Dexamethasone priming enhances stemness and immunomodulatory property of tissue-specific human mesenchymal stem cells

Background Human Mesenchymal Stem Cells (hMSCs) represent a promising cell source for cell-based therapy in autoimmune diseases and other degenerative disorders due to their immunosuppressive, anti-inflammatory and regenerative potentials. Belonging to a glucocorticoid family, Dexamethasone (Dex) is a powerful anti-inflammatory compound that is widely used as therapy in autoimmune disease conditions or allogeneic transplantation. However, minimal immunomodulatory effect of hMSCs may limit their therapeutic uses. Moreover, the effect of glucocorticoids on the immunomodulatory molecules or other regenerative properties of tissue-specific hMSCs remains unknown. Method Herein, we evaluated the in vitro effect of Dex at various dose concentrations and time intervals, 1000 ng/ml, 2000 ng/ml, 3000 ng/ml and 24 h, 48 h respectively, on the basic characteristics and immunomodulatory properties of Bone marrow derived MSC (BM-MSCs), Adipose tissue derived MSCs (AD-MSCs), Dental Pulp derived MSC (DP-MSCs) and Umbilical cord derived MSCs (UC-MSCs). Results The present study indicated that the concentration of Dex did not ramify the cellular morphology nor showed cytotoxicity as well as conserved the basic characteristics of tissue specific hMSCs including cell proliferation and surface marker profiling. However, quite interestingly it was observed that the stemness markers (Oct-4, Sox-2, Nanog and Klf-4) showed a significant upregulation in DP-MSCs and AD-MSCs followed by UC-MSCs and BM-MSCs. Additionally, immunomodulatory molecules, Prostaglandin E-2 (PGE-2), Indoleamine- 2,3-dioxygenase (IDO) and Human Leukocyte Antigen-G (HLA-G) were seen to be upregulated in a dose-dependent manner. Moreover, there was a differential response of tissue specific hMSCs after pre-conditioning with Dex during mixed lymphocyte reaction, wherein UC-MSCs and DP-MSCs showed enhanced immunosuppression as compared to AD-MSCs and BM-MSCs, thereby proving to be a better candidate for therapeutic applications in immune-related diseases. Conclusion Dex preconditioning improved the hMSCs immunomodulatory property and may have reduced the challenge associated with minimal potency and strengthen their therapeutic efficacy. Graphical Abstract Preconditioning of tissue specific hMSCs with dexamethasone biomanufacturers the enhanced potential hMSCs with better stemness and immunomodulatory properties for future therapeutics.

Comparative transcriptome analysis uncovers cell wall reorganization and repressed cell division during cotton fiber initiation

Background Tetraploid cotton plants serve as prime natural fiber source for the textile industry. Although various omics studies have revealed molecular basis for fiber development, a better understanding of transcriptional regulation mechanism regulating lint fiber initiation is necessary to meet global natural fiber demand. Results Here, we aimed to perform transcriptome sequencing to identify DEGs (differentially expressed genes) in ovules of the cotton variety Xu142 and its fibreless mutant Xu142fl during early lint fiber initiation period. Totally, 5516 DEGs including 1840 upregulated and 3676 downregulated were identified. GO enrichment analysis revealed that the downregulated DEGs were mainly associated with biological processes such as transcription related biosynthesis and metabolism, organic cyclic compound biosynthesis and metabolism, photosynthesis, and plant cell wall organization, with molecular functions involving transcription related binding, organic cyclic compound binding, and dioxygenase activity, while the upregulated DEGs were associated with DNA replication and phospholipid biosynthetic related processes. Among the 490 DEGs annotated as transcription factor genes, 86.5% were downregulated in the mutant including the Malvaceae-specific MMLs, expression patterns of which were confirmed during the central period of lint fiber initiation. Investigation of the 16 genes enriched in the cell wall organization revealed that 15 were EXPA coding genes. Conclusions Overall, our data indicate that lint fiber initiation is a complicated process involving cooperation of multiple transcription factor families, which might ultimately lead to the reorganization of the cell wall and terminated cell division of the differentiating fiber initials.

