene expression of fused cells. (A) Morphology of fused cells colonies in bright field and EGFP expression. Fused cells of rabbit, bovine, and zebra are shown above. Colonies of fused cells were positive for EGFP and showed the same morphology with mouse ESCs.(B) Fused cells of rabbit, bovine, and zebra stained positive for alkaline phosphatase activity. (C) The relative expression level of Tert. Data represent the mean ± SE. The different letters (aeb) indicate significant differences at P < 0.01. (D) RT-PCR analysis of rabbit fused cells. Expression of rabbit, bovine and zebra Oct3/4 was strongly induced after cell fusion.
electrofusion method. PEG-mediated cell fusion had long been used, but had been known as low efficiency with high cytotoxicity. In our study, we selected the electrofusion method because fusion efficiency and reproducibility is higher than that in PEG [15,16]. In consequence, EGFP signal derived from mouse ESCs was observed in the fused cells of all mammalian species used in this research, indicating that this electrofusion method has possibility of application for cell fusion of all mammalian species. Following stimulation of somatic cells of domestic or wild animals with mouse ESCs by cell fusion, two cell types emerged in fusion cells; fibroblast-like cells and naive pluripotent stem celllike colony. Based on appearance, only fused cells could be separated, collected, and culture, but un-fused cells were eliminated after cell fusion. Generally, hybridomas, one of fused cells, could be selectively cultured using a cell line deficient in Hprt gene. In the primary fibroblasts derived from domestic or wild animals, fused cells are efficiently purified using a combination of EGFP expression mouse ESCs [17] and puromycin resistant fibroblasts transduced with a retroviral vector; pMCs [18]. We first succeeded in establishing cell fusion between nonexperimental, domestic or wild animals, and mouse ESCs. The fused cells among experimental animals such as between mouse ESCs and mouse fibroblasts or between mouse haploid ESCs and rat
haploid ESCs has been reported which indicate that the fused cells show naive type cell colonies and maintained pluripotent state [10,11,19]. In our study, naive-type pluripotent stem cell-like colonies [20] were also emerged shortly after cell fusion between all fibroblasts of rabbit, large Japanese field mouse (Apodemus speciosis), squirrel monkey (Saimiri boliviensis), bovine, and zebra (Equus quagga) and mouse ESCs. In mouse iPSCs, four factors are essential for its establishment [21]. However, in non-experimental animals, it is still difficult to establish of pluripotent cells because essential factors converting cellular state from somatic state to pluripotent state are not well understood. In our study, we employed mouse ESCs as a whole cocktail of reprogramming factors, resulting in some mammalian fibroblasts being finally reprogrammed as establishment of iPSCs. However, only fibroblastlike cells were observed after cell fusion with mouse ESCs in some species of these animals, which indicates that the mouse reprogramming factors could be insufficient to convert cell state from somatic to pluripotent for specific animal species. It had been known that reprogramming highly depend on metabolic dynamics of original cells [22,23]. Furthermore, after cell fusion, DNA replication is required for the acquisition of pluripotency in the fused cells between mouse ESCs and mouse embryonic fibroblasts [24]. Both metabolic status and DNA replication might be important to
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
