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

Abstract

BackgroundAmniotic 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 FindingsFreshly 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/SignificanceThe 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.

Effect of scaffold morphology and cell co-culture on tenogenic differentiation of HADMSC on centrifugal melt electrospun poly (L‑lactic acid) fibrous meshes

“…The HADMSC and HUVEC co-culture system was demonstrated to promote in vitro HADMSC differentiation towards tenocytes on the aligned PLLA fibrous scaffold. Some other studies also employed a co-culture technique to promote MSC differentiation, and confirmed that a co-culture of MSC and tenocytes enhanced the tenogenic differentiation of MSC [66–69]. Our results indicate that the incorporation of EC may initiate the vascularization and promote tenogenesis of MSC to recapitulate the initial tendon healing process.…”

Ovarian cancer stem-like cells elicit the polarization of M2 macrophages

“…For the co-culture investigations, 2x10 5 OCSCs were seeded with an equal number of human PBMC-derived macrophages (2x10 5 ) on day 7 of culture and then cultured for a further 3 days in RPMI-1640 medium. For the Transwell co-culture, cultivation was performed, as described previously (23). Briefly, 0.4 µm pore-size Corning Transwell inserts (VWR International, Inc., West Chester, PA, USA) were placed into 6-well plates with 2x10 5 macrophages, initially seeded at the bottom of the chamber and 2x10 5 OCSCs seeded onto the inserts, followed by culture for a further 3 days (24).…”

Synthetic Bone Substitute Engineered with Amniotic Epithelial Cells Enhances Bone Regeneration after Maxillary Sinus Augmentation

“…cytokeratin 8 (epithelial marker) and alpha smooth muscle actin (α-SMA: mesodermal marker), expression by immunocytochemistry [46];…”

“…Immunocytochemical analyses were carried out on freshly isolated, expanded and thawed oAEC as previously described [46]. Briefly, cells were fixed in 4% paraformaldehyde in phosphate buffered saline solution (PBS) and permeabilized with 0.1% Triton X-100/PBS.…”