Later Stages Of Differentiation Research Articles

Development of Exhaustion and Acquisition of Regulatory Function by Infiltrating CD8+CD28- T Lymphocytes Dictate Clinical Outcome in Head and Neck Cancer.

Simple SummaryCD8+ T lymphocytes are among the immune cells reputed to kill tumor cells. Head and neck squamous cell carcinoma (HNSCC) has a poor clinical outcome despite the presence of a rich CD8+ T cell tumor infiltrate. This may be due to alterations of tumor infiltrating CD8+ T cells. Here, we performed a characterization of infiltrating CD8+ T cells in a cohort of 30 HNSCC patients. The results showed that differential intratumoral frequency of CD8+CD28+ T cells, CD8+CD28− T cells, and CD8+CD28−CD127−CD39+ Treg distinguished between HNSCC patients who did or did not respond to treatment. Moreover, we identified an intratumoral CD8+CD28- T cell subpopulation, which expressed markers of both exhausted (i.e., with impaired effector functions) and regulatory (i.e., exerting suppressive activities) cells. This suggests that in HNSCC effector T cells progressively undergo exhaustion and acquisition of regulatory properties, hampering their anti-tumor functions.Head and neck squamous cell carcinoma (HNSCC) has a poor clinical outcome despite the presence of a rich CD8+ T cell tumor infiltrate in the majority of patients. This may be due to alterations of tumor infiltrating CD8+ T cells. Here, we performed a characterization of HNSCC infiltrating CD8+ T cells in a cohort of 30 patients. The results showed that differential intratumoral frequency of CD8+CD28+ T cells, CD8+CD28− T cells, and CD8+CD28−CD127−CD39+ Treg distinguished between HNSCC patients who did or did not respond to treatment. Moreover, high PD1 expression identified a CD8+CD28− T cell subpopulation, phenotypically/functionally corresponding to CD8+CD28−CD127−CD39+ Treg, which showed a high expression of markers of exhaustion. This observation suggests that development of exhaustion and acquisition of regulatory properties may configure the late differentiation stage for intratumoral effector T cells, a phenomenon we define as effector-to-regulatory T cell transition.

Functional differences of Toll-like receptor 4 in osteogenesis, adipogenesis and chondrogenesis in human bone marrow-derived mesenchymal stem cells.

Multipotent human bone marrow‐derived mesenchymal stem cells (hMSCs) are promising candidates for bone and cartilage regeneration. Toll‐like receptor 4 (TLR4) is expressed by hMSCs and is a receptor for both exogenous and endogenous danger signals. TLRs have been shown to possess functional differences based on the species (human or mouse) they are isolated from therefore, the effects of knockdown of TLR4 were evaluated in humans during the differentiation of MSCs into bone, fat and chondrocyte cells in vitro. We investigated the expression profile of TLR4 during the differentiation of hMSCs into three different lineages on days 7, 14 and 21 and assessed the differentiation potential of the cells in the presence of lipopolysaccharide (LPS, as an exogenous agonist) and fibronectin fragment III‐1c (FnIII‐1c, as an endogenous agonist). TLR4 expression increased following the induction of hMSC differentiation into all three lineages. Alkaline phosphatase activity revealed that FnIII‐1c accelerated calcium deposition on day 7, whereas LPS increased calcium deposition on day 14. Chondrogenesis increased in the presence of LPS; however, FnIII‐1c acted as a reducer in the late stage. TLR4 silencing led to decreased osteogenesis and increased adipogenesis. Furthermore, Wnt5a expression was inversely related to chondrogenesis during the late stage of differentiation. We suggest that understanding the functionality of TLR4 (in the presence of pathogen or stress signal) during the differentiation of hMSCs into three lineages would be useful for MSC‐based treatments.

