Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation
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Abstract: Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells. A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs. Therefore, to achieve a transplantable organ in animals without rejection, creation of vascular endothelial cells derived from humans within the organ is necessary. In this study, to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals, we generated rat-mouse chimeras by injection of rat embryonic stem cells (rESCs) into mouse blastocysts with deficiency of Flk-1 protein, which is associated with endothelial and hematopoietic cell development. We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras. The whole yolk sac (YS) of Flk-1 rat-mouse chimera was full of rat blood vasculature. Rat genes related to vascular endothelial cells, arteries, and veins, blood vessels formation process, as well as hematopoietic cells, were highly expressed in the YS. Our results suggested that rat vascular endothelial cells could undergo proliferation, migration, and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells, T cells, and myeloid cells in rat-mouse chimeras, which was able to rescue early embryonic lethality caused byFlk-1 deficiency in mouse.
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Key words:
- Blastocyst complementation /
- Interspecies chimera /
- Intraspecies chimera /
- Vascular endothelial cell /
- Hematopoietic cell
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Fig. 1. Rescue of Flk-1 embryonic lethality at early stage in mouse-mouse chimeras by blastocyst complementation. A and B: Images of mouse-mouse chimeric EP (A) and YS (B) at E10.5 with injection of mESCs into Flk-1, Flk-1, and Flk-1 mouse blastocysts. Green fluorescence was host vascular endothelial cells with EGFP knockin in Flk-1 locus, while red fluorescence was donor cells with transgene of tdTomato. Scale bars, 3 mm. C: Images of Flk-1 mouse-mouse chimeric EP (top panel) and YS (bottom panel) at E15.5. The EP was red in the bright-field. No host vascular endothelial cells were EGFP positive. Red fluorescence was derived from donor cells, and blood vessels were obviously seen with tdTomato in the YS. Scale bar, 3 mm. D: Immunofluorescence analysis for the marker of vascular endothelial cells (CD31) of mouse-mouse chimeric EP at E10.5 with injection of mESCs into Flk-1, Flk-1 and Flk-1 mouse blastocysts. Sections were stained with antibody against tdTomato for donor cells, antibody against mouse CD31 for vascular endothelial cells, and DAPI for nuclear counterstaining. Scale bars, 200 μm. E: Fluorescence-field images of Flk-1, Flk-1, and Flk-1 mouse-mouse chimeric YS at E10.5. Host vascular endothelial cells were marked with EGFP, and donor cells were marked with tdTomato. Scale bar, 300 μm. F: Immunofluorescence analysis for the marker of vascular endothelial cells (CD31) of Flk-1 mouse-mouse chimeric YS at E10.5. Sections were stained with antibody against tdTomato for donor cells, antibody against mouse CD31 for vascular endothelial cells, and DAPI for nuclear counterstaining. Scale bars, 200 μm. G: Flow cytometry of Td+ or EGFP+ cells in the EP and YS of E10.5 of Flk-1 mouse-mouse chimeras or Flk-1 embryo, respectively. Data are presented as mean ± SEM. P < 0.05 was considered statistically significant. ∗∗,P < 0.01; n.s., not significant.Flk-1 YS: n = 4, Flk-1 EP: n = 4, Flk-1 YS: n = 4, Flk-1 EP: n = 6. mESCs, mouse embryonic stem cells; EP, embryo proper; YS, yolk sac.
Fig. 2. Generation of Flk-1 mouse-mouse chimeric mice by blastocyst complementation. A: Summary of Flk-1, Flk-1, and Flk-1 mouse-mouse chimeric mice generated by blastocyst complementation. B: Bright-field and red fluorescence images of full-term Flk-1 mouse-mouse chimeric mice. Dotted circles indicate Flk-1 chimeric mice. C and D: Immunofluorescence analysis for the marker of vascular endothelial cells (CD31) in the blood vessel (C) and lung (D) of Flk-1, Flk-1, and Flk-1 chimeric mice. Sections were stained with antibody against tdTomato for donor cells, antibody against mouse CD31 for vascular endothelial cells, and DAPI for nuclear counterstaining. Scale bars, 200 μm. E: Flow cytometry of Td+ or EGFP+ cells in the muscle, lung, heart, kidney, and intestine of Flk-1 mouse-mouse chimeras or Flk-1 heterozygote mice, respectively. Data are presented as mean ± SEM (n = 3).
