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Malignant mesothelioma (MM) is a very aggressive neoplasia with a short life expectancy and limited therapeutic options. Thus, the identification of novel molecular targets is a matter of great urgency. Kelch-like (KLHL) proteins play an important role in a number of physiological and pathological cell-regulatory processes. Among this family, the function of KLHL14 is still very poorly characterized. KLHL14 was originally identified as a gene involved in regulating the epithelial-mesenchymal transition (EMT) process. Here, we demonstrate that KLHL14 not only prevents EMT but also plays an anti-oncogenic role in MM. Indeed, KLHL14 depletion enhanced proliferation, motility, invasion and colony formation in MM cells. Importantly, we also demonstrated that KLHL14 mechanism of action is dependent on Transforming Growth Factor β (TGF-β). In fact, TGF-β promotes de novo synthesis, increases protein stability and induces nuclear-cytoplasmic shuttling of KLHL14. Collectively, this research is an important step further to decipher KLHLs mechanism of action and further contributes to the understanding of the molecular mechanisms regulating MM.

Regenerative medicine harnesses stem cells’ capacity to restore damaged tissues and organs. In vitro methods employing specific bioactive molecules, such as growth factors, bio-inductive scaffolds, 3D cultures, co-cultures, and mechanical stimuli, steer stem cells toward the desired differentiation pathways, mimicking their natural development. Chondrogenesis presents a challenge for regenerative medicine. This intricate process involves precise modulation of chondro-related transcription factors and pathways, critical for generating cartilage. Cartilage damage disrupts this process, impeding proper tissue healing due to its unique mechanical and anatomical characteristics. Consequently, the resultant tissue often forms fibrocartilage, which lacks adequate mechanical properties, posing a significant hurdle for effective regeneration. This review comprehensively explores studies showcasing the potential of amniotic mesenchymal stem cells (AMSCs) and amniotic epithelial cells (AECs) in chondrogenic differentiation. These cells exhibit innate characteristics that position them as promising candidates for regenerative medicine. Their capacity to differentiate toward chondrocytes offers a pathway for developing effective regenerative protocols. Understanding and leveraging the innate properties of AMSCs and AECs hold promise in addressing the challenges associated with cartilage repair, potentially offering superior outcomes in tissue regeneration.

The application of biomaterials on immune regenerative strategies to deal with unsolved pathologies is getting attention in the field of tissue engineering. In this context, graphene oxide (GO) has been proposed as an immune-mimetic material largely used for developing stem cell-based regenerative therapies, since it has shown to influence stem cell behavior and modulate their immune response. Similarly, amniotic epithelial stem cells (AECs) are getting an increasing clinical interest as source of stem cells due to their great plasticity and immunomodulatory paracrine activities, even though GO bio-mimetic effects still remain unknown. To this aim, GO-functionalized glass coverslips have been used for AECs culture. The results demonstrated how GO-coating is able to induce and accelerate the Epithelial-Mesenchymal Transition (EMT), in a process mediated by the intracellular activation of TGFβ1-SMAD2/3 signaling pathway. The trans-differentiation towards mesenchymal phenotype provides AECs of migratory ability and substantially changes the pattern of cytokines secretion upon inflammatory stimulus. Indeed, GO-exposed AECs enhance their pro-inflammatory interleukins production thus inducing a more efficient activation of macrophages and, at the same time, by slightly reducing their inhibitory action on peripheral blood mononuclear cells proliferation.Therefore, the adhesion of AECs on GO-functionalized surfaces might contribute to the generation of a tailored microenvironment useful to face both the phases of the inflammation, thereby fostering the regenerative process.

Summary: Epithelial-mesenchymal transition (EMT) changes cell phenotype by affecting immune properties of amniotic epithelial cells (AECs). The present study shows how the response to lipopolysaccharide of cells collected pre- (eAECs) and post-EMT (mAECs) induces changes in their transcriptomics profile. In fact, eAECs mainly upregulate genes involved in antigen-presenting response, whereas mAECs over-express soluble inflammatory mediator transcripts. Consistently, network analysis identifies CIITA and Nrf2 as main drivers of eAECs and mAECs immune response, respectively. As a consequence, the depletion of CIITA and Nrf2 impairs the ability of eAECs and mAECs to inhibit lymphocyte proliferation or macrophage-dependent IL-6 release, thus confirming their involvement in regulating immune response. Deciphering the mechanisms controlling the immune function of AECs pre- and post-EMT represents a step forward in understanding key physiological events wherein these cells are involved (pregnancy and labor). Moreover, controlling the immunomodulatory properties of eAECs and mAECs may be essential in developing potential strategies for regenerative medicine applications.

