IVM Advances for Early Antral Follicle-Enclosed Oocytes Coupling Reproductive Tissue Engineering to Inductive Influences of Human Chorionic Gonadotropin and Ovarian Surface Epithelium Coculture.

In vitro maturation (IVM) is not a routine assisted reproductive technology (ART) for oocytes collected from early antral (EA) follicles, a large source of potentially available gametes. Despite substantial improvements in IVM in the past decade, the outcomes remain low for EA-derived oocytes due to their reduced developmental competences. To optimize IVM for ovine EA-derived oocytes, a three-dimensional (3D) scaffold-mediated follicle-enclosed oocytes (FEO) system was compared with a validated cumulus-oocyte complex (COC) protocol. Gonadotropin stimulation (eCG and/or hCG) and/or somatic cell coculture (ovarian vs. extraovarian-cell source) were supplied to both systems. The maturation rate and parthenogenetic activation were significantly improved by combining hCG stimulation with ovarian surface epithelium (OSE) cells coculture exclusively on the FEO system. Based on the data, the paracrine factors released specifically from OSE enhanced the hCG-triggering of oocyte maturation mechanisms by acting through the mural compartment (positive effect on FEO and not on COC) by stimulating the EGFR signaling. Overall, the FEO system performed on a developed reproductive scaffold proved feasible and reliable in promoting a synergic cytoplasmatic and nuclear maturation, offering a novel cultural strategy to widen the availability of mature gametes for ART.

AEC and AFMSC Transplantation Preserves Fertility of Experimentally Induced Rat Varicocele by Expressing Differential Regenerative Mechanisms.

Amniotic membrane and amniotic fluid derived cells are regarded as a promising stem cell source for developing regenerative medicine techniques, although they have never been tested on male infertility diseases such as varicocele (VAR). The current study aimed to examine the effects of two distinct cell sources, human Amniotic Fluid Mesenchymal Stromal Cells (hAFMSCs) and amniotic epithelial cells (hAECs), on male fertility outcomes in a rat induced VAR model. To explain cell-dependent enhancement of reproductive outcomes in rats transplanted with hAECs and hAFMSCs, insights on testis morphology, endocannabinoid system (ECS) expression and inflammatory tissue response have been carried out alongside cell homing assessment. Both cell types survived 120 days post-transplantation by modulating the ECS main components, promoting proregenerative M2 macrophages (Mφ) recruitment and a favorable anti-inflammatory IL10 expression pattern. Of note, hAECs resulted to be more effective in restoring rat fertility rate by enhancing both structural and immunoresponse mechanisms. Moreover, immunofluorescence analysis revealed that hAECs contributed to CYP11A1 expression after transplantation, whereas hAFMSCs moved towards the expression of Sertoli cell marker, SOX9, confirming a different contribution into the mechanisms leading to testis homeostasis. These findings highlight, for the first time, a distinct role of amniotic membrane and amniotic fluid derived cells in male reproduction, thus proposing innovative targeted stem-based regenerative medicine protocols for remedying high-prevalence male infertility conditions such as VAR.

Profiling of mitochondrial heteroplasmy in single human oocytes by next-generation sequencing.

Mitochondrial DNA (mtDNA) plays a crucial role in the development of a competent oocyte. Indeed, mtDNA alterations may predispose to chromosome nondisjunction, resulting in infertility due to a reduced vitality and quality of oocytes and embryos. In this methods paper, the multiple displacement amplification approach was applied in combination with next-generation sequencing (NGS) to amplify and sequence, in single-end, the entire mtDNA of single human oocytes to directly construct genomic NGS libraries, and subsequently, to highlight and quantify the mutations they presented. The bioinformatic workflow was carried out with a specific ad hoc developed in-house software. This approach proved to be sensitive and specific, also highlighting the mutations present in heteroplasmy, showing deletion, insertion or substitution mutations in the genes involved in the respiratory chain, even if the found variants were benign or of uncertain meaning. The analysis of mtDNA mutations in the oocyte could provide a better understanding of specific genetic abnormalities and of their possible effect on oocyte developmental competence. This study shows how this approach, based on a massive parallel sequencing of clonally amplified DNA molecules, allows to sequence the entire mitochondrial genome of single oocytes in a short time and with a single analytical run and to verify mtDNA mutations.

