Derivation of Germline Competent Rat Embryonic Stem Cells from DA Rats

The laboratory rat was one of the earliest mammalian species for scientific research and used as animal disease models in physiology, toxicology, behavior, immunology, and tumor-biology for over 150 years （Jacob, 1999）. However, rat lags far behind mouse in generating human disease models and functional genomic studies because of the lack of authentic rat embryonic stem （ES） cells （Voigt and Serikawa, 2009）,

Individual blastomeres of 4-and 8-cell embryos have ability to develop into a full organism in mouse

Following fertilization in mammals, the zygote initiates the developmental program, which has a transient capacity to generate cell types of both embryonic and extraembryonic lineages, which is defined as totipotency (Condic, 2014). In mice,only zygotes and blastomeres of 2-cell stage embryos are considered totipotent, since they have the ability to develop into a full

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.

Generation of Tripotent Neural Progenitor Cells from Rat Embryonic Stem Cells

Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem （rES） cell lines have been successfully established and the molecular networks maintaining the self-renewing, undifferentiated state of rES cells have also been well uncovered. However, little is known about the differentiation strategies and the underlying mechanisms of how these authentic rat pluripotent stem ceils give rise to specific cell types. The aim of this study is to investigate the neural differentiation capacity of rES cells. By means of a modified procedure based on previous publications - combination of mitogen-activated protein kinase （MAPK） and glycogen synthase kinase 3 （GSK3） inhibitors （two inhibitors, ＂2i＂） with feeder-conditioned medium, we successfully obtained high- quality rat embryoid bodies （rEBs） from rES cells and then differentiated them to tripotent neural progenitors. These rES cell-derived neural progenitor cells （rNPCs） were capable of self-renewing and giving rise to all three neural lineages, including astrocytes, oligo- dendrocytes, and neurons. Besides, these rES cell-derived neurons stained positive for y-aminobutyric acid （GABA） and tyrosine hydroxylase （TH）. In summary, we develop an experimental system for differentiating rES cells to tripotent neural progenitors, which may provide a powerful tool for pharmacological test and a valuable platform for studying the pathogenesis of many neurodegenerative disorders such as Parkinson＇s disease and the development of rat nervous system.

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,wereport thatmousehaESCs 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 resultsdemonstrate durable pluripotency inmousehaESCs andhaEpiSCs,aswell asthe valuable potential of using these haploid pluripotent stem cells in high-throughput genetic screening.

Overexpression of Stella improves the efficiency of nuclear transfer reprogramming

In mammalians, the state of a somatic cell can be reversed from the terminal state to the totipotent state by means of somatic cell nuclear transfer （SCNT） （Gurdon, 1962） or induced pluripotent stem cells （iPSCs） （Takahashi and Yamanaka, 2006）. The DNA methylation and transcriptome profiles of embryonic stern cells （ESCs） derived from SCNT embryos （NT-ESCs） correspond closely to those of ESCs derived from in vitro fertilization embryos （IVF- ESCs）. In contrast, iPSCs differ from both NT-ESCs and IVF-ESCs in that they retain the residual DNA methylation patterns of their parental somatic cells. As SCNT can be used to faithfully reprogram human somatic cells to pluripotency, it is ideal for cell replacement therapies （Ma et al., 2014）. Following the successful production of the first human NT-ESCs （Tachibana et al., 2013） and the later gen- eration of human NT-ESCs based on cells from elderly adults or pa- tient cells （Chung et al., 2014; Yamada et al., 2014）, a version of the SCNT technique for human therapeutics comes closer to reality. However, no matter what animal species or donor cell types are used in the cloned process, the cloning efficiency remains undesir- able. Besides, there are many phenotypic abnormalities in cloned animals, containing frequent embryonic and perinatal death and placentomegaly, and the underlying mechanisms remain unclear （Yang et al, 2007）.

Generation of rat-mouse chimeras by introducing single cells of rat inner cell masses into mouse blastocysts

In the field of developmental biology and regenerative medicine,mammalian interspecific chimeras have been proved very useful for investigating early embryonic development and the immune system establishment,and extended to a promising potential for human organ generation（Rossant et al.,1982）.