Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells

Abstract

Background: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells.

Methodology/principal findings: In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed.

Conclusion/significance: Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.

Figure 1. Cumulative population doublings of MSC after culture in FBS, HPLF or HPLO.

MSC were cultured in culture media supplemented with 10% of FBS, platelet lysate from fresh platelet concentrates (HPLF) or lysate from expired platelet concentrates (HPLO). Population doubling assay was performed at the end of every passage for a total of six passages (P1–P6, n = 3). MSC cultured in either HPLF or HPLO consistently had higher numbers of population doublings at the end of every passage compared to MSC cultured in FBS. By the end of the sixth passage cumulative population doublings were 13.77±0.77 CPD for HPLO, 12.77±0.12 CPD for HPLF and 9.50±1.14 CPD for FBS supplemented cultures, respectively.

Figure 2. Mesenchymal stem cell morphology during expansion and differentiation.

MSC were stained with crystal violet after expansion in media containing 10% of FBS, HPLF or HPLO for three passages (A–C). MSC cultured in HPLF or HPLO developed more spindle shaped morphology than cells cultured in FBS, proliferated faster and left circular areas containing no cells between them. Differentiation towards osteoblasts, adipocytes and chondrocytes was initiated after expansion in the three supplements. After 28 days of osteogenic differentiation mineralization in the culture could be visualized with a Von Kossa staining (D–F). Successful adipogenic differentiation was confirmed with Oil red O staining (G–I) and after 28 days of chondrogenic pellet cultures, the pellets were sectioned and stained with toludine blue staining (J–L). Pictures representative of three experiments.

Figure 3. Reduction of mononuclear cell proliferation during co-culture with MSC.

Mononuclear cells (MNC) were stimulated with phytohemaglutinin (PHA) and then cultured with or without MSC that had been previously expanded in media containing 10% of FBS, HPLF or HPLO. MNC proliferation was significantly less when co-cultured with MSC than if cultured on their own (p≤0.05, n = 3). The reduced proliferation was seen irrespective of the culture conditions the MSC had previously been exposed to. * = p≤0.05.

Figure 4. Increased enzymatic activity of alkaline phosphatase during osteogenic differentiation.

MSC that had previously been cultured in media containing 10% of FBS, HPLF or HPLO underwent osteogenic differentiation. ALP activity was evaluated after 7 and 14 days of differentiation. ALP activity increased significantly from day 7 to day 14 independent of the type of culture conditions the MSC had been exposed to prior to osteogenic differentiation (p≤0.05, n = 3). * = p≤0.05.

Figure 5. Expression of osteogenic marker genes during osteogenic differentiation of MSC.

Osteogenic differentiation was initiated after MSC expansion in 10% of FBS, HPLF or HPLO. Expression of osteogenic marker genes was evaluated after 7 and 21 days of osteogenic differentiation and is represented with graphs for each gene (A–C, n = 3) and a heat-map (D) where green shades represent lower levels of expression and red shades higher levels of expression. ALP was evenly expressed throughout the differentiation (A,D) while the expression of SPP1 increased from day 7 to day 21 (B,D), irrespective of the culture supplements the MSC had previously been exposed to (p≤0.01). Expression of RUNX2 decreased from day 7 to day 21 for all cultures (C–D), still expression of RUNX2 in cultures originating from HPLO treated MSC was higher at both time-points compared to FBS and also at day 21 for HPLF compared to FBS (p≤0.05). * = p≤0.05, ** = p≤0.01.