Annunziata Mauro

PKCα-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells

We have previously suggested that PKCα has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCα mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCα, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating myogenic-related morphology in differentiated RD cells.

Isolation, characterization, and in vitro differentiation of ovine amniotic stem cells

Stem cell (SC) regenerative therapy represents an emerging strategy for the treatment of human diseases. Since amniotic fluid-derived cells have been recently proposed as a promising source of human SCs, the present research aimed to amplify in vitro and characterize ovine amniotic fluid-derived SCs collected from the membranes (AMSCs) or fluid (AFSCs). These cells were found to proliferate, express the pluripotent SC markers OCT-4 and TERT, and differentiate in both osteogenic and smooth muscle lineages in vitro. However, AMSCs presented an earlier down-regulation of SC markers and a faster rate of differentiation. Thus, AMSCs and AFSCs may represent sources of characterized pluripotent SCs that can be easily collected and amplified in vitro. These ovine SCs may be used in preclinical studies on large animals to develop future human therapies.

The role of Akt signalling in the mammalian ovary

The serine/threonine protein kinase Akt is involved in many cellular processes including cell growth, survival, proliferation and metabolism. Akt activity is modulated downstream of phosphatidylinositol-3-kinase (PI3K) in response to different extracellular stimuli. In the mammalian ovary, Akt collaborates with other kinases in the regulation of coordinate follicle and oocyte development. Akt determines the pool of primordial follicles and the transition from quiescent to growing phase. In addition, the kinase modulates granulosa cell apoptosis throughout folliculogenesis. In oocytes Akt participates in the control of meiosis resumption and, at metaphase II stage, regulates polar body emission and spindle organization. Its inhibition negatively affects preimplantation embryo development. As a consequence of such a central role, Akt dysregulation is associated with several human diseases including infertility and ovarian cancer.

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 cm3), alone or engineered with 1×106 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.

Indirect Co-Culture with Tendons or Tenocytes Can Program Amniotic Epithelial Cells towards Stepwise Tenogenic Differentiation

Background Amniotic epithelial cells (AEC) have potential applications in cell-based therapy. Thus far their ability to differentiate into tenocytes has not been investigated although a cell source providing a large supply of tenocytes remains a priority target of regenerative medicine in order to respond to the poor self-repair capability of adult tendons. Starting from this premise, the present research has been designed firstly to verify whether the co-culture with adult primary tenocytes could be exploited in order to induce tenogenic differentiation in AEC, as previously demonstrated in mesenchymal stem cells. Since the co-culture systems inducing cell differentiation takes advantage of specific soluble paracrine factors released by tenocytes, the research has been then addressed to study whether the co-culture could be improved by making use of the different cell populations present within tendon explants or of the high regenerative properties of fetal derived cell/tissue. Methodology/Principal Findings Freshly isolated AEC, obtained from ovine fetuses at mid-gestation, were co-incubated with explanted tendons or primary tenocytes obtained from fetal or adult calcaneal tendons. The morphological and functional analysis indicated that AEC possessed tenogenic differentiation potential. However, only AEC exposed to fetal-derived cell/tissues developed in vitro tendon-like three dimensional structures with an expression profile of matrix (COL1 and THSB4) and mesenchymal/tendon related genes (TNM, OCN and SCXB) similar to that recorded in native ovine tendons. The tendon-like structures displayed high levels of organization as documented by the cell morphology, the newly deposited matrix enriched in COL1 and widespread expression of gap junction proteins (Connexin 32 and 43). Conclusions/Significance The co-culture system improves its efficiency in promoting AEC differentiation by exploiting the inductive tenogenic soluble factors released by fetal tendon cells or explants. The co-cultural system can be proposed as a low cost and easy technique to engineer tendon for biological study and cell therapy approach.

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.

Achilles Tendon Regeneration can be Improved by Amniotic Epithelial Cell Allotransplantation

Amniotic epithelial cells (AECs) are ideal seed cells for tissue regeneration, but no research has yet been reported on their tendon regeneration potential. This study investigated the efficiency of AEC allotransplantation for tendon healing, as well as the mechanism involved. To this aim ovine AECs, characterized by specific surface and stemness markers (CD14-, CD31-, CD45-, CD49f, CD29, CD166, OCT4, SOX2, NANOG, TERT), were allotransplanted into experimentally induced tissue defects in sheep Achilles tendon. In situ tissue repair revealed that AEC-treated tendons had much better structural and mechanical recoveries than control ones during the early phase of healing. Immunohistochemical and biochemical analyses indicated that extracellular matrix remodeling was more rapid and that immature collagen fibers were completely replaced by mature ones in 28 days. Moreover, spatial–temporal analysis of cellularity, proliferation index, vascular area, and leukocyte infiltration revealed that AECs induced a specific centripetal healing process that first started in the tissue closer to the healthy portion of the tendons, where AECs rapidly migrated to then progress through the core of the lesion. This peculiar healing evolution could have been induced by the growth factor stimulatory influence (TGF-β1 and VEGF) and/or by the host progenitor cells recruitment, but also as the consequence of a direct tenogenic AEC differentiation resulting in the regeneration of new tendon matrix. These findings demonstrate that AECs can support tendon regeneration, and their effects may be used to develop future strategies to treat tendon disease characterized by a poor clinical outcome in veterinary medicine.