Msx1 haploinsufficiency modifies the Pax9-deficient cardiovascular phenotype

Background Successful embryogenesis relies on the coordinated interaction between genes and tissues. The transcription factors Pax9 and Msx1 genetically interact during mouse craniofacial morphogenesis, and mice deficient for either gene display abnormal tooth and palate development. Pax9 is expressed specifically in the pharyngeal endoderm at mid-embryogenesis, and mice deficient for Pax9 on a C57Bl/6 genetic background also have cardiovascular defects affecting the outflow tract and aortic arch arteries giving double-outlet right ventricle, absent common carotid arteries and interruption of the aortic arch. Results In this study we have investigated both the effect of a different genetic background and Msx1 haploinsufficiency on the presentation of the Pax9-deficient cardiovascular phenotype. Compared to mice on a C57Bl/6 background, congenic CD1-Pax9–/– mice displayed a significantly reduced incidence of outflow tract defects but aortic arch defects were unchanged. Pax9–/– mice with Msx1 haploinsufficiency, however, have a reduced incidence of interrupted aortic arch, but more cases with cervical origins of the right subclavian artery and aortic arch, than seen in Pax9–/– mice. This alteration in arch artery defects was accompanied by a rescue in third pharyngeal arch neural crest cell migration and smooth muscle cell coverage of the third pharyngeal arch arteries. Although this change in phenotype could theoretically be compatible with post-natal survival, using tissue-specific inactivation of Pax9 to maintain correct palate development whilst inducing the cardiovascular defects was unable to prevent postnatal death in the mutant mice. Hyoid bone and thyroid cartilage formation were abnormal in Pax9–/– mice. Conclusions Msx1 haploinsufficiency mitigates the arch artery defects in Pax9–/– mice, potentially by maintaining the survival of the 3rd arch artery through unimpaired migration of neural crest cells to the third pharyngeal arches. With the neural crest cell derived hyoid bone and thyroid cartilage also being defective in Pax9–/– mice, we speculate that the pharyngeal endoderm is a key signalling centre that impacts on neural crest cell behaviour highlighting the ability of cells in different tissues to act synergistically or antagonistically during embryo development.

Identification of reference genes for gene expression studies among different developmental stages of murine hearts

Background Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely-used standard assay for assessing gene expression. RT-qPCR data requires reference genes for normalization to make the results comparable. Therefore, the selected reference gene should be highly stable in its expression throughout the experimental datasets. So far, reports about the optimal set of reference genes in murine left ventricle (LV) across embryonic and postnatal stages are few. The objective of our research was to identify the appropriate reference genes in murine LV among different developmental stages. Methods We investigated the gene expression profiles of 21 widely used housekeeping genes in murine LV from 7 different developmental stages (almost throughout the whole period of the mouse lifespan). The stabilities of the potential reference genes were evaluated by five methods: GeNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder. Results We proposed a set of reliable reference genes for normalization of RT-qPCR experimental data in different conditions. Furthermore, our results showed that 6 genes (18S, Hmbs, Ubc, Psmb4, Tfrc and Actb) are not recommended to be used as reference genes in murine LV development studies. The data also suggested that the Rplp0 gene might serve as an optimal reference gene in gene expression analysis. Conclusions Our study investigated the expression stability of the commonly used reference genes in process of LV development and maturation. We proposed a set of optimal reference genes that are suitable for accurate normalization of RT-qPCR data in specific conditions. Our findings may be helpful in future studies for investigating the gene expression patterns and mechanism of mammalian heart development.

