Optimization of a nanoparticle uptake protocol applied to amniotic-derived cells: unlocking the therapeutic potential.

Stem cell-based therapy implementation relies heavily on advancements in cell tracking. The present research has been designed to develop a gold nanorod (AuNR) labeling protocol applied to amniotic epithelial cells (AECs) leveraging the pro-regenerative properties of this placental stem cell source which is widely used for both human and veterinary biomedical regenerative applications, although not yet exploited with tracking technologies. Ovine AECs, in native or induced mesenchymal (mAECs) phenotypes via epithelial-mesenchymal transition (EMT), served as the model. Initially, various uptake methods validated on other sources of mesenchymal stromal cells (MSCs) were assessed on mAECs before optimization for AECs. Furthermore, the protocol was implemented by adopting the biological strategy of MitoCeption to improve endocytosis. The results indicate that the most efficient, affordable, and easy protocol leading to internalization of AuNRs in living mAECs recognized the combination of the one-step uptake condition (cell in suspension), centrifugation-mediated internalization method (G-force) and MitoCeption (mitochondrial isolated from mAECs). This protocol produced labeled vital mAECs within minutes, suitable for preclinical and clinical trials. The optimized protocol has the potential to yield feasible labeled amniotic-derived cells for biomedical purposes: up to 10 million starting from a single amniotic membrane. Similar and even higher efficiency was found when the protocol was applied to ovine and human AECs, thereby demonstrating the transferability of the method to cells of different phenotypes and species-specificity, hence validating its great potential for the development of improved biomedical applications in cell-based therapy and diagnostic imaging.

Amniotic Membrane and Amniotic Epithelial Cell Culture.

Amniotic membrane (AM) is considered an important medical device for 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. To this regard, ovine amniotic epithelial cells (AECs) are a subset of placental stem cells with great regenerative and immunomodulatory properties. Indeed, either oAEC or AM have been object of intense study for regenerative medicine, thanks to several advantages in developing preclinical studies on a high value translational animal model, such as sheep. For this reason, a critical standardization of cultural practices is fundamental in order to maintain, on one hand, AM integrity and structure and, on the other hand, oAEC native properties, thus improving their in vivo therapeutic potential and clinical outcomes.In addition, freshly isolated AECs or AM can be exploited to produce enriched immunomodulatory secretomes that had been used with success into cell-free regenerative medicine procedures.To this aim, here is described an improved oAEC cultural protocol able to preserve their native epithelial phenotype also after the in vitro amplification and an innovative AM in vitro cultural protocol design to prolong the integrity and the biological properties of this tissue in order to collect stable conditioned media enriched with immunomodulatory factors.

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.

Hypoxia-Mimetic CoCl2 Agent Enhances Pro-Angiogenic Activities in Ovine Amniotic Epithelial Cells-Derived Conditioned Medium.

Amniotic epithelial stem cells (AECs) are largely studied for their pro-regenerative properties. However, it remains undetermined if low oxygen (O2) levels that AECs experience in vivo can be of value in maintaining their biological properties after isolation. To this aim, the present study has been designed to evaluate the effects of a hypoxia-mimetic agent, cobalt chloride (CoCl2), on AECs' stemness and angiogenic activities. First, a CoCl2 dose-effect was performed to select the concentration able to induce hypoxia, through HIF-1α stabilization, without promoting any cytotoxicity effect assessed through the analysis of cell vitality, proliferation, and apoptotic-related events. Then, the identified CoCl2 dose was evaluated on the expression and angiogenic properties of AECs' stemness markers (OCT-4, NANOG, SOX-2) by analysing VEGF expression, angiogenic chemokines' profiles, and AEC-derived conditioned media activity through an in vitro angiogenic xeno-assay. Results demonstrated that AECs are sensitive to the cytotoxicity effects of CoCl2. The unique concentration leading to HIF-1α stabilization and nuclear translocation was 10 µM, preserving cell viability and proliferation up to 48 h. CoCl2 exposure did not modulate stemness markers in AECs while progressively decreasing VEGF expression. On the contrary, CoCl2 treatment promoted a significant short-term release of angiogenic chemokines in culture media (CM). The enrichment in bio-active factors was confirmed by the ability of CoCl2-derived CM to induce HUVEC growth and the cells' organization in tubule-like structures. These findings demonstrate that an appropriate dose of CoCl2 can be adopted as a hypoxia-mimetic agent in AECs. The short-term, chemical-induced hypoxic condition can be targeted to enhance AECs' pro-angiogenic properties by providing a novel approach for stem cell-free therapy protocols.

