Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs

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.

Fig 1. hPL-ASC and SF-ASC appearance and stem cell surface marker patterns of expression.

(A) Representative micrographs of ASCs at passage 4 cultured in serum free medium conditions (hPL-ASCs and SF-ASCs) as well as of ASCs cultured in standard laboratory condition (SC-ASCs) using MEM-alpha as growth medium supplemented with 10% of FBS provided by Sigma Aldrich (optical microscopy: 10X; scale bar 200 μm). (B) Representative expression of the typical mesenchymal stem cell surface markers (CD13, CD44, CD90, CD73 and CD105), of the endothelial marker CD31 and of the hematopoietic antigen CD45 for both populations at passage 4 (red: isotypic control, green: ASCs). (C) Quantification of the above depicted markers, pooled for all lines (n = 4).

Fig 2. Morphological appearance and cell viability of hPL-ASCs and SF-ASCs during tenogenic induction.

(A) Cell morphology of CTRL and TENO hPL-ASCs and SF-ASCs at 3 days of differentiation is shown (optical microscopy 20x; scale bar 200 μm). (B) Percent of viable CTRL and TENO hPL and SF-ASCs at 1, 3, 7 and 14 days (n = 3). Data were expressed as average ± standard deviation of percentage of viable cells. (C) Cell viability of CTRL and TENO hPL and SF-ASCs at 1, 7 and 10 days of differentiation (n = 3). Data were expressed as average ± standard deviation of arbitrary fluorescence units (AFU). * p<0.05, **p<0.01 for TENO versus CTRL cells; # p<0.05, ## p<0–01 for hPL-ASCs vs SF-ASCs.

Fig 3. Gene expression of cell proliferation and embryonic stem cell markers in hPL-ASCs and SF-ASCs.

Evaluation of Ki67, PCNA, NANOG, OCT4, KLF4 gene expression determined by quantitative real-time PCR in CTRL and TENO hPL and SF-ASCs at 1, 3, 7 and 14 days of culture (n = 4). Data were normalized against the expression of the housekeeping GAPDH, GUSB and YWHAZ genes and expressed as relative to the calibrator (CAL). ## p<0.01 for hPL-ASCs vs SF-ASCs.

Fig 4. Gene expression of cell proliferation and embryonic stem cell markers after tenogenic differentiation.

(A) Effect of tenogenic induction on Ki67, PCNA, OCT4, KLF4 and NANOG gene expression in CTRL and TENO hPL and SF-ASCs at 1, 3, 7 and 14 days of differentiation (n = 4). Data were normalized against the expression of the housekeeping GAPDH, GUSB and YWHAZ genes and expressed as relative to the calibrator (CAL). * p<0.05 for TENO vs CTRL cells. (B) Normalized values of hPL-TENO and SF-TENO to their CTRL. Data expressed as average fold increase ± standard deviation compared with the respective CTRL cells (dashed line means equal).

Fig 5. Gene expression of tendon-related markers after tenogenic differentiation.

(A) Effect of tenogenic medium on SCX, TNC and DCN gene expression in hPL and SF at 1, 3, 7 and 14 days of differentiation (n = 4). Data were normalized against the expression of the housekeeping GAPDH, GUSB and YWHAZ genes and expressed as relative to the calibrator (CAL). * p<0.05, **p<0.01 for TENO versus CTRL cells. (B) Effect of serum-free media between hPL-TENO and SF-TENO. Data are expressed as average fold increase ± standard deviation compared with the respective CTRL cells (dashed line set at 1). # p<0.05 for hPL versus SF.

Fig 6. Gene expression of tendon extracellular-related marker after tenogenic differentiation.

(A) Effect of tenogenic medium on COL1A1, COL3A1 and COMP gene expression in hPL and SF at 1, 3, 7 and 14 days of differentiation (n = 4). Data were normalized against the expression of the housekeeping GAPDH, GUSB and YWHAZ genes and expressed as relative to the calibrator (CAL). * p<0.05, **p<0.01 for TENO vs CTRL cells; $ p<0.05 for differences between time-points. (B) Effect of serum-free media between hPL-TENO and SF-TENO. Data are expressed as average fold increase ± standard deviation compared with the respective CTRL cells (dashed line set at 1). # p<0.05 for hPL versus SF.

Fig 7. Gene expression of MMP3, MMP13 and TIMP2 after tenogenic differentiation.

(A) Effect of tenogenic medium on MMP3, MMP13 and TIMP2 gene expression in hPL and SF at 1, 3, 7 and 14 days of differentiation (n = 4). Data were normalized against the expression of the housekeeping GAPDH, GUSB and YWHAZ genes and expressed as relative to the calibrator (CAL). * p<0.05 for TENO vs CTRL cells; $ p<0.05 for differences between time-points. (B) Effect of serum-free media between hPL-TENO and SF-TENO. Data expressed as average fold increase ± standard deviation compared with the respective CTRL cells (dashed line set at 1). # p<0.05 for hPL versus SF.

Fig 8. Scleraxis expression and collagen matrix deposition after tenogenic differentiation.

Upper four panels show representative images of scleraxis expression (green) in CTRL and TENO cells at 3 days of differentiation (the nuclei were stained with DAPI, blue) captured by fluorescence microscope (40x; scale bar 20 μm); four panels on the button show representative images related to the collagen matrix deposition, stained by Sirius Red (10x; scale bar 100 μm), that occurred after 7 days differentiation in hPL-ASCs and SF-ASCs.

Fig 9. Tenomodulin expression on hPL-ASC and SF-ASC surfaces.

Representative histograms of percentage of the subpopulation of hPL-ASCs and SF-ASCs positive (green) and negative (red) for tenomodulin (TNMD) surface expression at 7 and 14 days of culture in CTRL and TENO medium. Data related to three ASC population are expressed as mean ± standard deviation (n = 3). * p<0.05 for TENO versus CTRL cells.