Gonadotropin Cell Transduction Mechanisms.

The intention of this Special Edition was to collect review and original research articles that illustrate and stimulate the growing efforts to highlight the mechanisms of action of gonadotropins, as well as deepen our understanding of their biological roles in health and disease, aiming at revealing novel therapeutic opportunities in reproductive and regenerative medicine [...].

Equine Chorionic Gonadotropin as an Effective FSH Replacement for In Vitro Ovine Follicle and Oocyte Development.

The use of assisted reproductive technologies (ART) still requires strategies through which to maximize individual fertility chances. In vitro folliculogenesis (ivF) may represent a valid option to convey the large source of immature oocytes in ART. Several efforts have been made to set up ivF cultural protocols in medium-sized mammals, starting with the identification of the most suitable gonadotropic stimulus. In this study, Equine Chorionic Gonadotropin (eCG) is proposed as an alternative to Follicle Stimulating Hormone (FSH) based on its long superovulation use, trans-species validation, long half-life, and low costs. The use of 3D ivF on single-ovine preantral (PA) follicles allowed us to compare the hormonal effects and to validate their influence under two different cultural conditions. The use of eCG helped to stimulate the in vitro growth of ovine PA follicles by maximizing its influence under FBS-free medium. Higher performance of follicular growth, antrum formation, steroidogenic activity and gap junction marker expression were recorded. In addition, eCG, promoted a positive effect on the germinal compartment, leading to a higher incidence of meiotic competent oocytes. These findings should help to widen the use of eCG to ivF as a valid and largely available hormonal support enabling a synchronized in vitro follicle and oocyte development.

Effects of P4 Antagonist RU486 on VEGF and Its Receptors' Signaling during the In Vivo Transition from the Preovulatory to Periovulatory Phase of Ovarian Follicles.

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P4) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P4 receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P4 antagonist RU486 inhibits follicular VEGF receptors' signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles' microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P4 in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase.

Clinically relevant radioresistant rhabdomyosarcoma cell lines: functional, molecular and immune-related characterization.

BACKGROUND: The probability of local tumor control after radiotherapy (RT) remains still miserably poor in pediatric rhabdomyosarcoma (RMS). Thus, understanding the molecular mechanisms responsible of tumor relapse is essential to identify personalized RT-based strategies. Contrary to what has been done so far, a correct characterization of cellular radioresistance should be performed comparing radioresistant and radiosensitive cells with the same isogenic background. METHODS: Clinically relevant radioresistant (RR) embryonal (RD) and alveolar (RH30) RMS cell lines have been developed by irradiating them with clinical-like hypo-fractionated schedule. RMS-RR cells were compared to parental isogenic counterpart (RMS-PR) and studied following the radiobiological concept of the "6Rs", which stand for repair, redistribution, repopulation, reoxygenation, intrinsic radioresistance and radio-immuno-biology. RESULTS: RMS-RR cell lines, characterized by a more aggressive and in vitro pro-metastatic phenotype, showed a higher ability to i) detoxify from reactive oxygen species; ii) repair DNA damage by differently activating non-homologous end joining and homologous recombination pathways; iii) counteract RT-induced G2/M cell cycle arrest by re-starting growth and repopulating after irradiation; iv) express cancer stem-like profile. Bioinformatic analyses, performed to assess the role of 41 cytokines after RT exposure and their network interactions, suggested TGF-ß, MIF, CCL2, CXCL5, CXCL8 and CXCL12 as master regulators of cancer immune escape in RMS tumors. CONCLUSIONS: These results suggest that RMS could sustain intrinsic and acquire radioresistance by different mechanisms and indicate potential targets for future combined radiosensitizing strategies.

