Population Size, Genetic Diversity and Molecular Evidence of a Recent Population Bottleneck in Hynobius chinensis, an Endangered Salamander Species

Severe population declines can reduce species to small populations, offering permissive conditions for deleterious processes. For example, following such events, species can become prone to inbreeding and genetic drift which can lead to a loss of genetic diversity and evolutionary potentials. Hynobius chinensis is a poorly studied very rare and declining endangered amphibian species endemic to China in Changyang County. We investigated adult census population size by monitoring breeding populations from 2015 to 2018, developed microsatellite markers from the transcriptome and used them to investigate genetic diversity, and a population bottleneck in this species. We found H. chinensis in 4 different localities in a total area of 2.18 km2 and estimated the overall adult census population size at 386–404 individuals. The adult census size (mean ± SE) per breeding pond ranged from 44 ± 6 to 141 ± 8 individuals and appeared smaller than that reported in closely related species in undisturbed habitats. We developed and characterized 13 microsatellite markers in total. Analysis of data at 7 loci (N = 118) in Hardy-Weinberg equilibrium gathered from the largest population showed that genetic diversity level was low. The average number of alleles per locus was 2.14. The observed and expected heterozygosities averaged 0.38 and 0.40, respectively. The inbreeding coefficient was –0.06. All tests performed to investigate a population bottleneck, i.e. The Garza-Williamson test, Heterozygosity excess test, Mode shift test of allele frequency, and effective population size estimates detected a population bottleneck. The contemporary and the historical effective population sizes were estimated at 36 and 234 individuals, respectively. We argue that as bottleneck effects, the studied population may have become prone to genetic drift and inbreeding, losing microsatellite alleles and heterozygosity. Our results suggest that populations of H. chinensis may have been extirpated in the study area

Growth inhibition of mouse embryonic stem (ES) cells on the feeders from domestic animals

Mouse embryonic stem cells (mESCs) can be propagated in vitro on the feeders of mouse embryonic fibroblasts. In this study, we found growth inhibition of mESCs cultured on embryonic fibroblast feeders derived from different livestock animals. Under the same condition, mESCs derived from mouse embryonic fibroblast feeders were seen on the mass-like colonies and round or oval images, and more significant growth in the total number of colonies (p<0.05) and viable cells in the colonies (p<0.01) than that from goat embryonic fibroblast feeders, and viable cells in the colonies (p<0.05) than that from porcine embryonic fibroblast feeders. The feeders from bovine embryonic fibroblasts also reduced viable cells in the colonies, but were not significantly different in the total number of colonies and viable cells in the colonies with mouse embryonic fibroblast feeders. mESCs on the different embryonic fibroblast feeders were expressed as stem cell-specific markers Oct 4 and stage-specific embryonic antigen 1 (SSEA 1). Here, our results indicate that the feeders from goat, porcine and bovine embryonic fibroblasts inhibit the proliferation of mESCs.Key words: Domestic animals, feeders, mouse embryonic stem cells (mESCs), growth

Characterization of Bovine Induced Pluripotent Stem Cells by Lentiviral Transduction of Reprogramming Factor Fusion Proteins

Pluripotent stem cells from domesticated animals have potential applications in transgenic breeding. Here, we describe induced pluripotent stem (iPS) cells derived from bovine fetal fibroblasts by lentiviral transduction of Oct4, Sox2, Klf4 and c-Myc defined-factor fusion proteins. Bovine iPS cells showed typical colony morphology, normal karyotypes, stained positively for alkaline phosphatase (AP) and expressed Oct4, Nanog and SSEA1. The CpG in the promoter regions of Oct4 and Nanog were highly unmethylated in bovine iPS cells compared to the fibroblasts. The cells were able to differentiate into cell types of all three germ layers in vitro and in vivo. In addition, these cells were induced into female germ cells under defined culture conditions and expressed early and late female germ cell-specific genes Vasa, Dazl, Gdf9, Nobox, Zp2, and Zp3. Our data suggest that bovine iPS cells were generated from bovine fetal fibroblasts with defined-factor fusion proteins mediated by lentivirus and have potential applications in bovine transgenic breeding and gene-modified animals

Efficient Reprogramming of Naïve-Like Induced Pluripotent Stem Cells from Porcine Adipose-Derived Stem Cells with a Feeder-Independent and Serum-Free System

<div><p>Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed by ectopic expression of transcription factors or small molecule treatment, which resemble embryonic stem cells (ESCs). They hold great promise for improving the generation of genetically modified large animals. However, few porcine iPSCs (piPSCs) lines obtained currently can support development of cloned embryos. Here, we generated iPSCs from porcine adipose-derived stem cells (pADSCs), using drug-inducible expression of defined human factors (Oct4, Sox2, c-Myc and Klf4). Reprogramming of iPSCs from pADSCs was more efficient than from fibroblasts, regardless of using feeder-independent or feeder-dependent manners. By addition of Lif-2i medium containing mouse Lif, CHIR99021 and PD0325901 (Lif-2i), naïve-like piPSCs were obtained under feeder-independent and serum-free conditions. These successfully reprogrammed piPSCs were characterized by short cell cycle intervals, alkaline phosphatase (AP) staining, expression of Oct4, Sox2, Nanog, SSEA3 and SSEA4, and normal karyotypes. The resemblance of piPSCs to naïve ESCs was confirmed by their packed dome morphology, growth after single-cell dissociation, Lif-dependency, up-regulation of Stella and Eras, low expression levels of TRA-1-60, TRA-1-81 and MHC I and activation of both X chromosomes. Full reprogramming of naïve-like piPSCs was evaluated by the significant up-regulation of Lin28, Esrrb, Utf1 and Dppa5, differentiating into cell types of all three germ layers <i>in vitro</i> and <i>in vivo</i>. Furthermore, nuclear transfer embryos from naïve-like piPSCs could develop to blastocysts with improved quality. Thus, we provided an efficient protocol for generating naïve-like piPSCs from pADSCs in a feeder-independent and serum-free system with controlled regulation of exogenous genes, which may facilitate optimization of culture media and the production of transgenic pigs.</p></div

