Rescuing ocular development in an anophthalmic pig by blastocyst complementation
2018; Springer Nature; Volume: 10; Issue: 12 Linguagem: Inglês
10.15252/emmm.201808861
ISSN1757-4684
AutoresHongyong Zhang, Jiaojiao Huang, Zechen Li, Guosong Qin, Nan Zhang, Tang Hai, Qianlong Hong, Qiantao Zheng, Ying Zhang, Ruigao Song, Jing Yao, Chunwei Cao, Jianguo Zhao, Qi Zhou,
Tópico(s)Ocular Disorders and Treatments
ResumoResearch Article16 November 2018Open Access Source DataTransparent process Rescuing ocular development in an anophthalmic pig by blastocyst complementation Hongyong Zhang Hongyong Zhang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jiaojiao Huang Jiaojiao Huang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zechen Li Zechen Li College of Life Sciences, Qufu Normal University, Qufu, China Search for more papers by this author Guosong Qin Guosong Qin State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Nan Zhang Nan Zhang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Tang Hai Tang Hai State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qianlong Hong Qianlong Hong State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qiantao Zheng Qiantao Zheng State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Ying Zhang Ying Zhang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Ruigao Song Ruigao Song State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jing Yao Jing Yao State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chunwei Cao Chunwei Cao State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jianguo Zhao Corresponding Author Jianguo Zhao [email protected] orcid.org/0000-0001-6587-4823 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qi Zhou Corresponding Author Qi Zhou [email protected] orcid.org/0000-0002-6549-9362 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Hongyong Zhang Hongyong Zhang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jiaojiao Huang Jiaojiao Huang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Zechen Li Zechen Li College of Life Sciences, Qufu Normal University, Qufu, China Search for more papers by this author Guosong Qin Guosong Qin State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Nan Zhang Nan Zhang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Tang Hai Tang Hai State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qianlong Hong Qianlong Hong State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qiantao Zheng Qiantao Zheng State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Ying Zhang Ying Zhang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Ruigao Song Ruigao Song State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jing Yao Jing Yao State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chunwei Cao Chunwei Cao State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jianguo Zhao Corresponding Author Jianguo Zhao [email protected] orcid.org/0000-0001-6587-4823 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Qi Zhou Corresponding Author Qi Zhou [email protected] orcid.org/0000-0002-6549-9362 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Author Information Hongyong Zhang1,2,3,‡, Jiaojiao Huang1,2,3,‡, Zechen Li4,‡, Guosong Qin1,2,3, Nan Zhang1, Tang Hai1,2,3, Qianlong Hong1, Qiantao Zheng1,2,3, Ying Zhang1,2,3, Ruigao Song1,2,3, Jing Yao1,2,3, Chunwei Cao1,2,3, Jianguo Zhao *,1,2,3 and Qi Zhou *,1,2,3 1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 2Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China 3Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China 4College of Life Sciences, Qufu Normal University, Qufu, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 10 64806259; E-mail: [email protected] *Corresponding author. Tel: +86 10 64806299; E-mail: [email protected] EMBO Mol Med (2018)10:e8861https://doi.org/10.15252/emmm.201808861 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Porcine-derived xenogeneic sources for transplantation are a promising alternative strategy for providing organs for treatment of end-stage organ failure in human patients because of the shortage of human donor organs. The recently developed blastocyst or pluripotent stem cell (PSC) complementation strategy opens a new route for regenerating allogenic organs in miniature pigs. Since the eye is a complicated organ with highly specialized constituent tissues derived from different primordial cell lineages, the development of an intact eye from allogenic cells is a challenging task. Here, combining somatic cell nuclear transfer