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.

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.

Therapeutic potential of hAECs for early Achilles tendon defect repair through regeneration.

Cell-based therapy holds great promise for tendon disorders, a widespread debilitating musculoskeletal condition. Even if the cell line remains to be defined, preliminary evidences have proven that amniotic-derived cells possess in vitro and in vivo a great tenogenic potential. This study investigated the efficacy of transplanted human amniotic epithelial cells (hAECs) by testing their early regenerative properties and mechanisms involved on a validated ovine Achilles tendon partial defect performed on 29 animals. The injured tendons treated with hAECs recovered rapidly, in 28 days, structural and biomechanical properties undertaking a programmed tissue regeneration, differently from the spontaneous healing tissues. hAECs remained viable within the host tendons establishing with the endogenous progenitor cells an active dialogue. Through the secretion of modulatory factors, hAECs inhibited the inflammatory cells infiltration, activated the M2 macrophage subpopulation early recruitment, and accelerated blood vessel as well as extracellular matrix remodelling. In parallel, some in situ differentiated hAECs displayed a tenocytelike phenotype. Both paracrine and direct hAECs stimulatory effects were confirmed analysing their genome profile before and after transplantation. The 49 human up-regulated transcripts recorded in transplanted hAECs belonged to tendon lineage differentiation (epithelial-mesenchymal transition, connective specific matrix components, and skeleton or muscle system development-related transcripts), as well as the in situ activation of paracrine signalling involved in inflammatory and immunomodulatory response. Altogether, these evidences support the hypothesis that hAECs are a practicable and efficient strategy for the acute treatment of tendinopathy, reinforcing the idea of a concrete use of amniotic epithelial cells towards the clinical practice.

Synthetic bone substitute engineered with amniotic epithelial cells enhances bone regeneration after maxillary sinus augmentation.

BACKGROUND: Evidence has been provided that a cell-based therapy combined with the use of bioactive materials may significantly improve bone regeneration prior to dental implant, although the identification of an ideal source of progenitor/stem cells remains to be determined. AIM: In the present research, the bone regenerative property of an emerging source of progenitor cells, the amniotic epithelial cells (AEC), loaded on a calcium-phosphate synthetic bone substitute, made by direct rapid prototyping (rPT) technique, was evaluated in an animal study. MATERIAL AND METHODS: Two blocks of synthetic bone substitute (∼0.14 cm(3)), alone or engineered with 1×10(6) ovine AEC (oAEC), were grafted bilaterally into maxillary sinuses of six adult sheep, an animal model chosen for its high translational value in dentistry. The sheep were then randomly divided into two groups and sacrificed at 45 and 90 days post implantation (p.i.). Tissue regeneration was evaluated in the sinus explants by micro-computer tomography (micro-CT), morphological, morphometric and biochemical analyses. RESULTS AND CONCLUSIONS: The obtained data suggest that scaffold integration and bone deposition are positively influenced by allotransplantated oAEC. Sinus explants derived from sheep grafted with oAEC engineered scaffolds displayed a reduced fibrotic reaction, a limited inflammatory response and an accelerated process of angiogenesis. In addition, the presence of oAEC significantly stimulated osteogenesis either by enhancing bone deposition or making more extent the foci of bone nucleation. Besides the modulatory role played by oAEC in the crucial events successfully guiding tissue regeneration (angiogenesis, vascular endothelial growth factor expression and inflammation), data provided herein show that oAEC were also able to directly participate in the process of bone deposition, as suggested by the presence of oAEC entrapped within the newly deposited osteoid matrix and by their ability to switch-on the expression of a specific bone-related protein (osteocalcin, OCN) when transplanted into host tissues.

