Spatially ordered embryo-like structures self-assembled from blastocyst-derived stem cells can be generated to mimic embryogenesis in vitro. However, the assembly system and developmental potential of such structures needs to be further studied. Here, we devise a nonadherent-suspension-shaking system to generate self-assembled embryo-like structures (ETX-embryoids) using mouse embryonic, trophoblast and extra-embryonic endoderm stem cells. When cultured together, the three cell types aggregate and sort into lineage-specific compartments. Signaling among these compartments results in molecular and morphogenic events that closely mimic those observed in wild-type embryos. These ETX-embryoids exhibit lumenogenesis, asymmetric patterns of gene expression for markers of mesoderm and primordial germ cell precursors, and formation of anterior visceral endoderm-like tissues. After transplantation into the pseudopregnant mouse uterus, ETX-embryoids efficiently initiate implantation and trigger the formation of decidual tissues. The ability of the three cell types to self-assemble into an embryo-like structure in vitro provides a powerful model system for studying embryogenesis.

Cultured meat production has emerged as a breakthrough technology for the global food industry with the potential to reduce challenges associated with environmental sustainability, global public health, animal welfare, and competition for food between humans and animals. The muscle stem cell lines currently used for cultured meat cannot be passaged in vitro for extended periods of time. Here, we develop a directional differentiation system of porcine pre-gastrulation epiblast stem cells (pgEpiSCs) with stable cellular features and achieve serum-free myogenic differentiation of the pgEpiSCs. We show that the pgEpiSCs-derived skeletal muscle progenitor cells and skeletal muscle fibers have typical muscle cell characteristics and display skeletal muscle transcriptional features during myogenic differentiation. Importantly, we establish a three-dimensional differentiation system for shaping cultured tissue by screening plant-based edible scaffolds of non-animal origin, followed by the generation of pgEpiSCs-derived cultured meat. These advances provide a technical approach for the development of cultured meat.

MicroRNAs (miRNAs) play an important role in regulating almost all biological processes. miRNAs bind to the 3′ untranslated region (UTR) of mRNAs by sequence matching. In a previous study, we demonstrated that miR-21 was differently expressed in alpaca skin with different hair color. However, the molecular and cellular mechanisms for miR-21 to regulate the coat color are not yet completely understood. In this study, we transfected miR-21a-5p into mouse melanocytes and demonstrated its function on melanogenesis of miR-21a-5p by targeting Sox5, which inhibits melanogenesis in mouse melanocytes. The results suggested that miR-21a-5p targeted Sox5 gene based on the binding site in 3′ UTR of Sox5 and overexpression of miR-21a-5p significantly down-regulated Sox5 mRNA and protein expression. Meanwhile, mRNA and protein expression of microphthalmia transcription factor (MITF) and Tyrosinase (TYR) were up-regulated, which subsequently make the melanin production in melanocytes increased. The results suggest that miR-21a-5p regulates melanogenesis via MITF by targeting Sox5.

MicroRNAs (miRNAs) play an essential role in the regulation of almost all the biological processes, including melanogenesis. MiR-27a-3p is nearly six times higher in white alpaca skin compared to brown skin, which indicates that miR-27a-3p may be a candidate regulator for melanogenesis. Wnt3a plays an important role in promoting melanoblasts to differentiate into melanocytes and melanogenesis. To confirm the function of miR-27a-3p to melanogenesis in mammals, miR-27a-3p mimic, inhibitor and their negative control were transfected into mouse melanocytes. As a result, miR-27a-3p inhibits melanogenesis by repressing Wnt3a at post-transcriptional level. A significant decrease in Wnt3a luciferase activity was observed in 293T cells co-transfected with the matched luciferase reporter vector and pre-miR-27a. Furthermore, the presence of exogenous miR-27a-3p significantly decreased Wnt3a protein expression rather than mRNA and reduced β-catenin mRNA levels in melanocytes. The over-expression of miR-27a-3p significantly increased the melanin content of melanocytes. However, miR-27a-3p inhibitor performs an opposite effect on melanogenesis. Wnt3a is one target of miR-27a-3p. MiR-27a-3p could inhibit Wnt3a protein amount by post-transcriptional regulation and melanogenesis in mouse melanocytes. Previous studies reported that Wnt3a promoted melanogenensis in mouse melanocytes. Thus, miR-27-3p inhibits melanogenesis by repressing Wnt3a protein expression.

To investigate whether the membrane-associated transporter protein SLC45A2 is differentially expressed in the skin of sheep with different coat colors and to determine its correlation with coat color establishment in sheep. The expression of SLC45A2 in sheep skin samples with different coat colors was qualitatively and quantitatively analyzed by PCR amplification, RT-PCR, immunohistochemical staining and Western blotting. A 193-bp SLC45A2 CDS sequence was successfully amplified from sheep skin samples with diverse coat colors. RT-PCR analysis revealed that SLC45A2 mRNA was expressed in all sheep skin samples tested, with relative expression levels of 512.74 ± 121.51 in black skin, 143.38 ± 119.31 and 1.36 ± 0.09 in black dots and white dots of piebald skin, respectively, and 1.02 ± 0.23 in white skin (p < 0.01**). Positive SLC45A2 protein bands were also detected in all skin samples by Western blot analysis, with relative expression levels of 0.85 ± ± 0.17** in black skin, 0.60 ± 0.05** and 0.34 ± 0.07 in black dots and white dots of piebald skin, respectively, and 0.20 ± 0.05 in white skin (p < 0.01**). Immunohistochemical assays revealed that SLC45A2 was expressed in the hair follicle matrix, the inner and outer root sheath, and the dermal papilla in the skin tissues with different coat colors. These patterns were quantified by optical density (OD) analysis, which yielded relative expression levels of 0.23 ± 0.11 in black skin, 0.19 ± 0.09 and 0.10 ± 0.03 in black dots and white dots of piebald skin, respectively, and 0.08 ± 0.01 in white skin (p < 0.05*). SLC45A2 is detectably expressed in sheep skin of all coat colors, though at significantly different levels. SLC45A2 may participate in the establishment of coat color by regulating the synthesis and trafficking of melanin.