technology (SCNT) and an anophthalmic pig model (MITFL247S/L247S), allogenic retinal pigmented epithelium cells (RPEs) were retrieved from an E60 chimeric fetus using blastocyst complementation. Furthermore, all structures were successfully regenerated in the intact eye from the injected donor blastomeres. These results clearly demonstrate that not only differentiated functional somatic cells but also a disabled organ with highly specialized constituent tissues can be generated from exogenous blastomeres when delivered to pig embryos with an empty organ niche. This system may also provide novel insights into ocular organogenesis. Synopsis Blastocyst complementation is a new route for regenerating allogeneic organs in pigs for xenotransplantation, but reconstituting complicated organs, like a whole eye, was never done up to now. This strategy offers potentials for generating personalized human patient-specific organs in large animals. Intact allogenic eyes can be regenerated using exogenous blastocyst complementation in an anophthalmic pig model. Porcine RPEs are isolated and cultured in vitro from 60-day chimeric foetuses. The chimeric pig with regenerated eyes was derived from the somatic cloned blastocyst complementation and developed to full term. Introduction Organ transplantation is the only therapy solution for those patients suffering from end-stage organ failure and fatal diseases (Denner, 2017). The shortage of donor organs has limited the number of patients that can obtain treatment, and many individuals die before the organs become available. Thus, the generation of transplantable organs is one important goal of stem cell-based regenerative medicine (Rami et al, 2017). Pluripotent stem cells (PSCs) have opened new avenues for the treatment of degenerative diseases using patient-specific stem cells to generate tissues and cells (Shi et al, 2017). However, the generation of a functional organ from PSCs has not been feasible because it remains difficult to mimic in vitro the sophisticated interactions among cells and tissues during organogenesis (Kobayashi & Nakauchi, 2011). This difficulty might be addressed by generating organs in vivo using a blastocyst complementation strategy, which was first reported by Chen et al (1993) to generate mature B and T lymphocytes. Recently, two studies from the Nakauchi laboratory have reported proof-of-principle findings to demonstrate that functional organs—kidney and pancreas—could be generated from PSCs in vivo using blastocyst complementation in organogenesis-disabled mouse embryos (Kobayashi et al, 2010; Usui et al, 2012). Furthermore, with rat iPS cell injection, blastocyst complementation successfully rescued pancreas, hearts, and eyes in organogenesis-disabled mouse offspring (Wu et al, 2017). Considering ethical issues, infectious disease concerns, and physiological characteristics and size, larger mammals are preferred for generating transferable human organs, and pigs are the most suitable choice (Niemann & Petersen, 2016). Studies from Matsunari et al (2013) confirmed that the blastocyst complementation strategy is feasible in a large-animal model, using apancreatic pigs to generate a functional pancreas with allogenic blastomeres. The eye is a complicated organ with highly specialized constituent tissues derived from different primordial cell lineages (Hayashi et al, 2016). Age-related ocular degenerative diseases, such as retinal degeneration or age-related macular degeneration (AMD) and retinitis pigmentosa, are difficult to cure and are characterized by the dysfunction and death of light-sensitive photoreceptors and RPE (Forest et al, 2015; Mellough et al, 2015). The diseases have shown significant promise in being treated with PSCs. Stem cell-based therapies, such as subretinal injection of human embryonic stem cell (hESC)-derived RPE cells, adult autologous induced pluripotent stem cell (iPSC)-RPE, neural stem cells, umbilical cord stem cells, or bone marrow stem cells, have been the most important tools used to treat AMD (Eiraku et al, 2011; Nakano et al, 2012; Reichman et al, 2014; Zhong et al, 2014; Forest et al, 2015; Mellough et al, 2015; Song et al, 2015). Several studies have reported that PSCs can be induced to differentiate along a retinal lineage, including differentiation into photoreceptors using specifically defined culture conditions (Ikeda et al, 2005; Lamba et al, 2006; Osakada et al, 2008; Meyer et al, 2009; Boucherie et al, 2013). Moreover, a three-dimensional optic cup can be formed in vitro