Magnesium isotopic fractionation during basalt differentiation as recorded by evolved magmas

Magnesium isotopic fractionation during basalt differentiation is commonly thought to be negligible at current levels of analytical precision. However, Mg isotopic variation of ∼0.4‰ (δ26Mg) can be observed in some highly evolved volcanic rocks with MgO contents of <5 wt.%, suggesting that detectable Mg isotopic fractionation may occur during late-stage basalt differentiation. Here we examine this possibility with a Mg isotopic study of a suite of well-characterized cogenetic alkaline volcanic rocks from St. Helena Island (South Atlantic), which vary from primitive nepheline-normative basalt to highly evolved trachyandesite with MgO contents decreasing from 15.72 wt.% to 0.81 wt.%. Our results show that the basalt samples which only experienced segregation of olivine and clinopyroxene have a narrow δ26Mg range (−0.23‰ to −0.32‰), while the evolved rocks saturated with Fe–Ti oxides (MgO < 5 wt.%) display larger Mg isotopic variation with δ26Mg vary to higher (−0.14‰) or lower (−0.36‰) values relative to mantle value (δ26Mg=−0.25±0.04‰). For most of the Fe–Ti oxide saturated samples, their δ26Mg values are positively correlated with TiO2 contents and Ti/Ti* ratios and negatively correlated with total-alkali contents. This indicates that detectable Mg isotopic fractionation occurred in MgO-poor samples, probably through fractional crystallization of Mg-bearing Fe–Ti oxides. Further Mg isotopic analysis of Fe–Ti oxide phenocrysts (titanomagnetite) in the evolved samples reveals that titanomagnetite has remarkably higher δ26Mg value (+0.15‰ to +0.52‰) than the corresponding bulk sample and silicate minerals. Fractional crystallization of such isotopically heavy titanomagnetite is thus expected to drive the residual magma progressively enriched in light Mg isotopes. In order to reproduce the observed Mg isotopic variation in St. Helena evolved samples, quantitative modeling requires a titanomagnetite-melt Mg isotopic fractionation factor (Δ26MgTi-Mgt−melt) of ∼0.6 ‰, which is among our measured δ26Mg difference value (0.41–0.73‰) between titanomagnetite and bulk samples. When combining our results with published data for alkaline lavas from the Antipodes volcano (New Zealand), contrasting patterns of Mg isotope fractionation emerge during late-stage basalt differentiation. Quantitative modeling shows that the positive and negative δ26Mg–MgO correlations shown by evolved lavas could be related to segregation of isotopically heavy titanomagnetite and isotopically light ilmenite, respectively, depending on redox state of the magma system. Therefore, this study highlights that basalt differentiation involving separation of Fe–Ti oxides may induce resolvable Mg isotopic fractionation, and the δ26Mg values of compositionally evolved rocks should be used with caution in studies of petrogenesis.

4-phenylbutyrate exerts stage-specific effects on cardiac differentiation via HDAC inhibition.

4-phenylbutyrate (4-PBA), a terminal aromatic substituted fatty acid, is used widely to specifically attenuate endoplasmic reticulum (ER) stress and inhibit histone deacetylases (HDACs). In this study, we investigated the effect of 4-PBA on cardiac differentiation of mouse embryonic stem (ES) cells. Herein, we found that 4-PBA regulated cardiac differentiation in a stage-specific manner just like trichostatin A (TSA), a well-known HDAC inhibitor. 4-PBA and TSA favored the early-stage differentiation, but inhibited the late-stage cardiac differentiation via acetylation. Mechanistic studies suggested that HDACs exhibited a temporal expression profiling during cardiomyogenesis. Hdac1 expression underwent a decrease at the early stage, while was upregulated at the late stage of cardiac induction. During the early stage of cardiac differentiation, acetylation favored the induction of Isl1 and Nkx2.5, two transcription factors of cardiac progenitors. During the late stage, histone acetylation induced by 4-PBA or TSA interrupted the gene silence of Oct4, a key determinant of self-renewal and pluripotency. Thereby, 4-PBA and TSA at the late stage hindered the exit from pluripotency, and attenuated the expression of cardiac-specific contractile proteins. Overexpression of HDAC1 and p300 exerted different effects at the distinct stages of cardiac induction. Collectively, our study shows that timely manipulation of HDACs exhibits distinct effects on cardiac differentiation. And the context-dependent effects of HDAC inhibitors depend on cell differentiation states marked by the temporal expression of pluripotency-associated genes.

