21 research outputs found
Effect of long-term different physical activity training on human LHCM-M2 myoblast differentiation
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
Long-term effect of Progesterone in preservation of the epithelioid phenotype of amniotic epithelial cells in vitro
Progesterone prevents epithelial-mesenchymal transition of ovine amniotic epithelial cells and enhances their immunomodulatory properties
Progesterone prevents the epithelial-to-mesenchymal transition of amniotic epithelial cells
Progesterone prevents epithelial-mesenchymal transition of ovine amniotic epithelial cells and enhances their immunomodulatory properties
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. P4supplementation 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 P4still 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, P4promoted 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 P4supplementation 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
Progesterone Prolongs Viability and Anti-inflammatory Functions of Explanted Preterm Ovine Amniotic Membrane
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.
FUNDING
This work was supported by Tercas Foundation and by PRIN 2015 (PRIN C42F15000180001) financed by the Ministry of Education, University and Research (M.I.U.R.), Rome, Italy. BB was the author who received this funding. This study was in part supported by the Commonwealth of Pennsylvania, Department of Health for Sbarro Health Research Organization, S.H.R.O. (www.shro.org). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript
Protocol for the Ovine Amniotic Epithelial Cell In Vitro Culture and Differentiation
Ovine Amniotic epithelial cells (oAEC) are a subject of placental stem cells with great regenerative and immunomodulatory properties. Indeed, oAEC are object of intense study for regenerative medicine thanks to the several advantages in developing pre-clinical studies on a high value translational animal model, such as sheep. For this reason, a critical standardization of in vitro culture practices is fundamental in order to maintain during amplification the oAEC native phenotype, improving both oAEC in vivo therapeutic potential and clinical outcomes. Here, is described an oAEC culture protocol with supplementation of Progesterone and Estradiol hormones, able to modulate the native epithelial phenotype during the in vitro amplification. In addition, it is described the culture protocol that is able to differentiate oAECs towards osteogenic and chondrogenic lineage