PS1/γ‐Secretase‐Mediated Cadherin Cleavage Induces β‐Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

Abstract

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

The Manufacture of GMP-Grade Bone Marrow Stromal Cells with Validated In Vivo Bone-Forming Potential in an Orthopedic Clinical Center in Brazil

“…The cumulative PD (cPD) was calculated by adding PD 1 and PD 2 . The doubling time (dT) was determined by dividing the time in days required for total cell expansion by the cumulative PD (dT = Δ t /cPD) [51].…”

“…BMSCs were plated at a density of 1.3 × 10 4 cells/cm 2 in triplicate in α -MEM supplemented with 20% FBS and allowed to grow until 100% confluence was reached. Osteogenic differentiation was induced by incubation with α -MEM containing 10 mM β -glycerophosphate, 5 μ g/mL ascorbic acid 2-phosphate, and 10 −6 M dexamethasone (all from Sigma) supplemented with 20% FBS for 21 days, and the medium was changed every 3 days [51]. To assess the mineralization, monolayers were stained with Von Kossa.…”

“…BMSCs were plated as described above. After reaching 100% confluence, the cells were incubated with α -MEM containing 0.5 mM isobutylmethylxanthine, 200 mM indomethacin, 10 −6 M dexamethasone (all from Sigma), and 10 mM insulin (Humulin®, Lilly, São Paulo, SP, BRA) supplemented with 20% FBS for 21 days, and the medium was changed every 3 days [51]. To confirm the lipidic composition of the cell vacuoles, BMSCs were fixed in 4% paraformaldehyde for 10 minutes at room temperature, washed with propylene glycol PA (Vetec Quimica, Rio de Janeiro, RJ, BRA), and incubated with 0.5% Oil Red O solution (Sigma) in propylene glycol for 20 minutes.…”

Atrophic nonunion stromal cells form bone and recreate the bone marrow environment in vivo

“…BMSC were isolated as previously described, with minor modifications. [ 21 , 22 ] Briefly, iliac crest trabecular bone fragments were mechanically triturated; the bone marrow was resuspended and homogenized in PBS, and then allowed to stand for 30 seconds to enable sedimentation of bone spicules. The supernatant was collected and centrifuged at 300× g for 10 minutes.…”

“…Osteogenic differentiation was induced by incubation with DMEM 10% FBS and antibiotics, containing 10 mM β-glicerophosphate, 5 μg/mL ascorbic acid 2-phosphate, and 10 −6 M dexamethasone (all from Sigma) for 21 days, with medium change at every 3 days. [ 22 ] After this period, cell monolayers were fixed with 4% paraformaldehyde for 10 minutes at room temperature and incubated with a 2% silver nitrate aqueous solution for 1 hour, protected from light. Cells were washed 5 times with distilled water and exposed to UV light for 10 minutes.…”