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impart pluripotency to somatic cells through cell fusion with pluripotent cells. Among domestic or wild animal cells, cell fusion with mouse ESCs was successfully purified and cultured for further analysis in rabbit, bovine, and zebra. It is unclear why the fused cells using large Japanese field mouse and squirrel monkey failed to proceed cell culture after purification. In previous reports, cell fusion affect cell cycle and epigenome status in fusion cells among embryonic cells [25,26], cell division and cell proliferation in fusion cells between normal fibroblasts and immortalized cells [27]. Abnormal genome modifications or cell cycles might be provoked in large Japanese field mouse and squirrel monkey, resulting in unavailability for expansion culture and furthermore analysis. It would be necessary for detailed analysis after cell fusion to trace the fusion process at the single cell level by time-lapse observation with multi-colored fluorescence labeled cells. Even after cell fusion, expression of mouse pluripotent marker genes, Oct3/4, Nanog, Sox2 were detected and expression level of Tert in fusion cells was similar to that in mouse ESCs. These results suggested that the nuclei of the fused cells derived from mouse ESCs have been maintained stem cell state after fusion with domestic or animal fibroblast cells. Although rabbit and zebra Nanog were not detected in this present study, we could not determine whether these genes were not expressed or the sequence data used for primer design was inconsistent to detect mRNA of these fused cells. Pluripotent marker genes, Oct3/4, Klf4 and Nanog that are unique to each domestic and wild animal were expressed in the purified fused cells, indicating that pluripotent factors of mouse ESCs induced somatic cell nuclear reprogramming in each animal fibroblasts. Establishment of iPSCs has been reported in domestic and companion animals such as dog, bovine, horse, and wild animals such as white rhinoceros and Amami spiny rat (Tokudaia osimensis) [28e32]. Somatic cell reprogramming is partially observed in these non-model organisms [33]; however, these cells are unstable to maintain pluripotent state and show different gene expression pattern from well-established mouse and human pluripotent stem cells [34]. In this study, the fused cells using several animals formed naive type of colonies and expressed pluripotent marker genes, suggesting that the fused cells might successfully mimic original embryonic stem cells of individual animals. As pluripotency maintenance mechanisms is different between mouse and human [35,36], clarification of the mechanisms in each mammalian species would be essential for establishing and maintaining pluripotent stem cells. Comprehensive analysis of transcriptome or genome modification of fused cells might clarify pluripotency maintenance mechanism that is unique to each mammalian species. However, for this purpose, highly accurate reference to sequences of nucleic acid of each species would be necessary. In the future, highly reprogrammed pluripotent stem cells could be established without cell fusion method by elucidating pluripotency maintenance mechanisms in various mammalian species based on the fused cells in this study. Furthermore, production of germ cells could be expected from high-quality pluripotent stem cells [37,38]. The progress of our present study could establish pluripotent stem cells that maintain undifferentiated state in various animal species. Furthermore, our study might contribute to promote researches on genetic resources of endangered mammals through establishment of pluripotent stem cells from these animals [39]. In this study, we succeeded to convert cellular status somatic to pluripotent in various wild or domestic mammalian fibroblasts through cell fusion with mouse ESCs. Further study using cell fusion method might help establishment of highly reprogrammed stem
cells in non-experimental animals. Acknowledgments The authors thank Dr. Nobuhide Kido (Kanazawa Zoological Gardens) for the gift of an aborted placenta of black rhinoceros, Taiki Matsuo and Prof. Naomi Wada (Veterinary System Physiology, Yamaguchi University) for autopsied tissues, and Prof. Toshio Kitamura (Institute of Medical Science, the University of Tokyo) for pMCs-puro. This work was supported by JSPS KAKENHI; Grant Numbers JP25660254, JP15K14880, JP17J07902, grants-in-aid from the Foundation for Growth Science and grant for Joint Research Project of Institute of Medical Science, the University of Tokyo; Number 3015. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.026. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.026. References [1] G. Vassilopoulos, P.R. Wang, D.W. Russell, Transplanted bone marrow regenerates liver by cell fusion, Nature 422 (6934) (2003) 901e904. [2] A.W. Duncan, M.H. Taylor, R.D. Hickey, A.E. Hanlon Newell, M.L. Lenzi, S.B. Olson, et al., The ploidy conveyor of mature hepatocytes as a source of genetic variation [Internet], Nature 467 (7316) (2010) 707e710, https:// doi.org/10.1038/nature09414. Available from:. [3] A. Moffett, C. Loke, Immunology of placentation in eutherian mammals, Nat. Rev. Immunol. 6 (8) (2006) 584e594. [4] C. Laowtammathron, E.C. Cheng, P.-H. Cheng, B.R. Snyder, S.-H. Yang, Z. Johnson, et al., Monkey hybrid stem cells develop cellular features of Huntington’s disease, BMC Cell Biol. 11 (2010) 12. [5] O. Sugawara, M. Oshimura, M. Koi, L.A. Annab, J.C. Barrett, Induction of cellular senescence in immortalized cells by human chromosome 1, Science 247 (4943) (1990) 707e710, 80-. [6] D. Peter, M. van Vliet, A. Bardoel, T. Kievits, N. Kuipers-Dijkshoorn, L.P. Pearson, et al., Frequent somatic imbalance of marker alleles for chromosome 1 in human primary breast carcinoma, Cancer Res. 51 (3) (1991) 1020e1025. [7] H. Harris, O.J. Miller, G. Klein, P. Worst, T. Tachibana, Suppression of malignancy by cell fusion [Internet], Nature 223 (1969 Jul 26) 363, https://doi.org/ 10.1038/223363a0. Available from:. €hler, C. Milstein, Continuous cultures of fused cells secreting antibody of [8] G. Ko predefined specificity [Internet], Nature 256 (1975 Aug 7) 495, https://doi.org/ 10.1038/256495a0. Available from:. nek, E.F. Wagner, Generation of completely em[9] Z.Q. Wang, F. Kiefer, P. Urba bryonic stem cell-derived mutant mice using tetraploid blastocyst injection, Mech. Dev. 62 (2) (1997) 137e145. [10] A.A. Kruglova, E.A. Kizilova, A.I. Zhelezova, M.M. Gridina, A.N. Golubitsa, O.L. Serov, Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras, Cell Tissue Res. 334 (3) (2008) 371e380. [11] X. Li, X.L. Cui, J.Q. Wang, Y.K. Wang, Y.F. Li, L.Y. Wang, et al., Generation and application of mouse-rat allodiploid embryonic stem cells, Cell 164 (1e2) (2016) 279e292. [12] H. Imai, K. Kano, W. Fujii, K. Takasawa, S. Wakitani, M. Hiyama, et al., Tetraploid embryonic stem cells maintain pluripotency and differentiation potency into three germ layers [Internet], PLoS One 10 (6) (2015), e0130585. Available from: http://dx.plos.org/10.1371/journal.pone.0130585. [13] C.C. Steiner, A. Mitelberg, R. Tursi, O.A. Ryder, Molecular phylogeny of extant equids and effects of ancestral polymorphism in resolving species-level phylogenies [Internet], Mol. Phylogenet. Evol. 65 (2) (2012) 573e581, https://doi.org/10.1016/j.ympev.2012.07.010. Available from:. [14] M.H. Hsieh, Y.T. Chen, Y.T. Chen, Y.H. Lee, J. Lu, C.L. Chien, et al., PARP1 controls KLF4-mediated telomerase expression in stem cells and cancer cells, Nucleic Acids Res. 45 (18) (2017) 10492e10503. [15] X. Yu, P.A. McGraw, F.S. House, J.E. Crowe, An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies, J. Immunol. Methods 336 (2) (2008) 142e151. [16] J. Vienken, U. Zimmermann, An improved electrofusion technique for
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026
H. Imai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx production of mouse hybridoma cells, FEBS Lett. 182 (2) (1985) 278e280. [17] T. Abe, H. Kiyonari, G. Shioi, K.I. Inoue, K. Nakao, S. Aizawa, et al., Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging, Genesis 49 (7) (2011) 579e590. [18] T. Kitamura, M. Onishi, S. Kinoshita, a Shibuya, a Miyajima, G.P. Nolan, Efficient screening of retroviral cDNA expression libraries, Proc. Natl. Acad. Sci. U. S. A. 92 (20) (1995) 9146e9150. [19] N.R. Battulin, I.E. Pristyazhnyuk, N.M. Matveeva, V.S. Fishman, A.A. Vasilkova, O.L. Serov, Allelic expression and DNA methylation profiles of promoters at the parental Oct4 and Nanog genes in Mus musculus ES cell/Mus caroli splenocyte hybrid cells, Cell Tissue Res. 337 (3) (2009) 439e448. [20] J. Nichols, A. Smith, Naive and primed pluripotent states [Internet], Cell Stem Cell 4 (6) (2009) 487e492, https://doi.org/10.1016/j.stem.2009.05.015. Available from:. [21] K. Takahashi, S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell 126 (4) (2006) 663e676. [22] N.M. Vacanti, C.M. Metallo, Exploring metabolic pathways that contribute to the stem cell phenotype [Internet], Biochim. Biophys. Acta Gen. Subj. 1830 (2) (2013) 2361e2369, https://doi.org/10.1016/j.bbagen.2012.08.007. Available from:. [23] M. Sone, N. Morone, T. Nakamura, A. Tanaka, K. Okita, K. Woltjen, et al., Hybrid cellular metabolism coordinated by Zic3 and Esrrb synergistically Enhances induction of naive pluripotency [Internet], Cell Metab. 