Generation of mouse-human chimeric embryos.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse-human chimeric embryos. Here, we describe a protocol for generating mouse-human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3-4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1-4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals.

Fitness selection in human pluripotent stem cells and interspecies chimeras: Implications for human development and regenerative medicine.

A small number of pluripotent cells within early embryo gives rise to all cells in the adult body, including germ cells. Hence, any mutations occurring in the pluripotent cell population are at risk of being propagated to their daughter cells and could lead to congenital defects or embryonic lethality and pose a risk of being transmitted to future generations. The observation that genetic errors are relatively common in preimplantation embryos, but their levels reduce as development progresses, suggests the existence of mechanisms for clearance of aberrant, unfit or damaged cells. Although early human embryogenesis is largely experimentally inaccessible, pluripotent stem cell (PSC) lines can be derived either from the inner cell mass (ICM) of a blastocyst or by reprogramming somatic cells into an embryonic stem cell-like state. PSCs retain the ability to differentiate into any cell type in vitro and, hence, they represent a unique and powerful tool for studying otherwise intractable stages of human development. The advent of PSCs has also opened up a possibility of developing regenerative medicine therapies, either through PSC differentiation in vitro or by creating interspecies chimeras for organ replacement. Here, we discuss the emerging evidence of cell selection in human PSC populations in vivo and in vitro and we highlight the implications of understanding this phenomenon for human development and regenerative medicine.

Characterization of muscle development and gene expression in early embryos of chicken, quail, and their hybrids.

Myogenesis is a complex, regulated process that involves myoblast proliferation, migration, adhesion, and fusion into myotubes. To investigate early development of embryonic muscles and the expression of regulatory genes during myogenesis in chicken, quail and their hybrids, meat-breeding cocks and egg-breeding cocks were selected as male parents, quails were used as female parents. Their offspring were meat and egg hybrids via Artificial insemination. We measured expression of MUSTN1, IGF-1, and PDK4 using qRT-PCR. We examined muscle fiber diameter using scanning electron microscopy. The results showed that muscle development was two days slower in chicken, egg hybrid, and meat hybrid than in quail. Muscle fiber spacing was the largest in chicken, followed by meat hybrid, egg hybrid, and quail. A similar trend was obtained for muscle fiber diameter. Additionally, muscle fiber diameter increased with embryogenesis. The sarcomere was present on day 17 of incubation in quail, but not in the other species. MUSTN1 could up-regulated IGF-1 by activating PI3K/Akt. IGF-1 expression was consistent with myoblast proliferation and myotube fusion. PDK4 was expressed from E7 to E17. The first peak was reached on E10, egg hybrid and meat hybrid reached their peak at E15. PDK4 is involved in the early proliferation and differentiation of muscle, thereby affecting muscle growth and development. Our findings demonstrated that MUSTN1, IGF-1 and PDK4 genes are expressed to varying levels in breast muscle of chicken, quail, egg hybrid and meat hybrid during the embryonic period. Interestingly, with increasing embryonic age, muscle development was approximately 48 h faster in quail than in other species. We speculated that MUSTN1, IGF-1 and PDK4 genes may be the main candidate genes that cause differences in poultry muscle traits, but the molecular regulation mechanisms need to be further studied. Our findings shed some light on the avian embryo muscle formation and molecular breeding of poultry muscle traits, which provide theoretical basis for poultry breeding.

Diverse Routes toward Early Somites in the Mouse Embryo.

Somite formation is foundational to creating the vertebrate segmental body plan. Here, we describe three transcriptional trajectories toward somite formation in the early mouse embryo. Precursors of the anterior-most somites ingress through the primitive streak before E7 and migrate anteriorly by E7.5, while a second wave of more posterior somites develops in the vicinity of the streak. Finally, neuromesodermal progenitors (NMPs) are set aside for subsequent trunk somitogenesis. Single-cell profiling of T-/- chimeric embryos shows that the anterior somites develop in the absence of T and suggests a cell-autonomous function of T as a gatekeeper between paraxial mesoderm production and the building of the NMP pool. Moreover, we identify putative regulators of early T-independent somites and challenge the T-Sox2 cross-antagonism model in early NMPs. Our study highlights the concept of molecular flexibility during early cell-type specification, with broad relevance for pluripotent stem cell differentiation and disease modeling.