Integrated transcriptomic and proteomic analysis reveals the complex molecular mechanisms underlying stone cell formation in Korla pear

Korla pear (Pyrus sinkiangensis Yü) is a landrace selected from a hybrid pear species in the Xinjiang Autonomous Region in China. In recent years, pericarp roughening has been one of the major factors that adversely affects fruit quality. Compared with regular fruits, rough-skin fruits have a greater stone cell content. Stone cells compose sclerenchyma tissue that is formed by secondary thickening of parenchyma cell walls. In this work, we determined the main components of stone cells by isolating them from the pulp of rough-skin fruits at the ripening stage. Stone cell staining and apoptosis detection were then performed on fruit samples that were collected at three different developmental stages (20, 50 and 80 days after flowering (DAF)) representing the prime, late and stationary stages of stone cell differentiation, respectively. The same batches of samples were used for parallel transcriptomic and proteomic analysis to identify candidate genes and proteins that are related to SCW biogenesis in Korla pear fruits. The results showed that stone cells are mainly composed of cellulose (52%), hemicellulose (23%), lignin (20%) and a small amount of polysaccharides (3%). The periods of stone cell differentiation and cell apoptosis were synchronous and primarily occurred from 0 to 50 DAF. The stone cell components increased abundantly at 20 DAF but then decreased gradually. A total of 24,268 differentially expressed genes (DEGs) and 1011 differentially accumulated proteins (DAPs) were identified from the transcriptomic and proteomic data, respectively. We screened the DEGs and DAPs that were enriched in SCW-related pathways, including those associated with lignin biosynthesis (94 DEGs and 31 DAPs), cellulose and xylan biosynthesis (46 DEGs and 18 DAPs), S-adenosylmethionine (SAM) metabolic processes (10 DEGs and 3 DAPs), apoplastic ROS production (16 DEGs and 2 DAPs), and cell death (14 DEGs and 6 DAPs). Among the identified DEGs and DAPs, 63 significantly changed at both the transcript and protein levels during the experimental periods. In addition, the majority of these identified genes and proteins were expressed the most at the prime stage of stone cell differentiation, but their levels gradually decreased at the later stages.

Post-Aire Medullary Thymic Epithelial Cells and Hassall's Corpuscles as Inducers of Tonic Pro-Inflammatory Microenvironment.

While there is convincing evidence on the role of Aire-positive medullary thymic epithelial cells (mTEC) in the induction of central tolerance, the nature and function of post-Aire mTECs and Hassall’s corpuscles have remained enigmatic. Here we summarize the existing data on these late stages of mTEC differentiation with special focus on their potential to contribute to central tolerance induction by triggering the unique pro-inflammatory microenvironment in the thymus. In order to complement the existing evidence that has been obtained from mouse models, we performed proteomic analysis on microdissected samples from human thymic medullary areas at different differentiation stages. The analysis confirms that at the post-Aire stages, the mTECs lose their nuclei but maintain machinery required for translation and exocytosis and also upregulate proteins specific to keratinocyte differentiation and cornification. In addition, at the late stages of differentiation, the human mTECs display a distinct pro-inflammatory signature, including upregulation of the potent endogenous TLR4 agonist S100A8/S100A9. Collectively, the study suggests a novel mechanism by which the post-Aire mTECs and Hassall’s corpuscles contribute to the thymic microenvironment with potential cues on the induction of central tolerance.

