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Abstract
Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFβ-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFβ3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells.
Highlights
Tendons are ubiquitous, dense fibrous connective tissue made up primarily of collagenous fibers, with the essential role of transmitting contractile forces from muscle to the bone making movement of the body possible
In order to confirm the maintenance of the mesenchymal stem cell (MSC) nature of cultured Adipose-derived stem cells (ASCs), immunophenotyping was performed in both human platelet lysate (hPL) and serum free medium (SF) ASC populations according to the International Society for Cell Therapy (ISCT) standards [33, 34]
The cell viability associated with the metabolic activity of SF-CTRL was higher of what observed in hPL-CTRL cells and resulted in increases of +105% (p
Summary
Dense fibrous connective tissue made up primarily of collagenous fibers, with the essential role of transmitting contractile forces from muscle to the bone making movement of the body possible. Cultured ASCs exhibit differentiative potential toward several cell lineages, as well as possess immunomodulatory properties, the ability to express anti-inflammatory cytokines and to prolongate allotransplant survival [5,6,7,8,9,10] These favorable regenerative and paracrine abilities make ASCs currently under investigation for a high number of clinical therapeutic applications even if compared to bone- and cartilage-related pathologies, the use of MSCs in tendon related disorders has been investigated very little, so far [11,12,13,14,15]. An important scientific and technological goal that must be achieved is the development of an ideal culture system suitable for cellular therapy represented by xenogenic- and serum-free medium with a chemically defined composition Based on these purposes, the aim of this study was to evaluate for the first time the tenogenic differentiation potential of ASCs using a defined serum free medium (SF) or a xenogenic-free medium supplemented with human platelet lysate (hPL). The second medium here used was a commercial hPL supplemented medium, obtained by pooling more than 300
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