Search

Blastocyst complementation by pluripotent stem cell (PSC) injection is believed to be the most promising method to generate xenogeneic organs. However, ethical issues prevent the study of human chimeras in the late embryonic stage of development. Primate embryonic stem cells (ESCs), which have similar pluripotency to human ESCs, are a good model for studying interspecies chimerism and organ generation. However, whether primate ESCs can be used in xenogenous grafts remains unclear. In this study, we evaluated the chimeric ability of cynomolgus monkey (Macaca fascicularis) ESCs (cmESCs) in pigs, which are excellent hosts because of their many similarities to humans. We report an optimized culture medium that enhanced the anti-apoptotic ability of cmESCs and improved the development of chimeric embryos, in which domesticated cmESCs (D-ESCs) injected into pig blastocysts differentiated into cells of all three germ layers. In addition, we obtained two neonatal interspecies chimeras, in which we observed tissue-specific D-ESC differentiation. Taken together, the results demonstrate the capability of D-ESCs to integrate and differentiate into functional cells in a porcine model, with a chimeric ratio of 0.001-0.0001 in different neonate tissues. We believe this work will facilitate future developments in xenogeneic organogenesis, bringing us one step closer to producing tissue-specific functional cells and organs in a large animal model through interspecies blastocyst complementation.

Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR-Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3-3072 site using CRISPR-Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.

(F) Expression of Nanos3, Dazl, Ddx4, Stra8, Uchl1 in Nanos3-/- male pigs (G), and of Nanos3, Dazl, Ddx4, Lhx8, Zp3 in Nanos3-/- female pigs. The anatomy and physiological characteristics of pigs are closer to those of humans than those of other non-primate animals. (E) Genotyping Nanos3-/- #7, Nanos3-/- #1 and Nanos3-/- #51 single cell lines using polymerase chain reaction (PCR). [Extracted from the article]

Leukocytes of the mother sow were also shown a few GFP positive cells, and the second column showed that most of these GFP positive cells were CD3+ cells. We found that 0.043% of GFP positive cells were present in PBMC and 88.3% of these cells were CD3 SP + sp T cells (Figure 1G). To find out whether foetal cells could also circulate through the blood to other organs of the surrogates, we euthanized the sows after delivery and examined the presence of GFP positive cells in other organs by freezing sections (Method S5). The presence of a tiny population of foreign cells or DNA in an organism is known as microchimerism. [Extracted from the article]

Background: Mice with humanized livers are important models to study drug toxicology testing, development of hepatitis virus treatments, and hepatocyte transplantation therapy. However, the huge difference between mouse and human in size and anatomy limited the application of humanized mice in investigating human diseases. Therefore, it is urgent to construct humanized livers in pigs to precisely investigate hepatocyte regeneration and human hepatocyte therapy. CRISPR/Cas9 system and somatic cell cloning technology were used to generate two pig models with FAH deficiency and exhibiting severe immunodeficiency (FAH/RAG1 and FAH/RAG1/IL2RG deficiency). Human primary hepatocytes were then successfully transplanted into the FG pig model and constructed two pigs with human liver. Results: The constructed FAH/RAG1/IL2RG triple-knockout pig models were characterized by chronic liver injury and severe immunodeficiency. Importantly, the FG pigs transplanted with primary human hepatocytes produced human albumin in a time dependent manner as early as 1 week after transplantation. Furthermore, the colonization of human hepatocytes was confirmed by immunochemistry staining. Conclusions: We successfully generated pig models with severe immunodeficiency that could construct human liver tissues. [ABSTRACT FROM AUTHOR]