Interspecies Chimerism with Mammalian Pluripotent Stem Cells
Jun Wu, Aida Platero-Luengo, Masahiro Sakurai, Atsushi Sugawara, Maria Antonia Gil, Takayoshi Yamauchi, Keiichiro Suzuki, Yanina Soledad Bogliotti, Cristina Cuello, Mariana Morales Valencia, Daiji Okumura7, Jingping Luo, Marcela Vilariño, Inmaculada Parrilla, Delia Alba Soto, Cristina A. Martinez, Tomoaki Hishida, Sonia Sánchez-Bautista, M. Llanos Martinez-Martinez, Huili Wang, Alicia Nohalez, Emi Aizawa, Paloma Martinez-Redondo, Alejandro Ocampo, Pradeep Reddy, Jordi Roca, Elizabeth A. Maga, Concepcion Rodriguez Esteban, W. Travis Berggren, Estrella Nuñez Delicado, Jeronimo Lajara, Isabel Guillen, Pedro Guillen, Josep M. Campistol, Emilio A. Martinez, Pablo Juan Ross, Juan Carlos Izpisua Belmonte8.
7Present address: Graduate School of Agriculture, Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
8Lead Contact
Highlights
• Naive rat PSCs robustly contribute to live rat-mouse chimeras
• A versatile CRISPR-Cas9 mediated interspecies blastocyst complementation system
• Naive rodent PSCs show no chimeric contribution to post-implantation pig embryos
• Chimerism is observed with some human iPSCs in post-implantation pig embryos
Summary
Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.
Author Contributions
J.W. and J.C.I.B. conceived the study. J.W. generated and characterized all naive and intermediate hiPSC lines. K.S. generated and characterized primed hiPSCs. J.W. and T.H. generated rat iPSCs. J.W., A.P.-L., T.Y., M.M.V., D.O., A.O., P.R., C.R.E., J.W., and P.M.R. performed immunohistochemistry analyses of mouse and pig embryos. K.S., T.Y., E.S., A.P.-L., and M.M.V. performed genotyping, genomic PCR, and genomic qPCR analyses. A.S., M.S., and J.P.L. performed mouse Cas9/sgRNA injection, blastocyst injection, and embryo transfer. Y.S.B., M.S., and M.V. prepared hiPSCs, performed morulae and blastocyst injections, and analyzed hiPSC contribution to cattle and ppig ICMs. H.W. produced parthenogenetic pig embryos. D.A.S., Y.S.B., and M.V. produced cattle embryos. Work at UC Davis and University of Murcia was performed under the supervision of P.J.R. and E.A.M., respectively. E.A.M., M.A.G., C.C., I.P., C.A.M., S.S.B., A.N., and J.R. designed, coordinated, performed, and analyzed data related to pig embryo collection, embryo culture, blastocyst injection, embryo transfer, and embryo recover. E.N.D., J.L., I.G., P.G., T.B., M.L.M.-M., and J.M.C. coordinated work between Salk, and University of Murcia. J.W., P.J.R., and J.C.I.B. wrote the manuscript.
Acknowledgments
J.C.I.B. dedicates this paper to Dr. Rafael Matesanz, Director of the Spain’s National Organ Transplant Organization. Rafael’s work has helped save thousands of patients in need of an organ. He constitutes a relentless inspiration for those of us trying to understand and alleviate human disease. The authors are grateful to Xiomara Lucas, Maria Dolores Ortega, Moises Gonzalvez, Jose Antonio Godinez, and Jesus Gomis for their assistance throughout this work. We thank the staff of the Agropor S.A. and Porcisan S.A. piggeries (Murcia, Spain) for the help and excellent management of animals. We thank Joan Rowe, Bret McNabb, Aaron Prinz, and Kent Parker and their crews for excellent assistance with embryo transfers and pig care at UC Davis. We thank Mako Yamamoto for help with mouse embryo dissection. We would like to thank Uri Manor of the Salk Waitt Advanced Biophotonics Core for technical advice on imaging analysis. We would like to thank the Salk Stem Cell Core for providing cell culture reagents. We would like to thank May Schwarz and Peter Schwarz for administrative help. We thank David O’Keefe for critical reading and editing of the manuscript. This experimental study was supported by The Fundación Séneca ( GERM 19892/GERM/15 ), Murcia, Spain. The MINECO is acknowledged for their grant-based support ( BES-2013-064087 and BES-2013-064069 ) (to C.A.M and A.N.). P.J.R was supported by a UC Davis Academic Senate New Research grant. Work in the laboratory of J.C.I.B. was supported by the UCAM, Fundacion Dr. Pedro Guillen, G. Harold and Leila Y. Mathers Charitable Foundation, and The Moxie Foundation.
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NOTA DESTE BLOGGER:
Os cientistas NÃO FUNDIRAM o DNA de um humano e um porco.
Eles apenas deixaram crescer um embrião de porco normal.
Depois eles injetaram um número "baixo" de células-tronco humanas no embrião de porco.
Eles então removeram as células-tronco humanas.
Após isso, não havia células que tinham uma mistura de DNA humano e de porco.
Eles NÃO DEMONSTRARAM a compatibilidade do DNA humano com o DNA de proco, e nem demonstraram ser o embrião de porco seria viável até o nascimento com as células humanas.
+++++
NOTA DESTE BLOGGER:
Os cientistas NÃO FUNDIRAM o DNA de um humano e um porco.
Eles apenas deixaram crescer um embrião de porco normal.
Depois eles injetaram um número "baixo" de células-tronco humanas no embrião de porco.
Eles então removeram as células-tronco humanas.
Após isso, não havia células que tinham uma mistura de DNA humano e de porco.
Eles NÃO DEMONSTRARAM a compatibilidade do DNA humano com o DNA de proco, e nem demonstraram ser o embrião de porco seria viável até o nascimento com as células humanas.