Lithium isotope behavior during magmatic differentiation and fluid exsolution in the Jiajika granite–pegmatite deposit, Sichuan, China

The Jiajika lithium (Li) deposit in Sichuan Province is the largest pegmatite-type Li deposit in China. The petrogenesis and metallogenesis of the Jiajika granitic pegmatites remain debatable. This study presents the Li isotopic compositions of two-mica granites, Li-rich and Li-poor pegmatites, and muscovites in Li-poor and Li-rich pegmatites to unravel the Li enrichment mechanism in the Jiajika deposit. The two-mica granites have a lower average δ7Li values than those of the Li-rich and Li-poor pegmatites. The Li contents of the two-mica granites are almost the same as those of the Li-poor pegmatites but are much lower than those of the Li-rich pegmatites. Muscovites in the Li-rich pegmatites have higher average Li contents and δ7Li values than those of the Li-poor pegmatites. All these results indicate the Jiajika granitic pegmatites were the final products of the extreme fractional crystallization of two-mica granitic magmas rather than direct anatectic melts, and that the Li-poor pegmatites derived from early differentiation of the two-mica granitic magmas before they evolved into Li-rich pegmatites during the late stage of magmatic differentiation. The lower average δ7Li values of the more evolved Li-rich pegmatites compared with the Li-poor pegmatites may have been caused by fluid exsolution and kinetic diffusive fractionation during melt-fluid separation. We discovered that fluid exsolution during melt-fluid separation can cause significant Li isotopic fractionation, with 7Li enriched in a H2O-poor silicate-rich melt system. Considering the crystallization ages of the two-mica granites and granitic pegmatites and other geochemical evidence (e.g., major- and trace-element compositions), the magmatic differentiation and fluid exsolution in the late stage of the granitic magma evolution together with the Li-rich surrounding strata led to the multistage enrichments in lithium, thus contributing to the formation of the Jiajika large Li deposit.

RGMa can induce skeletal muscle cell hyperplasia via association with neogenin signalling pathway.

Although originally discovered inducing important biological functions in the nervous system, repulsive guidance molecule a (RGMa) has now been identified as a player in many other processes and diseases, including in myogenesis. RGMa is known to be expressed in skeletal muscle cells, from somites to the adult. Functional in vitro studies have revealed that RGMa overexpression could promote skeletal muscle cell hypertrophy and hyperplasia, as higher efficiency in cell fusion was observed. Here, we extend the potential role of RGMa during C2C12 cell differentiation in vitro. Our results showed that RGMa administrated as a recombinant protein during late stages of C2C12 myogenic differentiation could induce myoblast cell fusion and the downregulation of different myogenic markers, while its administration at early stages induced the expression of myogenic markers with no detectable morphological effects. We also found that RGMa effects on skeletal muscle hyperplasia are performed via neogenin receptor, possibly as part of a complex with other proteins. Additionally, we observed that RGMa-neogenin is not playing a role as an inhibitor of the BMP signalling in skeletal muscle cells. This work contributes to placing RGMa as a component of the mechanisms that determine skeletal cell fusion via neogenin receptor.

Osteogenic differentiation system based on biopolymer nanoparticles for stem cells in simulated microgravity

An efficient long-term intracellular growth factor release system in simulated microgravity for osteogenic differentiation was prepared based on polylactic acid (PLA) and polyhydroxyalkanoate (PHA) nanoparticles (NPs) for loading of bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein 7 (BMP7) (defined as sB2-PLA-NPs and sB7-PHA-NPs), respectively, associated with osteogenic differentiation of human adipose derived stem cells (hADSCs). On account of soybean lecithin (SL) as biosurfactants, sB2-PLA-NPs and sB7-PHA-NPs had a high encapsulation efficiency (>80%) of BMPs and uniform small size (<100 nm), and showed a different slow-release to provide BMP2 in early stage and BMP7 in late stages of osteogenic differentiation within 20 d, due to degradation rate of PLA and PHA in cells. After uptake into hADSCs, by comparison with single sB2-PLA-NPs or sB7-PHA-NPs, the Mixture NPs compound of sB2-PLA-NP and sB7-PHA-NP with a mass ratio of 1:1, can well-promote ALP activity, expression of OPN and upregulated related osteo-genes. Directed osteo-differentiation of mixture NPs was similar to result of sustained free-BMP2 and BMP7-supplying (sFree-B2&B7) in simulated microgravity, which demonstrated the reliability and stability of Mixture NPs as a long-term osteogenic differentiation system in space medicine and biology in future.