Fig. 3. Generation of rat vascular endothelial and hematopoietic cells in rat-mouse chimeric YS and EP. A: rESCs with red fluorescence were transfected with tdTomato plasmid. Scale bar, 100 μm. B and C: Rat-mouse chimeras at E10.5 and E11.5 of EP (B) and YS (C). Scale bars, 3 mm. D: Immunofluorescence analysis of rat-mouse chimeric EP and YS. Scale bar, 300 μm. E: Flow cytometry of Td+ cells at E10.5 rat-mouse chimeric EP and YS. Data are presented as mean ± SEM. P < 0.05 was considered statistically significant. ∗,P < 0.05,n = 9. F: Full-term rat-mouse chimeric mice. Dotted circles indicate rat-mouse chimeric mice. G and H: Immunofluorescence analysis for marker of vascular endothelial cells (CD31) at E10.5 rat-mouse chimeric EP (G) and YS (H). Scale bars, 200 μm. I and J: Flow cytometry analysis of rat hematopoietic cells at E10.5 rat-mouse chimeric EP (I) and YS (J). Cells were stained with antibody against rat CD45 (n = 6). EP, embryo proper; YS, yolk sac; rESCs, rat embryonic stem cells.
Fig. 4. Generation of rat blood vasculature and hematopoietic cells inFlk-1 rat-mouse chimeras. A: Images of Flk-1, Flk-1 and Flk-1 rat-mouse chimeric YS at E10.5. Green fluorescence represented host vascular endothelial cells with EGFP knockin in Flk-1 locus, while red fluorescence represented donor cells with transgene of tdTomato. Scale bars, 3 mm. B: Immunofluorescence analysis of Flk-1, Flk-1, and Flk-1 rat-mouse chimeric YS at E10.5. Red fluorescence represented donor cells with transgene of tdTomato, and blue fluorescence represented DAPI for nuclear counterstaining. Scale bar, 300 μm. C: Images of Flk-1, Flk-1, and Flk-1 rat-mouse chimeric EP at E10.5. Green fluorescence represented host cells with EGFP knockin in Flk-1 locus, while red fluorescence represented donor cells with transgene of tdTomato. Scale bars, 3 mm. D: Flow cytometry analysis of chimerism of Td+ cells in Flk-1 and Flk-1 rat-mouse chimeric EP and YS of E10.5. Data are presented as mean ± SEM. P < 0.05 was considered statistically significant. ∗,P < 0.05; n.s., not significant.Flk-1 YS: n = 4, Flk-1 EP: n = 4, Flk-1 YS: n = 8, Flk-1 EP: n = 7. E and F: Immunofluorescence analysis for marker of vascular endothelial cells (CD31) of Flk-1, Flk-1, and Flk-1 rat-mouse chimeric EP (E) and YS (F) at E10.5. Sections were stained with antibody against tdTomato for donor cells, antibody against CD31 for vascular endothelial cells, and DAPI for nuclear counterstaining. Scale bars, 200 μm.G and H: Flow cytometry analysis of donor cell–derived hematopoietic cells of Flk-1 and Flk-1 rat-mouse chimeric YS (G) and EP (H) at E10.5. Cells were stained with antibody against rat CD45 (n = 5). I: Bright-field and red fluorescence images of full-term Flk-1 rat-mouse chimeric mice. J: Immunofluorescence analysis for the marker of vascular endothelial cells (CD31) in the blood vessel of Flk-1 chimeric mice. Sections were stained with antibody against tdTomato for donor cells, antibody against CD31 for vascular endothelial cells, and DAPI for nuclear counterstaining. Scale bars, 200 μm. EP, embryo proper; YS, yolk sac.
Fig. 5. Bulk RNA sequencing of Flk-1 rat-mouse chimeric YS at E10.5. A: Hierarchical cluster analysis of rat cells harvested from Flk-1 rat-mouse chimeric YS at E10.5 and rESCs as control. Red and purple represent higher and lower gene expression levels, respectively. B, C, D, and F: Histogram (left panels) and hierarchical cluster analysis (right panels) of representative upregulated genes related to vascular endothelial cells (B), arteries and veins (C), blood vessels formation (D), and hematopoietic cell lineage (F). E: Gene ontology analysis of the upregulated and downregulated genes enriched on biological processes for rESCs-derived cells from Flk-1 rat-mouse chimeric YS at E10.5. YS, yolk sac; rESCs, rat embryonic stem cells.