from mouse or human embryonic stem cells and can develop into a structure that remarkably resembles the embryonic vertebrate eye (Nakano et al, 2012; Boucherie et al, 2013). However, recently two studies reported less compelling results in patients with respect to stem cell-based therapy for AMD (Kuriyan et al, 2017; Mandai et al, 2017) and Kuriyan et al (2017) reported that three patients encountered severe bilateral visual loss that developed after they received intravitreal injections of autologous adipose tissue-derived "stem cells" at a private clinic in the United States. Other clinical trials also failed to show functional improvements in macular degeneration patients, possibly because of immune rejection and graft failure (Kimbrel & Lanza, 2015; Song et al, 2015). The failure of PSC-based therapy suggests that in vitro culture system cannot mimic the in vivo environment completely and it is unclear to what extent hPSCs can recapitulate the cellular and molecular features of native RPE in vitro. Thus, our results suggest that high-quality characterized RPE cells from in vivo differentiation systems or intact eyes might provide alternative solutions to address the safety and technical challenges of stem cell-based therapies for ocular degenerative diseases. In the current study, we demonstrate that intact eyes can be regenerated from allogenic blastomeres in vivo using complementation of organogenesis-disabled pig embryos. The regenerated eyes in the chimeric pig show normal configuration and function. In addition, allogenic-characterized RPEs can be generated from E60 fetuses, which enable the organ-defective fetus to be a niche for in vivo differentiation. Blastocyst complementation, using somatic cloned, organ-defective pig embryos, may thus permit the use of a large animal to generate functional and complex organs such as eyes from xenogenic PSCs. Results Generation of porcine chimeric embryos in vitro by blastocyst complementation To generate allogenic chimeric pigs, we first explored the possibility of blastocyst complementation in vitro using cloned embryos derived from pig embryonic fibroblast cells (PEFs; Fig EV1A). PEFs derived from Bama miniature pigs were labeled with either red fluorescence protein (RFP) or green fluorescence protein (GFP) and then used as donors for SCNT (Fig EV1B). Somatic cloned embryos derived from RFP-positive PEFs at the 4-cell or 8-cell stage (day 3) were used as host embryos, and ~ 5 GFP-labeled blastomeres (day 4) were injected as donors for the generation of the chimeric embryos (Fig EV1C). The reconstructed embryos were further cultured for 3–4 days and then assessed for blastocyst formation and genotyping (Fig EV1D). The injection of donor blastomeres did not affect the developmental competency of reconstructed embryos as evidenced by the similar blastocyst rates between complemented embryos and non-injected SCNT embryos (23.47% ± 1.685 vs. 18.57% ± 1.434, P = 0.09; Table 1). In the observed blastocysts, 9 of 10 (90%) expressed both RFP and GFP, indicating successful chimerism in vitro (Fig EV2A). To further confirm the feasibility of blastocyst complementation in PEFs with a different genetic background, somatic cloned embryos derived from PEFs that carried a lysine-to-serine substitution (L247S) in the microphthalmia-associated transcription factor (MITF) (termed as MITFL247S/L247S) were injected with Large White (LW) GFP-positive blastomeres. Our results showed that most injected MITFL247S/L247S embryos expressed GFP during blastocyst formation, consistent with our previous findings (Fig EV2B). Restriction fragment length polymorphism (RFLP) analysis with single blastocyst PCR amplification was used to characterize the MITFL247S/L247S mutation and substantiate the chimerism. Results showed that 7 of 10 blastocysts were chimeric (Figs EV2C and EV3A). The findings confirm that blastocyst complementation is feasible in vitro with embryos derived from PEFs. Click here to expand this figure. Figure EV1. Generation of GFP- and RFP-labeled PEFs and chimeric porcine blastocysts A. Schematic procedures for the generation of chimeric fetuses and pigs. B. The PEFs were confirmed by expression of GFP and RFP, which were then used for SCNT. Scale bars, 100 μm. C. Complementation of cloned host embryos, derived from the Bama RFP-labeled PEFs, with injection of donor blastomeres, derived from Bama GFP-labeled PEFs. Scale bar, 100 μm. D. The chimeric blastocysts. Scale bar, 100 μm. Download figure Download PowerPoint Table 1. The