Effects of targeting the transcription factors Ikaros and Aiolos on B cell activation and differentiation in systemic lupus erythematosus

ObjectiveTo evaluate the effects of targeting Ikaros and Aiolos by cereblon modulator iberdomide on the activation and differentiation of B-cells from patients with systemic lupus erythematosus (SLE).MethodsCD19+ B-cells isolated from...

Identification of characteristic proteins at late-stage erythroid differentiation in vitro.

The production of red blood cells in vitro, which is useful for basic or clinical research, has been improved. Further optimization of culture protocols may facilitate erythroid differentiation from hematopoietic stem cells to red blood cells. However, the details of erythropoiesis, particularly regarding the behaviors of differentiation-related proteins, remain unclear. Here, we performed erythroid differentiation using two independent bone marrow- or cord blood-derived CD34+ cell sources and identified proteins showing reproducible differential expression in all groups. Notably, most of the proteins expressed at the early stage were downregulated during erythroid differentiation. However, seven proteins showed upregulated expression in both bone marrow cells and cord blood cells. These proteins included alpha-synuclein and selenium-binding protein 1, the roles of which have not been clarified in erythropoiesis. There is a possibility that these factors contribute to erythroid differentiation as they maintained a high expression level. These findings provide a foundation for further mechanistic studies on erythropoiesis.

Investigating the Involvement of Cytoskeletal Proteins MreB and FtsZ in the Origin of Legume-Rhizobial Symbiosis

Rhizobia are rod-shaped bacteria that form nitrogen-fixing root nodules on leguminous plants, but they don't carry MreB, a key determinant of rod-like cell shape. Here, we introduced an actin-like mreB homologue from pseudomonad into Mesorhizobium huakuii 7653R (a microsymbiont of Astragalus sinicus L.) and examined the molecular, cellular and symbiotic phenotypes of the resultant mutant. Exogenous mreB caused an enlarged cell size and slower growth in laboratory medium. However, the mutant formed small ineffective nodules on A. sinicus (Nod+ Fix-), and rhizobial cells in the infection zone were unable to differentiate into bacteroids. RNA-seq analysis also revealed minor effects of mreB on global gene expression in free-living cells but larger effects for cells grown in planta. Differentially expressed nodule-specific genes include cell cycle regulators such as the tubulin-like ftsZ1 and ftsZ2. Unlike the ubiquitous FtsZ1, FtsZ2 was commonly found in Rhizobium, Sinorhizobium and Mesorhizobium, but not in closely related non-symbiotic species. Bacterial Two-Hybrid analysis revealed that MreB interacts with FtsZ1 and FtsZ2, which are targeted by the host-derived NCR peptides. Significantly, MreB mutation D283A disrupted the protein-protein interactions and restored the aforementioned phenotypic defects caused by MreB in M. huakuii. Together, our data indicate that MreB is detrimental for modern rhizobia and its interaction with FtsZ1/FtsZ2 cause the symbiotic process to cease at the late stage of bacteroid differentiation. These findings led to a hypothesis that loss of mreB in the common ancestor of Rhizobiales and subsequent acquisition of ftsZ2 are critical evolutionary steps leading to legume-rhizobial symbiosis.

Early B-cell Factor1 (Ebf1) promotes early osteoblast differentiation but suppresses osteoblast function.

