Ling Shuai

Androgenetic haploid embryonic stem cells produce live transgenic mice

Haploids and double haploids are important resources for studying recessive traits and have large impacts on crop breeding, but natural haploids are rare in animals. Mammalian haploids are restricted to germline cells and are occasionally found in tumours with massive chromosome loss. Recent success in establishing haploid embryonic stem (ES) cells in medaka fish and mice raised the possibility of using engineered mammalian haploid cells in genetic studies. However, the availability and functional characterization of mammalian haploid ES cells are still limited. Here we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into an enucleated oocyte. The ahES cells maintain haploidy and stable growth over 30 passages, express pluripotent markers, possess the ability to differentiate into all three germ layers in vitro and in vivo, and contribute to germlines of chimaeras when injected into blastocysts. Although epigenetically distinct from sperm cells, the ahES cells can produce viable and fertile progenies after intracytoplasmic injection into mature oocytes. The oocyte-injection procedure can also produce viable transgenic mice from genetically engineered ahES cells. Our findings show the developmental pluripotency of androgenentic haploids and provide a new tool to quickly produce genetic models for recessive traits. They may also shed new light on assisted reproduction.

Genetic modification and screening in rat using haploid embryonic stem cells.

The rat is an important animal model in biomedical research, but practical limitations to genetic manipulation have restricted the application of genetic analysis. Here we report the derivation of rat androgenetic haploid embryonic stem cells (RahESCs) as a tool to facilitate such studies. Our approach is based on removal of the maternal pronucleus from zygotes to generate androgenetic embryos followed by derivation of ESCs. The resulting RahESCs have 21 chromosomes, express pluripotency markers, differentiate into three germ layer cells, and contribute to the germline. Homozygous mutations can be introduced by both large-scale gene trapping and precise gene targeting via homologous recombination or the CRISPR-Cas system. RahESCs can also produce fertile rats after intracytoplasmic injection into oocytes and are therefore able to transmit genetic modifications to offspring. Overall, RahESCs represent a practical tool for functional genetic studies and production of transgenic lines in rat.

Generation and Application of Mouse-Rat Allodiploid Embryonic Stem Cells

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

Induced Pluripotent Stem–Induced Cells Show Better Constitutive Heterochromatin Remodeling and Developmental Potential After Nuclear Transfer Than Their Parental Cells

Recently, reprogramming of somatic cells from a differentiated to pluripotent state by overexpression of specific external transcription factors has been accomplished. It has been widely speculated that an undifferentiated state may make donor cells more efficient for nuclear transfer. To test this hypothesis, we derived induced pluripotent stem cells (iPS cells) from several somatic cell lines: mouse embryonic fibroblast (MEF), adult tail tip fibroblast (TTF), and brain neural stem cells (NSCs). Three dimensional (3D)-fluorescent in situ hybridization (FISH) and quantitative-FISH (Q-FISH) were then used to evaluate constitutive (pericentric and telomeric) heterochromatin organization in these iPS cells and in their parental differentiated cells. Here, we show that important nuclear remodeling and telomeres rejuvenation occur in these iPS cells regardless of their parental origin. When we used these cells as donors for nuclear transfer, we produced live-born cloned mice at much higher rates with the iPS-induced cells than with the parental cell lines. Interestingly, we noticed that developmental potential after nuclear transfer could be correlated with telomere length of the donor cells. Altogether, our findings suggest that constitutive heterochromatin organization from differentiated somatic cells can be reprogrammed to the pluripotent state by induction of iPS cells, which in turn support nuclear transfer procedure quite efficiently.

Efficient Production of Fluorescent Transgenic Rats using the piggyBac Transposon

Rats with fluorescent markers are of great value for studies that trace lineage-specific development, particularly those assessing the differentiation potential of embryonic stem cells (ESCs). The piggyBac (PB) transposon is widely used for the efficient introduction of genetic modifications into genomes, and has already been successfully used to produce transgenic mice and rats. Here, we generated transgenic rats carrying either the desRed fluorescent protein (RFP) gene or the enhanced green fluorescent protein (eGFP) gene by injecting pronuclei with PB plasmids. We showed that the transgenic rats expressed the RFP or eGFP gene in many organs and had the capability to transmit the marker gene to the next generation through germline integration. In addition, rat embryonic stem cells (ESCs) carrying an RFP reporter gene can be derived from the blastocysts of the transgenic rats. Moreover, the RFP gene can be detected in chimeras derived from RFP ESCs via blastocyst injection. This work suggests that PB-mediated transgenesis is a powerful tool to generate transgenic rats expressing fluorescent proteins with high efficiency, and this technique can be used to derive rat ESCs expressing a reporter protein.

