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Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR mice

Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR... DEVELOPMENTAL BIOLOGY ANIMAL CELLS AND SYSTEMS 2020, VOL. 24, NO. 2, 91–98 https://doi.org/10.1080/19768354.2020.1752306 Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR mice a a a b c b Na Rae Han , Song Baek , Hwa-Young Kim , Kwon Young Lee , Jung Im Yun , Jung Hoon Choi , b a,d a,d,e Eunsong Lee , Choon-Keun Park and Seung Tae Lee a b Department of Animal Life Science, Kangwon National University, Chuncheon, Korea; College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea; Institute of Animal Resources, Kangwon National University, Chuncheon, d e Korea; Department of Applied Animal Science, Kangwon National University, Chuncheon, Korea; KustoGen Inc., Chuncheon, Korea ABSTRACT ARTICLE HISTORY Received 30 December 2019 Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics Revised 3 March 2020 similar to humans have been requested for developing stem cell-based bioengineering techniques Accepted 18 March 2020 directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old KEYWORDS male outbred ICR mice ( ESCs). Similar to those from 129/Ola mouse blastocysts ( ESCs), the ICR E14 Embryonic stem cells; established ESCs showed inherent characteristics of ESCs except for partial and weak protein ICR outbred; ICR; establishment; expression and activity of alkaline phosphatase. Moreover, ESCs were not originated from ICR B6CBAF1 strain-derived embryonic germ cells or pluripotent cells that may co-exist in outbred ICR strain-derived mouse mouse embryonic fibroblasts embryonic fibroblasts ( MEFs) used for deriving colonies from inner cell mass of outbred ICR ICR mouse blastocysts. Furthermore, instead of outbred MEFs, hybrid MEFs as feeder cells ICR B6CBAF1 could sufficiently support in vitro maintenance of ESC self-renewal. Additionally, ESC-specific ICR ICR characteristics (self-renewal, pluripotency, and chromosomal normality) were observed in ESCs ICR cultured for 40th subpassages (164 days) on MEFs without any alterations. These results B6CBAF1 confirmed the successful establishment of ESCs derived from outbred ICR mice, and indicated that self-renewal and pluripotency of the established ESCs could be maintained on ICR MEFs in culture. B6CBAF1 Introduction In the early stages of stem cell research, ESCs derived from blastocysts of mice with a variety of Embryonic stem cells (ESCs) with self-renewal and pluri- genetic backgrounds were widely used for the devel- potency properties have attracted a great deal of interest opment of stem cell-related techniques (Arufe et al. as a model of organogenesis during embryogenesis in 2006; Ouyang et al. 2007). Commencing with gener- developmental biology (Dvash et al. 2006; Prajumwongs ation of the first mouse ESCs derived from the et al. 2016) and as a source of cells for cell therapies in 129SvE strain in 1981 (Evans and Kaufman 1981; regenerative medicine (Trounson and McDonald 2015; Martin 1981), attempts have been made to establish Duncan and Valenzuela 2017). Furthermore, they have mouse ESCs (mESCs) derived from a variety of strains been used for the generation of genetically modified (Schoonjans et al. 2003; Tanimoto et al. 2008; Nichols animals, screening of drugs without clinical experiments, and Smith 2011). However, successful establishment and for the development of personalized drug treatment of mESC lines have been limited to a few permissive regimens (Kawamata and Ochiya 2010; Lou and Liang strains, such as 129 and C57BL/6 sub-strains (Tanimoto 2011; Lee et al. 2020). Therefore, they have been actively et al. 2008; Nichols and Smith 2011). Simultaneously, applied not only in basic research in the fields of regen- the generation of mESCs derived from non-permissive erative medicine, transgenic animal research, and phar- strains that are refractory to ESC generation, such as maceutics (Prajumwongs et al. 2016; Ukai et al. 2017), ICR, CBA, NOD, DBA, and BALB/c, showed extremely but also in clinical research (Ilic et al. 2015; Duncan and low efficiency (Kawase et al. 1994). Valenzuela 2017). CONTACT Seung Tae Lee stlee76@kangwon.ac.kr Department of Animal Life Science, Kangwon National University, Chuncheon 24341, Korea; Department of Applied Animal Science, Kangwon National University, Chuncheon 24341, Korea; KustoGen Inc., Chuncheon 24341, Korea Supplemental data for this article can be accessed at https://doi.org/10.1080/19768354.2020.1752306. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 92 N. R. HAN ET AL. As the genetic identity between mice and humans is ICR mice, and their identity was confirmed based on approximately 99%, various laboratory mouse strains, parameters related to self-renewal and differentiation including inbred, hybrid, and outbred mice, have been potential. widely used for research purposes (Fox et al. 2006). Con- sistent results can be obtained from inbred mice with Materials and methods good genetic and phenotypic stability (Yoshiki and Mor- iwaki 2006; Choi et al. 2017), whereas the impaired Detailed information of all experimental procedures and homeostasis regulation-related genes are trouble in statistical analysis performed in this study can be found recovery because of their genetic homology at chromo- in the supplementary information. somes (Fox et al. 2006). Hybrid mice generated by delib- erately crossing mice of two inbred strains maintain genetic and phenotypic uniformity, similar to inbred Results stains (National Research Council 1999). However, acqui- Establishment of ESCs ICR sition of data related to genetic background may be difficult (Schauwecker 2011). On the other hand, Establishment of ESCs was performed according to the ICR outbred mice have genetic characteristics similar to procedure presented in Figure 1. Of the 218 blastocysts humans, including undefined genetics and phenotypic produced from outbred ICR mice, 115 blastocysts were variation, and a high degree of heterogeneity (Chia adherent to MEF feeder cells and 106 colonies grew ICR et al. 2005; Jensen et al. 2016). They have also been out from the inner cell masses of the 115 MEF-adher- ICR shown to be useful as base populations for selection in ent blastocysts. However, in establishing ESCs from 106 producing new or improved humanized mouse models outgrown colonies, only one ESC was successfully main- (Zuluaga et al. 2006). The results obtained from tained over the 14th subpassage. Subsequently, the outbred stocks are generally considered more valuable established ESCs were characterized between the ICR than those from inbred or hybrid strains for application 15th and 20th subpassages. Similar to ESC colonies E14 of the results to humans (Shin et al. 2017). Therefore, (upper right in Figure 2(A)), colonies of ESCs showed ICR toxicology, pharmacology, and fundamental biomedical well-defined boundaries and dome-shaped morphology research continue to be performed using outbred mice (Figure 2(A)). AP protein expression (Figure 2(B)) and (Chia et al. 2005). activity (Figure 2(C)) were observed partially and