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. 2016 Aug;22(15-16):1036-46.
doi: 10.1089/ten.TEA.2016.0102.

Fibroblast Growth Factor Ligand Dependent Proliferation and Chondrogenic Differentiation of Synovium-Derived Stem Cells and Concomitant Adaptation of Wnt/Mitogen-Activated Protein Kinase Signals

Tyler Pizzute  1   2 , Jingting Li  1   2 , Ying Zhang  1   3 , Mary E Davis  4 , Ming Pei  1   2   3
Affiliations

Fibroblast Growth Factor Ligand Dependent Proliferation and Chondrogenic Differentiation of Synovium-Derived Stem Cells and Concomitant Adaptation of Wnt/Mitogen-Activated Protein Kinase Signals

Tyler Pizzute et al. Tissue Eng Part A. 2016 Aug.

Abstract

Cell expansion techniques commonly utilize exogenous factors to increase cell proliferation and create a larger cell population for use in cell-based therapies. One strategy for cartilage regenerative therapies is autologous stem cell expansion and fibroblast growth factor (FGF) supplementation during cell expansion, particularly FGF-2. However, it is unknown whether FGF-10, another FGF implicated in limb and skeletal development, can elicit the same rejuvenation responses in terms of proliferation and differentiation of human synovium-derived stem cells (SDSCs). In this study, we expanded SDSCs in either FGF-2 or FGF-10 for 7 days; a control group had no treatment. FGF-2 and FGF-10 supplementation was also exclusively tested during the differentiation phase. Expanded SDSCs were evaluated for their ability to successfully engage in chondrogenic and osteogenic differentiation. We found that FGF-2 supplementation during proliferation, but not differentiation, was able to increase glycosaminoglycan deposition, pellet size, and chondrogenic gene expression following chondrogenic induction, as well as increased calcium deposition, alkaline phosphatase activity, and expression of vital osteogenic differentiation genes following osteogenic induction. FGF-10 did not elicit a similar preconditioning effect. We also observed changes of both Wnt signals and mitogen-activated protein kinase expression during SDSC chondrogenesis, which occurred in a manner dependent upon the supplementation phase of FGF-2 administration. These results indicated that FGF-2, but not FGF-10, may be supplemented during stem cell expansion to prime cells for successful chondrogenesis and osteogenesis.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Gene expression of FGF ligands in cell expansion phase and chondrogenic markers in chondrogenic induction phase following the dECM pretreatment. The study design was detailed previously. Briefly, human SDSCs were expanded on either dECM or Plastic for one passage followed by a 2-week chondrogenic induction. Microarray analysis was used to evaluate FGF ligand genes in expanded cells (A) and chondrogenic marker genes in differentiated cells (B). The raw data were uploaded into Partek (St. Louis, MO) software for initial analysis. After raw intensity was background-subtracted, robust multiarray analysis was normalized, log transformed, and fold changes were determined. Heatmap.2 in R was used to show the effects on FGF ligand and chondrogenic genes, annotated in Ingenuity Pathway Analysis (IPA, Redwood City, CA) as affecting cell proliferation and chondrogenic differentiation, respectively. dECM expanded cells were referred to as Ec, while Plastic expanded cells were referred to as Pc (A). The pellets from dECM expanded cells were referred to as Ep, while those from Plastic expanded cells were referred to as Pp (B). FGF, fibroblast growth factor; dECM, decellularized extracellular matrix; SDSC, synovium-derived stem cell. Color images available online at www.liebertpub.com/tea
<b>FIG. 2.</b>
FIG. 2.
FGF ligand mediated human SDSC proliferation. (A) Experimental design. Five groups in total: FGF-2 and FGF-10 were only included in a 7-day cell expansion phase (F2P and F10P, respectively) or in a 21-day differentiation phase (F2D and F10D, respectively) with non-FGF treatment as a control (CNTL). (B) Flow cytometry was used to measure proliferation (prolif.) index of expanded SDSCs. (C) Cell number was counted ( × 106) after a 7-day cell expansion on 175 cm2 flasks (n = 6). (D). Flow cytometry was used to measure both percentage and median fluorescence intensity of mesenchymal stem cell surface markers (CD29, CD90, CD105, and SSEA4) of expanded SDSCs. SSEA4, stage-specific embryonic antigen 4. Color images available online at www.liebertpub.com/tea
<b>FIG. 3.</b>
FIG. 3.
FGF ligand mediated senescence and differentiation-related gene expression in expanded SDSCs. TaqMan® real-time PCR was used to quantify mRNA levels of senescent genes (A): P16, P21, and TP53, chondrogenic genes (B): SOX9, ACAN, and COL2A1, and osteogenic genes (C): RUNX2, SP7, SPP1, IBSP, and BGLAP. Data are shown as average ± SD for n = 4. Groups not connected by the same letter are significantly different (p < 0.05). PCR, polymerase chain reaction; SD, standard deviation; SOX9, SRY (sex determining region Y)-box 9; ACAN, aggrecan; COL2A1, type II collagen; RUNX2, Runt-related transcription factor 2; SP7, Osterix; SPP1, secreted phosphoprotein 1; IBSP, bone sialoprotein; BGLAP, bone gamma-carboxyglutamate (Gla) protein.
<b>FIG. 4.</b>
FIG. 4.
FGF ligand mediated SDSC chondrogenic differentiation. After a 21-day chondrogenic induction, SDSC pellets were evaluated using Alcian blue staining for sulfated GAGs and immunohistochemical staining for Col2 (A), biochemical analysis for both DNA and GAG amounts in a pellet (B), and TaqMan real-time PCR for mRNA levels of chondrogenic genes (C): COL1A1, COL2A1, and ACAN and hypertrophic genes (D): COL10A1 and ALP. Data are shown as average ± SD for n = 4. Groups not connected by the same letter are significantly different (p < 0.05). GAG, glycosaminoglycan; Col2, type II collagen; ALP, alkaline phosphatase. Color images available online at www.liebertpub.com/tea
<b>FIG. 5.</b>
FIG. 5.
FGF ligand mediated Wnt and MAPK signal changes in SDSC chondrogenesis. During cell expansion (A–C) and chondrogenic induction (D–F), Wnt signals were evaluated using both TaqMan real-time PCR (A, D) and Western blot (B, E), while MAPK signals were evaluated using Western blot (C, F). GAPDH was used as an internal control. Data are shown as average ± SD for n = 4. Groups not connected by the same letter are significantly different (p < 0.05). MAPK, mitogen-activated protein kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
<b>FIG. 6.</b>
FIG. 6.
FGF ligand mediated SDSC osteogenic differentiation. After a 21-day osteogenic induction, both ARS (A) and ALP staining (C) were used to evaluate osteogenic differentiation. ImageJ software was used to semiquantify the density of staining (B, D, respectively). Data are shown as average ± SD for n = 4. Groups not connected by the same letter are significantly different (p < 0.05). ARS, Alizarin Red S. Color images available online at www.liebertpub.com/tea
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