In Vitro and In Vivo Interspecies Chimera Assay Using Early Pig Embryos.

Chimeric pigs harboring organs derived from human stem cells are promising for patient-specific regenerative therapies. Induced pluripotent stem cells (iPSCs) can contribute to all cell types of the fetus, including germline after injection into embryos. However, ethical concerns prohibit testing human iPSCs in chimera assays. Here, we evaluated porcine embryos as hosts for an interspecies chimera assay using iPSCs from either cynomolgus monkeys (cyiPSCs) or mouse (miPSCs). To establish an in vitro culture system compatible for cyiPSCs and porcine embryos, we determined blastocyst development in eight different stem cell media. The highest developmental rates of blastocysts were achieved in Knockout Dulbecco's modified Eagle's medium with 20% knockout serum replacement. We found that cyiPSCs injected into porcine embryos survived in vitro and were mostly located in the trophectoderm (TE). Instead, when miPSCs were injected into porcine embryos, the cells rapidly proliferated. The behavior of chimeras developed in vitro was recapitulated in vivo; cyiPSCs were observed in the TE, but not in the porcine epiblast. However, when miPSCs were injected into in vivo derived porcine embryos, mouse cells were found in both, the epiblast and TE. These results demonstrate that porcine embryos could be useful for evaluating the interspecies chimera-forming ability of iPSCs from different species.

Intravital imaging of mouse embryos.

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.

Embryo-derived and induced pluripotent stem cells: Towards naive pluripotency and chimeric competency in rabbits.

Both embryo-derived (ESC) and induced pluripotent stem cell (iPSC) lines have been established in rabbit. They exhibit the essential characteristics of primed pluripotency. In this review, we described their characteristic features at both molecular and functional levels. We also described the attempts to reprogram rabbit pluripotent stem cells (rbPSCs) toward the naive state of pluripotency using methods established previously to capture this state in rodents and primates. In the last section, we described and discussed our current knowledge of rabbit embryo development pertaining to the mechanisms of early lineage segregation. We argued that the molecular signature of naive-state pluripotency differs between mice and rabbits. We finally discussed some of the key issues to be addressed for capturing the naive state in rbPSCs, including the generation of embryo/PSC chimeras.

Domesticated cynomolgus monkey embryonic stem cells allow the generation of neonatal interspecies chimeric pigs.

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.

Chick Models and Human-Chick Organizer Grafts.

The combination of affordability, large size, and ease of access at almost every stage of development renders the chick an excellent model organism for studying vertebrate development. Not only is it a great system in and of itself, but these qualities make it a great host for interspecies chimera experiments. In this chapter we highlight some notable examples of mammalian-chick chimeras, and show how one can for instance use the chick to push mammalian stem cell experiments further to learn about the behavior and capabilities of these cells in vivo. In particular, here we present the methodology necessary for transplantation of human embryonic stem cell (hESC)-derived "gastruloids" stimulated to generate a human organizer into the chick embryo. In these human-chick chimeras, the human organizer cells self-organize to contribute directly to notochord-like tissue and indirectly induce host chick cells to generate neural tissue.

Embryonic Chimeras with Human Pluripotent Stem Cells.