blastocyst rate between the SCNT embryo and complementation embryo Treatment No. of replications No. of embryos No. of blastocysts (%) P-value Non-injected SCNT embryo 3 371 69 (18.57 ± 1.434)a Complementation embryo 3 380 89 (23.47 ± 1.685)a 0.09 a t-test indicates that values are not significantly different (P = 0.09), the results are presented as means ± SEM. A P-value of < 0.05 was considered statistically significant. Click here to expand this figure. Figure EV2. The chimeric contributions in vitro were detected by immunofluorescence and RFLP analysis simultaneously A. Representative immunofluorescence images show the expression of RFP and GFP simultaneously in chimeric embryos. GFP-labeled and RFP-labeled blastocysts are shown as negative controls. Scale bar, 50 μm. B. Representative immunofluorescence images showed the expression of GFP in the chimeric blastocyst. Scale bar, 50 μm. C. Genotyping for porcine blastocysts derived from injected embryos. Porcine MITF-specific primers were used for the detection of chimeric contribution. Restriction enzymes DraI was used for digestion of PCR products. NC, negative control with no genomic DNA loaded. Mut, Bama MITFL247S/L247S blastocyst DNA loaded. WT, Bama wild-type blastocyst DNA loaded. B1-B10, collected the injected blastocysts DNA loaded. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Genotyping for the chimeric contributions in fetuses and full-term chimeric pig A. PCR amplification of MITF in the blastocysts. Mut, Bama MITFL247S/L247S blastocyst DNA loaded. WT1-WT2, DNA of Bama WT number 1 and 2 blastocysts loaded. B1-B10, DNA of the injected blastocysts from number 1 to number 10 loaded. B. PCR amplification of MITF in the E44 fetuses. NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. C. PCR amplification of MITF and PCR-RFLP analysis of MITF in E60 fetuses. NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, Bama genomic DNA loaded. NW-5-NW-11, fetuses at E60. D. PCR amplification of MITF and digestion of the PCR products in the multiple organs of the NW-7 and NW-8 fetuses. NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, Bama genomic DNA loaded. E. PCR amplification of MITF and PCR-RFLP analysis of MITF in the full-term chimeric pig. NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, Bama GFP-labeled genomic DNA loaded. F. PCR amplification of MITF in multiple organs of the full-term chimeric pig. NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, Bama GFP-labeled genomic DNA loaded. Download figure Download PowerPoint The anophthalmic phenotype was repaired in E44 MITFL247S/L247S porcine fetus MITFL247S/L247S confers an eye developmental defect and can be observed as early as embryonic day 28 (E28; Hai et al, 2017b). To further investigate the developmental competency of pig embryos derived from the blastocyst complementation, cloned embryos derived from MITFL247S/L247S male PEFs were used as the host and blastomeres of cloned embryos derived from female LW PEFs were used as donors for complementation. Reconstructed embryos at day 5 were transferred to surrogates for further development evaluation. A total of 3,754 embryos were transferred to 16 surrogate sows, and three became pregnant (Appendix Table S2). Of the three pregnancies, two terminated during early pregnancy and four fetuses (named NW-1, NW-2, NW-3, and NW-4) were retrieved at E44 by Caesarean section from one litter (Fig 1A). The four fetuses demonstrated normal shape and size compared with control wild-type (WT) and MITFL247S/L247S cohorts produced by natural mating (Fig 1A). Genotyping results showed that the NW-2 fetus was chimeric in all the tissues tested, whereas the NW-4 fetus was derived from donor embryos and the NW-1 and NW-3 fetuses were derived from host embryos (Figs 1B and EV3B). The chimerism of NW-2 was further confirmed by the characterization of an LW-specific KIT allele. A KIT duplication is seen in LW pigs but not in Bama pigs, so a pair of primers was designed to generate a 152-bp PCR product spanning the 3′–5′ breakpoint as a diagnostic test for the duplication of KIT in the chimeric fetus (Giuffra et al, 2002). Results further confirmed that the LW-specific fragment could be detected in the NW-2 and NW-4 fetuses but not in the NW-1 and NW-3 fetuses (Fig 1B). Figure 1. Generation of E44 chimeric porcine fetus in vivo by complementation of MITFL247S/L247S embryos with donor blastomeres derived from LW PEFs A. Four fetuses (named NW-1, NW-2, NW-3, and NW-4) with different eye morphology were retrieved