Recently, the research on stemness and multilineage differentiation mechanisms has greatly increased its value due to the potential therapeutic impact of stem cell-based approaches. Stem cells modulate their self-renewing and differentiation capacities in response to endogenous and/or extrinsic factors that can control stem cell fate. One key factor controlling stem cell phenotype is oxygen (O₂). Several pieces of evidence demonstrated that the complexity of reproducing O₂ physiological tensions and gradients in culture is responsible for defective stem cell behavior in vitro and after transplantation. This evidence is still worsened by considering that stem cells are conventionally incubated under non-physiological air O₂ tension (21%). Therefore, the study of mechanisms and signaling activated at lower O₂ tension, such as those existing under native microenvironments (referred to as hypoxia), represent an effective strategy to define if O₂ is essential in preserving naïve stemness potential as well as in modulating their differentiation. Starting from this premise, the goal of the present review is to report the status of the art about the link existing between hypoxia and stemness providing insight into the factors/molecules involved, to design targeted strategies that, recapitulating naïve O₂ signals, enable towards the therapeutic use of stem cell for tissue engineering and regenerative medicine.

Glioblastoma multiforme (GBM) is a lethal disease characterized by an overall survival of about 1 year, making it one of the most aggressive tumours, with very limited therapeutic possibilities. Specific biomarkers for early diagnosis as well as innovative therapeutic strategies are urgently needed to improve the management of this deadly disease. In this work, we demonstrated that vesicular galectin‐3‐binding protein (LGALS3BP), a glycosylated protein overexpressed in a variety of human malignancies, is a potential GBM disease marker and can be efficiently targeted by a specific antibody–drug conjugate (ADC). Immunohistochemical analysis on patient tissues showed that LGALS3BP is highly expressed in GBM and, compared with healthy donors, the amount of vesicular but not total circulating protein is increased. Moreover, analysis of plasma‐derived extracellular vesicles from mice harbouring human GBM revealed that LGALS3BP can be used for liquid biopsy as a marker of disease. Finally, an ADC targeting LGALS3BP, named 1959‐sss/DM4, specifically accumulates in tumour tissue, producing a potent and dose‐dependent antitumor activity. In conclusion, our work provides evidence that vesicular LGALS3BP is a potential novel GBM diagnostic biomarker and therapeutic target deserving further preclinical and clinical validation.

KDEL receptors (KDELRs) are ubiquitous seven-transmembrane domain proteins encoded by three mammalian genes. They bind to and retro-transport endoplasmic reticulum (ER)-resident proteins with a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence or variants thereof. In doing this, KDELR participates in the ER quality control of newly synthesized proteins and the unfolded protein response. The binding of KDEL proteins to KDELR initiates signaling cascades involving three alpha subunits of heterotrimeric G proteins, Src family kinases, protein kinases A (PKAs), and mitogen-activated protein kinases (MAPKs). These signaling pathways coordinate membrane trafficking flows between secretory compartments and control the degradation of the extracellular matrix (ECM), an important step in cancer progression. Considering the basic cellular functions performed by KDELRs, their association with various diseases is not surprising. KDELR mutants unable to bind the collagen-specific chaperon heat-shock protein 47 (HSP47) cause the osteogenesis imperfecta. Moreover, the overexpression of KDELRs appears to be linked to neurodegenerative diseases that share pathological ER-stress and activation of the unfolded protein response (UPR). Even immune function requires a functional KDELR1, as its mutants reduce the number of T lymphocytes and impair antiviral immunity. Several studies have also brought to light the exploitation of the shuttle activity of KDELR during the intoxication and maturation/exit of viral particles. Based on the above, KDELRs can be considered potential targets for the development of novel therapeutic strategies for a variety of diseases involving proteostasis disruption, cancer progression, and infectious disease. However, no drugs targeting KDELR functions are available to date; rather, KDELR has been leveraged to deliver drugs efficiently into cells or improve antigen presentation.