Durable pluripotency and haploidy in epiblast stem cells derived from haploid embryonic stem cells in vitro

Haploid pluripotent stem cells, such as haploid embryonic stem cells (haESCs), facilitate the genetic study of recessive traits. In vitro, fish haESCs maintain haploidy in both undifferentiated and differentiated states, but whether mammalian haESCs can preserve pluripotency in the haploid state has not been tested. Here, we report that mouse haESCs can differentiate in vitro into haploid epiblast stem cells (haEpiSCs), which maintain an intact haploid genome, unlimited self-renewal potential, and durable pluripotency to differentiate into various tissues in vitro and in vivo. Mechanistically, the maintenance of self-renewal potential depends on the Activin/bFGF pathway. We further show that haEpiSCs can differentiate in vitro into haploid progenitor-like cells. When injected into the cytoplasm of an oocyte, androgenetic haEpiSC (ahaEpiSCs) can support embryonic development until midgestation (E12.5). Together, these results demonstrate durable pluripotency in mouse haESCs and haEpiSCs, as well as the valuable potential of using these haploid pluripotent stem cells in high-throughput genetic screening.

Haploid embryonic stem cells serve as a new tool for mammalian genetic study

In mammals, all somatic cells carry two sets of chromosomes while haploids are restricted only to gametes and are occasionally found in tumors with genome instability. Mammalian haploid embryonic stem (ES) cells have recently been established successfully in mice and monkeys, from either parthenogenetic or androgenetic haploid embryos. These haploid ES cells maintain haploidy and stable growth during extensive in vitro culture, express pluripotent markers, and possess the ability to differentiate into all three germ layers in vitro and in vivo. The mouse haploid ES cells can also contribute to germlines of chimeras. Moreover, the mouse androgenetic haploid ES cells can produce fertile progenies after intracytoplasmic injection into mature oocytes, and the mouse parthenogenetic haploid ES cells can also achieve this by substitution of the maternal genome, albeit at a lower efficiency. These distinct features of mammalian haploid ES cells empower themselves not only as a valuable tool for genetic screening at a cellular level, but also as a new tool for genome-modified animal production and genetic studies at the animal level. Here we review the current progress on mammalian haploid ES cell research, describe in detail their characteristics, and discuss their potential applications. These achievements may provide a new but powerful tool for mammalian genetic studies, and may also shed light on the some interesting questions regarding genome ploidy maintenance and genomic imprinting.

Three dimensional collagen scaffolds promote iPSC induction with higher pluripotency.

