Human platelet lysate as an alternative to fetal bovine serum for culture and endothelial differentiation of human amniotic fluid mesenchymal stem cells.

Human amniotic fluid (hAF) mesenchymal stem cells (MSCs) are commonly cultured in medium containing FBS. However, there are concerns about using animal serum in therapeutic applications due to the potential for immunogenic reactions and the risk of transmission of pathogens. For safety reasons, human platelet lysate (hPL) has been suggested as a replacement for FBS because it appears to be a natural source of growth factors. In this present study, it was investigated whether FBS could be substituted with hPL in hAF­MSCs culture without affecting their properties. Pooled hPL was generated by the freeze­thaw method. The concentration of hPL was selected after evaluation by MTT assay. The hAF­MSCs were cultured in FBS­ or hPL­supplemented conditions and shared a fibroblast­like morphology. Cell proliferation assays showed that the growth characteristic of hAF­MSCs cultured in 10% hPL­supplemented media was similar to those cultured in 10% FBS­supplemented media. The expression of MSC markers did not differ between the cells cultured in the different conditions. The endothelial differentiation potential was also investigated. Reverse transcription­quantitative (RT­q)PCR revealed that induced cells supplemented with hPL showed an increase level of endothelial specific gene expression compared to the FBS­supplemented cells. Immunofluorescence analysis showed specific protein localization in both induced cell groups. Additionally, induced cells supplemented with hPL had the potential to form networks on Matrigel. This present study indicated that hPL could be used to culture and enhance the endothelial differentiation potential of hAF­MSCs.

Amniotic fluid: Source of valuable mesenchymal stem cells and alternatively used as cryopreserved solution.

Mesenchymal stem cells (MSCs), which possess remarkable capabilities, are found in amniotic fluid (AF). The findings of several studies have shown the potential benefits of these cells in applications of regenerative medicine. In clinical applications, an over-period of time is required in a preparation process that makes cell collection become more necessary. Herein, the aim of this study was to preserve and characterize the cell's properties after cell cryopreservation into an appropriate cryogenic medium. The results illustrated that the highest hAF-MSCs viability was found when the cells were conserved in a solution of 5% DMSO + 10% FBS in AF. However, no statistical differences were identified in a chromosomal aberration of the post-thawed cells when compared to the non-frozen cells. These cells could also maintain their MSC features through the ability to express cell prolific quality, illustrating the typical MSC markers and immune privilege properties of CD44, CD73, CD90 and HLA-ABC. Additionally, post-thawed cells were able to differentiate into chondrogenic lineage by exhibiting chondrogenic related genes (SOX9, AGC, COL2A1) and proteins (transcription factor SOX9 protein (SOX9), cartilage oligomeric matrix protein (COMP) and aggrecan core protein (AGC)), as well as to present sGAGs accumulation. Interestingly, the use of a transmission electron microscope (TEM) uncovered the enrichment of the rough endoplasmic reticulum (rER) that coincided with euchromatin and the prominent nucleolus in the chondrogenic-induced cells that are normally found in the cells of natural cartilage. All in all, this study manifested that AF can be a major consideration and applied for use as a co-mixture of cryogenic medium.

Sesamin encouraging effects on chondrogenic differentiation of human amniotic fluid-derived mesenchymal stem cells.

Worldwide, the most recognized musculoskeletal degenerative disease is osteoarthritis (OA). Sesamin, a major abundant lignan compound present in Sesamun Indicum Linn, has been described for its various pharmacological effects and health benefits. However, the promoting effects of sesamin on chondrogenic differentiation have not yet been observed. Herein, the aim of this study was to investigate the effects of sesamin on cell cytotoxicity and the potent supporting effects on chondrogenic differentiation of human amniotic fluid-derived mesenchymal stem cells (hAF-MSCs). The results indicated that sesamin was not toxic to hAF-MSCs after sesamin treatment. When treating the cells with a combination of sesamin and inducing factors, sesamin was able to up-regulate the expression level of specific genes which play an essential role during the cartilage development process, including SOX9, AGC, COL2A1, COL11A1, and COMP and also simultaneously promote the cartilage extracellular protein synthesis, aggrecan and type II collagen. Additionally, histological analysis revealed a high amount of accumulated sGAG staining inside the porous scaffold in the sesamin co-treating group. In conclusion, the results of this study have indicated that sesamin can be considered a chondrogenic inducing factor and a beneficial dietary supplement for cartilage repair.