RESUMEN
Heterologous organ transplantation is an effective way of replacing organ function but is limited by severe organ shortage. Although generating human organs in other large mammals through embryo complementation would be a groundbreaking solution, it faces many challenges, especially the poor integration of human cells into the recipient tissues. To produce human cells with superior intra-niche competitiveness, we combined optimized pluripotent stem cell culture conditions with the inducible overexpression of two pro-survival genes (MYCN and BCL2). The resulting cells had substantially enhanced viability in the xeno-environment of interspecies chimeric blastocyst and successfully formed organized human-pig chimeric middle-stage kidney (mesonephros) structures up to embryonic day 28 inside nephric-defective pig embryos lacking SIX1 and SALL1. Our findings demonstrate proof of principle of the possibility of generating a humanized primordial organ in organogenesis-disabled pigs, opening an exciting avenue for regenerative medicine and an artificial window for studying human kidney development.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Humanos , Porcinos , Animales , Mesonefro , Embrión de Mamíferos , Blastocisto , Mamíferos , Proteínas de HomeodominioRESUMEN
Currently, there is a significant shortage of transplantable organs for patients in need. Interspecies chimerism and blastocyst complementation are alternatives for generating transplantable human organs in host animals such as pigs to meet this shortage. While successful interspecies chimerism and organ generation have been observed between evolutionarily close species such as rat and mouse, barriers still exist for more distant species pairs such as human-mouse, marmoset-mouse, human-pig, and others. One of the proposed barriers to chimerism is the difference in developmental stages between the donor cells and the host embryo at the time the cells are introduced into the host embryo. Hence, there is a logical effort to stage-match the donor cells with the host embryos for enhancing interspecies chimerism. In this study, we used an in silico approach to simultaneously stage-match the early developing embryos of four species, including human, marmoset, mouse, and pig based on transcriptome similarities. We used an unsupervised clustering algorithm to simultaneously stage-match all four species as well as Spearman's correlation analyses to stage-match pairs of donor-host species. From our stage-matching analyses, we found that the four stages that best matched with each other are the human blastocyst (E6/E7), the gastrulating mouse embryo (E6-E6.75), the marmoset late inner cell mass, and the pig late blastocyst. We further demonstrated that human pluripotent stem cells best matched with the mouse post-implantation stages. We also performed ontology analysis of the genes upregulated and commonly expressed between donor-host species pairs at their best matched stages. The stage-matching results predicted by this study will inform in vivo and in vitro interspecies chimerism and blastocyst complementation studies and can be used to match donor cells with host embryos between multiple species pairs to enhance chimerism for organogenesis.
Asunto(s)
Callithrix , Células Madre Pluripotentes , Porcinos , Ratones , Animales , Humanos , Ratas , Quimerismo , Embrión de Mamíferos , BlastocistoRESUMEN
Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful complementation of the male germline in adult chimeras following injection of mouse or rat PSCs into mouse blastocysts carrying a mutation in Tsc22d3, an essential gene for spermatozoa production. Injection of mouse PSCs into Tsc22d3-Knockout (KO) blastocysts gave rise to intraspecies chimeras exclusively embodying PSC-derived functional spermatozoa. In addition, injection of rat embryonic stem cells (rESCs) into Tsc22d3-KO embryos produced interspecies mouse-rat chimeras solely harboring rat spermatids and spermatozoa capable of fertilizing oocytes. Furthermore, using single-cell RNA sequencing, we deconstructed rat spermatogenesis occurring in a mouse-rat chimera testis. Collectively, this study details a method for exclusive xenogeneic germ cell production in vivo, with implications that may extend to rat transgenesis, or endangered animal species conservation efforts.
Asunto(s)
Células Madre Pluripotentes , Animales , Blastocisto , Quimera , Células Madre Embrionarias , Masculino , Ratones , Ratones Noqueados , Ratas , EspermatozoidesRESUMEN
Obtaining functional human cells through interspecies chimerism with human pluripotent stem cells (hPSCs) remains unsuccessful due to its extremely low efficiency. Here, we show that hPSCs failed to differentiate and contribute teratoma in the presence of mouse PSCs (mPSCs), while MYCN, a pro-growth factor, dramatically promotes hPSC contributions in teratoma co-formation by hPSCs/mPSCs. MYCN combined with BCL2 (M/B) greatly enhanced conventional hPSCs to integrate into pre-implantation embryos of different species, such as mice, rabbits, and pigs, and substantially contributed to mouse post-implantation chimera in embryonic and extra-embryonic tissues. Strikingly, M/B-hPSCs injected into pre-implantation Flk-1+/- mouse embryos show further enhanced chimerism that allows for obtaining live human CD34+ blood progenitor cells from chimeras through cell sorting. The chimera-derived human CD34+ cells further gave rise to various subtype blood cells in a typical colony-forming unit (CFU) assay. Thus, we provide proof of concept to obtain functional human cells through enhanced interspecies chimerism with hPSCs.
Asunto(s)
Células Madre Pluripotentes , Teratoma , Animales , Diferenciación Celular , Quimera , Quimerismo , Humanos , Ratones , Proteína Proto-Oncogénica N-Myc , Conejos , PorcinosRESUMEN
A reliable source of human hepatocytes and transplantable livers is needed. Interspecies embryo complementation, which involves implanting donor human stem cells into early morula/blastocyst stage animal embryos, is an emerging solution to the shortage of transplantable livers. We review proposed mutations in the recipient embryo to disable hepatogenesis, and discuss the advantages of using fumarylacetoacetate hydrolase knockouts and other genetic modifications to disable hepatogenesis. Interspecies blastocyst complementation using porcine recipients for primate donors has been achieved, although percentages of chimerism remain persistently low. Recent investigation into the dynamic transcriptomes of pigs and primates have created new opportunities to intimately match the stage of developing animal embryos with one of the many varieties of human induced pluripotent stem cell. We discuss techniques for decreasing donor cell apoptosis, targeting donor tissue to endodermal structures to avoid neural or germline chimerism, and decreasing the immunogenicity of chimeric organs by generating donor endothelium.
Asunto(s)
Edición Génica/métodos , Hidrolasas/genética , Trasplante de Hígado/métodos , Donadores Vivos , Quimera por Trasplante/genética , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Hidrolasas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Quimera por Trasplante/metabolismoRESUMEN
Pluripotent stem cells (PSCs) are cells that can differentiate into any type of cells in the body, therefore have valuable promise in regenerative medicine of cell replacement therapies and tissue/organ engineering. PSCs can be derived either from early embryos or directly from somatic cells by epigenetic reprogramming that result in customized cells from patients. Here we summarize the methods of deriving PSCs, the various types of PSCs generated with different status, and their versatile applications in both clinical and embryonic development studies. We also discuss an intriguing potential application of PSCs in constructing tissues/organs in large animals by interspecies chimerism. All these emerging findings are likely to contribute to the breakthroughs in biological research and the prosperous prospects of regenerative medicine.