In Vitro Innovation of Tendon Tissue Engineering Strategies.

Tendinopathy is the term used to refer to tendon disorders. Spontaneous adult tendon healing results in scar tissue formation and fibrosis with suboptimal biomechanical properties, often resulting in poor and painful mobility. The biomechanical properties of the tissue are negatively affected. Adult tendons have a limited natural healing capacity, and often respond poorly to current treatments that frequently are focused on exercise, drug delivery, and surgical procedures. Therefore, it is of great importance to identify key molecular and cellular processes involved in the progression of tendinopathies to develop effective therapeutic strategies and drive the tissue toward regeneration. To treat tendon diseases and support tendon regeneration, cell-based therapy as well as tissue engineering approaches are considered options, though none can yet be considered conclusive in their reproduction of a safe and successful long-term solution for full microarchitecture and biomechanical tissue recovery. In vitro differentiation techniques are not yet fully validated. This review aims to compare different available tendon in vitro differentiation strategies to clarify the state of art regarding the differentiation process.

Fabrication and Plasma Surface Activation of Aligned Electrospun PLGA Fiber Fleeces with Improved Adhesion and Infiltration of Amniotic Epithelial Stem Cells Maintaining their Teno-inductive Potential.

Electrospun PLGA microfibers with adequate intrinsic physical features (fiber alignment and diameter) have been shown to boost teno-differentiation and may represent a promising solution for tendon tissue engineering. However, the hydrophobic properties of PLGA may be adjusted through specific treatments to improve cell biodisponibility. In this study, electrospun PLGA with highly aligned microfibers were cold atmospheric plasma (CAP)-treated by varying the treatment exposure time (30, 60, and 90 s) and the working distance (1.3 and 1.7 cm) and characterized by their physicochemical, mechanical and bioactive properties on ovine amniotic epithelial cells (oAECs). CAP improved the hydrophilic properties of the treated materials due to the incorporation of new oxygen polar functionalities on the microfibers' surface especially when increasing treatment exposure time and lowering working distance. The mechanical properties, though, were affected by the treatment exposure time where the optimum performance was obtained after 60 s. Furthermore, CAP treatment did not alter oAECs' biocompatibility and improved cell adhesion and infiltration onto the microfibers especially those treated from a distance of 1.3 cm. Moreover, teno-inductive potential of highly aligned PLGA electrospun microfibers was maintained. Indeed, cells cultured onto the untreated and CAP treated microfibers differentiated towards the tenogenic lineage expressing tenomodulin, a mature tendon marker, in their cytoplasm. In conclusion, CAP treatment on PLGA microfibers conducted at 1.3 cm working distance represent the optimum conditions to activate PLGA surface by improving their hydrophilicity and cell bio-responsiveness. Since for tendon tissue engineering purposes, both high cell adhesion and mechanical parameters are crucial, PLGA treated for 60 s at 1.3 cm was identified as the optimal construct.

Wharton's jelly mesenchymal stromal cells have contrasting effects on proliferation and phenotype of cancer stem cells from different subtypes of lung cancer.

Studies on the role of multipotent mesenchymal stromal cells (MSC) on tumor growth have reported both a tumor promoting and a suppressive effect. The aim of the present study was to determine the effect of MSC isolated from Wharton's jelly of umbilical cord (WJMSC) on lung cancer stem cells (LCSC) derived from human lung tumors: two adenocarcinomas (AC) and two squamous cell carcinomas (SCC). LCSC derived from SCC and AC expressed, to varying extents, the more relevant stem cell markers. The effect of WJMSC on LCSC was investigated in vitro using conditioned medium (WJ-CM): a proliferation increase in AC-LCSC was observed, with an increase in the ALDH+ and in the CD133+ cell population. By contrast, WJ-CM hampered the growth of SCC-LCSC, with an increase in the pre-G1 phase indicating the induction of apoptosis. Furthermore, the ALDH+ and CD133+ population was also reduced. In vivo, subcutaneous co-transplantation of AC-LCSC/WJMSC generated larger tumors than AC-LCSC alone, characterized by an increased percentage of CD133+ and CD166+ cells. By contrast, co-transplantation of WJMSC and SCC-LCSC did not affect the tumor size. Our results strongly suggest that WJMSC exert, both in vitro and in vivo, contrasting effects on LCSC derived from different lung tumor subtypes.

