Transient Canonical Wnt Stimulation Enriches Human Bone Marrow Mononuclear Cell Isolates for Osteoprogenitors

Abstract

Activation of the canonical Wnt signaling pathway is an attractive anabolic therapeutic strategy for bone. Emerging data suggest that activation of the Wnt signaling pathway promotes bone mineral accrual in osteoporotic patients. The effect of Wnt stimulation in fracture healing is less clear as Wnt signaling has both stimulatory and inhibitory effects on osteogenesis. Here, we tested the hypothesis that transient Wnt stimulation promotes the expansion and osteogenesis of a Wnt‐responsive stem cell population present in human bone marrow. Bone marrow mononuclear cells (BMMNCs) were isolated from patients undergoing hip arthroplasty and exposed to Wnt3A protein. The effect of Wnt pathway stimulation was determined by measuring the frequency of stem cells within the BMMNC populations by fluorescence‐activated cell sorting and colony forming unit fibroblast (CFU‐F) assays, before determining their osteogenic capacity in in vitro differentiation experiments. We found that putative skeletal stem cells in BMMNC isolates exhibited elevated Wnt pathway activity compared with the population as whole. Wnt stimulation resulted in an increase in the frequency of skeletal stem cells marked by the STRO‐1bright/Glycophorin A− phenotype. Osteogenesis was elevated in stromal cell populations arising from BMMNCs transiently stimulated by Wnt3A protein, but sustained stimulation inhibited osteogenesis in a concentration‐dependent manner. These results demonstrate that Wnt stimulation could be used as a therapeutic approach by transient targeting of stem cell populations during early fracture healing, but that inappropriate stimulation may prevent osteogenesis. Stem Cells 2016;34:418–430

PEGylated liposomes associate with Wnt3A protein and expand putative stem cells in human bone marrow populations

“…Bone marrow mononuclear cells (BMMNCs) were isolated from femoral samples obtained from hematologically normal individuals undergoing hip replacement surgery at Southampton General Hospital or Spire Hospital Southampton, with the approval of the appropriate Local Research Ethics Committee (LREC 194/99/1), as previously described [33]. Cells were maintained in basal medium (α-MEM containing 10% FBS and 100 μg/ml penicillin/streptomycin) or osteogenic medium (basal medium supplemented with 100 μM ascorbate-2-phosphate, 10 nM dexamethasone and 5 mM β-glycerophosphate), with the exception of when exposed to Wnt3A protein (5036-WN, R&D Systems, McKinley, MN, USA) or liposomes, where serum concentration in the media was 5%.…”

“…Uptake of DiO-labeled or AF647-labeled liposomes was studied on fresh bone marrow isolates after 3 h of incubation in suspension with approximately 10 6 liposomes/cell. Following this, immunostaining for CD14, STRO-1 and Glycophorin A (GPA) was carried out as described previously [25]. Cells were assayed on an FACS Canto II cytometer (BD Biosciences, NJ, USA) or Image Stream (Amnis, WA, USA).…”

“…All flow cytometry experiments were analyzed using FlowJo v10 software. Data were acquired and analyzed as described previously [25].…”

“…We have previously shown that the 'monocyte' gated cell population includes stem and progenitor cells, which are the intended cellular targets of Wnt activation in bone marrow and at injury sites [25]. To determine the relative association of liposomes with these different cell populations we immunostained dyelabeled liposome-exposed cells for the cell surface antigens CD14, STRO-1 and GPA.…”

“…Several humanized monoclonal antibodies which bind to and inactivate inhibitors of Wnt signaling, such as DKK1 [20] or SOST [21] are currently in clinical trials, with recent data suggesting efficacy in promoting bone formation in osteoporotic patients [22,23]. The efficacy of these monoclonal antibodies in fracture healing is less clear, however, which may be related to their prolonged in vivo activity (with half-lives of > 4 weeks [24]), and the fact that it is known that elevated Wnt signaling can have both positive and negative effects depending on the timing of delivery [25]. In these cases, delivery of short-acting molecules such as Wnt proteins rather than antibodies will provide much better control of temporal activation of signaling, as well as the potential of selective localization of the molecule to the injury site.…”