Fig. 6. De novo angiogenesis and hematopoiesis of rESCs in rat-mouse chimeras. De novo angiogenesis and hematopoiesis of rESCs in Flk-1 mutation mouse embryo.
Table 1. Generation of rat-mouse chimeras at E10.5 by blastocyst complementation.
Injected cell line/mouse Number of embryos transferred Chimerism of E10.5 chimeras/embryos Flk-1 genotype Flk-1 +/+ chimeras/embryos Flk-1 +/EGFP chimeras/embryos Flk-1 EGFP/EGFP chimeras/embryos DA-rESCs 342 64/112 (57%) 16/30 (25%) 34/62 (53%) 14/20 (22%) Flk-1 +/EGFP – – 10/39 (26%) 25/39 (64%) 4/39 (10%) 
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[1] Bandopadhyay, R., Orte, C., Lawrenson, J.G., Reid, A.R., De, S.S., and Allt, G. 2001. Contractile proteins in pericytes at the blood-brain and blood-retinal barriers. J. Neurocytol. 30, 35-44. [2] Carmeliet, P. 2000a. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6, 389-395. [3] Carmeliet, P. 2000b. Developmental biology. One cell, two fates. Nature 408, 43-45. [4] Cooney, A.J., Tsukiyama, T., Kato-Itoh, M., Nakauchi, H., and Ohinata, Y. 2014. A comprehensive system for generation and evaluation of induced pluripotent stem cells using piggyBac transposition. PloS One 9, e92973. [5] Costa, G., Kouskoff, V., and Lacaud, G. 2012. Origin of blood cells and HSC production in the embryo. Trends Immunol. 33, 215-223. [6] Crisan, M., Corselli, M., Chen, W.C., and Peault, B. 2012. Perivascular cells for regenerative medicine. J. Cell Mol. Med. 16, 2851-2860. [7] Crisan, M., Yap, S., Casteilla, L., Chen, C.W., Corselli, M., Park, T.S., Andriolo, G., Sun, B., Zheng, B., Zhang, L., Norotte, C., Teng, P.N., Traas, J., Schugar, R., Deasy, B.M., Badylak, S., Buhring, H.J., Giacobino, J.P., Lazzari, L., Huard, J., and Peault, B. 2008. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3, 301-313. [8] Cross, M.J., Dixelius, J., Matsumoto, T., and Claesson-Welsh, L. 2003. VEGF-receptor signal transduction. Trends Biochem. Sci. 28, 488-494. [9] Du, C., Narayanan, K., Leong, M.F., Ibrahim, M.S., Chua, Y.P., Khoo, V.M., and Wan, A.C. 2016. Functional kidney bioengineering with pluripotent stem-cell-derived renal progenitor cells and decellularized kidney scaffolds. Adv Healthc Mater 5, 2080-2091. [10] Gomes, M.E., Rodrigues, M.T., Domingues, R.M.A., and Reis, R.L. 2017. Tissue engineering and regenerative medicine: new trends and directions-a year in review. Tissue Eng. B Rev. 23, 211-224. [11] Goto, T., Hara, H., Sanbo, M., Masaki, H., Sato, H., Yamaguchi, T., Hochi, S., Kobayashi, T., Nakauchi, H., and Hirabayashi, M. 2019. Generation of pluripotent stem cell-derived mouse kidneys in Sall1-targeted anephric rats. Nat. Commun. 10, 451. [12] Hall, A.P. 2006. Review of the pericyte during angiogenesis and its role in cancer and diabetic retinopathy. Toxicol. Pathol. 34, 763-775. [13] Hamanaka, S., Umino, A., Sato, H., Hayama, T., Yanagida, A., Mizuno, N., Kobayashi, T., Kasai, M., Suchy, F.P., Yamazaki, S., Masaki, H., Yamaguchi, T., and Nakauchi, H. 2018. Generation of vascular endothelial cells and hematopoietic cells by blastocyst complementation. Stem Cell Rep 11, 988-997. [14] Huang, K., Zhu, Y., Ma, Y., Zhao, B., Fan, N., Li, Y., Song, H., Chu, S., Ouyang, Z., Zhang, Q., Xing, Q., Lai, C., Li, N., Zhang, T., Gu, J., Kang, B., Shan, Y., Lai, K., Huang, W., Mai, Y., Wang, Q., Li, J., Lin, A., Zhang, Y., Zhong, X., Liao, B., Lai, L., Chen, J., Pei, D., and Pan, G. 2018. BMI1 enables interspecies chimerism with human pluripotent stem cells. Nat. Commun. 