Cellular and molecular maturation in fetal and adult ovine calcaneal tendons.

Processes of development during fetal life profoundly transform tendons from a plastic tissue into a highly differentiated structure, characterised by a very low ability to regenerate after injury in adulthood. Sheep tendon is frequently used as a translational model to investigate cell-based regenerative approaches. However, in contrast to other species, analytical and comparative baseline studies on the normal developmental maturation of sheep tendons from fetal through to adult life are not currently available. Thus, a detailed morphological and biochemical study was designed to characterise tissue maturation during mid- (2 months of pregnancy: 14 cm of length) and late fetal (4 months: 40 cm of length) life, through to adulthood. The results confirm that ovine tendon morphology undergoes profound transformations during this period. Endotenon was more developed in fetal tendons than in adult tissues, and its cell phenotype changed through tendon maturation. Indeed, groups of large rounded cells laying on smaller and more compacted ones expressing osteocalcin, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were identified exclusively in fetal mid-stage tissues, and not in late fetal or adult tendons. VEGF, NGF as well as blood vessels and nerve fibers showed decreased expression during tendon development. Moreover, the endotenon of mid- and late fetuses contained identifiable cells that expressed several pluripotent stem cell markers [Telomerase Reverse Transcriptase (TERT), SRY Determining Region Y Box-2 (SOX2), Nanog Homeobox (NANOG) and Octamer Binding Transcription Factor-4A (OCT-4A)]. These cells were not identifiable in adult specimens. Ovine tendon development was also accompanied by morphological modifications to cell nuclei, and a progressive decrease in cellularity, proliferation index and expression of connexins 43 and 32. Tendon maturation was similarly characterised by modulation of several other gene expression profiles, including Collagen type I, Collagen type III, Scleraxis B, Tenomodulin, Trombospondin 4 and Osteocalcin. These gene profiles underwent a dramatic reduction in adult tissues. Transforming growth factor-ß~1 expression (involved in collagen synthesis) underwent a similar decrease. In conclusion, these morphological studies carried out on sheep tendons at different stages of development and aging offer normal structural and molecular baseline data to allow accurate evaluation of data from subsequent interventional studies investigating tendon healing and regeneration in ovine experimental models.

Stem-Cell-Driven Chondrogenesis: Perspectives on Amnion-Derived Cells.

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.

Assessing the functional potential of conditioned media derived from amniotic epithelial stem cells engineered on 3D biomimetic scaffolds: An in vitro model for tendon regeneration.

Tendon diseases pose a significant challenge in regenerative medicine due to the limited healing capacity of this tissue. Successful tendon regeneration requires a combination of angiogenesis, immune response, and tenogenesis processes. An effective tendon engineering (TE) strategy must finely tune this systems' interplay toward homeostasis. This study explores in vitro the paracrine influence of amniotic epithelial stem cells (AECs) engineered on a validated 3D electrospun PLGA scaffolds on HUVECs (angiogenesis), PBMCs/Jurkat (immune response), and AECs (tenogenic stem cell activation). The results revealed the role of scaffold's topology and topography in significantly modulating the paracrine profile of the cells. In detail, AECs basal release of bioactive molecules was boosted in the cells engineered on 3D scaffolds, in particular VEGF-D, b-FGF, RANTES, and PDGF-BB (p < 0.0001 vs. CMCTR). Moreover, biological tests demonstrated 3D scaffolds' proactive role in potentiating AECs' paracrine inhibition on PBMCs proliferation (CM3Dvs. CTR, p < 0.001) and LPS-mediated Jurkat activation with respect to controls (CM3D and CM2Dvs. CTR, p < 0.01 and p < 0.05, respectively), without exerting any in vitro pro-angiogenic role in promoting HUVECs proliferation and tubule formation. Teno-inductive paracrine ability of AECs engineered on 3D scaffolds was assessed on co-cultured ones, which formed tendon-like structures. These latter demonstrated an upregulation of tendon-related genes (SCX, THBS4, COL1, and TNMD) and the expression TNMD and COL1 proteins. Overall, this research underscores the pivotal role of the 3D topology and topography of PLGA tendon mimetic scaffolds in orchestrating effective tendon regeneration through modulating cell behavior and crosstalk between engineered stem cells and different subpopulations in the damaged tendon.