Human pluripotent stem (PS) cells can be isolated from preimplantation embryos or by reprogramming of somatic cells or germline progenitors. Human PS cells are considered the "holy grail" of regenerative medicine because they have the potential to form all cell types of the adult body. Because of their similarity to humans, nonhuman primate (NHP) PS cells are also important models for studying human biology and disease, as well as for developing therapeutic strategies and test bed for cell replacement therapy. This chapter describes adjusted methods for cultivation of PS cells from different primate species, including African green monkey, rhesus monkey, chimpanzee, and human. Supplementation of E8 medium and inhibitors of the Tankyrase and GSK3 kinases to various primate PS cell media reduce line-dependent predisposition for spontaneous differentiation in conventional PS cell cultures. We provide methods for basic characterization of primate PS cell lines, which include immunostaining for pluripotency markers such as OCT4 and TRA-1-60, as well as in vivo teratoma formation assay. We provide methods for generating alternative PS cells including region-selective primed PS cells, two different versions of naïve-like cells, and recently reported extended pluripotent stem (EPS) cells. These derivations are achieved by acclimation of conventional PS cells to target media, episomal reprogramming of somatic cells, or resetting conventional PS cells to a naïve-like state by overexpression of KLF2 and NANOG. We also provide methods for isolation of PS cells from human blastocysts. We describe how to generate interspecies primate-mouse chimeras at the blastocyst and postimplantation embryo stages. Systematic evaluation of the chimeric competency of human and primate PS cells will aid in efforts to overcome species barriers and achieve higher grade chimerism in postimplantation conceptuses that could enable organ-specific enrichment of human xenogeneic PS cell derivatives in large animals such as pigs and sheep.

Deer antler stem cells are a novel type of cells that sustain full regeneration of a mammalian organ-deer antler.

Deer antlers are extraordinary mammalian organs that can fully regenerate annually. Antler renewal is a stem cell-based epimorphic process and antler stem (AS) cells can initiate de novo generation of antlers in postnatal mammals. However, although being called stem cells, the AS cells have not been characterized at molecular level based on the stem cell criteria. Comprehensive characterization of the AS cells would undoubtedly help to decipher the mechanism underlying the full regeneration of deer antlers, the only case of stem cell-based epimorphic regeneration in mammals. In the present study, three types of AS cells (antlerogenic periosteal cells APCs, for initial pedicle and first antler formation; pedicle periosteal cells PPC, for annual antler regeneration; and reserve mesenchyme cells RMCs, for rapid antler growth), were isolated for comprehensive molecular characterization. A horn-growth-related gene, RXFP2, was found to be expressed only in AS cells lineages but not in the facial periosteal cells (FPCs, locates geographically in the vicinity of the APCs or PPCs), suggesting the RXFP2 might be a specific marker for the AS cell lineage in deer. Our results demonstrated that AS cells expressed classic MSC markers including surface markers CD73, CD90, CD105 and Stro-1. They also expressed some of the markers including Tert, Nestin, S100A4, nucleostemin and C-Myc, suggesting that they have some attributes of the ESCs. Microinjection of male APC into deer blastocysts resulted in one female foetus (110 days gestation) recovered with obvious pedicle primordia with both male and female genotype detected in the ovary. In conclusion, the AS cells should be defined as MSCs but with partial attributes of ESCs.

Nicole Le Douarin and the use of quail-chick chimeras to study the developmental fate of neural crest and hematopoietic cells.

The quail-chick chimera marking system, devised in 1969, gave a new impetus to the analysis of cell migrations and interactions in the developing nervous, immune and hematopoietic systems. The method is based on the observation that the constitutive heterochromatin in all embryonic and adult cells of the quail is condensed in one large mass in the centre of the nucleus and is associated with the nucleolus, making this organelle strongly stained with the Feulgen-Rossenbeck reaction. The association of cells or rudiments from two avian species, advocated as a means to identify cells that migrate during embryogenesis, was rapidly recognized in this context as a useful tool for the study of many developmental biology problems. This article summarizes the fundamental contribution of Nicole Le Douarin to the discovery and the application of this technique over the last 40 years.

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example.

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology. Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively. The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development). Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

A single-cell molecular map of mouse gastrulation and early organogenesis.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.