To date, there have been a few reports regarding weakly in a portion of ESC colonies, unlike ESCs ICR E14 the establishment of ESC lines derived from outbred showing strong AP protein expression (upper right in ICR mouse blastocysts (Meng et al. 2003; Lee et al. Figure 2(B)) and activity (upper right in Figure 2(C)) 2012). These establishment of ESC lines derived from throughout the colonies. Additionally, the established denuded intact embryos or blastomeres of ICR mice ESCs showed the same expression pattern as ESCs ICR E14 was mainly conducted under microenvironments with regard to the transcription and translation of self- specialized by the addition of diverse extrinsic renewal-related genes. With the successful transcrip- factors such as knockout serum replacement (KSR), tional expression of Oct4, Sox2, Nanog, Tert, and AP differentiation inhibitors and proliferation stimulators (Figure 2(D)), positive expression of Oct4 (Figure 2(E)), as an alternative for enhancing derivation efficiency Sox2 (Figure 2(F)), and Nanog (Figure 2(G)), and negative (Lee et al. 2012). However, any characterization and expression of Tra-1-60 (Figure 2(H)) and Tra-1-81 (Figure long-term culture of the established ICR mice-derived 2(I)) were detected in both the established ESCs ICR ESCs have not been reported (Lee et al. 2012). In (Figure 2(E–I)) and the ESCs (upper right in Figure 2 E14 addition, the usage of extrinsic factors in the establish- (E–I)). In addition, the EBs formed from ESCs (Figure ICR ment of ESCs resulted in reduction of ESC viability 2(J)) showed lineage-specificdifferentiation into endo- (Naujok et al. 2014) and alteration of ESC character- derm, mesoderm, and ectoderm. The spontaneously istics (Wu et al. 2015). Therefore, with establishment differentiated EBs showed positive staining for neurofila- of ESC lines derived from outbred ICR mice under ments as an ectodermal marker (Figure 2(K)), α-smooth extrinsic factors-free microenvironments, their charac- muscle actin as a mesodermal marker (Figure 2(L)), and terization and long-term culture system development cytokeratin 18 as an endodermal marker (Figure 2(M)). should be required for enhancing their usability. The teratomas formed from ESCs transplanted into ICR Here, we report the establishment of ESC lines nude mice included ducts with simple columnar epi- derived from outbred stocks of ICR mice. ICR stock thelial cells (endodermal lineage; Figure 2(N1)), blood mESCs were isolated and cultured in vitro from the vessels (endodermal lineage; Figure 2(N2)), simple cuboi- inner cell mass of blastocysts derived from outbred dal cells (endodermal lineage; Figure 2(N3)), chondrocyte ANIMAL CELLS AND SYSTEMS 93 Figure 1. Schematic diagram depicting the procedure to establish embryonic stem cells (ESCs) from outbred ICR mice blastocysts ( ICR- ESCs). Nine-week-old female ICR mice superovulated by injection with pregnant mare serum gonadotropin and human chorionic gon- adotropin were mated with nine-week-old male ICR mice. Next, zygotes with a 2nd polar body and pronucleus were obtained from oviduct of female ICR mice and in-vitro-cultured for inducing generation of blastocysts. Subsequently, inner cell mass was retrieved from collected blastocysts and cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) feeder cells derived from fetuses of outbred ICR mice ( MEFs) for generation of ESCs. ICR ICR (mesodermal lineage; Figure 2(N4)), adipocytes (meso- renewal was determined by analyzing the doubling dermal lineage; Figure 2(N5)), muscle cells (mesodermal time, colony size, and number, and self-renewal-related lineage; Figure 2(N6)), neural tubes (ectodermal protein expression among ESCs cultured on MEF ICR lineage; Figure 2(N7)), germinal hair bulb-like structures feeder cells derived from outbred ICR, inbred C57BL/6, with pigmented cells in the core region (ectodermal and hybrid B6CBAF1 mice. The results indicated that lineage; Figure 2(N8)), and nervous tissue (ectodermal ESCs maintained on MEFs showed significantly ICR C57BL/6 lineage; Figure 2(N9)). Differentiation of ESCs into longer doubling time (Supplementary Figure S2A), ICR germ cells induced successful generation of oocyte-like smaller colony size (Supplementary Figure S2B), and cells with ZP (Figure 2(O), arrowhead). The established fewer colonies (Supplementary Figure S2C) than those ESCs had a normal diploid karyotype of 40 (Figure 2 on MEFs and MEFs, which did not differ signifi- ICR ICR B6CBAF1 (P)) and their sex was confirmed as female by identifying cantly from each other in each of the above parameters. the presence of X-chromosome-specific Xist and the Moreover, there were no significant differences in absence of Y-chromosome-specific Zfy1 in the genome expression of self-renewal-related proteins (Oct4, Sox2, (Figure 2(Q)). Subsequently, to determine whether and Nanog) among ESCs cultured on MEFs, ICR ICR C57BL/ ESCs originated from embryonic germ cells or pluripo- MEFs, and MEFs (Supplementary Figure S2D). ICR 6 B6CBAF1 tent cells that may co-exist in MEF feeder cells, MEF These results indicated that MEF feeder cells derived ICR ICR feeder cells used in the process of ESC establishment from ICR and B6CBAF1 mice were useful for maintaining were cultured for 14 days in standard ESC culture the self-renewal of ESCs derived from ICR mice. Further- medium. Throughout the culture period, no dome- more, as the genetic background of feeder cells used for shaped colonies were formed on the cultured MEF in vitro culture should be different from the cultured ESCs ICR feeder cells (Supplementary Figure S1A) and the yield for eliminating cellular contamination derived from of cells positive for pluripotent stem cell-specific proteins feeder cells, we confirmed that the usage of MEF (Oct4, Sox2, and Nanog) and embryonic germ cell- feeder cells derived from B6CBAF1 mice in the in vitro specific protein (VASA) was extremely low (< 1%) in the culture of ESCs was the best choice for maintenance ICR cultured MEFs (Supplementary Figure S1B), indicating of their self-renewal capability. Subsequently, culture of ICR that the established ESCs were not derived from plur- ESCs was conducted on hybrid MEFs from the ICR ICR B6CBAF1 ipotent stem cells or embryonic germ cells in the MEF 21st subpassage. ICR feeder cell population. These results confirmed that the ESCs with self-renewal ability and pluripotency could ICR be successfully established from the inner cell mass of Characterization of long-term cultured ESCs in ICR blastocysts derived from outbred ICR mice. the ESC-optimized MEF feeder cell-based ICR culture system To examine the usefulness of the ESC-optimized MEF ICR Establishment of MEF feeder cell-based culture feeder cell-based culture system for long-term mainten- system customized to ESCs ICR ance of ESCs, the ESCs at the 21st subpassage were ICR ICR th Subsequently, the strain of MEF feeder cells sufficiently cultured on hybrid MEFs until the 34 subpassage B6CBAF1 supporting the in vitro maintenance of ESC self- and long-term cultured ESCs were characterized ICR ICR 94 N. R. HAN ET AL. Figure 2. Characterization of embryonic stem