Proteomic characterisation reveals active Wnt-signalling by human multipotent stromal cells as a key regulator of beta cell survival and proliferation

Abstract

Aims/hypothesis Novel strategies to stimulate the expansion of beta cell mass in situ are warranted for diabetes therapy. The aim of this study was to elucidate the secretome of human bone marrow (BM)-derived multipotent stromal cells (MSCs) with documented islet regenerative paracrine function. We hypothesised that regenerative MSCs will secrete a unique combination of protein factors that augment islet regeneration. Methods Human BM-derived MSCs were examined for glucose-lowering capacity after transplantation into streptozotocin-treated NOD/severe combined immunodeficiency (SCID) mice and segregated into samples with regenerative (MSC R ) vs nonregenerative (MSC NR ) capacity. Secreted proteins associated with islet regenerative function were identified using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics. To functionally validate the importance of active Wnt signalling, we stimulated the Wnt-signalling pathway in MSC NR samples during ex vivo expansion using glycogen synthase kinase 3 (GSK3) inhibition (CHIR99201), and the conditioned culture media (CM) generated was tested for the capacity to support cultured human islet cell survival and proliferation in vitro. Results MSC R showed increased secretion of proteins associated with cell growth, matrix remodelling, immunosuppressive and proangiogenic properties. In contrast, MSC NR uniquely secreted proteins known to promote inflammation and negatively regulate angiogenesis. Most notably, MSC R maintained Wnt signalling via Wnt5A/B (~2.5-fold increase) autocrine activity during ex vivo culture, while MSC NR repressed Wnt signalling via Dickkopf-related protein (DKK)1 (~2.5-fold increase) and DKK3 secretion. Inhibition of GSK3 activity in MSC NR samples increased the accumulation of nuclear β-catenin and generated CM that augmented beta cell survival (13% increases) and proliferation when exposed to cultured human islets. Conclusions/interpretation Maintenance of active Wnt signalling within human MSCs promotes the secretion of matricellular and proangiogenic proteins that formulate a niche for islet regeneration.

Characterization of a Vimentinhigh/Nestinhigh proteome and tissue regenerative secretome generated by human pancreas-derived mesenchymal stromal cells

“…Digested peptides were resuspended in 0.1% formic acid prior to label‐free LC/MS‐MS. Approximately 1 μg of each sample was injected onto a Waters M‐Class nanoAcquity HPLC system (Waters, Milford, Massachusetts) coupled to a Q Exactive Plus Orbitrap (Thermo Fisher Scientific, Waltham, Massachusetts) operating in positive mode, as previously described . Full MS parameters are outlined in the Table S3.…”

Human Multipotent Stromal Cell Secreted Effectors Accelerate Islet Regeneration

“…We have recently established active Wnt‐signaling as a characteristic of islet regenerative hMSC . To investigate whether pharmacological modulation of Wnt‐signaling during CM generation could further improve glycemic control after iPan CM‐injection, we sought to activate or inhibit basal Wnt‐signaling in hMSC using small molecules CHIR99201 or IWR‐1, respectively .…”

“…In our previous studies on islet regeneration, transplantation of human bone marrow‐derived multipotent stromal cells (hMSC) induced the emergence of small, recipient‐derived islet‐like structures associated with the ductal epithelial niche . Detailed proteomic analysis of secretory factors deposited by hMSC revealed Wnt‐signaling as a central pathway in islet regenerative hMSC . Indeed, active Wnt‐signaling in hMSC generated conditioned media (CM) that increased β‐cell survival and proliferation in cultured human islets.…”

Ultrafiltration Segregates Tissue Regenerative Stimuli Harboured Within and Independent of Extracellular Vesicles

“…A collection of studies has demonstrated the potential of utilizing MSC as EV-generating biofactories and engineering the secretome of MSC to meet therapeutic needs 37,41,67 . Our previous studies have supported this progression of thought and we envision the creation of a cell-free biotherapeutics 'tailored' to combat a given pathology.…”

“…Methodological details of protein precipitation, digestion, liquid chromatography-tandem mass spectrometry, and proteomic data base analysis are described thoroughly in previous studies 13,36,37 . LC-MS/MS settings are outlined in Supplemental Table 2.…”