The effect of platelet rich plasma on amniotic membrane derived stem cells

Stem cells become very important apparatus in this cutting edge technology in the field of biomedical science. Amniotic membrane is one of the vital sources of stem cells that give the cells with lesser immunogenicity having the strength more than multipotency. Thus equine amniotic membrane derived stem cells were isolated, cultured and propagated till passages ten. Along with these osteogenic, adipogenic, chondrogenic differentiation as well as neurospheres formation were also performed. In proliferative study, doubling time revealed that passage three (P3) to passage six (P6) had the higher growth and passage four (P4) was the highest proliferation in both epithelial (AEC) and mesenchymal stem cells (AMC). In AECs and AMCs, growth curve analysis showed that passage five (P5) was the best for growth kinetics properties. A handy double centrifuged laboratory technique was developed to yield a PRP from horse blood with higher platelets (717.17±43.34×109 /L) concentration. Four types of PRP was made such as fresh and inactivated (PRP-1), fresh and CaCl2 activated (PRP-2), stored and CaCl2 activated (PRP-3) as well as stored and freeze-thaw activated (PRP-4). Epidermal Growth Factor (EGF) concentration was recorded in higher concentration having without any significant difference among different types of PRPs. Platelet-derived growth factor (PDGF) concentration was the highest in the freeze-thaw activated PRP. MTT procedure showed that PRP activated by freezing-thawing method was related with higher propagation of both AECs and AMCs. Viable cells counting also favoured the freeze-thaw activated PRP in terms of growth of cells. In case of different dosages of PRP, 5% concentration of freeze-thaw PRP had over all improved relation with all cellular growth indexes for both AECs and AMCs although 10% and 15% concentration of PRP had the close results. In differentiation study both types of stem cells were successfully differentiated into osteogenic, adipogenic and chondrogenic lineage that were confirmed by alizarin red stain, oil red O stain and alcian blue stain, respectively. In neurospheres generation, both cells proved their ability to produce progenitor cells for nerve tissue by creating well defined, spheroid, three dimensional structures. In PCR analysis, strong expression of two pluripotent markers (SOX 2 and OCT 4) was recorded. Besides these, multipotent stem cell markers like CD 14, CD 44, CD 73, CD 90 and CD 105 had good expression in this study (P0, P3, P5 and P7) for AECs and AMCs. On the contrary hemopoietic cell marker (CD 45) was negative for both cell types. In addition, MHC-II marker (CD 74) had also negative. These results have shown that the amniotic membrane derived stem cells which have both of multipotent and pluripotent characteristics are an important source of stem cells for allogenic treatments. Key words: Amniotic membrane derived stem cell, PRP, PDGF, EGF, horse