Prolonged in vitro expansion partially affects phenotypic features and osteogenic potential of ovine amniotic fluid-derived mesenchymal stromal cells.

BACKGROUND AIMS: Ovine amniotic fluid mesenchymal stromal cells (oAFMSCs) are an emerging alternative source of stem cells to develop pre-clinical cell replacement protocols. For tissue engineering purposes, oAFMSCs can be used either immediately after isolation or after in vitro expansion. However, detailed studies are still required to investigate the advantages and drawbacks of their in vitro expansion. METHODS: The phenotype and osteogenic differentiation potential of oAFMSCs were analyzed in relation to in vitro expansion that was carried out for 20 consecutive passages. Expanded oAFMSCs were analyzed for proliferation index, expression profiles of several surface, pluripotency-associated and HLA antigens, global DNA methylation, telomere length and karyotype. The osteogenic differentiation ability of expanded oAFMSCs was assessed by qualitative and quantitative methods. RESULTS: Expanded oAFMSCs reduced their proliferative activity after 10 passages and partially modified the expression of surface antigens and the intracellular distribution of pluripotency-associated markers (NANOG, SOX2 and TERT) after 20 passages. The phenotypic alteration of cultured oAFMSCs was associated with a reduction of in vitro osteogenic plasticity. In detail, after 20 passages of cellular expansion, oAFMSCs lost the ability to increase osteocalcin and decreased collagen type I messenger RNA expression. Also, a lower percentage of cells displayed intracellular calcium release after stimulation with salmon calcitonin. CONCLUSIONS: The results presented here suggest that long-term in vitro expansion may cause significant alterations in phenotypic features and plasticity of oAFMSCs, suggesting a careful re-evaluation of in vitro cultural and temporal conditions before employing expanded oAFMSCs for therapeutic purposes.

Graphene oxide accelerates TGFß-mediated epithelial-mesenchymal transition and stimulates pro-inflammatory immune response in amniotic epithelial cells.

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 TGFß1-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.

Mammal comparative tendon biology: advances in regulatory mechanisms through a computational modeling.

There is high clinical demand for the resolution of tendinopathies, which affect mainly adult individuals and animals. Tendon damage resolution during the adult lifetime is not as effective as in earlier stages where complete restoration of tendon structure and property occurs. However, the molecular mechanisms underlying tendon regeneration remain unknown, limiting the development of targeted therapies. The research aim was to draw a comparative map of molecules that control tenogenesis and to exploit systems biology to model their signaling cascades and physiological paths. Using current literature data on molecular interactions in early tendon development, species-specific data collections were created. Then, computational analysis was used to construct Tendon NETworks in which information flow and molecular links were traced, prioritized, and enriched. Species-specific Tendon NETworks generated a data-driven computational framework based on three operative levels and a stage-dependent set of molecules and interactions (embryo-fetal or prepubertal) responsible, respectively, for signaling differentiation and morphogenesis, shaping tendon transcriptional program and downstream modeling of its fibrillogenesis toward a mature tissue. The computational network enrichment unveiled a more complex hierarchical organization of molecule interactions assigning a central role to neuro and endocrine axes which are novel and only partially explored systems for tenogenesis. Overall, this study emphasizes the value of system biology in linking the currently available disjointed molecular data, by establishing the direction and priority of signaling flows. Simultaneously, computational enrichment was critical in revealing new nodes and pathways to watch out for in promoting biomedical advances in tendon healing and developing targeted therapeutic strategies to improve current clinical interventions.