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.

Ovarian follicle vascularization in fasted pig.

The authors have investigated in the different classes of ovarian follicles the vascular area, the blood vessel distribution, the vascular endothelial growth factor (VEGF) mRNA expression and the VEGF secretion during equine chorionic gonadotropin (eCG) induced follicle growth in prepubertal gilts fed ad libitum or fasted. Immunohistochemistry staining of Von Willebrand factor showed that fasting caused a dramatic increase in the vascular area of medium-large tertiary follicles. The increase involved the two concentric vessel networks and the area between them that, becoming crossed by several anastomosis, modified the whole vessel architecture. Both in situ hybridization and in vitro culture experiments demonstrate that granulosa cells from medium-large follicles are engaged in a copious VEGF production upon eCG stimulation both in gilts fed ad libitum or fasted. More surprisingly, the production of VEGF becomes diffuse amongst theca cells of fasted animals thus recruiting a compartment that in condition of normal feeding regimen appears nearly quiescent. In conclusion, the data presented describe a local angiogenic process that develops in the follicle wall of growing antral follicle in case of acute severe food restriction. The mechanism, essentially confined to follicles that potentially approach ovulation, appears to assume the meaning of a local compensatory mechanism that may help maintaining adequate nutrient delivery to follicles that undergo ovulation.

Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation.

BACKGROUND: The possibility to predict the ability of a germ cell to properly sustain embryo development in vitro or in vivo as early as possible is undoubtedly the main problem of reproductive technologies. To date, only the achievement of nuclear maturation and cumulus expansion is feasible, as all the studies on cytoplasmic maturation are too invasive and have been complicated by the death of the cells analyzed. The authors studied the possibility to test the cytoplasmic quality of pig oocytes by evaluating their ability to produce steroidogenesis enabling factor(s). To this aim, oocytes matured under different culture conditions that allowed to obtain gradable level of cytoplasmic maturation, were used to produce conditioned media (OCM). The secretion of the factor(s) in conditioned media was then recorded by evaluating the ability of the spent media to direct granulosa cells (GC) steroidogenesis. METHODS: In order to obtain germ cells characterized by a different degree of developmental competence, selected pig oocytes from prepubertal gilts ovaries were cultured under different IVM protocols; part of the matured oocytes were used to produce OCM, while those remaining were submitted to in vitro fertilization trials to confirm their ability to sustain male pronuclear decondensation. The OCM collected were finally used on cumulus cells grown as monolayers for 5 days. The demonstration that oocytes secreted factor(s) can influence GC steroidogenesis in the pig was confirmed in our lab by studying E2 and P4 production by cumulus cells monolayers using a radioimmunoassay technique. RESULTS: Monolayers obtained by growing GC surrounding the oocytes for five days represent a tool, which is practical, stable and available in most laboratories; by using this bioassay, we detected the antiluteal effect of immature oocytes, and for the first time, demonstrated that properly matured germ cells are able to direct cumulus cells steroidogenesis by inhibiting E2 production (P < 0.01). Nevertheless, only fully competent oocytes were able to suppress estrogens production, while those cultured under unfavourable conditions were unable to exert any inhibitory effect on the functions of cumulus cells (P < 0.01). CONCLUSION: These results demonstrated that good quality oocytes can be easily selected on the basis of their ability to affect granulosa cell steroidogenesis thus reducing failures in reproductive technologies due to the transfer of fertilized oocytes with a scarce ability to sustain embryo development.