Characterization of naïve-like piPSCs produced by DOX-inducible system.

<p>(A) AP staining of naïve-like piPSCs, scale bar = 50 µm. (B) Pluripotency of naïve-like piPSCs was demonstrated by immunofluorescence staining of Sox2, Nanog, SSEA1, SSEA3, SSEA4, TRA-1-60 and TRA-1-81, scale bar = 100 µm. (C) Real-time PCR analysis of expression level of pluripotency genes in pADSCs, C4-6 NpiPSCs and C4-30 NpiPSCs. (D) DNA methylation analysis of the Nanog promoter in C4-6 NpiPSCs and C4-30 NpiPSCs. (E) Karyotype analysis of naïve-like piPSCs.</p

NpiPSCs for nuclear transfer.

<p>(A) Blastocyst rate of nuclear transfer embryos from NpiPSCs and pADSCs. Blastocyst rate I  =  No. blastocyst/No. cultured embryos (a); Blastocyst rate II  =  No. blastocyst/No. cleaved embryos (b). Different superscripts above the bars denote significant difference (P<0.05). (B) Total cell number of blastocyst from NpiPSCs and pADSCs. (C) Spindle-like morphology of cells 24h after the withdrawal of DOX. (D) Reverse transcription PCR analysis of expression of exogenous genes 6 days after the withdrawal of DOX.</p

Identification of pADSCs.

<p>(A) Morphology of pADSCs at passage 3, scale bar = 100 µm. (B) Multi-lineage differentiation of pADSCs, mature adipocytes were detected by Oil Red O staining (a), scale bar = 50 µm; osteogenesis was analysis by Alizarin Red S staining (c), scale bar = 200 µm. Cells cultured in the corresponding proliferation medium served as negative controls, respectively (b, scale bar = 50 µm; d, scale bar = 200 µm). (C) Expression of cell surface markers in pADSCs at passage 3 including CD29, CD44, CD90, CD105,CD31, CD45 and HLA-DR. Positive cells were gated based on staining with isotype antibody controls.</p

Characterization of fully reprogrammed porcine naïve-like iPSCs.

<p>(A) Real-time PCR analysis of expression levels of genes associated with fully reprogramming in pADSCs, C4-6 NpiPSCs and C4-30 NpiPSCs. (B) Reverse transcription PCR analysis of differentiation markers for the three germ layers in the EBs. (C) Hematoxylin and eosin staining of naïve-like piPSCs-derived teratoma (C4-30 NpiPSCs), the tumor was differentiated into the tissues of three germ layers, including cuticulated epithelium (a, ectoderm), adipose tissue (b, mesoderm) and gut-like epithelium (c, endoderm). Black arrows and square denote the specific structure of ectoderm mesoderm and endoderm, scale bar = 100 µm.</p

Evidences of naïve-like state of piPSCs.

<p>(A) Morphology of naïve-like piPSCs at phase contrast (a, scale bar = 50 µm) and immunofluorescence (GFP) imaging (b, scale bar = 50 µm). (B) AP staining of C4-6 NpiPSCs, when Lif was present (a, scale bar = 500 µm) and withdrawn (b, scale bar = 500 µm). (C) mRNA levels of MHC I in pADSCs, C4-6 NpiPSCs and C4-30 NpiPSCs. (D) mRNA levels of stella and Eras in pADSCs, C4-6 NpiPSCs and C4-30 NpiPSCs. (E) Immunofluorescence staining of H3K27me3 in pADSCs and naïve-like piPSCs (C4-30 NpiPSCs), nuclei surrounded by squares were magnified (right panels), arrows indicate H3K27me3-positive areas, scale bar = 50 µm.</p

Generation of piPSCs from pADSCs.

<p>(A) AP staining of colonies which were reprogrammed from pADSCs and pEFs in the presence of feeders and feeder-independent condition. The rate of AP positive colonies was compared between the groups, different superscripts above the bars denote significant difference (<i>P</i><0.05). (B) Schematic of the reprogramming strategy and the, morphology of cells at day 3, 6, 8 and 10. (C) Expression levels of genes associated with reprogramming, including Oct4, Sox2, c-Myc, Klf4, Lin28 and Nanog were evaluated in pADSCs and pEFs. (D) Expression levels of 5-mC and 5-hmC were analyzed in pADSCs and pEFs by immunofluorescence staining, *<i>P</i><0.05, **<i>P</i><0.01.</p