Unraveling the link: locomotor activity exerts a dual role in predicting Achilles tendon healing and boosting regeneration in mice.

Introduction: Tendon disorders present significant challenges in the realm of musculoskeletal diseases, affecting locomotor activity and causing pain. Current treatments often fall short of achieving complete functional recovery of the tendon. It is crucial to explore, in preclinical research, the pathways governing the loss of tissue homeostasis and its regeneration. In this context, this study aimed to establish a correlation between the unbiased locomotor activity pattern of CRL:CD1 (ICR) mice exposed to uni- or bilateral Achilles tendon (AT) experimental injuries and the key histomorphometric parameters that influence tissue microarchitecture recovery. Methods: The study involved the phenotyping of spontaneous and voluntary locomotor activity patterns in male mice using digital ventilated cages (DVC®) with access to running wheels either granted or blocked. The mice underwent non-intrusive 24/7 long-term activity monitoring for the entire study period. This period included 7 days of pre-injury habituation followed by 28 days post-injury. Results and discussion: The results revealed significant variations in activity levels based on the type of tendon injury and access to running wheels. Notably, mice with bilateral lesions and unrestricted wheel access exhibited significantly higher activity after surgery. Extracellular matrix (ECM) remodeling, including COL1 deposition and organization, blood vessel remodeling, and metaplasia, as well as cytological tendon parameters, such as cell alignment and angle deviation were enhanced in surgical (bilateral lesion) and husbandry (free access to wheels) groups. Interestingly, correlation matrix analysis uncovered a strong relationship between locomotion and microarchitecture recovery (cell alignment and angle deviation) during tendon healing. Overall, this study highlights the potential of using mice activity metrics obtained from a home-cage monitoring system to predict tendon microarchitecture recovery at both cellular and ECM levels. This provides a scalable experimental setup to address the challenging topic of tendon regeneration using innovative and animal welfare-compliant strategies.

Unveiling the immunomodulatory shift: Epithelial-mesenchymal transition Alters immune mechanisms of amniotic epithelial cells.

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.

Stemness characteristics and osteogenic potential of sheep amniotic epithelial cells.

We set out to characterize stemness properties and osteogenic potential of sheep AEC (amniotic epithelial cells). AEC were isolated from 3-month-old fetuses and expanded in vitro for 12 passages. The morphology, surface markers, stemness markers and osteogenic differentiation were inspected after 1, 6 and 12 passages of expansion, with an average doubling time of 24 h. AEC clearly expressed the stemness markers Oct-3/4 (octamer-binding protein-3/4), Nanog, Sox2 and TERT (telomerase reverse transcriptase) and displayed low levels of global DNA methylation. Culture had moderate effects on cell conditions; some adhesion molecules progressively disappeared from the cell surface, and the expression of Sox2 and TERT was slightly reduced while Nanog increased. No changes occurred in the levels of DNA methylation. Cells organized in 3D spheroids were used for IVD (in vitro differentiation). Within these structures the cells developed a complex intercellular organization that involved extensive intercellular coupling despite continuous cell migration. Marked deposition of calcein in the ECM (extracellular matrix), increased ALP (alkaline phosphatase) activity, expression of bone-related genes (osteocalcin) and the matrix mineralization shown by Alizarin Red staining demonstrate that AEC can undergo rapid and extensive osteogenic differentiation. AEC introduced in experimental bone lesions survived in the site of implantation for 45 days and supported consistent bone neoformation, thus showing promising potential applications in osteogenic regenerative medicine.

Tendon Healing Response Is Dependent on Epithelial-Mesenchymal-Tendon Transition State of Amniotic Epithelial Stem Cells.

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.