Combination of human platelet lysate and 3D gelatin scaffolds to enhance osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells.

Bone disorders are major health issues requiring specialized care; however, the traditional bone grafting method had several limitations. Thus, bone tissue engineering has become a potential alternative. In therapeutic treatments, using fetal bovine serum (FBS) as a culture supplement may result in the risk of contamination and host immunological response; therefore, human platelet lysate (hPL) has been considered a viable alternative source. This study attempted to compare the effectiveness and safety of different culture supplements, either FBS or hPL, on the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid (hAF-MSCs) under a three-dimensional gelatin scaffold. The results indicate that hAF-MSCs have the potential to be used in clinical applications as they meet the criteria for mesenchymal stem cells based on their morphology, the expression of a particular surface antigen, their proliferation ability, and their capacity for multipotent differentiation. After evaluation by MTT and Alamar blue proliferation assay, 10% of hPL was selected. The osteogenic differentiation of hAF-MSCs under three-dimensional gelatin scaffold using osteogenic-induced media supplemented with hPL was achievable and markedly stimulated osteoblast differentiation. Moreover, the expressions of osteoblastogenic related genes, including OCN, ALP, and COL1A1, exhibited the highest degree of expression under hPL-supplemented circumstances when compared with the control and the FBS-supplemented group. The induced cells under hPL-supplemented conditions also presented the highest ALP activity level and the greatest degree of calcium accumulation. These outcomes would indicate that hPL is a suitable substitute for animal derived serum. Importantly, osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells using hPL-supplemented media and three-dimensional scaffolds may open the door to developing an alternative construct for repairing bone defects.

Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine.

Human amniotic fluid mesenchymal stem cells (hAF-MSCs) have been shown to be effective in the treatment of many diseases. Platelet lysate (PL) contains multiple growth and differentiation factors; therefore, it can be used as a differentiation inducer. In this study, we attempted to evaluate the efficiency of human platelet lysate (hPL) on cell viability and the effects on cardiomyogenic differentiation of hAF-MSCs. When treating the cells with hPL, the result showed an increase in cell viability. Expressions of cardiomyogenic specific genes, including GATA4, cTnT, Cx43 and Nkx2.5, were higher in the combined treatment groups of 5-azacytidine (5-aza) and hPL than the expressions of cardiomyogenic specific genes in the control group and in the 5-aza treatment group. In terms of the results of immunofluorescence and immunoenzymatic staining, the highest expressions of cardiomyogenic specific proteins were revealed in combined treatment groups. It can be summarized that hPL may be an effective supporting cardiomyogenic supplementary factor for cardiomyogenic differentiation in hAF-MSCs.

The effects of human platelet lysate versus commercial endothelial growth medium on the endothelial differentiation potential of human amniotic fluid mesenchymal stem cells.

To differentiate stem cells into endothelial cells, vascular endothelia growth factors (VEGF) serve as the major signal for stimulating the cells. However, there are other cytokines or growth factors associated with endothelial cell development and differentiation. Human platelet lysate (hPL) has been a promising reagent in cell-based therapy since it is considered as a source of bioactive molecules and growth factors. The aim of this study was to investigate the in vitro differentiation of human amniotic fluid mesenchymal stem cells (hAF-MSCs) into endothelial-like cells under hPL together with VEGF or endothelial cell growth medium 2 (EGM-2), a commercially induced medium. In this study, hAF-MSCs were isolated from human amniotic fluid cells (hAFCs) using the direct adherence method. The cells expressed CD44, CD73, CD90, and HLA-ABC at high levels and expressed Oct-4 (octamer-binding transcription factor 4) at low levels. The cells were negative for CD31, CD34, CD45, CD105 and HLA-DR. This study found that hAF-MSCs induced with hPL and VEGF had the ability to differentiate into endothelial-like cells by presenting endothelial specific markers (vWF, VEGFR2 and eNOS), forming a network-like structure on Matrigel, and producing nitric oxide (NO). This outcome was similar to those of experiments involving EGM-2 induced cells. The present findings indicate that hPL + VEGF can induce hAF-MSCs to express endothelial cell characteristics. Our findings represent an important step forward in the development of a clinically compliant process for the production of endothelial cell-derived hAF-MSCs, and their subsequent testing in future clinical trials.

Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells.

The aim of this study was to evaluate the efficiency of ascorbic acid (AA) on cell viability, cytotoxicity and the effects on cardiomyogenic differentiation of the human amniotic fluid mesenchymal stem cells (hAF-MSCs). The results of methylthiazole tetrazolium (MTT) assay and cell apoptosis assay indicated that after 24, 48 and 72 h of treatment, AA had no effect on cells viability and cytotoxicity. After treating the hAF-MSCs with 5-azacytidine (5-aza) and a combination of AA and 5-aza, the alamar blue cells proliferation assay showed the normal growth characteristic similar to control group. Especially, the morphological changes were observed between day 0 and day 21, and it was revealed that the hAF-MSCs exhibited myotube-like morphology after 7 days of cell culturing. Moreover, the treatment with a combination of AA and 5-aza was able to up-regulate the cardiomyogenic specific gene levels, which are known to play an important role in cardiomyogenesis. This was specifically notable with the results of immunofluorescence and immunoenzymatic staining in the AA combined with 5-aza treatment group, the highest expression of cardiomyogenic specific proteins was revealed including for GATA4, cTnT, Cx43 and Nkx2.5. It could be concluded that AA may be a good alternative cardiomyogenic inducing factor for hAF-MSCs and may open new insights into future biomedical applications for a clinically treatment.

