Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum

Abstract

Introduction: Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several mesenchymal lineages, classically derived from bone marrow (BM) but potentially from umbilical cord blood (UCB). Although they are becoming a good tool for regenerative medicine, they usually need to be expanded in fetal bovine serum (FBS)-supplemented media. Human platelet lysate (HPL) has recently been proposed as substitute for safety reasons, but it is not yet clear how this supplement influences the properties of expanded MSCs.

Methods: In the present study, we compared the effect of various media combining autologous HPL with or without FBS on phenotypic, proliferative and functional (differentiation, cytokine secretion profile) characteristics of human BM-derived MSCs.

Results: Despite less expression of adipogenic and osteogenic markers, MSCs cultured in HPL-supplemented media fully differentiated along osteoblastic, adipogenic, chondrogenic and vascular smooth muscle lineages. The analyses of particular specific proteins expressed during osteogenic differentiation (calcium-sensing receptor (CaSR) and parathormone receptor (PTHR)) showed their decrease at D0 before any induction for MSC cultured with HPL mostly at high percentage (10%HPL). The cytokine dosage showed a clear increase of proliferation capacity and interleukin (IL)-6 and IL-8 secretion.

Conclusions: This study shows that MSCs can be expanded in media supplemented with HPL that can totally replace FBS. HPL-supplemented media not only preserves their phenotype as well as their differentiation capacity, but also shortens culture time by increasing their growth rate.

Figure 1

Morphological characterization of bone marrow (BM)-derived mesenchymal stem cells (MSCs) cultured in different media at passage 2 (P2). The media were M1 (10% FBS + FGF2), M2 (10% FBS + 5% HPL), M3 (10% HPL), and M4 (5% HPL). Bar represents 100 μm. FBS, fetal bovine serum; FGF2, fibroblast growth factor 2; HPL, human platelet lysate. Representative results from 13 experiments are shown.

Figure 2

Representative flow cytometry analysis of BM-derived MSCs cultured in different expansion media. Comparison of membrane antigen expression of MSCs cultured at passage 2 (P2) in four different media: M1 (10% FBS + FGF2) (n = 4), M2 (10% FBS + 5% HPL) (n = 5), M3 (10% HPL) (n = 6), and M4 (5% HPL) (n = 5). The histogram plots represent flow cytometry analysis of the cells with directly labeled monoclonal antibodies (histograms colored inside) or exposed to isotype-matched non-immune directly labeled immunoglobulins (IgG1FITC, IgG1PE, and IgG1PerCP). MSCs were negative for hematopoietic markers CD45, CD34, CD133, and CD14 (A) but were positive for CD90, CD73, CD105, and CD106 and weakly positive for CD49e (B). (A) Hematopoietic and immaturity markers. (B) Stromal cell markers.

Figure 3

Osteogenic, chondrogenic, adipogenic, and vascular smooth muscular differentiation capacity assessed by staining of BM-derived MSCs cultured in different expansion media. The media are M1 (10% FBS + FGF2), M2 (10% FBS + 5% HPL), M3 (10% HPL), and M4 (5% HPL). Results are of one representative experiment. A, adipogenic differentiation; ASMA, alpha smooth muscle actin; C, chondrogenic differentiation; D0, before differentiation; D14, after 14 days of differentiation; O, osteogenic differentiation; VSM, vascular smooth muscle.

Figure 4

mRNA expression by BM-derived MSCs cultured in different expansion media. Reverse transcription-polymerase chain reaction (RT-PCR) for positive control GAPDH (A), osteogenic (B), adipogenic (C), and vascular smooth muscle (D) gene expression of BM-derived MSCs cultured in four media: M1 (10% FBS + FGF2), M2 (10% FBS + 5% HPL), M3 (10% HPL), and M4 (5% HPL). Total RNA was extracted from undifferentiated and differentiated MSCs previously cultured in four expansion media (M1, M2, M3, and M4). The cDNA obtained was used for PCR assays to evaluate the expression of the different genes studied. One representative experiment out of four experiments is shown. For GAPDH gene expression (A): 1, at D0 before any differentiation; 2, after osteogenic differentiation (D14); 3, after adipogenic differentiation (D14); 4, after vascular smooth muscle differentiation (D14). For osteogenic (RUNX2 and ALP) (B), adipogenic (PPARγ and LPL) (C), and vascular smooth muscle (ASMA) (D) gene expression: 1, D0 before any differentiation; 2, after differentiation induction (D14). ALP, alkaline phosphatase; ASMA, alpha-smooth muscle actin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LPL, lipoprotein lipase; PPARγ, peroxisome proliferator-activated receptor-γ; RUNX2, runt-related transcription factor 2.

Figure 5

Western blot analysis showing differentiation markers of osteogenic, vascular smooth muscle, and adipogenic lineages expressed by BM-derived MSCs cultured in different expansion media. The media are M1 (10% FBS + FGF2), M2 (10% FBS + 5% HPL), M3 (10% HPL), and M4 (5% HPL). The experiments were performed in triplicate, and one representative blot is shown here. Beta-actin expression was used as positive control. Osteogenic proteins studied are calcium-sensing receptor (CaSR) and parathormone receptor (PTHR). Adipogenic protein studied is Leptin. Vascular smooth muscle proteins studied are smooth muscle 22alpha (SM22α) and alpha-smooth muscle actin (ASMA). D0, day 0 (before induction); D14, day 14 (after induction).

Figure 6

Effect of expansion media on colony-forming unit-fibroblast (CFU-F) counts per 10⁶ bone marrow mononuclear cells. The media are M1 (10% FBS + FGF2), M2 (10% FBS + 5% HPL), M3 (10% HPL), and M4 (5% HPL). Data shown represent mean ± standard error of the mean from the indicated number of experiments. ^#No significant difference; P > 0.05.

Figure 7

Population doubling time (PDT) in days of adherent cells cultured in different expansion media between passage 1 (P1) and passage 2 (P2). The media are M1 (10% FBS + FGF2), M2 (10% FBS + 5% HPL), M3 (10% HPL), and M4 (5% HPL). Data shown represent mean ± standard error of the mean from the indicated number of experiments. *Significant difference; P < 0.0007. ^#No significant difference; P > 0.05.

Figure 8

Cytokine expression profile of mesenchymal stem cells (MSCs) cultured in different expansion media. Cytokine concentrations (expressed in picograms per 10⁶ cells) were evaluated in MSC supernatants at passage 2 (P2) after culture in M1 (10% FBS + FGF2) (n = 4), M2 (10% FBS + 5% HPL) (n = 6), M3 (10% HPL) (n = 4), and M4 (5% HPL) (n = 3). (A) IL-6 (*P = 005; **P = 0.01). (B) IL-8 (*P < 0.05). (C) VEGF (*P < 0.01). IL, interleukin; VEGF, vascular endothelial growth factor.