Early B cell factor 1 (Ebf1) is a transcription factor that regulates B cell, neuronal cell and adipocyte differentiation. We and others have shown that Ebf1 is expressed in osteoblasts and that global deletion of Ebf1 results in increased bone formation in vivo. However, as Ebf1 is expressed in multiple tissues and cell types, it has remained unclear, which of the phenotypic changes in bone are derived from bone cells. The aim of this study was to determine the cell-autonomous and differentiation stage-specific roles of Ebf1 in osteoblasts. In vitro, haploinsufficient Ebf1+/- calvarial cells showed impaired osteoblastic differentiation indicated by lower alkaline phosphatase (ALP) activity and reduced mRNA expression of osteoblastic genes, while overexpression of Ebf1 in wild type mouse calvarial cells led to enhanced osteoblast differentiation with increased expression of Osterix (Osx). We identified a putative Ebf1 binding site in the Osterix promoter by ChIP assay in MC3T3-E1 osteoblasts and showed that Ebf1 was able to activate Osx-luc reporter construct that included this Ebf1 binding site, suggesting that Ebf1 indeed regulates osteoblast differentiation by inducing Osterix expression. To reconcile our previous data and that of others with our novel findings, we hypothesized that Ebf1 could have a dual role in osteoblast differentiation promoting early but inhibiting late stages of differentiation and osteoblast function. To test this hypothesis in vivo, we generated conditional Ebf1 knockout mice, in which Ebf1 deletion was targeted to early or late osteoblasts by crossing Ebf1fl/fl mice with Osx- or Osteocalcin (hOC)-Cre mouse lines, respectively. Deletion of Ebf1 in early Ebf1Osx-/- osteoblasts resulted in significantly increased bone volume and trabecular number at 12weeks by μCT analysis, while Ebf1hOC-/- mice did not have a bone phenotype. To conclude, our data demonstrate that Ebf1 promotes early osteoblast differentiation by regulating Osterix expression. However, Ebf1 inhibits bone accrual in the Osterix expressing osteoblasts in vivo but it is redundant in the maintenance of mature osteoblast function.

Gelatin Methacryloyl-Riboflavin (GelMA-RF) Hydrogels for Bone Regeneration.

Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been used in various biomedical fields. UV light is commonly used to photocrosslink such materials; however, its use has raised several biosafety concerns. We investigated the mechanical and biological properties of a visible-wavelength (VW)-light-crosslinked gelatin-based hydrogel to evaluate its viability as a scaffold for bone regeneration in bone-destructive disease treatment. Irgacure2959 or riboflavin was added as a photoinitiator to create GelMA solutions. GelMA solutions were poured into a mold and exposed to either UV or VW light. KUSA-A1 cell-laden GelMA hydrogels were crosslinked and then cultured. Mechanical characterization revealed that the stiffness range of GelMA–RF hydrogel was suitable for osteoblast differentiation. KUSA-A1 cells encapsulated in GelMA hydrogels photopolymerized with VW light displayed significantly higher cell viability than cells encapsulated in hydrogels photopolymerized with UV light. We also show that the expression of osteogenesis-related genes at a late stage of osteoblast differentiation in osteoblasts encapsulated in GelMA–RF hydrogel was markedly increased under osteoblast differentiation-inducing conditions. The GelMA–RF hydrogel served as an excellent scaffold for the encapsulation of osteoblasts. GelMA–RF hydrogel-encapsulated osteoblasts have the potential not only to help regenerate bone mass but also to treat complex bone defects associated with bone-destructive diseases such as periodontitis.

A proportion of CD4+ T cells from patients with chronic Chagas disease undergo a dysfunctional process, which is partially reversed by benznidazole treatment

Signs of senescence and the late stages of differentiation associated with the more severe forms of Chagas disease have been described in the Trypanosoma cruzi antigen-specific CD4+ T-cell population. However, the mechanisms involved in these functions are not fully known. To date, little is known about the possible impact of benznidazole treatment on the T. cruzi-specific functional response of CD4+ T cells. The functional capacity of CD4+ T cells was analyzed by cytometric assays in chronic Chagas disease patients, with indeterminate form (IND) and cardiac alterations (CCC) (25 and 15, respectively) before and after benznidazole treatment. An increase in the multifunctional capacity (expression of IFN-γ, IL-2, TNF-α, perforin and/or granzyme B) of the antigen-specific CD4+ T cells was observed in indeterminate versus cardiac patients, which was associated with the reduced coexpression of inhibitory receptors (2B4, CD160, CTLA-4, PD-1 and/or TIM-3). The functional profile of these cells shows statistically significant differences between IND and CCC (p<0.001), with a higher proportion of CD4+ T cells coexpressing 2 and 3 molecules in IND (54.4% versus 23.1% and 4.1% versus 2.4%, respectively). A significant decrease in the frequencies of CD4+ T cells that coexpress 2, 3 and 4 inhibitory receptors was observed in IND after 24-48 months of treatment (p<0.05, p<0.01 and p<0.05, respectively), which was associated with an increase in antigen-specific multifunctional activity. The IND group showed, at 9-12 months after treatment, an increase in the CD4+ T cell subset coproducing three molecules, which were mainly granzyme B+, perforin+ and IFN-γ+ (1.4% versus 4.5%). A CD4+ T cell dysfunctional process was detected in chronic Chagas disease patients, being more exacerbated in those patients with cardiac symptoms. After short-term benznidazole treatment (9-12 months), indeterminate patients showed a significant increase in the frequency of multifunctional antigen-specific CD4+ T cells.