cells established from outbred ICR mice blastocysts. The ESCs were established and ICR maintained on mitotically inactivated MEFs derived from ICR mice and cell subpassage was conducted every 4 days. Moreover, charac- terization of ESCs was performed between passages 15 and 20. (A) Colony morphology of ESCs (n = 3). Dome-shaped morphology ICR ICR of ESC colonies was maintained during culture, similar to ESC colonies (upper right in A). Scale bar, 200 μm. (B and C) Alkaline ICR E14 phosphatase (AP) protein expression and activity (n = 3). Both AP protein expression (B) and activity (C) were identified partially in the colonies of ESCs, whereas the colonies of ESCs showed strong AP protein expression (upper right in B) and activity (upper ICR E14 right in C). Scale bar, 200 μm. (D) Transcriptional expression of self-renewal-related genes (n = 3). Transcripts of self-renewal-related genes, Oct4, Sox2, Nanog, Tert, and AP, were detected in ESCs, similar to ESCs. (E–I) Translational expression of self-renewal- ICR E14 related genes (n = 3). Like ESCs (upper right in E–I), ESCs showed positive staining for Oct4, Sox2, and Nanog and negative staining E14 ICR for Tra-1-60 and Tra-1-81. Scale bar, 200 μm. (J) Embryoid body (EB) formation (n = 3). ESCs were cultured for 3 days in feeder- and ICR leukemia inhibitory factor (LIF)-free environment to allow differentiation into EBs, and EBs with spherical morphology were observed. Scale bar, 200 μm. (K–M) Spontaneous in vitro differentiation into three germ layers (n = 3). EBs were differentiated further by culturing for 7 days without feeder cells and LIF, and cells derived from the differentiated EBs showed immunoreactivity for neurofilaments (ecto- derm; K), α-smooth muscle actin (mesoderm; L), and cytokeratin 18 (endoderm; M). Scale bar, 200 μm. (N) In vivo differentiation into three germ layers (n = 3). Transplantation of ESCs into nude mice showed successful teratoma formation at 5 weeks. Teratoma ICR stained with hematoxylin and eosin contained endoderm (ducts with simple columnar epithelial cells, blood vessels, and simple cuboi- dal cells), mesoderm (chondrocytes, adipocytes, and muscle cells), and ectoderm (neural tubes, germinal hair bulb-like structure with pigmented cells in the core region, and nervous tissue). Scale bar, 50 μm. (O) Differentiation into oocytes (n = 3). Oocyte-like cells with zona pellucida (arrowhead) were derived from large-sized germ cells differentiated from the ESCs. Scale bar, 50 μm. (P) Karyotyping ICR (n = 3). A normal diploid karyotype of 40 was identified in the established ESCs. (Q) Sex determination (n = 3). Sexing was performed ICR by checking for the presence of X-chromosome-specific Xist or Y-chromosome-specific Zfy1 in the genome. Xist and Zfy1 were present and absent in the genome of ESCs, respectively, indicating that they were female. ICR between the 35th and 40th subpassages. All colonies transcriptional level in ESCs cultured for a long time ICR derived from ESCs (upper right in Figure 3(A)) and on MEFs compared to ESCs at an early subpas- E14 B6CBAF1 ICR ESCs (Figure 3(A)) had well-defined boundaries and sage (Figure 3(D)). The long-term cultured ESCs (Figure ICR ICR dome-shaped morphology. Strong AP protein expression 3(E–I)) showed an equivalent expression pattern to (upper right in Figure 3(B)) and activity (upper right in ESCs with regard to Oct4, Sox2, Nanog, Tra-1-60, and E14 Figure 3(C)) were detected throughout all colonies Tra-1-81 (upper right in Figure 3(E–I)) as follows: positive derived from ESCs, whereas a portion of the colonies for Oct4 (Figure 3(E)), Sox2 (Figure 3(F)), and Nanog E14 derived from ESCs showed partial and weak AP (Figure 3(G)), and negative for Tra-1-60 (Figure 3(H)) ICR protein expression (Figure 3(B)) and activity (Figure 3 and Tra-1-81 (Figure 3(I)). With successful formation of (C)). Moreover, no significant differences were observed EBs from long-term cultured ESCs (Figure 3(J)), neurofi- ICR in Oct4, Sox2, Nanog, Tert, and AP expression at the laments as an ectodermal marker (Figure 3(K)), α-smooth ANIMAL CELLS AND SYSTEMS 95 Figure 3. Characterization of long-term cultured ESCs on MEFs. The ESCs at the 20th subpassage were cultured on mito- ICR B6CBAF1 ICR tically inactivated MEFs and subpassaged at intervals of 4 days. Subsequently, characterization of the cultured ESCs was per- B6CBAF1 ICR formed at passages between 35 and 40. (A) Colony morphology of ESCs (n = 3). The ESCs showed a dome-shaped morphology, ICR ICR similar to ESCs (upper right in A). Scale bar, 200 μm. (B and C) AP protein expression and activity (n = 3). Partial weak AP protein E14 expression (B) and activity (C) were detected in the colonies of ESCs, whereas strong AP protein expression (upper right in B) ICR and activity (upper right in C) were identified in the ESCs. Scale bar, 200 μm. (D) Transcriptional expression of self-renewal- E14 related genes (n = 3). Levels of Oct4, Sox2, Nanog, Tert, and AP transcripts in long-term (above 35th subpassage) cultured ESCs ICR were not significantly different from those in short-term (below 20th subpassage) cultured ESCs. (E–I) Translational expression of ICR self-renewal-related genes (n = 3). The ESCs stained positively for Oct4, Sox2, and Nanog and negatively for Tra-1-60 and Tra-1- ICR 81, similar to ESCs (upper right in E–I). Scale bar, 200 μm. (J) Embryoid body (EB) formation (n = 3). EBs with spherical colony mor- E14 phology were successfully generated in feeder- and LIF-free environment for 3 days. Scale bar, 200 μm. (K–M) Spontaneous in vitro differentiation into three germ layers (n = 3). EBs were differentiated for 7 days in feeder- and LIF-free environment and cells differ- entiated from EBs showed immunoreactivity to neurofilaments (ectoderm; K), α-smooth muscle actin (mesoderm; L), and cytokeratin 18 (endoderm; M). Scale bar, 200 μm. (N) In vivo differentiation into three germ layers (n = 3). Transplantation of ESCs into nude mice ICR showed successful teratoma formation at 5 weeks. Staining of the teratomas with hematoxylin and eosin showed endodermal (gut epithelium, double-layered apocrine ducts, and ducts consisting of simple cuboidal cells), mesodermal (adipocytes, striated muscle, and smooth muscle cells), and ectodermal (epithelium with keratinization, nerve bundles, and neural epithelium) tissues. Scale bar, 50 μm. (O) Differentiation into oocytes (n = 3). Oocyte-like cells with zona pellucida (arrowhead) were derived from large-sized germ cells differentiated from the ESCs. Scale bar, 50 μm. (P) Karyotyping (n = 3). The long-term cultured ESCs showed a ICR ICR normal diploid karyotype of 40. (Q) Sex determination (n = 3). Xist was present and Zfy1 was absent in the genome of the long- term cultured ESCs, indicating that they were female. (R) Analysis of microsatellite DNA (n = 3). Alleles of D1Mit16 and ICR D17Mit124 markers were identical between MEFs and ESCs. By contrast, alleles of D1Mit16 and D17Mit124 markers in ESCs ICR ICR ICR were different from those of MEFs and MEFs. (S) Methylation analysis of Oct4 and Nanog promoter regions. Open and C57BL/6 B6CBAF1 filled circles represent