9, 4649. [15] Isotani, A., Hatayama, H., Kaseda, K., Ikawa, M., and Okabe, M. 2011. Formation of a thymus from rat ES cells in xenogeneic nude mouse<-->rat ES chimeras. Gene Cell. 16, 397-405. [16] Kitahara, H., Yagi, H., Tajima, K., Okamoto, K., Yoshitake, A., Aeba, R., Kudo, M., Kashima, I., Kawaguchi, S., Hirano, A., Kasai, M., Akamatsu, Y., Oka, H., Kitagawa, Y., and Shimizu, H. 2016. Heterotopic transplantation of a decellularized and recellularized whole porcine heart. Interact. Cardiovasc. Thorac. Surg. 22, 571-579. [17] Kobayashi, T., Yamaguchi, T., Hamanaka, S., Kato-Itoh, M., Yamazaki, Y., Ibata, M., Sato, H., Lee, Y.S., Usui, J., Knisely, A.S., Hirabayashi, M., and Nakauchi, H. 2010. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142, 787-799. [18] Kubis, N., and Levy, B.I. 2003. Vasculogenesis and angiogenesis: molecular and cellular controls. Part 1: growth factors. Intervent Neuroradiol. 9, 227-237. [19] Lacaud, G., and Kouskoff, V. 2017. Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis. Exp. Hematol. 49, 19-24. [20] Lux, C.T., Yoshimoto, M., McGrath, K., Conway, S.J., Palis, J., and Yoder, M.C. 2008. All primitive and definitive hematopoietic progenitor cells emerging before E10 in the mouse embryo are products of the yolk sac. Blood 111, 3435-3438. [21] Mascetti, V.L., and Pedersen, R.A. 2016. Human-mouse chimerism validates human stem cell pluripotency. Cell Stem Cell 18, 67-72. [22] Mazzini, L., Gelati, M., Profico, D.C., Sgaravizzi, G., Projetti Pensi, M., Muzi, G., Ricciolini, C., Rota Nodari, L., Carletti, S., Giorgi, C., Spera, C., Domenico, F., Bersano, E., Petruzzelli, F., Cisari, C., Maglione, A., Sarnelli, M.F., Stecco, A., Querin, G., Masiero, S., Cantello, R., Ferrari, D., Zalfa, C., Binda, E., Visioli, A., Trombetta, D., Novelli, A., Torres, B., Bernardini, L., Carriero, A., Prandi, P., Servo, S., Cerino, A., Cima, V., Gaiani, A., Nasuelli, N., Massara, M., Glass, J., Soraru, G., Boulis, N.M., and Vescovi, A.L. 2015. Human neural stem cell transplantation in ALS: initial results from a phase I trial. J. Transl. Med. 13, 17. [23] Ozerdem, U., and Stallcup, W.B. 2003. Early contribution of pericytes to angiogenic sprouting and tube formation. Angiogenesis 6, 241-249. [24] Palis, J., Robertson, S., Kennedy, M., Wall, C., and Keller, G. 1999. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126, 5073-5084. [25] Patel, J.J., Srivastava, S., and Siow, R.C. 2016. Isolation, culture, and characterization of vascular smooth muscle cells. Methods Mol. Biol. 1430, 91-105. [26] Pettinato, G., Lehoux, S., Ramanathan, R., Salem, M.M., He, L.X., Muse, O., Flaumenhaft, R., Thompson, M.T., Rouse, E.A., Cummings, R.D., Wen, X., and Fisher, R.A. 2019. Generation of fully functional hepatocyte-like organoids from human induced pluripotent stem cells mixed with Endothelial Cells. Sci. Rep. 9, 8920. [27] Picelli, S., Faridani, O.R., Bjorklund, A.K., Winberg, G., Sagasser, S., and Sandberg, R. 2014. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171-181. [28] Raza, A., Franklin, M.J., and Dudek, A.Z. 2010. Pericytes and vessel maturation during tumor angiogenesis and metastasis. Am. J. Hematol. 