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BackgroundThree-dimensional (3D) cultivation with biomaterials was proposed to facilitate stem cell epithelial differentiation for wound healing. However, whether human adipose-derived stem cells (hASCs) on collagen sponge scaffold (CSS) better differentiate to keratinocytes remains unclear.Methods3D cultivation with CSS on hASC epidermal differentiation co-cultured with HaCaT cells at air-liquid interface (ALI) was compared with two-dimensional (2D) form and cultivation without “co-culture” or “ALI.” Cellular morphology, cell adhesion, and growth condition were evaluated, followed by the protein and gene expression of keratin 14 (K14, keratinocyte specific marker).ResultsTypical cobblestone morphology of keratinocytes was remarkably observed in co-cultured hASCs at ALI, but those seeded on the CSS exhibited more keratinocyte-like cells under an invert microscope and scanning electron microscope. Desired cell adhesion and proliferation were confirmed in 3D differentiation groups by rhodamine-labeled phalloidin staining, consistent with H&E staining. Compared with those cultured in 2D culture system or without “ALI,” immunofluorescence staining and gene expression analysis revealed hASCs co-cultured over CSS expressed K14 at higher levels at day 15.ConclusionsCSS is positive to promote epithelial differentiation of hASCs, which will foster a deeper understanding of artificial dermis in skin wound healing and regeneration.

A comparative transcriptome analysis of Poncirus trifoliata and four herbaceous model plants identifies a core set of arbuscular mycorrhiza-induced genes that are likely to play key roles in symbiosis., The perennial woody plants of citrus are one of the most important fruit crops in the world and largely depends on arbuscular mycorrhizal symbiosis (AMS) to obtain essential nutrients from soil. However, the molecular aspects of AMS in citrus and perennial woody plants in general have largely been understudied. We used RNA-sequencing to identify differentially expressed genes in roots of Poncirus trifoliata upon mycorrhization by the AM fungus Glomus versiforme and evaluated their conservation by comparative transcriptome analyses with four herbaceous model plants. We identified 282 differentially expressed genes in P. trifoliata, including orthologs of 21 genes with characterized roles in AMS and 83 genes that are considered to be conserved in AM-host plants. Comparative transcriptome analysis revealed a ‘core set’ of 156 genes from P. trifoliata whose orthologous genes from at least three of the five species also exhibited similar transcriptional changes during AMS. Functional analysis of one of these conserved AM-induced genes, a 3-keto-acyl-ACP reductase (FatG) involved in fatty acid biosynthesis, confirmed its involvement in AMS in Medicago truncatula. Our results identify a core transcriptional program for AMS that is largely conserved between P. trifoliata and other plants. The comparative transcriptomics approach adds to previous phylogenomics studies to identify conserved genes required for AMS.

Arbuscular mycorrhiza (AM) is a widespread endosymbiosis between terrestrial plants and AM fungi belonging to sub-phylum Glomeromycotina (Mycologia 108: 1028–1046, 2016). To date, many plant genes involved in establishing the AM symbiosis have been identified. Yet, the precise mechanisms governing the early signaling process are still not well understood. In this study, we employed the isotope tags for relative and absolute quantification (iTRAQ) and LC–MS/MS analysis to investigate the phosphoproteomic changes in the root cells of Poncirus trifoliata (L.) Raf. (a common citrus rootstock) during the establishment of the AM symbiosis. A total of 1920 unique phosphopeptides derived from 1016 phosphoproteins were identified, which collectively contained 2308 phosphorylation sites. Motif-X analysis of all the detected phosphopeptides showed that 25 phosphoserine motifs and 4 phosphothreonine motifs were overrepresented in phosphorylation processes. Among the phosphoserine motifs, [SDXE] and [PXSP] showed the highest fold increase upon colonization by Rhizophagus intraradices, suggesting that they may play potential roles in AM symbiosis. At 1 week post-inoculation of R. intraradices, the phosphorylation levels of 65 phosphopeptides were significantly changed (p value 1.35), with 39 upregulated and 26 downregulated, implying that at least some of the 65 phosphoproteins may be involved in the signal transduction during early events of the AM symbiosis. This study provides a comprehensive phosphoproteomic analysis in a citrus rootstock and the phosphoproteomic changes should shed light onto the signaling mechanisms involved in the establishment of AM symbiosis.

Plants form a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, which facilitates the acquisition of scarce minerals from the soil. In return, the host plants provide sugars and lipids to its fungal partner. However, the mechanism by which the AM fungi obtain sugars from the plant has remained elusive. In this study we investigated the role of potential SWEET family sugar exporters in AM symbiosis in Medicago truncatula. We show that M. truncatula SWEET1b transporter is strongly upregulated in arbuscule-containing cells compared to roots and localizes to the peri-arbuscular membrane, across which nutrient exchange takes place. Heterologous expression of MtSWEET1b in a yeast hexose transport mutant showed that it mainly transports glucose. Overexpression of MtSWEET1b in M. truncatula roots promoted the growth of intraradical mycelium during AM symbiosis. Surprisingly, two independent Mtsweet1b mutants, which are predicted to produce truncated protein variants impaired in glucose transport, exhibited no significant defects in AM symbiosis. However, arbuscule-specific overexpression of MtSWEET1bY57A/G58D , which are considered to act in a dominant-negative manner, resulted in enhanced collapse of arbuscules. Taken together, our results reveal a (redundant) role for MtSWEET1b in the transport of glucose across the peri-arbuscular membrane to maintain arbuscules for a healthy mutually beneficial symbiosis.