The miR-200 family in normal mammary gland development

The miR-200 family of microRNAs plays a significant role in inhibiting mammary tumor growth and progression, and its members are being investigated as therapeutic targets. Additionally, if future studies can prove that miR-200s prevent mammary tumor initiation, the microRNA family could also offer a preventative strategy. Before utilizing miR-200s in a therapeutic setting, understanding how they regulate normal mammary development is necessary. No studies investigating the role of miR-200s in embryonic ductal development could be found, and only two studies examined the impact of miR-200s on pubertal ductal morphogenesis. These studies showed that miR-200s are expressed at low levels in virgin mammary glands, and elevated expression of miR-200s have the potential to impair ductal morphogenesis. In contrast to virgin mammary glands, miR-200s are expressed at high levels in mammary glands during late pregnancy and lactation. miR-200s are also found in the milk of several mammalian species, including humans. However, the relevance of miR-200s in milk remains unclear. The increase in miR-200 expression in late pregnancy and lactation suggests a role for miR-200s in the development of alveoli and/or regulating milk production. Therefore, studies investigating the consequence of miR-200 overexpression or knockdown are needed to identify the function of miR-200s in alveolar development and lactation.

Development of the indirect flight muscles of Aedes aegypti, a main arbovirus vector

Background Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available. Methods A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy. Results At L3–L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal–ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure. Conclusions A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages.

Wdpcp regulates cellular proliferation and differentiation in the developing limb via hedgehog signaling

Background Mice with a loss of function mutation in Wdpcp were described previously to display severe birth defects in the developing heart, neural tube, and limb buds. Further characterization of the skeletal phenotype of Wdpcp null mice was limited by perinatal lethality. Results We utilized Prx1-Cre mice to generate limb bud mesenchyme specific deletion of Wdpcp. These mice recapitulated the appendicular skeletal phenotype of the Wdpcp null mice including polydactyl and limb bud signaling defects. Examination of late stages of limb development demonstrated decreased size of cartilage anlagen, delayed calcification, and abnormal growth plates. Utilizing in vitro assays, we demonstrated that loss of Wdpcp in skeletal progenitors lead to loss of hedgehog signaling responsiveness and associated proliferative response. In vitro chondrogenesis assays showed this loss of hedgehog and proliferative response was associated with decreased expression of early chondrogenic marker N-Cadherin. E14.5 forelimbs demonstrated delayed ossification and expression of osteoblast markers Runx2 and Sp7. P0 growth plates demonstrated loss of hedgehog signaling markers and expansion of the hypertrophic zones of the growth plate. In vitro osteogenesis assays demonstrated decreased osteogenic differentiation of Wdpcp null mesenchymal progenitors in response to hedgehog stimulation. Conclusions These findings demonstrate how Wdpcp and associated regulation of the hedgehog signaling pathway plays an important role at multiple stages of skeletal development. Wdpcp is necessary for positive regulation of hedgehog signaling and associated proliferation is key to the initiation of chondrogenesis. At later stages, Wdpcp facilitates the robust hedgehog response necessary for chondrocyte hypertrophy and osteogenic differentiation.

Changes in the expression levels of elastic fibres in yak lungs at different growth stages

Background Yaks have a strong adaptability to the plateau environment, which can be attributed to the effective oxygen utilization rate of their lung tissue. Elastic fibre confers an important adaptive structure to the alveolar tissues in yaks. However, little research has been focused on the structural development of lung tissues and the expression levels of elastic fibres in yaks after birth. Therefore, this study aimed to investigate the morphological changes of elastic fibers and expression profiles of fibre-formation genes in yak lungs at different growth stages and the relationship between these changes and plateau adaptation. Results Histological staining was employed to observe the morphological changes in the lung tissue structure of yaks at four different ages: 1 day old, 30 days old, 180 days old and adult. There was no significant difference in the area of a single alveolus between the 1-day-old and 30-day-old groups (P-value > 0.05). However, the single alveolar area was gradually increased with an increase in age (P-value < 0.05). Elastic fibre staining revealed that the amount of elastic fibres in alveolar tissue was increased significantly from the ages of 30 days to 180 days (P-value < 0.05) and stabilized during the adult stage. Transcriptome analysis indicated that the highest levels of differentially expressed genes were found between 30 days of age and 180 days of age. KEGG analysis showed that PI3K-Akt signalling pathway and MAPK pathway, which are involved in fibre formation, accounted for the largest proportion of differentially expressed genes between 30 days of age and 180 days of age. The expression levels of 36 genes related to elastic fibre formation and collagen fibre formation were also analysed, and most of these genes were highly expressed in 30-day-old and 180-day-old yaks. Conclusions The content of elastic fibres in the alveolar tissue of yaks increases significantly after birth, but this change occurs only from 30 days of age to 180 days of age. Our study indicates that elastic fibres can improve the efficiency of oxygen utilization in yaks under harsh environmental conditions.