Epithelial-mesenchymal plasticity (EMP) refers to the ability of cells to dynamically interconvert between epithelial (E) and mesenchymal (M) phenotypes, thus generating an array of hybrid E/M intermediates with mixed E and M features. Recent findings have demonstrated how these hybrid E/M rather than fully M cells play key roles in most of physiological and pathological processes involving EMT. To this regard, the onset of hybrid E/M state coincides with the highest stemness gene expression and is involved in differentiation of either normal and cancer stem cells. Moreover, hybrid E/M cells are responsible for wound healing and create a favorable immunosuppressive environment for tissue regeneration. Nevertheless, hybrid state is responsible of metastatic process and of the increasing of survival, apoptosis and therapy resistance in cancer cells. The present review aims to describe the main features and the emerging concepts regulating EMP and the formation of E/M hybrid intermediates by describing differences and similarities between cancer and normal hybrid stem cells. In particular, the comprehension of hybrid E/M cells biology will surely advance our understanding of their features and how they could be exploited to improve tissue regeneration and repair.

Tendinopathies are at the frontier of advanced responses to health challenges and sectoral policy targets. Cell-based therapy holds great promise for tendon disorder resolution. To verify the role of stepwise trans-differentiation of amniotic epithelial stem cells (AECs) in tendon regeneration, in the present research three different AEC subsets displaying an epithelial (eAECs), mesenchymal (mAECs), and tendon-like (tdAECs) phenotype were allotransplanted in a validated experimental sheep Achilles tendon injury model. Tissue healing was analyzed adopting a comparative approach at two early healing endpoints (14 and 28 days). All three subsets of transplanted cells were able to accelerate regeneration: mAECs with a lesser extent than eAECs and tdAECs as indicated in the summary of the total histological scores (TSH), where at day 28 eAECs and tdAECs had better significant scores with respect to mAEC-treated tendons (p < 0.0001). In addition, the immunomodulatory response at day 14 showed in eAEC-transplanted tendons an upregulation of pro-regenerative M2 macrophages with respect to mAECs and tdAECs (p < 0.0001). In addition, in all allotransplanted tendons there was a favorable IL10/IL12 compared to CTR (p < 0.001). The eAECs and tdAECs displayed two different underlying regenerative mechanisms in the tendon. The eAECs positively influenced regeneration mainly through their greater ability to convey in the host tissue the shift from pro-inflammatory to pro-regenerative responses, leading to an ordered extracellular matrix (ECM) deposition and blood vessel remodeling. On the other hand, the transplantation of tdAECs acted mainly on the proliferative phase by impacting the density of ECM and by supporting a prompt recovery, inducing a low cellularity and angle alignment of the host cell compartment. These results support the idea that AECs lay the groundwork for production of different cell phenotypes that can orient tendon regeneration through a crosstalk with the host tissue. In particular, the obtained evidence suggests that eAECs are a practicable and efficient strategy for the treatment of acute tendinopathies, thus reinforcing the grounds to move their use towards clinical practice.

The usefulness of nanoparticles (NPs) in the diagnostic and/or therapeutic sector is derived from their aptitude for navigating intra- and extracellular barriers successfully and to be spatiotemporally targeted. In this context, the optimization of NP delivery platforms is technologically related to the exploitation of the mechanisms involved in the NP–cell interaction. This review provides a detailed overview of the available technologies focusing on cell–NP interaction/detection by describing their applications in the fields of cancer and regenerative medicine. Specifically, a literature survey has been performed to analyze the key nanocarrier-impacting elements, such as NP typology and functionalization, the ability to tune cell interaction mechanisms under in vitro and in vivo conditions by framing, and at the same time, the imaging devices supporting NP delivery assessment, and consideration of their specificity and sensitivity. Although the large amount of literature information on the designs and applications of cell membrane-coated NPs has reached the extent at which it could be considered a mature branch of nanomedicine ready to be translated to the clinic, the technology applied to the biomimetic functionalization strategy of the design of NPs for directing cell labelling and intracellular retention appears less advanced. These approaches, if properly scaled up, will present diverse biomedical applications and make a positive impact on human health.