25 (5) (2017) 1103e1117, https://doi.org/10.1016/j.cmet.2017.04.017. e6. Available from:. [24] T. Tsubouchi, J. Soza-Ried, K. Brown, F.M. Piccolo, I. Cantone, D. Landeira, et al., DNA synthesis is required for reprogramming mediated by stem cell fusion [Internet], Cell 152 (4) (2013) 873e883, https://doi.org/10.1016/ j.cell.2013.01.012. Available from:. [25] J. Kawaguchi, K. Kano, K. Naito, Expression profiling of tetraploid mouse embryos in the developmental stages using a cDNA microarray analysis [Internet], J. Reprod. Dev. 55 (6) (2009) 670e675. Available from: http://www. ncbi.nlm.nih.gov/pubmed/19789425. [26] B.J. Wu, L.X. Zhao, C.C. Zhu, Y.L. Chen, M.Y. Wei, S.Q. Bao, et al., Altered apoptosis/autophagy and epigenetic modifications cause the impaired postimplantation octaploid embryonic development in mice, Cell Cycle 16 (1) (2017) 82e90. [27] S. Ohshima, A. Seyama, Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts, Genes Chromosomes
7
Cancer 47 (April) (2008) 238e246. [28] A.E.G. Baird, T. Barsby, D.J. Guest, Derivation of canine induced pluripotent stem cells, Reprod. Domest. Anim. 50 (4) (2015) 669e676. [29] T. Kawaguchi, T. Tsukiyama, K. Kimura, S. Matsuyama, N. Minami, M. Yamada, et al., Generation of naïve bovine induced pluripotent stem cells using piggybac transposition of doxycycline-inducible transcription factors, PLoS One 10 (8) (2015) 1e18. [30] T. Nishimura, S. Hatoya, R. Kanegi, D.P.H. Wijesekera, K. Sanno, E. Tanaka, et al., Feeder-independent canine induced pluripotent stem cells maintained under serum-free conditions, Mol. Reprod. Dev. 84 (4) (2017) 329e339. [31] I. Friedrich Ben-Nun, S.C. Montague, M.L. Houck, H.T. Tran, I. Garitaonandia, T.R. Leonardo, et al., Induced pluripotent stem cells from highly endangered species, Nat. Methods 8 (10) (2011) 829e831. [32] A. Honda, N. Choijookhuu, H. Izu, Y. Kawano, M. Inokuchi, K. Honsho, et al., Flexible adaptation of male germ cells from female iPSCs of endangered Tokudaia osimensis, Sci. Adv. 3 (5) (2017) 1e10. , C.C. Chen, R. Dai, J.T. Howard, U. Hochgeschwender, E.D. Jarvis, [33] R.A. Rossello Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species, Elife 2013 (2) (2013) 1e24. [34] Y.Z. Paterson, C. Kafarnik, D.J. Guest, Characterization of companion animal pluripotent stem cells, Cytometry A 93 (1) (2018) 137e148. [35] J. Deng, R. Shoemaker, B. Xie, A. Gore, E.M. Leproust, J. Antosiewicz-Bourget, et al., Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming, Nat. Biotechnol. 27 (4) (2009) 353e360. [36] B. Papp, K. Plath, Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape [Internet], Cell. Res. 21 (3) (2011) 486e501, https:// doi.org/10.1038/cr.2011.28. Available from:. [37] K. Hayashi, S. Ogushi, K. Kurimoto, S. Shimamoto, H. Ohta, M. Saitou, Offspring from oocytes derived from in vitro primordial germ cellelike cells in mice Katsuhiko [Internet], Science 80 (2012) 971 (2012):10e5. Available from: http://science.sciencemag.org/content/338/6109/971.full. [38] O. Hikabe, N. Hamazaki, G. Nagamatsu, Y. Obata, Y. Hirao, N. Hamada, et al., Reconstitution in vitro of the entire cycle of the mouse female germ line [Internet], Nature 539 (7628) (2016) 299e303, https://doi.org/10.1038/nature20104. Available from:. [39] J. Saragusty, S. Diecke, M. Drukker, B. Durrant, I. Friedrich Ben-Nun, C. Galli, et al., Rewinding the process of mammalian extinction, Zoo Biol. 35 (4) (2016) 280e292.
Please cite this article as: H. Imai et al., Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.026