The migratory pathways of the cells that form the endocardium, dorsal aortae, and head vasculature in the mouse embryo

BackgroundVasculogenesis in amniotes is often viewed as two spatially and temporally distinct processes, occurring in the yolk sac and in the embryo. However, the spatial origins of the cells that form the primary intra-embryonic vasculature remain uncertain. In particular, do they obtain their haemato-endothelial cell fate in situ, or do they migrate from elsewhere? Recently developed imaging techniques, together with new Tal1 and existing Flk1 reporter mouse lines, have allowed us to investigate this question directly, by visualising cell trajectories live and in three dimensions.ResultsWe describe the pathways that cells follow to form the primary embryonic circulatory system in the mouse embryo. In particular, we show that Tal1-positive cells migrate from within the yolk sac, at its distal border, to contribute to the endocardium, dorsal aortae and head vasculature. Other Tal1 positive cells, similarly activated within the yolk sac, contribute to the yolk sac vasculature. Using single-cell transcriptomics and our imaging, we identify VEGF and Apela as potential chemo-attractants that may regulate the migration into the embryo. The dorsal aortae and head vasculature are known sites of secondary haematopoiesis; given the common origins that we observe, we investigate whether this is also the case for the endocardium. We discover cells budding from the wall of the endocardium with high Tal1 expression and diminished Flk1 expression, indicative of an endothelial to haematopoietic transition.ConclusionsIn contrast to the view that the yolk sac and embryonic circulatory systems form by two separate processes, our results indicate that Tal1-positive cells from the yolk sac contribute to both vascular systems. It may be that initial Tal1 activation in these cells is through a common mechanism.

Knockdown of hspg2 is associated with abnormal mandibular joint formation and neural crest cell dysfunction in zebrafish

Background Heparan sulfate proteoglycan 2 (HSPG2) encodes for perlecan, a large proteoglycan that plays an important role in cartilage formation, cell adhesion, and basement membrane stability. Mutations in HSPG2 have been associated with Schwartz-Jampel Syndrome (SJS) and Dyssegmental Dysplasia Silverman-Handmaker Type (DDSH), two disorders characterized by skeletal abnormalities. These data indicate a function for HSPG2 in cartilage development/maintenance. However, the mechanisms in which HSPG2 regulates cartilage development are not completely understood. Here, we explored the relationship between this gene and craniofacial development through morpholino-mediated knockdown of hspg2 using zebrafish. Results Knockdown of hspg2 resulted in abnormal development of the mandibular jaw joint at 5 days post fertilization (DPF). We surmised that defects in mandible development were a consequence of neural crest cell (NCC) dysfunction, as these multipotent progenitors produce the cartilage of the head. Early NCC development was normal in morphant animals as measured by distal-less homeobox 2a (dlx2a) and SRY-box transcription factor 10 (sox10) expression at 1 DPF. However, subsequent analysis at later stages of development (4 DPF) revealed a decrease in the number of Sox10 + and Collagen, type II, alpha 1a (Col2a1a)+ cells within the mandibular jaw joint region of morphants relative to random control injected embryos. Concurrently, morphants showed a decreased expression of nkx3.2, a marker of jaw joint formation, at 4 DPF. Conclusions Collectively, these data suggest a complex role for hspg2 in jaw joint formation and late stage NCC differentiation.