unmethylated and methylated CpGs, respectively. The status of Oct4 and Nanog promoter regions in ESCs ICR was largely unmethylated. muscle actin as a mesodermal marker (Figure 3(L)), and (mesodermal lineage; Figure 3(N4)), striated muscle cytokeratin 18 as an endodermal marker (Figure 3(M)) (mesodermal lineage; Figure 3(N5)), smooth muscle were detected in spontaneously differentiated EBs. cells (mesodermal lineage; Figure 3(N6)), epithelium Additionally, following transplantation into nude mice, with keratinization (ectodermal lineage; Figure 3(N7)), the long-term cultured ESC-derived teratomas nerve bundles (ectodermal lineage; Figure 3(N8)), and ICR showed gut epithelium (endodermal lineage; Figure 3 neural epithelium (ectodermal lineage; Figure 3(N9)). (N1)), double-layered apocrine duct (endodermal Differentiation of long-term cultured ESCs into germ ICR lineage; Figure 3(N2)), ducts consisting of simple cuboi- cells induced successful generation of oocyte-like cells dal cells (endodermal lineage; Figure 3(N3)), adipocytes with ZP (Figure 3(O), arrowhead). The long-term cultured 96 N. R. HAN ET AL. ESCs showed a normal diploid karyotype of 40 (Figure be decreased more than other ESCs, resulting in weak ICR 3(P)), and the presence of X-chromosome-specific Xist protein expression and activity of AP. Subsequently, for and the absence of Y-chromosome-specific Zfy1 in their verifying the hypothesis, we quantified expression of genome (Figure 3(Q)), indicating that their sex was p38 MAPK proteins, and there was no significant differ- female. Moreover, alleles detected in ESCs by the ence in the amount of p38 MAPK proteins expressed in ICR microsatellite markers D1Mit16 and D17Mit124 were between the ESCs and the ESCs (Supplementary E14 ICR equally observed in MEFs (Figure 3(R)), whereas Figure S3). These results demonstrate that weak ICR alleles of ESCs were different from those of protein expression and activity of AP in ESCs result ICR C57BL/ ICR MEFs and MEFs (Figure 3(R)). The hypomethy- from not expression of p38 MAPK proteins but another 6 B6CBAF1 lated status of Oct4 and Nanog promoter regions were unknown factors. Of course, studies on the another observed in the long-term cultured ESCs (Figure 3 unknown factors should be conducted in the future. ICR (S)), indicating that these gene promoters in ESCs are Although human ESCs differentiated into numerous ICR active and the ESCs retain pluripotency. These results types of cells are considered valuable tools for cell ICR clearly indicated that ESCs were derived from blasto- therapy (Gerecht-Nir and Itskovitz-Eldor 2004; Ryu et al. ICR cysts of outbred ICR mice. Despite long-term culture of 2019), their maintenance and manipulation are costly ESCs on MEFs, self-renewal, pluripotency, and and difficult, and require high-end facilities of good man- ICR B6CBAF1 chromosomal normality could be successfully main- ufacturing practice (GMP) level (McKee and Chaudhry tained without any alterations, indicating that in vitro 2017; Ye et al. 2017). By contrast, maintenance and culture of ESCs on MEFs is a MEF feeder cell- manipulation of the established ESCs are cheaper ICR B6CBAF1 ICR based culture system customized to established ESCs. and easier than those of human ESCs and advanced ICR facilities are not required. Additionally, the results derived from ESCs with similar genetic characteristics ICR Discussion or heterogeneity to humans (Choi and He 2015) could In this study, ESCs were successfully derived from one be used directly for clinical applications without any pre- ICR of 218 blastocysts retrieved from outbred ICR mice. Unu- clinical tests. Therefore, ESCs have a great deal of ICR sually, weak AP protein expression and activity were potential for significantly reducing the costs and time observed in the derived ESCs. Nevertheless, the required for specific clinical applications, such as toxicity ICR derived ESCs had a normal female karyotype and evaluation or development of pharmaceuticals and stem ICR showed characteristics of stem cells: colonies with well- cell therapy. defined boundaries and dome-shaped morphology, In conclusion, we established ESCs derived from the transcriptional and translational expression of self- inner cell mass of blastocysts derived from outbred ICR renewal-related genes, in vitro and in vivo differentiation mice as well as a culture system specific for the estab- into three germ layers, and germ cell differentiation. lished ESCs. The established ESCs will contribute ICR ICR Moreover, ESCs maintained for a long time on to studies related to unknown characteristics of ESCs ICR B6CBAF1- MEFs showed no loss of stem cell-related characteristics derived from outbred ICR mice and will yield results com- and no abnormalities of female karyotype. Therefore, we parable to human ESCs in preclinical studies alone. established ESCs derived from blastocysts of outbred ICR mice and developed a system for culture of ESCs using ICR Acknowledgements MEFs. A pathway for deriving more human-similar B6CBAF1 This work was supported by the Korea Institute of Planning and results in a mouse model could be developed. Evaluation for Technology in Food, Agriculture, Forestry, and In addition to Oct4, Sox2, and Nanog, AP is conven- Fisheries (IPET) through Agri-Bioindustry Technology Develop- tionally used as an ESC-specific marker regardless of ment Program, funded by the Ministry of Agriculture, Food, species (Tielens et al. 2006). Interestingly, as shown in and Rural Affairs (MAFRA) (IPET 312060-05 and IPET117042-3). Figures 2(B,C) and 3(B,C), weak protein expression and activity of AP were detected in ESCs in comparison ICR to ESCs, which is a widely used ESC line. In previous Disclosure statement E14 studies, the regulation of tissue nonspecific AP was No potential conflict of interest was reported by the author(s). shown to be affected by p38 mitogen-activated protein kinase (MAPK) (Suzuki et al. 2002; Rey et al. 2007), and Funding decreased protein level and activity were detected in −/− p38 mouse ESCs with no changes in pluripotent This work was supported by the Korea Institute of Planning and marker expression (Štefková et al. 2015). Accordingly, it Evaluation for Technology in Food, Agriculture, Forestry, and is possible that expression of p38 kinase in ESCs may Fisheries (IPET) through Agri-Bioindustry Technology ICR ANIMAL CELLS AND SYSTEMS 97 Development Program, funded by the Ministry of Agriculture, mouse embryonic stem (ES) cell lines. Int J Dev Biol. Food, and Rural Affairs (MAFRA) [IPET 312060-05 and 38:385–390. IPET117042-3]. Lee KH, Chuang CK, Guo SF, Tu CF. 2012. 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Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR mice

Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR mice

Abstract

Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics similar to humans have been requested for developing stem cell-based bioengineering techniques directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old male outbred ICR mice (ICRESCs). Similar to those from 129/Ola mouse blastocysts (E14ESCs), the established...