85, 593-598. [29] Sakurai, Y., Ohgimoto, K., Kataoka, Y., Yoshida, N., and Shibuya, M. 2004. Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice. Proc. Natl. Acad. Sci. U. S. A. 102, 1076-1081. [30] Sakurai, Y., Ohgimoto, K., Kataoka, Y., Yoshida, N., and Shibuya, M. 2005. Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice. Proc. Natl. Acad. Sci. U. S. A. 102, 1076-1081. [31] Salaris, F., and Rosa, A. 2019. Construction of 3D in vitro models by bioprinting human pluripotent stem cells: challenges and opportunities. Brain Res. 1723, 146393. [32] Shalaby, F., Ho, J., Stanford, W.L., Fischer, K.D., Schuh, A.C., Schwartz, L., Bernstein, A., and Rossant, J. 1997a. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 89, 981-990. [33] Shalaby, F., Rossant, J., Yamaguchi, T.P., Gertsenstein, M., Wu, X.F., Breitman, M.L., and Schuh, A.C. 1995. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376, 62-66. [34] Shalaby F., Ho J., Stanford W.L., Fischer K.D., Schuh A.C., Schwartz L., Bernstein A., and J., R. 1997b. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 89, 981-990. [35] van Dijk, C.G., Nieuweboer, F.E., Pei, J.Y., Xu, Y.J., Burgisser, P., van Mulligen, E., el Azzouzi, H., Duncker, D.J., Verhaar, M.C., and Cheng, C. 2015. The complex mural cell: pericyte function in health and disease. Int. J. Cardiol. 190, 75-89. [36] Wang, X., Li, T., Cui, T., Yu, D., Liu, C., Jiang, L., Feng, G., Wang, L., Fu, R., Zhang, X., Hao, J., Wang, Y., Wang, L., Zhou, Q., Li, W., and Hu, B. 2018. Human embryonic stem cells contribute to embryonic and extraembryonic lineages in mouse embryos upon inhibition of apoptosis. Cell Res. 28, 126-129. [37] Wu, J., Platero-Luengo, A., Sakurai, M., Sugawara, A., Gil, M.A., Yamauchi, T., Suzuki, K., Bogliotti, Y.S., Cuello, C., Morales Valencia, M., Okumura, D., Luo, J., Vilarino, M., Parrilla, I., Soto, D.A., Martinez, C.A., Hishida, T., Sanchez-Bautista, S., Martinez-Martinez, M.L., Wang, H., Nohalez, A., Aizawa, E., Martinez-Redondo, P., Ocampo, A., Reddy, P., Roca, J., Maga, E.A., Esteban, C.R., Berggren, W.T., Nunez Delicado, E., Lajara, J., Guillen, I., Guillen, P., Campistol, J.M., Martinez, E.A., Ross, P.J., and Izpisua Belmonte, J.C. 2017. Interspecies chimerism with mammalian pluripotent stem cells. Cell 168, 473-486. [38] Yamaguchi, T., Sato, H., Kato-Itoh, M., Goto, T., Hara, H., Sanbo, M., Mizuno, N., Kobayashi, T., Yanagida, A., Umino, A., Ota, Y., Hamanaka, S., Masaki, H., Rashid, S.T., Hirabayashi, M., and Nakauchi, H. 2017. Interspecies organogenesis generates autologous functional islets. Nature 542, 191-196. [39] Yang, Y., Liu, B., Xu, J., Wang, J., Wu, J., Shi, C., Xu, Y., Dong, J., Wang, C., Lai, W., Zhu, J., Xiong, L., Zhu, D., Li, X., Yang, W., Yamauchi, T., Sugawara, A., Li, Z., Sun, F., Li, X., Li, C., He, A., Du, Y., Wang, T., Zhao, C., Li, H., Chi, X., Zhang, H., Liu, Y., Li, C., Duo, S., Yin, M., Shen, H., Belmonte, J.C.I., and Deng, H. 2017. Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell 169, 243-257. [40] Zhou, Q., Li, L., and Li, J. 2015. Stem cells with decellularized liver scaffolds in liver regeneration and their potential clinical applications. Liver Int. 35, 687-694. -
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