An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors

Dental Anomalies in Ciliopathies: Lessons from Patients with <i>BBS2</i>, <i>BBS7,</i> and <i>EVC2</i> Mutations

Objective: To investigate dental anomalies and the molecular etiology of a patient with Ellis–van Creveld syndrome and two patients with Bardet–Biedl syndrome, two examples of ciliopathies. Patients and Methods: Clinical examination, radiographic evaluation, whole exome sequencing, and Sanger direct sequencing were performed. Results: Patient 1 had Ellis–van Creveld syndrome with delayed dental development or tooth agenesis, and multiple frenula, the feature found only in patients with mutations in ciliary genes. A novel homozygous mutation in EVC2 (c.703G>C; p.Ala235Pro) was identified. Patient 2 had Bardet–Biedl syndrome with a homozygous frameshift mutation (c.389_390delAC; p.Asn130ThrfsTer4) in BBS7. Patient 3 had Bardet–Biedl syndrome and carried a heterozygous mutation (c.389_390delAC; p.Asn130ThrfsTer4) in BBS7 and a homozygous mutation in BBS2 (c.209G>A; p.Ser70Asn). Her clinical findings included global developmental delay, disproportionate short stature, myopia, retinitis pigmentosa, obesity, pyometra with vaginal atresia, bilateral hydronephrosis with ureteropelvic junction obstruction, bilateral genu valgus, post-axial polydactyly feet, and small and thin fingernails and toenails, tooth agenesis, microdontia, taurodontism, and impaired dentin formation. Conclusions: EVC2, BBS2, and BBS7 mutations found in our patients were implicated in malformation syndromes with dental anomalies including tooth agenesis, microdontia, taurodontism, and impaired dentin formation

CID induced osteoclastogenesis in RAW264.7+iRANK cells is OPG-independent.

<p>TRAP-positive multinucleated cells (MuNC) after treatment of RAW264.7+iRANK cells with 10 nM AP20187 (A) or RAW264.7 cells with 1 nM RANKL (B) in the presence of increasing concentrations of OPG. TRAP-positive multinucleated cells (MuNC) were counted over 4 high power fields of view and averaged over 3 wells. *p<0.05 compared to 0 nM OPG.</p

Schematic representation of CID-inducible cytoplasmic RANK (iRANK) lentiviral construct and Western blot detecting construct.

<p>A) LTR = long terminal repeat; RRE = rev response element; cPPT = central polypurine tract; EF1a = elongation factor 1-Alpha; EGFP = green fluorescent protein; I = IRES; M = myristoylation; F36V = FKBP12; F36V' = modified FKBP12; cRANK = cytoplasmic domain of RANK; WPRE = WHP posttranscriptional regulatory element. B) Western blot probed with an antibody for FKBP12 showing overexpression of the iRANK construct in RAW264.7+iRANK cells migrating around 70 kDa. Cells transduced just with the F36V' domains (RAW264.7+F2) show a band around 30 kDa. No bands appear in the untransduced RAW264.7 cells.</p

Cell survival study.

<p>RAW264.7 cells and RAW264.7+iRANK cells were treated with either RANKL (40 ng/ml) or AP20187 (50 nM) for 4 days to allow osteoclasts to form. The supplemented media was removed, and cells were cultured for additional 0, 3 or 5 days in the presence (A) or absence (B) of inducers, and the number of TRAP-positive multinucleated cells (MuNC) per well was counted and averaged over 4 wells. *p<0.05.</p

CID-responsiveness of the iRANK construct.

<p>RAW264.7+iRANK cells were cultured in medium containing vehicle (EtOH), RANKL, or 0.1–50 nM AP20187 for 4 days and the cells were stained for TRAP. RANKL and AP20187 induced multinucleated TRAP-positive cells were observed (purple staining) (scale bars = 100 µm).</p

CID induced osteoclasts resorbed a three-dimensional mineralized substrate.

<p>(A) Mineralized fibrin scaffolds were seeded with control RAW264.7 cells (white bars) or iRANK transduced RAW264.7 cells (black bars), or no cells (hatched bar). The scaffolds were weighed at various time points (days 2, 5, 8 and 11). The scaffolds without cells were incubated in media for 11 days. Mass loss was calculated by subtracting the final mass from the initial mass. *p <0.05. (B) RAW264.7+iRANK were cultured with AP20187 in the fibrin scaffolds for 8 days. H&E staining (left panel) and adjacent TRAP staining (right panel) indicate the differentiation of osteoclasts within the scaffolds (arrows) (scale bars = 50 µm).</p

CID induced osteoclasts resorbed a two-dimensional mineralized substrate.

<p>(A) RAW264.7+iRANK cells were treated with either RANKL (100 ng/ml) or AP20187 (100 nM) on Osteologic discs. After 10 days, resorption lacunae were visualized by von Kossa staining. (B) The percent resorbed area per disc was measured and analyzed using ImageJ. Three experiments were averaged. Y-axis shows the % resorption per disc. *p<0.05.</p