Marinesco–Sjogren syndrome (MSS) is a rare multisystem pediatric disorder, caused by loss-of-function mutations in the gene encoding the endoplasmic reticulum cochaperone SIL1. SIL1 acts as a nucleotide exchange factor for BiP, which plays a central role in secretory protein folding. SIL1 mutant cells have reduced BiP-assisted protein folding, cannot fulfil their protein needs, and experience chronic activation of the unfolded protein response (UPR). Maladaptive UPR may explain the cerebellar and skeletal muscle degeneration responsible for the ataxia and muscle weakness typical of MSS. However, the cause of other more variable, clinical manifestations, such as mild to severe mental retardation, hypogonadism, short stature, and skeletal deformities, is less clear. To gain insights into the pathogenic mechanisms and/or adaptive responses to SIL1 loss, we carried out cell biological and proteomic investigations in skin fibroblasts derived from a young patient carrying the SIL1 R111X mutation. Despite fibroblasts not being overtly affected in MSS, we found morphological and biochemical changes indicative of UPR activation and altered cell metabolism. All the cell machineries involved in RNA splicing and translation were strongly downregulated, while protein degradation via lysosome-based structures was boosted, consistent with an attempt of the cell to reduce the workload of the endoplasmic reticulum and dispose of misfolded proteins. Cell metabolism was extensively affected as we observed a reduction in lipid synthesis, an increase in beta oxidation, and an enhancement of the tricarboxylic acid cycle, with upregulation of eight of its enzymes. Finally, the catabolic pathways of various amino acids, including valine, leucine, isoleucine, tryptophan, lysine, aspartate, and phenylalanine, were enhanced, while the biosynthetic pathways of arginine, serine, glycine, and cysteine were reduced. These results indicate that, in addition to UPR activation and increased protein degradation, MSS fibroblasts have profound metabolic alterations, which may help them cope with the absence of SIL1.

Bisphenol A (BPA) is an endocrine disruptor that negatively affects spermatogenesis, a process where Sertoli cells play a central role. Thus, in the present study we sought to ascertain whether BPA could modulate the endocannabinoid (eCB) system in exposed mouse primary Sertoli cells. Under our experimental conditions, BPA turned out to be cytotoxic to Sertoli cells with an half-maximal inhibitory concentration (IC₅₀) of ~6.0 µM. Exposure to a non-cytotoxic dose of BPA (i.e., 0.5 μM for 48 h) increased the expression levels of specific components of the eCB system, namely: type-1 cannabinoid (CB₁) receptor and diacylglycerol lipase-α (DAGL-α), at mRNA level, type-2 cannabinoid (CB₂) receptor, transient receptor potential vanilloid 1 (TRPV1) receptors, and DAGL-β, at protein level. Interestingly, BPA also increased the production of inhibin B, but not that of transferrin, and blockade of either CB₂ receptor or TRPV1 receptor further enhanced the BPA effect. Altogether, our study provides unprecedented evidence that BPA deranges the eCB system of Sertoli cells towards CB₂- and TRPV1-dependent signal transduction, both receptors being engaged in modulating BPA effects on inhibin B production. These findings add CB₂ and TRPV1 receptors, and hence the eCB signaling, to the other molecular targets of BPA already known in mammalian cells.

Tendon tissue engineering aims to develop effective implantable scaffolds, with ideally the native tissue’s characteristics, able to drive tissue regeneration. This research focused on fabricating tendon-like PLGA 3D biomimetic scaffolds with highly aligned fibers and verifying their influence on the biological potential of amniotic epithelial stem cells (AECs), in terms of tenodifferentiation and immunomodulation, with respect to fleeces. The produced 3D scaffolds better resemble native tendon tissue, both macroscopically, microscopically, and biomechanically. From a biological point of view, these constructs were able to instruct AECs genotypically and phenotypically. In fact, cells engineered on 3D scaffolds acquired an elongated tenocyte-like morphology; this was different from control AECs, which retained their polygonal morphology. The boosted AECs tenodifferentiation by 3D scaffolds was confirmed by the upregulation of tendon-related genes (SCX, COL1 and TNMD) and TNMD protein expression. The produced constructs also prompted AECs’ immunomodulatory potential, both at the gene and paracrine level. This enhanced immunomodulatory profile was confirmed by a greater stimulatory effect on THP-1-activated macrophages. These biological effects have been related to the mechanotransducer YAP activation evidenced by its nuclear translocation. Overall, these results support the biomimicry of PLGA 3D scaffolds, revealing that not only fiber alignment but also scaffold topology provide an in vitro favorable tenodifferentiative and immunomodulatory microenvironment for AECs that could potentially stimulate tendon regeneration.