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10.1080/19768354.2020.1752306
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DEVELOPMENTAL BIOLOGY ANIMAL CELLS AND SYSTEMS 2020, VOL. 24, NO. 2, 91–98 https://doi.org/10.1080/19768354.2020.1752306 Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR mice a a a b c b Na Rae Han , Song Baek , Hwa-Young Kim , Kwon Young Lee , Jung Im Yun , Jung Hoon Choi , b a,d a,d,e Eunsong Lee , Choon-Keun Park and Seung Tae Lee a b Department of Animal Life Science, Kangwon National University, Chuncheon, Korea; College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea; Institute of Animal Resources, Kangwon National University, Chuncheon, d e Korea; Department of Applied Animal Science, Kangwon National University, Chuncheon, Korea; KustoGen Inc., Chuncheon, Korea ABSTRACT ARTICLE HISTORY Received 30 December 2019 Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics Revised 3 March 2020 similar to humans have been requested for developing stem cell-based bioengineering techniques Accepted 18 March 2020 directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old KEYWORDS male outbred ICR mice ( ESCs). Similar to those from 129/Ola mouse blastocysts ( ESCs), the ICR E14 Embryonic stem cells; established ESCs showed inherent characteristics of ESCs except for partial and weak protein ICR outbred; ICR; establishment; expression and activity of alkaline phosphatase. Moreover, ESCs were not originated from ICR B6CBAF1 strain-derived embryonic germ cells or pluripotent cells that may co-exist in outbred ICR strain-derived mouse mouse embryonic fibroblasts embryonic fibroblasts ( MEFs) used for deriving colonies from inner cell mass of outbred ICR ICR mouse blastocysts. Furthermore, instead of outbred MEFs, hybrid MEFs as feeder cells ICR B6CBAF1 could sufficiently support in vitro maintenance of ESC self-renewal. Additionally, ESC-specific ICR ICR characteristics (self-renewal, pluripotency, and chromosomal normality) were observed in ESCs ICR cultured for 40th subpassages (164 days) on MEFs without any alterations. These results B6CBAF1 confirmed the successful establishment of ESCs derived from outbred ICR mice, and indicated that self-renewal and pluripotency of the established ESCs could be maintained on ICR MEFs in culture. B6CBAF1 Introduction In the early stages of stem cell research, ESCs derived from blastocysts of mice with a variety of Embryonic stem cells (ESCs) with self-renewal and pluri- genetic backgrounds were widely used for the devel- potency properties have attracted a great deal of interest opment of stem cell-related techniques (Arufe et al. as a model of organogenesis during embryogenesis in 2006; Ouyang et al. 2007). Commencing with gener- developmental biology (Dvash et al. 2006; Prajumwongs ation of the first mouse ESCs derived from the et al. 2016) and as a source of cells for cell therapies in 129SvE strain in 1981 (Evans and Kaufman 1981; regenerative medicine (Trounson and McDonald 2015; Martin 1981), attempts have been made to establish Duncan and Valenzuela 2017). Furthermore, they have mouse ESCs (mESCs) derived from a variety of strains been used for the generation of genetically modified (Schoonjans et al. 2003; Tanimoto et al. 2008; Nichols animals, screening of drugs without clinical experiments, and Smith 2011). However, successful establishment and for the development of personalized drug treatment of mESC lines have been limited to a few permissive regimens (Kawamata and Ochiya 2010; Lou and Liang strains, such as 129 and C57BL/6 sub-strains (Tanimoto 2011; Lee et al. 2020). Therefore, they have been actively et al. 2008; Nichols and Smith 2011). Simultaneously, applied not only in basic research in the fields of regen- the generation of mESCs derived from non-permissive erative medicine, transgenic animal research, and phar- strains that are refractory to ESC generation, such as maceutics (Prajumwongs et al. 2016; Ukai et al. 2017), ICR, CBA, NOD, DBA, and BALB/c, showed extremely but also in clinical research (Ilic et al. 2015; Duncan and low efficiency (Kawase et al. 1994). Valenzuela 2017). CONTACT Seung Tae Lee stlee76@kangwon.ac.kr Department of Animal Life Science, Kangwon National University, Chuncheon 24341, Korea; Department of Applied Animal Science, Kangwon National University, Chuncheon 24341, Korea; KustoGen Inc., Chuncheon 24341, Korea Supplemental data for this article can be accessed at https://doi.org/10.1080/19768354.2020.1752306. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 92 N. R. HAN ET AL. As the genetic identity between mice and humans is ICR mice, and their identity was confirmed based on approximately 99%, various laboratory mouse strains, parameters related to self-renewal and differentiation including inbred, hybrid, and outbred mice, have been potential. widely used for research purposes (Fox et al. 2006). Con- sistent results can be obtained from inbred mice with Materials and methods good genetic and phenotypic stability (Yoshiki and Mor- iwaki 2006; Choi et al. 2017), whereas the impaired Detailed information of all experimental procedures and homeostasis regulation-related genes are trouble in statistical analysis performed in this study can be found recovery because of their genetic homology at chromo- in the supplementary information. somes (Fox et al. 2006). Hybrid mice generated by delib- erately crossing mice of two inbred strains maintain genetic and phenotypic uniformity, similar to inbred Results stains (National Research Council 1999). However, acqui- Establishment of ESCs ICR sition of data related to genetic background may be difficult (Schauwecker 2011). On the other hand, Establishment of ESCs was performed according to the ICR outbred mice have genetic characteristics similar to procedure presented in Figure 1. Of the 218 blastocysts humans, including undefined genetics and phenotypic produced from outbred ICR mice, 115 blastocysts were variation, and a high degree of heterogeneity (Chia adherent to MEF feeder cells and 106 colonies grew ICR et al. 2005; Jensen et al. 2016). They have also been out from the inner cell masses of the 115 MEF-adher- ICR shown to be useful as base populations for selection in ent blastocysts. However, in establishing ESCs from 106 producing new or improved humanized mouse models outgrown colonies, only one ESC was successfully main- (Zuluaga et al. 2006). The results obtained from tained over the 14th subpassage. Subsequently, the outbred stocks are generally considered more valuable established ESCs were characterized between the ICR than those from inbred or hybrid strains for application 15th and 20th subpassages. Similar to ESC colonies E14 of the results to humans (Shin et al. 2017). Therefore, (upper right in Figure 2(A)), colonies of ESCs showed ICR toxicology, pharmacology, and fundamental biomedical well-defined boundaries and dome-shaped morphology research continue to be performed using outbred mice (Figure 2(A)). AP protein expression (Figure 2(B)) and (Chia et al. 2005). activity (Figure 2(C)) were observed partially and To date, there have been a few reports regarding weakly in a portion of ESC colonies, unlike ESCs ICR E14 the establishment of ESC lines derived from outbred showing strong AP protein expression (upper right in ICR mouse blastocysts (Meng et al. 2003; Lee et al. Figure 2(B)) and activity (upper right in Figure 2(C)) 2012). These establishment of ESC lines derived from throughout the colonies. Additionally, the established denuded intact embryos or blastomeres of ICR mice ESCs showed the same expression pattern as ESCs ICR E14 was mainly conducted under microenvironments with regard to the transcription and translation of self- specialized by the addition of diverse extrinsic renewal-related genes. With the successful transcrip- factors such as knockout serum replacement (KSR), tional expression of Oct4, Sox2, Nanog, Tert, and AP differentiation inhibitors and proliferation stimulators (Figure 2(D)), positive expression of Oct4 (Figure 2(E)), as an alternative for enhancing derivation efficiency Sox2 (Figure 2(F)), and Nanog (Figure 2(G)), and negative (Lee et al. 2012). However, any characterization and expression of Tra-1-60 (Figure 2(H)) and Tra-1-81 (Figure long-term culture of the established ICR mice-derived 2(I)) were detected in both the established ESCs ICR ESCs have not been reported (Lee et al. 2012). In (Figure 2(E–I)) and the ESCs (upper right in Figure 2 E14 addition, the usage of extrinsic factors in the establish- (E–I)). In addition, the EBs formed from ESCs (Figure ICR ment of ESCs resulted in reduction of ESC viability 2(J)) showed lineage-specificdifferentiation into endo- (Naujok et al. 2014) and alteration of ESC character- derm, mesoderm, and ectoderm. The spontaneously istics (Wu et al. 2015). Therefore, with establishment differentiated EBs showed positive staining for neurofila- of ESC lines derived from outbred ICR mice under ments as an ectodermal marker (Figure 2(K)), α-smooth extrinsic factors-free microenvironments, their charac- muscle actin as a mesodermal marker (Figure 2(L)), and terization and long-term culture system development cytokeratin 18 as an endodermal marker (Figure 2(M)). should be required for enhancing their usability. The teratomas formed from ESCs transplanted into ICR Here, we report the establishment of ESC lines nude mice included ducts with simple columnar epi- derived from outbred stocks of ICR mice. ICR stock thelial cells (endodermal lineage; Figure 2(N1)), blood mESCs were isolated and cultured in vitro from the vessels (endodermal lineage; Figure 2(N2)), simple cuboi- inner cell mass of blastocysts derived from outbred dal cells (endodermal lineage; Figure 2(N3)), chondrocyte ANIMAL CELLS AND SYSTEMS 93 Figure 1. Schematic diagram depicting the procedure to establish embryonic stem cells (ESCs) from outbred ICR mice blastocysts ( ICR- ESCs). Nine-week-old female ICR mice superovulated by injection with pregnant mare serum gonadotropin and human chorionic gon- adotropin were mated with nine-week-old male ICR mice. Next, zygotes with a 2nd polar body and pronucleus were obtained from oviduct of female ICR mice and in-vitro-cultured for inducing generation of blastocysts. Subsequently, inner cell mass was retrieved from collected blastocysts and cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) feeder cells derived from fetuses of outbred ICR mice ( MEFs) for generation of ESCs. ICR ICR (mesodermal lineage; Figure 2(N4)), adipocytes (meso- renewal was determined by analyzing the doubling dermal lineage; Figure 2(N5)), muscle cells (mesodermal time, colony size, and number, and self-renewal-related lineage; Figure 2(N6)), neural tubes (ectodermal protein expression among ESCs cultured on MEF ICR lineage; Figure 2(N7)), germinal hair bulb-like structures feeder cells derived from outbred ICR, inbred C57BL/6, with pigmented cells in the core region (ectodermal and hybrid B6CBAF1 mice. The results indicated that lineage; Figure 2(N8)), and nervous tissue (ectodermal ESCs maintained on MEFs showed significantly ICR C57BL/6 lineage; Figure 2(N9)). Differentiation of ESCs into longer doubling time (Supplementary Figure S2A), ICR germ cells induced successful generation of oocyte-like smaller colony size (Supplementary Figure S2B), and cells with ZP (Figure 2(O), arrowhead). The established fewer colonies (Supplementary Figure S2C) than those ESCs had a normal diploid karyotype of 40 (Figure 2 on MEFs and MEFs, which did not differ signifi- ICR ICR B6CBAF1 (P)) and their sex was confirmed as female by identifying cantly from each other in each of the above parameters. the presence of X-chromosome-specific Xist and the Moreover, there were no significant differences in absence of Y-chromosome-specific Zfy1 in the genome expression of self-renewal-related proteins (Oct4, Sox2, (Figure 2(Q)). Subsequently, to determine whether and Nanog) among ESCs cultured on MEFs, ICR ICR C57BL/ ESCs originated from embryonic germ cells or pluripo- MEFs, and MEFs (Supplementary Figure S2D). ICR 6 B6CBAF1 tent cells that may co-exist in MEF feeder cells, MEF These results indicated that MEF feeder cells derived ICR ICR feeder cells used in the process of ESC establishment from ICR and B6CBAF1 mice were useful for maintaining were cultured for 14 days in standard ESC culture the self-renewal of ESCs derived from ICR mice. Further- medium. Throughout the culture period, no dome- more, as the genetic background of feeder cells used for shaped colonies were formed on the cultured MEF in vitro culture should be different from the cultured ESCs ICR feeder cells (Supplementary Figure S1A) and the yield for eliminating cellular contamination derived from of cells positive for pluripotent stem cell-specific proteins feeder cells, we confirmed that the usage of MEF (Oct4, Sox2, and Nanog) and embryonic germ cell- feeder cells derived from B6CBAF1 mice in the in vitro specific protein (VASA) was extremely low (< 1%) in the culture of ESCs was the best choice for maintenance ICR cultured MEFs (Supplementary Figure S1B), indicating of their self-renewal capability. Subsequently, culture of ICR that the established ESCs were not derived from plur- ESCs was conducted on hybrid MEFs from the ICR ICR B6CBAF1 ipotent stem cells or embryonic germ cells in the MEF 21st subpassage. ICR feeder cell population. These results confirmed that the ESCs with self-renewal ability and pluripotency could ICR be successfully established from the inner cell mass of Characterization of long-term cultured ESCs in ICR blastocysts derived from outbred ICR mice. the ESC-optimized MEF feeder cell-based ICR culture system To examine the usefulness of the ESC-optimized MEF ICR Establishment of MEF feeder cell-based culture feeder cell-based culture system for long-term mainten- system customized to ESCs ICR ance of ESCs, the ESCs at the 21st subpassage were ICR ICR th Subsequently, the strain of MEF feeder cells sufficiently cultured on hybrid MEFs until the 34 subpassage B6CBAF1 supporting the in vitro maintenance of ESC self- and