Impact of Single-Walled Carbon Nanotubes on Neural Progenitors: From Endocytosis to Differentiation

Carbon nanotubes are often lauded for their utility as delivery agents for drug and gene therapeutics. Their size and tunable functionalization allows them to clear the blood brain barrier and gain access to the central nervous system (CNS). Our previous studies of the effect of (GT)20 DNA-wrapped single walled carbon nanotubes (SWNT) on C17.2 neural progenitor cells (NPCs) have shown that endocytosis of nanomolar concentrations of SWNTs triggers an increase in differentiation yield of NPCs. Biophysical prompts at the extracellular membrane will generate a mechanotransduction-based response in the intracellular space, setting off a signaling cascade and ending in the alteration of cellular fate processes. This biophysical stimulus of the cell can result in biochemical downstream alterations of cell division, proliferation and differentiation. While the later stages of differentiation (14-21 days) of NPCs have been studied, the early induction of differentiation as a result of nanomaterial entry into the cell is not widely understood. This study aims to identify markers associated with early differentiation of NPCs within the first 1-3 days post-nanomaterial internalization. Preceding work on receptor/clathrin-mediated endocytosis (RME) as the primary method of DNA-SWNT entry, lead to detailed pathway analysis and identification of doublecortin (DCX), Notch1/numb, clathrin/AP-2 complex and NeuroD1 to be among the proteins crucial to test for connecting the RME pathway to the onset of NPC differentiation. A mechanistic understanding of the path from endocytosis to neural differentiation, and how nanotubes alter stem cell decision-making machinery, can aid in designing natural differentiation augmentation as a potential therapeutic against neurodegenerative diseases.

IGFBPs mediate IGF-1's functions in retinal lamination and photoreceptor development during pluripotent stem cell differentiation to retinal organoids.

Development of the retina is regulated by growth factors, such as insulin-like growth factors 1 and 2 (IGF-1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF-1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF-1, by making it available or sequestering IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage-specific manner to mediate IGF-1 functions. Our data show that IGF-1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF-1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF-1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI-31772) and IGF-1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid-stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF-1's role in retinal lamination and photoreceptor development in a stage-specific manner.

MiR-101-loaded exosomes secreted by bone marrow mesenchymal stem cells requires the FBXW7/HIF1α/FOXP3 axis, facilitating osteogenic differentiation.

Exosomes derived from mesenchymal stem cells (MSCs) have emerged as significant mediators of intercellular communication, with studies highlighting their role in the transmission of biological signals between cells. Dominant microRNA (miRNA)-mediated translational repression of messenger RNAs has been extensively investigated in regard to its influence in orchestrating osteogenic differentiation. In the current study, we sought to ascertain the contributory role of miRNA-101 (miR-101) encapsulated in the process of bone marrow mesenchymal stem cell (BMSC)-derived exosomes in osteogenic differentiation. Exosomes were initially extracted from BMSCs at Days 0, 3, 12, and 21 of osteogenic differentiation by ultracentrifugation. Artificial modulation of miR-101 and FBXW7 (silencing and overexpression) were performed in the BMSCs to identify its effects on osteogenic factors, alkaline phosphatase activity, and osteogenic differentiation. Mechanistic exploration was performed to evaluate the binding affinity between miR-101 and FBXW7, the FBXW7-mediated HIF1α ubiquitination, and the HIF1α enrichment in the FOXP3 promoter region. Exosomes from MSCs in the late stage of osteogenic differentiation exhibited enhanced osteogenic differentiation. Upregulated miR-101 in MSC-derived exosomes was detected during osteogenic differentiation, while diminished levels of FBXW7 expression was noted. Importantly, miR-101 was found to specifically bind to the 3'-untranslated region of FBXW7. Meanwhile, data was obtained indicating that FBXW7 could ubiquitinate and degrade HIF1α to repress its upregulation during osteogenic differentiation. HIF1α bound to the promoter region of FOXP3 to facilitate osteogenic differentiation. Ultimately, the findings of the current study demonstrate that BMSC-derived exosomal miR-101 augments osteogenic differentiation in MSCs by inhibiting FBXW7 to regulate the HIF1α/FOXP3 axis.