Amniotic membrane (AM) is considered an important medical device with many applications in regenerative medicine. The therapeutic properties of AM are due to its resistant extracellular matrix and to the large number of bioactive molecules released by its cells. An important goal that still remains to be achieved is the identification of cultural and preservation protocols able to maintain in time the membrane morphology and the biological properties of its cells. Recently, our research group demonstrated that progesterone (P4) is crucial in preventing the loss of the epithelial phenotype of amniotic epithelial cells in vitro. Followed by this premise, it has been evaluated whether P4 may also affect AM properties in a short-term culture. Results confirm that P4 preserves AM integrity and architecture with respect to untreated AM, which showed alterations in morphology. Transmission electron microscopy (TEM) analyses demonstrate that P4 also maintains unaltered cell–cell junctions, nuclear status, and intracellular organelles. On the contrary, an untreated AM experienced an extensive cell death and a strong reduction of immunomodulatory properties, measured in terms of anti-inflammatory cytokine expression and secretion. Overall, these results could open to new strategies to ameliorate the protocols for cryopreservation and tissue culture, which represent preliminary stages of AM application in regenerative medicine.

Abstract The in vitro expansion is detrimental to therapeutic applications of amniotic epithelial cells (AEC), an emerging source of fetal stem cells. This study provides molecular evidences of progesterone (P4) role in preventing epithelial-mesenchymal transition (EMT) in ovine AEC (oAEC). oAEC amplified under standard conditions spontaneously acquired mesenchymal properties through the up-regulation of EMT-transcription factors. P4 supplementation prevented phenotype shift by inhibiting the EMT-inducing mechanism such as the autocrine production of TGF-β and the activation of intracellular-related signaling. The effect of P4 still persisted for one passage after steroid removal from culture as well as steroid supplementation promptly reversed mesenchymal phenotype in oAEC which have experienced EMT during amplification. Furthermore, P4 promoted an acute up-regulation of pluripotent genes whereas enhanced basal and LPS-induced oAEC anti-inflammatory response with an increase in anti-inflammatory and a decrease in pro-inflammatory cytokines expression. Altogether, these results indicate that P4 supplementation is crucial to preserve epithelial phenotype and to enhance biological properties in expanded oAEC. Therefore, an innovative cultural approach is proposed in order to improve therapeutic potential of this promising source of epithelial stem cells.

Amniotic epithelial cells (AECs), an emerging source of extrafoetal stem cells, have recently attracted attention for their great regenerative potential. Since AEC amplifications are accompanied by the loss of their native epithelial phenotype and by the progressive reduction of relevant biological properties, the issue to be addressed is the development of effective culture protocols. In this context, recently, it has been demonstrated that progesterone (P4) supplementation during ovine AEC (oAEC) expansion could prevent the undesirable epithelial-mesenchymal transition (EMT). In contrast, there is no information to date on the role of the other pregnancy steroids in culture. With this aim, the present study has been designed to clarify the impact of estradiol (E2), alone or in combination with P4 (12.5 μM and 25 μM), during oAEC amplification. Steroid supplementations were assessed by testing oAEC proliferation, stemness, EMT, and osteogenic or chondrogenic plasticity. The results indicated that EMT can be prevented exclusively in the presence of high doses of P4, while it occurred rapidly in cells exposed to E2 as denoted by protein (cytokeratin-8 and alpha-SMA) and gene expression (vimentin and snail) profiles. Moreover, steroid exposure was able to influence highly oAEC plasticity. Particularly, P4-treated cells displayed a precommitment towards osteogenic lineage, confirmed by the upregulation of OCN, RUNX2, and the greater deposition of calcium nodules. Conversely, P4 exposure inhibited oAEC chondrogenic differentiation, which was induced in E2-treated cells as confirmed by the upregulation of chondrogenesis-related genes (SOX9, ACAN, and COL2A1) and by the accumulation of Alcian blue-positive extracellular matrix. Simultaneously, E2-treated cells remained unresponsive to osteogenic inductive stimuli. In conclusion, media supplementation with high doses of steroids may be adopted to modulate phenotype and plasticity during oAEC amplification. Relevantly, the osteo or chondro steroid-induced precommitment may open unprecedented cell-based therapies to face the unsolved orthopaedic issues related to osteochondral regeneration.