Formation of cartilage and synovial tissue by human gingival stem cells

Abstract

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis.

FIG. 1.

Characterization of gingival-connective-tissue-derived cells. Analysis of growth kinetics showed an increase in cell numbers with a constant doubling time over 3 weeks (A). When incubated in the appropriate differentiation medium, cells showed osteogenic-like differentiation with deposition of mineralized matrix (B, alizarin red S staining) and differentiation into adipocyte-like cells (C, oil red O staining). Osteogenic differentiation at low (P≤3) and high passages (P≥6) was further tested with three HGSC lines from three different donors and isolated by the explant or enzymatic digestion method. All low-passage cell lines showed similar osteogenic response (D), while cells at high passage showed a decreased differentiation potential. Gene expression analysis of key genes involved in osteodifferentiation was also followed over time in osteogenic medium. Results from real-time PCR showed that the master genes were upregulated during this process and followed classical kinetics over time (E). The results show mRNA expression relative to GAPDH as housekeeping gene according to the 2ΔΔCt method. Each QPCR was performed in triplicate. Results show mean±standard deviation from four parallel cell lines (*P<0.05; **P<0.01). NS, no statistically significant differences. HGSCs, human gingival stem cells; QPCR, quantitative polymerase chain reaction. Color images available online at www.liebertpub.com/scd

FIG. 2.

Analysis of chondrogenic differentiation. After 21 days of micromass culture in chondrogenic induction medium, cultures generated cartilage-like tissue. (A) Alcian-blue-stained (pH=1) histological sections from the cultures that displayed a cartilage-like ECM particularly at the center of the micromass. Positive area is outlined by the arrow. (B) Picrosirius red staining showed collagen-rich (red) areas in both periphery and center of the culture. (C) Observation under polarized light of the periphery of the culture revealed collagen fibers that were aligned parallel to the micromass surface, and showed a more intense green color, indicating a difference in the diameter of the collagens fibers in the peripheral and central areas. Cultures showed also positive immunostaining for type II collagen (D), while the staining for type X collagen was mostly negative, with some areas closest to the outer surface showing a weak staining (E). (F) Negative control staining without primary antibody showed no positive immunoreaction. Representative images from three experiments performed with three parallel HGSC lines are shown. Magnification bar: (B, C) 200 μm; (D, E) 100 μm; and (F, G) 30 μm. (G) In the micromass cultures, expression of type II collagen, aggrecan, and SOX9 mRNA was strongly upregulated after 14 days of culture in chondrogenic medium, and was then downregulated after 1 week under hypoxia (corresponding to day 28 after initiation of the experiment). Results show QPCR analysis of mRNA expression relative to GAPDH (2ΔΔCt method). ECM, extracellular matrix. Color images available online at www.liebertpub.com/scd

FIG. 3.

Analysis of chondrocyte maturation. After 3 weeks of chondrogenic differentiation, followed by 2 weeks of hypoxic conditions, chondrogenic cells acquired a hypertrophic phenotype. Histological sections stained with picrosirius red showed that cells inside the micromass cultures were surrounded by a mature cartilage-like fibrillar collagen network (A) and (D) associated with a toluidine-blue-positive ECM (B). (C–F) Examination with higher magnification revealed a typical organization of mature cartilage. Many cells displayed morphology reminiscent of hypertrophic chondrocytes when examined by SEM analysis (C), and were surrounded by a collagen network composed of type II (E) and type X collagens (F). However, no mineralization of the matrix was noted (data not shown). Representative images from three experiments performed with three parallel HGSC lines are shown. Magnification bar: (B) 200 μm; (C) 100 μm; and (E–G) 20 μm. (G) QPCR analysis showed an upregulation of key genes associated or involved in chondrocyte hypertrophy after 1 week of hypoxia (corresponding to day 28 after initiation of the experiment). Results show mRNA expression relative to GAPDH (2ΔΔCt method). IHH, Indian Hedgehog; COL10A1, collagen type X alpha 1; VEGFA, vascular endothelial growth factor α; MMP13, matrix metalloproteinase 13; RUNX2, Runt-related transcription factor 2; COL1A1, collagen type I alpha 1. Results show mean of relative mRNA expression from two parallel HGSC lines. SEM, scanning electron microscopy. Color images available online at www.liebertpub.com/scd

FIG. 4.

Fibroblast-like synoviocyte differentiation. (A–D) SEM analysis of the histological sections from the micromass cultures at day 21 showed a distinct peripheral lining cell layer that was formed on the outer surface of the micromass culture. Hematoxylin staining (E) and tubulin immunostaining (F) showed that the cells in the peripheral layer were polarized and oriented perpendicularly to the underlying layer that was aligned more parallel to the outer surface of the micromass culture. Immunostaining showed that the peripheral cell lining was positive for cadherin-11 (CDH-11) (G, J) and hyaluronic acid (HA) (H, K). Inhibition of cadherin-11 by siRNA transfection showed an inhibition of the formation of the peripheral cell lining (I, L). Representative images from experiments with three parallel HGSC lines are shown. Magnification bar: (E) 20 μm; (F) 50 μm; (G) 100 μm; and (H–J) 50 μm. Color images available online at www.liebertpub.com/scd

FIG. 5.

SOX9 regulates morphogenesis of cartilage-like micromass cultures. (A) Real-time PCR of SOX9 mRNA expression in SOX9 siRNA (siSOX9)– and control siRNA (mock)–transfected cells 1 day after siRNA transfection showed a downregulation of SOX9 expression. Results show mean and standard deviation from two experiments performed with two parallel HGSC lines. iSOX9a, first duplex against SOX9; iSOX9b, second duplex; iSOX9c, third duplex; N, cells treated without siRNA; Scr, transfection with a scrambled iSOX9a siRNA; iSOX9mix, cells were transfected with all three SOX9 siRNAs (iSOX9a, iSOX9b, and iSOX9c) at the same time. (B) Hematoxylin-eosin staining shows the presence of an early compartmentalization of micromass cultures at day 7 with the presence of a cell-free area at the center. (C) Staining of apoptotic cells by TUNEL assay at day 7. Control cultures showed areas that contained abundantly apoptotic cells especially at the center. However, apoptosis was not evident in SOX9-siRNA samples (B, C). (D) Analysis of cell proliferation by Ki67 immunostaining at day 7 showed that proliferating cells were present in the peripheral area for both mock and siSOX9 micromass cultures. They were also present in the center of SOX9-siRNA cultures, but missing from the control cultures. (E, F) Immunostaining of α-SMA (red fluorescence) at day 14 showed immunoreactivity only in SOX9-siRNA-treated samples. Blue color indicates nuclear staining with DAPI. (G, H) Toluidine blue staining shows the presence of round cells in SOX9-siRNA micromass cultures, but not in mock conditions. Toluidine blue metachromasia (purple staining) was mainly observed in SOX9-siRNA conditions. Magnification bar: (B, D, E, F) 100 μm; (C, G, H) 50 μm. The siRNA experiment was performed twice on two different cell strains. Color images available online at www.liebertpub.com/scd