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.

Osteoblastic differentiation potential of human amniotic fluid-derived mesenchymal stem cells in different culture conditions.

Osteoporosis is a bone degenerative disease characterized by a decrease in bone strength and an alteration in the osseous micro-architecture causing an increase in the risk of fractures. These diseases usually happen in post-menopausal women and elderly men. The most common treatment involves anti-resorptive agent drugs. However, the inhibition of bone resorption alone is not adequate for recovery in patients at the severe stage of osteoporosis who already have a fracture. Therefore, the combination of utilizing osteoblast micro mimetic scaffold in cultivation with the stimulation of osteoblastic differentiations to regain bone formation is a treatment strategy of considerable interest. The aims of this current study are to investigate the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid and to compare the monolayer culture and scaffold culture conditions. The results showed the morphology of cells in human amniotic fluid as f-type, which is a typical cell shape of mesenchymal stem cells. In addition, the proliferation rate of cells in human amniotic fluid reached the highest peak after 14 days of culturing. After which time, the growth rate slowly decreased. Moreover, the positive expression of specific mesenchymal cell surface markers including CD44, CD73, CD90, and also HLA-ABC (MHC class I) were recorded. On the other hand, the negative expressions of the endothelial stem cells markers (CD31), the hematopoietic stem cells markers (CD34, 45), the amniotic stem cells markers (CD117), and also the HLA-DR (MHC class II) were also recorded. The expressions of osteoblastogenic related genes including OCN, COL1A1, and ALP were higher in the osteogenic-induced group when compared to the control group. Interestingly, the osteoblastogenic related gene expressions that occurred under scaffold culture conditions were superior to the monolayer culture conditions. Additionally, higher ALP activity and greater calcium deposition were recorded in the extracellular matrix in the osteogenic-induced group than in the culture in the scaffold group. In summary, the mesenchymal stem cells derived from human amniotic fluid can be induced to be differentiated into osteoblastic-like cells and can promote osteoblastic differentiation using the applied scaffold.

Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte­like cells.

Ischemic heart disease (IHD) is a major factor influencing worldwide mortality rates. Furthermore, IHD has become a significant health problem among the Thai population. Stem cell therapy using mesenchymal stem cells (MSCs) is an alternative therapeutic method that has been applied to improve the quality of life of patients. Amniotic fluid (AF) contains a heterogeneous cell population, including MSCs, which are multipotent stem cells that have the capability to differentiate into mesenchymal lineages. The purpose of the present study was to evaluate the MSC characteristics of human (h)AF and determine its potency regarding cardiogenic differentiation. MSC characterization following flow cytometric analysis revealed that the cells expressed MSC markers, cluster of differentiation (CD)44, CD90, human leukocyte antigen­ABC and CD73. The results of the alamar blue assay demonstrated that cell proliferation rate continuously increased from the early cultivation phase up to 5­fold during days 1 to 5 of cell culturing. The highest rate of cell proliferation was observed on day 17 with a 30­fold increase compared with that on day 1. During the cardiogenic induction stage, morphological changes were observed between day 0 and day 21, and it was revealed that the hAF derived­MSCs in the cardiogenic­induced group exhibited myotube­like morphology after 7 days of cell culturing. Following cardiogenic induction, immunohistochemistry staining was performed on day 21, and reverse transcription­quantitative polymerase chain reaction on day 7 and 21. These steps were performed to detect the protein and gene expression levels of cardiac specific proteins (GATA4, cardiac troponin T, Nkx2.5 and Connexin43). The results of the present study indicated that hAF­MSCs possess the potential to differentiate into cardiomyocyte­like cells. Thus, it was concluded that hAF may be a suitable source of MSCs for stem cell therapy and tissue engineering.

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.

Differentiation of mesenchymal stem cells from human amniotic fluid to vascular endothelial cells.

Endothelial dysfunction is a principle feature of vascular-related disease. Endothelial cells have been acquired for the purposes of the restoration of damaged tissue in therapeutic angiogenesis. However, their use is limited by expansion capacity and the small amount of cells that are obtained. Human amniotic fluid mesenchymal stem cells (hAF-MSCs) are considered an important source for vascular tissue engineering. In this study, hAF-MSCs were characterized and then induced in order to differentiate into the endothelial-like cells. Human amniotic fluid cells (hAFCs) were obtained from amniocentesis at the second trimester of gestation. The cells were characterized as mesenchymal stem cells by flow cytometry. The results showed that the cells were positive for mesenchymal stem cell markers CD44, CD73, CD90 and HLA-ABC, and negative for CD31, Amniotic fluid stem cells marker: CD117, anti-human fibroblasts, HLA-DR and hematopoietic differentiation markers CD34 and CD45. The hAF-MSCs were differentiated into endothelial cells under the induction of vascular endothelial growth factor (VEGF) and analyzed for the expression of the endothelial-specific markers and function. The expression of the endothelial-specific markers was determined by reverse transcriptase-quantitative PCR (RT-qPCR), while immunofluorescent analysis demonstrated that the induced hAF-MSCs expressed von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and endothelial nitric oxide synthase (eNOS). The network formation assay showed that the induced hAF-MSCs formed partial networks. All results indicated that hAF-MSCs have the potential to be differentiated into endothelial-like cells, while human amniotic fluid might be a suitable source of MSCs for vascularized tissue engineering.