long-term cultured ESCs were characterized ICR ICR 94 N. R. HAN ET AL. Figure 2. Characterization of embryonic stem cells established from outbred ICR mice blastocysts. The ESCs were established and ICR maintained on mitotically inactivated MEFs derived from ICR mice and cell subpassage was conducted every 4 days. Moreover, charac- terization of ESCs was performed between passages 15 and 20. (A) Colony morphology of ESCs (n = 3). Dome-shaped morphology ICR ICR of ESC colonies was maintained during culture, similar to ESC colonies (upper right in A). Scale bar, 200 μm. (B and C) Alkaline ICR E14 phosphatase (AP) protein expression and activity (n = 3). Both AP protein expression (B) and activity (C) were identified partially in the colonies of ESCs, whereas the colonies of ESCs showed strong AP protein expression (upper right in B) and activity (upper ICR E14 right in C). Scale bar, 200 μm. (D) Transcriptional expression of self-renewal-related genes (n = 3). Transcripts of self-renewal-related genes, Oct4, Sox2, Nanog, Tert, and AP, were detected in ESCs, similar to ESCs. (E–I) Translational expression of self-renewal- ICR E14 related genes (n = 3). Like ESCs (upper right in E–I), ESCs showed positive staining for Oct4, Sox2, and Nanog and negative staining E14 ICR for Tra-1-60 and Tra-1-81. Scale bar, 200 μm. (J) Embryoid body (EB) formation (n = 3). ESCs were cultured for 3 days in feeder- and ICR leukemia inhibitory factor (LIF)-free environment to allow differentiation into EBs, and EBs with spherical morphology were observed. Scale bar, 200 μm. (K–M) Spontaneous in vitro differentiation into three germ layers (n = 3). EBs were differentiated further by culturing for 7 days without feeder cells and LIF, and cells derived from the differentiated EBs showed immunoreactivity for neurofilaments (ecto- derm; K), α-smooth muscle actin (mesoderm; L), and cytokeratin 18 (endoderm; M). Scale bar, 200 μm. (N) In vivo differentiation into three germ layers (n = 3). Transplantation of ESCs into nude mice showed successful teratoma formation at 5 weeks. Teratoma ICR stained with hematoxylin and eosin contained endoderm (ducts with simple columnar epithelial cells, blood vessels, and simple cuboi- dal cells), mesoderm (chondrocytes, adipocytes, and muscle cells), and ectoderm (neural tubes, germinal hair bulb-like structure with pigmented cells in the core region, and nervous tissue). Scale bar, 50 μm. (O) Differentiation into oocytes (n = 3). Oocyte-like cells with zona pellucida (arrowhead) were derived from large-sized germ cells differentiated from the ESCs. Scale bar, 50 μm. (P) Karyotyping ICR (n = 3). A normal diploid karyotype of 40 was identified in the established ESCs. (Q) Sex determination (n = 3). Sexing was performed ICR by checking for the presence of X-chromosome-specific Xist or Y-chromosome-specific Zfy1 in the genome. Xist and Zfy1 were present and absent in the genome of ESCs, respectively, indicating that they were female. ICR between the 35th and 40th subpassages. All colonies transcriptional level in ESCs cultured for a long time ICR derived from ESCs (upper right in Figure 3(A)) and on MEFs compared to ESCs at an early subpas- E14 B6CBAF1 ICR ESCs (Figure 3(A)) had well-defined boundaries and sage (Figure 3(D)). The long-term cultured ESCs (Figure ICR ICR dome-shaped morphology. Strong AP protein expression 3(E–I)) showed an equivalent expression pattern to (upper right in Figure 3(B)) and activity (upper right in ESCs with regard to Oct4, Sox2, Nanog, Tra-1-60, and E14 Figure 3(C)) were detected throughout all colonies Tra-1-81 (upper right in Figure 3(E–I)) as follows: positive derived from ESCs, whereas a portion of the colonies for Oct4 (Figure 3(E)), Sox2 (Figure 3(F)), and Nanog E14 derived from ESCs showed partial and weak AP (Figure 3(G)), and negative for Tra-1-60 (Figure 3(H)) ICR protein expression (Figure 3(B)) and activity (Figure 3 and Tra-1-81 (Figure 3(I)). With successful formation of (C)). Moreover, no significant differences were observed EBs from long-term cultured ESCs (Figure 3(J)), neurofi- ICR in Oct4, Sox2, Nanog, Tert, and AP expression at the laments as an ectodermal marker (Figure 3(K)), α-smooth ANIMAL CELLS AND SYSTEMS 95 Figure 3. Characterization of long-term cultured ESCs on MEFs. The ESCs at the 20th subpassage were cultured on mito- ICR B6CBAF1 ICR tically inactivated MEFs and subpassaged at intervals of 4 days. Subsequently, characterization of the cultured ESCs was per- B6CBAF1 ICR formed at passages between 35 and 40. (A) Colony morphology of ESCs (n = 3). The ESCs showed a dome-shaped morphology, ICR ICR similar to ESCs (upper right in A). Scale bar, 200 μm. (B and C) AP protein expression and activity (n = 3). Partial weak AP protein E14 expression (B) and activity (C) were detected in the colonies of ESCs, whereas strong AP protein expression (upper right in B) ICR and activity (upper right in C) were identified in the ESCs. Scale bar, 200 μm. (D) Transcriptional expression of self-renewal- E14 related genes (n = 3). Levels of Oct4, Sox2, Nanog, Tert, and AP transcripts in long-term (above 35th subpassage) cultured ESCs ICR were not significantly different from those in short-term (below 20th subpassage) cultured ESCs. (E–I) Translational expression of ICR self-renewal-related genes (n = 3). The ESCs stained positively for Oct4, Sox2, and Nanog and negatively for Tra-1-60 and Tra-1- ICR 81, similar to ESCs (upper right in E–I). Scale bar, 200 μm. (J) Embryoid body (EB) formation (n = 3). EBs with spherical colony mor- E14 phology were successfully generated in feeder- and LIF-free environment for 3 days. Scale bar, 200 μm. (K–M) Spontaneous in vitro differentiation into three germ layers (n = 3). EBs were differentiated for 7 days in feeder- and LIF-free environment and cells differ- entiated from EBs showed immunoreactivity to neurofilaments (ectoderm; K), α-smooth muscle actin (mesoderm; L), and cytokeratin 18 (endoderm; M). Scale bar, 200 μm. (N) In vivo differentiation into three germ layers (n = 3). Transplantation of ESCs into nude mice ICR showed successful teratoma formation at 5 weeks. Staining of the teratomas with hematoxylin and eosin showed endodermal (gut epithelium, double-layered apocrine ducts, and ducts consisting of simple cuboidal cells), mesodermal (adipocytes, striated muscle, and smooth muscle cells), and ectodermal (epithelium with keratinization, nerve bundles, and neural epithelium) tissues. Scale bar, 50 μm. (O) Differentiation into oocytes (n = 3). Oocyte-like cells with zona pellucida (arrowhead) were derived from large-sized germ cells differentiated from the ESCs. Scale bar, 50 μm. (P) Karyotyping (n = 3). The long-term cultured ESCs showed a ICR ICR normal diploid karyotype of 40. (Q) Sex determination (n = 3). Xist was present and Zfy1 was absent in the genome of the long- term cultured ESCs, indicating that they were female. (R) Analysis of microsatellite DNA (n = 3). Alleles of D1Mit16 and ICR D17Mit124 markers were identical between MEFs and ESCs. By contrast, alleles of D1Mit16 and