Gestational stage affects amniotic epithelial cells phenotype, methylation status, immunomodulatory and stemness properties

Stem cells isolated from amniotic epithelium (AECs) have shown great potential in cell-based regenerative therapies. Because of their fetal origin, these cells exhibit elevated proliferation rates and plasticity, as well as, immune tolerance and anti-inflammatory properties. These inherent attitudes make AECs well-suited for both allogenic and xenogenic cellular transplants in animal models. Since in human only at term amnion is easily obtainable after childbirth, limited information are so far available concerning the phenotypic and functional difference between AECs isolated from early and late amnia. To this regard, the sheep animal model offers an undoubted advantage in allowing the easy collection of both types of AECs in large quantity. The aim of this study was to determine the effect of gestational age on ovine AECs (oAECs) phenotype, immunomodulatory properties, global DNA methylation status and pluripotent differentiation ability towards mesodermic and ectodermic lineages. The immunomodulatory property of oAECs in inhibiting lymphocyte proliferation was mainly unaffected by gestational age. Conversely, gestation considerably affected the expression of surface markers, as well the expression and localization of pluripotency markers. In detail, with progression of gestation the mRNA expression of NANOG and SOX2 markers was reduced, while the ones of TERT and OCT4A was unaltered; but at the end of gestation NANOG, SOX2 and TERT proteins mainly localized outside the nuclear compartment. Regarding the differentiation ability, LPL (adipogenic-specific gene) mRNA content significantly increased in oAECs isolated from early amnia, while OCN (osteogenic-specific gene) and NEFM (neurogenic-specific gene) mRNA content significantly increased in oAECs isolated from late amnia, suggesting that gestational stage affected cell plasticity. Finally, the degree of global DNA methylation increased with gestational age. All these results indicate that gestational age is a key factor capable of influencing morphological and functional properties of oAECs, and thus probably affecting the outcome of cell transplantation therapies.

Ovine amniotic epithelial cells: in vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects.

In vitro expanded and frosted ovine amniotic epithelial cells (oAECs) were evaluated for their phenotype, stemness and attitude to differentiate into tenocytes. Fifteen horses with acute tendon lesions were treated with one intralesional injection of oAECs. Tendon recovery under controlled training was monitored. In vitro expanded oAECs showed a constant proliferative ability, a conserved phenotype and stable expression profile of stemness markers. Differentiation into tenocytes was also regularly documented. US controls showed the infilling of the defect and early good alignment of the fibers and 12 horses resumed their previous activity. Histological and immunohistochemical examinations in an explanted tendon demonstrated the low immunogenicity of oAECs that were able to survive in the healing site. In addition, oAECs supported the regenerative process producing ovine collagen type I amongst the equine collagen fibers. Considering our results, oAECs can be proposed as a new approach for the treatment of spontaneous equine tendon injuries.

Characterization, GFP Gene Nucleofection, and Allotransplantation in Injured Tendons of Ovine Amniotic Fluid-Derived Stem Cells:

Amniotic fluid has drawn increasing attention in the recent past as a cost-effective and accessible source of fetal stem cells. Amniotic fluid-derived mesenchymal stem cells (AFMSCs) that display high proliferation rate, large spectrum of differentiation potential, and immunosuppressive features are considered optimal candidates for allogeneic repair of mesenchymal damaged tissues. In this study, ovine AFMSCs (oAFMSCs) isolated from 3-month-old sheep fetuses were characterized for their proliferation rate, specific surface antigen and pluripotency marker expression, genomic stability, and mesenchymal lineage differentiation during their in vitro expansion (12 passages) and after nucleofection. The high proliferation rate of oAFMSCs gradually decreased during the first six subculture passages while the expression of surface molecules (CD29, CD58, CD166) and of pluripotency-associated markers (OCT4, TERT, NANOG, SOX2), the in vitro osteogenic differentiation potential, and a normal karyotype were maintained. Afterwards, oAFMSCs were nucleofected with a selectable plasmid coding for green fluorescent protein (GFP) using two different programs, U23 and C17, previously optimized for human mesenchymal stem cells. Transfection efficiencies were ~63% and ~37%, while cell recoveries were ~10% and ~22%, respectively. Nucleofected oAFMSCs expressing the GFP transgene conserved their pluripotency marker profile and retained a normal karyotype and the osteogenic differentiation ability. Seven single clones with a GFP expression ranging from 80% to 97% were then isolated and expanded over 1 month, thus providing stably transfected cells with long-term therapeutic potential. The in vivo behavior of GFP-labeled oAFMSCs was tested on a previously validated preclinical model of experimentally induced Achilles tendon defect. The allotransplanted oAFMSCs were able to survive within the host tissue for 1 month enhancing the early phase of tendon healing as indicated by morphological and biomechanical results. Altogether these data suggest that genetically modified oAFMSCs might represent a valuable tool for in vivo preclinical studies in a highly valid translational model.