at E44 by Caesarean section. The arrows identify the melanin in the eye. B. DraI digestion of PCR products from the multiple organs of the four fetuses (top). NC, negative control with no genomic DNA loaded. Mut, MITFL247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (bottom). Mut, MITFL247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Source data are available online for this figure. Source Data for Figure 1 [emmm201808861-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint The MITFL247S/L247S E44 fetus displayed an anophthalmic phenotype and showed a loss of RPE cells (Steingrimsson et al, 2004; Hai et al, 2017b; Fig 1A). The eyes of NW-2 showed normal morphologically and were similar to those of the wild-type (WT) fetus (Fig 1A). Hematoxylin and eosin (H&E) staining showed that the RPE cells of NW-2 were normal and pigmented, whereas the RPEs of MITFL247S/L247S fetus were hypopigmented and disorganized (Fig 2A). Moreover, positive staining of RPE-specific markers, MITF, Pax6, and Bestrophin (Song et al, 2015), were observed in the regenerated RPE of NW-2 fetus, suggesting characterized RPE in NW-2 (Fig 2B). Interestingly, we found that MITFL247S/L247S mutant showed disordered subcellular distribution of Pax6 and MITF and negative expression of Bestrophin. In the Hai et al manuscript, they also found that the MITF mutation affected the subcellular distribution of MITF protein. Finally, using next-generation sequencing of liver, lung, kidney, and eye, we examined the chimerism contributions and showed the percentage of chimerism was 27.85, 28.06, 27.12, and 27.14%, respectively (Fig EV4A). Our data demonstrate that using blastocyst complementation, allogenic eyes with RPE cells can be regenerated in pigs at E44. Figure 2. Allogenic contribution and rescue RPEs in the E44 chimeric fetus A. Representative microscopic appearances of the retina and RPE cells (arrowhead) of a chimeric, a WT, and a Mut fetus at the same gestational age by H&E staining. WT, Bama WT fetus. Mut, Bama MITFL247S/L247S fetus. NW-2, the chimeric fetus. Scale bars, 100 μm. B. Representative immunofluorescence images showed the expression of multiple RPE markers Pax6, MITF, and Bestrophin in the chimeric fetuses. Blue, Hoechst 33342. WT, Bama WT fetus as positive control. NW-2, the chimeric fetus. MITFL247S/L247S fetus as negative control. Scale bars, 50 μm. Source data are available online for this figure. Source Data for Figure 2 [emmm201808861-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Identification of the chimeric contributions in tissues from E44 fetus and piglet NW-16 A. Next-generation sequencing analysis of the origin of MITF in multiple tissues of E44 fetus and piglet NW-16. The PCR product of MITF+/L247S was as control. WT, MITF+/+ allele. Mut, MITFL247S/L247S allele. B. The chimeric contributions were determined in the multiple tissues of the chimeric pig by immunohistochemistry analysis of GFP. Scale bar, 90 μm. Download figure Download PowerPoint RPE cells isolated and characterized from the E60 chimeric fetus It had been previously thought that functional human RPEs could be differentiated from either PSCs (Liao et al, 2010; Fields et al, 2016; Sugita et al, 2016) or cultured human fetal RPE cells directly in vitro (Maminishkis et al, 2006; Sonoda et al, 2009) for cell therapy. However, the in vitro differentiation of PSCs into RPEs still needs to be optimized to address safety and effectiveness concern, and human fetal RPE cells are still limiting, due to a lack of donor resources. Thus, we explored whether allogenic RPE cells could be isolated from chimeric fetuses. The allogenic fetuses constructed from MITFL247S/L247S PEF-derived embryos injected with Bama WT blastomeres were collected at E60 and used to generate RPE cells. At this stage, RPE cells are formed, mature, and easily peeled away from the choroid. A total of 821 embryos were transferred to three surrogate sows, and one sow became pregnant. In one pregnancy, seven fetuses were retrieved and one fetus (NW-7, 1/7, 14.29%) was proved to be chimeric. The other five fetuses (NW-5, NW-6, NW-9, NW-10, NW-11) were derived from host embryos, and 1 (NW-8) was derived from donor embryos (Fig EV3C). The contribution of the donor cells was identified in various tissues in the chimeric fetus (NW-7), for which the NW-8 fetus was used as control (Fig EV3D). RPE cells were isolated from NW-7 and NW-8 and characterized as successfully hexagonal mosaic (Fig 3A). RP
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