D17Mit124 markers in ESCs ICR ICR ICR were different from those of MEFs and MEFs. (S) Methylation analysis of Oct4 and Nanog promoter regions. Open and C57BL/6 B6CBAF1 filled circles represent unmethylated and methylated CpGs, respectively. The status of Oct4 and Nanog promoter regions in ESCs ICR was largely unmethylated. muscle actin as a mesodermal marker (Figure 3(L)), and (mesodermal lineage; Figure 3(N4)), striated muscle cytokeratin 18 as an endodermal marker (Figure 3(M)) (mesodermal lineage; Figure 3(N5)), smooth muscle were detected in spontaneously differentiated EBs. cells (mesodermal lineage; Figure 3(N6)), epithelium Additionally, following transplantation into nude mice, with keratinization (ectodermal lineage; Figure 3(N7)), the long-term cultured ESC-derived teratomas nerve bundles (ectodermal lineage; Figure 3(N8)), and ICR showed gut epithelium (endodermal lineage; Figure 3 neural epithelium (ectodermal lineage; Figure 3(N9)). (N1)), double-layered apocrine duct (endodermal Differentiation of long-term cultured ESCs into germ ICR lineage; Figure 3(N2)), ducts consisting of simple cuboi- cells induced successful generation of oocyte-like cells dal cells (endodermal lineage; Figure 3(N3)), adipocytes with ZP (Figure 3(O), arrowhead). The long-term cultured 96 N. R. HAN ET AL. ESCs showed a normal diploid karyotype of 40 (Figure be decreased more than other ESCs, resulting in weak ICR 3(P)), and the presence of X-chromosome-specific Xist protein expression and activity of AP. Subsequently, for and the absence of Y-chromosome-specific Zfy1 in their verifying the hypothesis, we quantified expression of genome (Figure 3(Q)), indicating that their sex was p38 MAPK proteins, and there was no significant differ- female. Moreover, alleles detected in ESCs by the ence in the amount of p38 MAPK proteins expressed in ICR microsatellite markers D1Mit16 and D17Mit124 were between the ESCs and the ESCs (Supplementary E14 ICR equally observed in MEFs (Figure 3(R)), whereas Figure S3). These results demonstrate that weak ICR alleles of ESCs were different from those of protein expression and activity of AP in ESCs result ICR C57BL/ ICR MEFs and MEFs (Figure 3(R)). The hypomethy- from not expression of p38 MAPK proteins but another 6 B6CBAF1 lated status of Oct4 and Nanog promoter regions were unknown factors. Of course, studies on the another observed in the long-term cultured ESCs (Figure 3 unknown factors should be conducted in the future. ICR (S)), indicating that these gene promoters in ESCs are Although human ESCs differentiated into numerous ICR active and the ESCs retain pluripotency. These results types of cells are considered valuable tools for cell ICR clearly indicated that ESCs were derived from blasto- therapy (Gerecht-Nir and Itskovitz-Eldor 2004; Ryu et al. ICR cysts of outbred ICR mice. Despite long-term culture of 2019), their maintenance and manipulation are costly ESCs on MEFs, self-renewal, pluripotency, and and difficult, and require high-end facilities of good man- ICR B6CBAF1 chromosomal normality could be successfully main- ufacturing practice (GMP) level (McKee and Chaudhry tained without any alterations, indicating that in vitro 2017; Ye et al. 2017). By contrast, maintenance and culture of ESCs on MEFs is a MEF feeder cell- manipulation of the established ESCs are cheaper ICR B6CBAF1 ICR based culture system customized to established ESCs. and easier than those of human ESCs and advanced ICR facilities are not required. Additionally, the results derived from ESCs with similar genetic characteristics ICR Discussion or heterogeneity to humans (Choi and He 2015) could In this study, ESCs were successfully derived from one be used directly for clinical applications without any pre- ICR of 218 blastocysts retrieved from outbred ICR mice. Unu- clinical tests. Therefore, ESCs have a great deal of ICR sually, weak AP protein expression and activity were potential for significantly reducing the costs and time observed in the derived ESCs. Nevertheless, the required for specific clinical applications, such as toxicity ICR derived ESCs had a normal female karyotype and evaluation or development of pharmaceuticals and stem ICR showed characteristics of stem cells: colonies with well- cell therapy. defined boundaries and dome-shaped morphology, In conclusion, we established ESCs derived from the transcriptional and translational expression of self- inner cell mass of blastocysts derived from outbred ICR renewal-related genes, in vitro and in vivo differentiation mice as well as a culture system specific for the estab- into three germ layers, and germ cell differentiation. lished ESCs. The established ESCs will contribute ICR ICR Moreover, ESCs maintained for a long time on to studies related to unknown characteristics of ESCs ICR B6CBAF1- MEFs showed no loss of stem cell-related characteristics derived from outbred ICR mice and will yield results com- and no abnormalities of female karyotype. Therefore, we parable to human ESCs in preclinical studies alone. established ESCs derived from blastocysts of outbred ICR mice and developed a system for culture of ESCs using ICR Acknowledgements MEFs. A pathway for deriving more human-similar B6CBAF1 This work was supported by the Korea Institute of Planning and results in a mouse model could be developed. Evaluation for Technology in Food, Agriculture, Forestry, and In addition to Oct4, Sox2, and Nanog, AP is conven- Fisheries (IPET) through Agri-Bioindustry Technology Develop- tionally used as an ESC-specific marker regardless of ment Program, funded by the Ministry of Agriculture, Food, species (Tielens et al. 2006). Interestingly, as shown in and Rural Affairs (MAFRA) (IPET 312060-05 and IPET117042-3). Figures 2(B,C) and 3(B,C), weak protein expression and activity of AP were detected in ESCs in comparison ICR to ESCs, which is a widely used ESC line. In previous Disclosure statement E14 studies, the regulation of tissue nonspecific AP was No potential conflict of interest was reported by the author(s). shown to be affected by p38 mitogen-activated protein kinase (MAPK) (Suzuki et al. 2002; Rey et al. 2007), and Funding decreased protein level and activity were detected in −/− p38 mouse ESCs with no changes in pluripotent This work was supported by the Korea Institute of Planning and marker expression (Štefková et al. 2015). Accordingly, it Evaluation for Technology in Food, Agriculture, Forestry, and is possible that expression of p38 kinase in ESCs may Fisheries (IPET) through Agri-Bioindustry Technology ICR ANIMAL CELLS AND SYSTEMS 97 Development Program, funded by the Ministry of Agriculture, mouse embryonic stem (ES) cell lines. Int J Dev Biol. Food, and Rural Affairs (MAFRA) [IPET 312060-05 and 38:385–390. IPET117042-3]. Lee KH, Chuang CK, Guo SF, Tu CF. 2012. 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Journal

Animal Cells and SystemsTaylor & Francis

Published: Mar 3, 2020

Keywords: Embryonic stem cells; outbred; ICR; establishment; B6CBAF1 strain-derived mouse embryonic fibroblasts

References