Extracellular environment plays a role in regulating stem cell fates and three dimensional (3D) scaffolds can be utilized to mimic the internal environment in vitro. Currently, many types of cells have been cultured in 3D conditions but only few studies have focused on reprogramming in a 3D environment. 3D culture systems provide circumstances that can better simulate native conditions which are comprised of distinctive cell morphology, oxygen levels, extracellular matrix secretion and concentration gradients of signaling factors (Keung et al., 2010; Gu et al., 2016). Herein, we used collagen, the major composition of the extracellular matrix (Di Lullo et al., 2002), that serves as scaffolds to offer porous 3D surrounding to mimic in vivo environments (Song et al., 2015) and to explore the role of 3D conditions in reprogramming. In this study, we investigated the effect of 3D collagen scaffolds on the reprogramming of mouse embryonic fibroblasts (MEFs) and pig embryonic fibroblasts (PEFs).MEFs could be successfully converted into mouse induced pluripotent stem cells (iPSCs) in 3D collagen scaffolds. After long time incubation, the results demonstrated that 3D conditions increased reprogramming efficiency with high levelsof pluripotency in comparisonwith the conventional 2Dmethod. Another reprogramming method, nuclear transfer (NT), was also detected with high improved efficiency when using the MEFs from 3D as nuclear donor. In addition, reprogramming inhibitors namely p21 andB-cell translocation gene 2 (Btg2), were suppressed during cultivation in 3D collagen scaffolds. Our first experiment is to investigate the effects of 3D collagen scaffolds for fibroblast cell viability. MEFswere respectively seeded on 2D cell plates and in 3D collagen scaffolds at the same time.After5daysculture, thecells in3Dcollagenscaffolds were characterized by SEM which showed different patterns from 2D (Fig. 1A). AlarmBlue cell viability assay showed that MEFscultured in3Dcollagenscaffoldshadabetter viability than those cultured in 2D (Fig. 1B). To further verify the role of cells culture in different conditions, MEFs were collected for qPCR analysis after 5 days culture. MEFs cultured in 3D collagen scaffolds showed lower expression of senescence markers, p21 and Btg2, compared to the MEFs cultured in 2D (Fig. 1C). The down-regulation of p21 and Btg2 might promote the metabolism of G1 phase cells and speed up the cell multiplication (Tirone, 2001). Moreover, p21 and Btg2 have been also known as two reprogramming inhibitors, the down-regulation of them might boost reprogramming (Bao et al., 2015). To study the effect of 3D collagen scaffolds on reprogramming, therefore, we used two approaches to reprogram MEFs into iPSCs (Fig. 1D). Firstly, MEFs were grown in 3D collagen scaffolds. Four days later, one part of MEFs was directly reprogrammed in 3D collagen scaffolds (3D), another part of MEFs was digested and seeded on 2D cell plates then reprogrammed (3D/2D). Consequently, the group of 3D and 3D/2D showed a higher efficiency and higher colony numbers compared to those in 2D conditions (Fig. 1E and 1F). When the iPSCs were re-seeded in the 3D scaffolds, the colony formed a grape-like cluster within the pores of 3D collagen scaffolds (Fig. 2A). The limited space on 2Dplates inhibited growth of the mouse iPSCs due to the cell-cell connections on the third day whereas the cells grewconsistently in 3Dscaffolds for at least 6 days (Fig. 2B). The mRNA expression results showed that Oct4, Zfp42, Gata4, Sox2 and Klf4 were significantly up-regulated except for c-Myc inmouse iPSCs in 3D collagen scaffolds than those cultured on 2D cell plates (Fig. 2C). These results indicated that 3D collagen scaffolds could enhance cell proliferation andstemnessofmouse iPSCs. Thehigher pluripotency demonstrates its future developmental ability (Jiang et al., 2011). Down-regulation of c-Myc will reduce risks of tumor formation in grafting experiments (Baudino et al., 2002). To further confirm the role of 3D collagen scaffolds in reprogramming, pig iPSCs, commonly difficult to silent their exogenous activation, were derived in 3D conditions from PEFs. PEFs were directly reprogrammed in 3D collagen scaffolds and typical colonies can be observed by SEM (Fig. S1A). Pig iPSCs were cultured in 3D conditions and longer lasting cell viability was observed in 3Dcompared to 2D conditions (Fig. S1B). The core problem of pig iPSCs is the persistent expression of transgenic genes (Petkov et al., 2015). Our result suggested that the expressions of exogenous,Oct4 and c-Myc, were down-regulated compared to 2D condition (Figure S1C). The endogenous genes of stemness, Oct4, Sox2, Rex1 and Nanog, were up-regulated when cells were reprogrammed in 3D collagen scaffolds (Figure S1D). The down-regulation of the exogenous genes and up-regulation of the endogenous genes (Fig. S1C andS1D)may bring

Generation of Mammalian offspring by haploid embryonic stem cells microinjection.

In this unit we introduce the derivation and genetic modification of mouse haploid embryonic stem (ES) cells. We detail how to produce haploid embryos and the subsequent ES derivation and cell culture. We further introduce readers to the intracytoplasmic injection processes of two types of haploid ES cells [androgenetic haploid ES (ahES) and parthenogenetic ES (phES)], both of which possess potential to produce fertile progenies by microinjection. This unit will be interesting to researchers who focus on recessive screens and transgenic animal model production with haploid stem cells.