Vaccination and Infection of Swine With Salmonella Typhimurium Induces a Systemic and Local Multifunctional CD4+ T-Cell Response.

The gram-negative facultative intracellular bacteria Salmonella Typhimurium (STM) often leads to subclinical infections in pigs, but can also cause severe enterocolitis in this species. Due to its high zoonotic potential, the pathogen is likewise dangerous for humans. Vaccination with a live attenuated STM strain (Salmoporc) is regarded as an effective method to control STM infections in affected pig herds. However, information on the cellular immune response of swine against STM is still scarce. In this study, we investigated the T-cell immune response in pigs that were vaccinated twice with Salmoporc followed by a challenge infection with a virulent STM strain. Blood- and organ-derived lymphocytes (spleen, tonsils, jejunal and ileocolic lymph nodes, jejunum, ileum) were stimulated in vitro with heat-inactivated STM. Subsequently, CD4+ T cells present in these cell preparations were analyzed for the production of IFN-γ, TNF-α, and IL-17A by flow cytometry and Boolean gating. Highest frequencies of STM-specific cytokine-producing CD4+ T cells were found in lamina propria lymphocytes of jejunum and ileum. Significant differences of the relative abundance of cytokine-producing phenotypes between control group and vaccinated + infected animals were detected in most organs, but dominated in gut and lymph node-residing CD4+ T cells. IL-17A producing CD4+ T cells dominated in gut and gut-draining lymph nodes, whereas IFN-γ/TNF-α co-producing CD4+ T cells were present in all locations. Additionally, the majority of cytokine-producing CD4+ T cells had a CD8α+CD27- phenotype, indicative of a late effector or effector memory stage of differentiation. In summary, we show that Salmonella-specific multifunctional CD4+ T cells exist in vaccinated and infected pigs, dominate in the gut and most likely contribute to protective immunity against STM in the pig.

Sprouty1 regulates gonadal white adipose tissue growth through a PDGFRα/β-Akt pathway

ABSTRACT Expansion of visceral white adipose tissue (vWAT) occurs in response to nutrient excess, and is a risk factor for metabolic disease. SPRY1, a feedback inhibitor of receptor tyrosine kinase (RTK) signaling, is expressed in PDGFRa+ adipocyte progenitor cells (APC) in vivo. Global deficiency of Spry1 in mice results in disproportionate postnatal growth of gonadal WAT (gWAT), while iWAT and BAT were similar in size between Spry1KO and WT mice. Spry1 deficiency increased the number of PDGFRa+ stromal vascular fraction (SVF) cells in gWAT and showed increased proliferation and fibrosis. Spry1KO gWAT had increased collagen deposition and elevated expression of markers of inflammation. In vitro, SPRY1 was transiently down regulated during early adipocyte differentiation of SVF cells, with levels increasing at later stages of differentiation. SPRY1 deficiency enhances PDGF-AA and PDGF-BB induced proliferation of SVF cells. Increased proliferation of SVF from Spry1KO gWAT accompanies an increase in AKT activation. PDGF-AA stimulated a transient down regulation of SPRY1 in wild type SVF, whereas PDGF-BB stimulated a sustained down regulation of SPRY1 in wild type SVF. Collectively, our data suggest that SPRY1 is critical for regulating postnatal growth of gWAT by restraining APC proliferation and differentiation in part by regulation of PDGFRa/b-AKT signaling.