Human Multipotent Stromal Cell Secreted Effectors Accelerate Islet Regeneration

Abstract

Human multipotent stromal cells (hMSC) can induce islet regeneration after transplantation via the secretion of proteins that establish an islet regenerative niche. However, the identity of hMSCsecreted signals and the mechanisms by which pancreatic islet regeneration is induced remain unknown. Recently, mammalian pancreatic α-cells have been shown to possess considerable plasticity, and differentiate into β-like cells after near complete β-cell loss or overexpression of key transcriptional regulators. These studies have generated new excitement that islet regeneration during diabetes may be possible if we can identify clinically applicable stimuli to modulate these key regulatory pathways. Herein, we demonstrate that intrapancreatic-injection of concentrated hMSC-conditioned media (CM) stimulated islet regeneration without requiring cell transfer. hMSC CM-injection significantly reduced hyperglycemia, increased circulating serum insulin concentration, and improved glucose tolerance in streptozotocin-treated mice. The rate and extent of endogenous β-cell mass recovery was dependent on total protein dose administered and was further augmented by the activation of Wnt-signaling using GSK3-inhibition during CM generation. Intrapancreatic hMSC CM-injection immediately set in motion a cascade of regenerative events that included the emergence of proliferating insulin + clusters adjacent to ducts, NKX6.1 expression in glucagon + cells at days 1-4 suggesting the acquisition of β-cell phenotype by α-cells, and accelerated β-cell maturation with increased MAFA-expression for >1 month postinjection. Discovery and validation of islet regenerative hMSC-secreted protein may lead to the development of cell-free regenerative therapies able to tip the balance in favor of β-cell regeneration versus destruction during diabetes. STEM CELLS 2019;37:516-528 SIGNIFICANCE STATEMENTCell-based therapies using human multipotent stromal cells (hMSC) to dampen autoimmunity while inducing islet regeneration represents a promising approach to treat diabetes. However, poor survival and retention of transplanted hMSC in the damaged pancreas complicates clinical translation. Here, it is demonstrated that hMSC secrete an array of islet-regenerative proteins and intrapancreatic delivery of hMSC-conditioned media (CM) could stimulate islet regeneration without the need for cell transfer. Wnt-pathway stimulated hMSC CM-injection set in motion a cascade of events consistent with the emergence of functional islets. Discovery of hMSC-secreted, islet-regenerative effectors may lead to the development of cell-free therapies to combat diabetes.

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

“…Previous studies have identified MSC‐like populations similar to our Panc‐MSCs; however, the therapeutic potential of these cells remains currently undetermined . We consider BM‐MSCs the gold‐standard of therapeutic MSCs, as a result of documented secretory function . Using label‐free mass spectrometry, we detected >1700 proteins in the secretome of both MSC types (Figure A), and 540 known secreted effectors based on GO terminologies.…”

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.…”

“…Animal procedures were approved by the Animal Care Committee at the University of Western Ontario according to guidelines of the Animal Use Protocol (2015-033).Pancreatic β-cell ablation and resting hyperglycemia was induced in NOD/SCID mice aged 8-to 10-week-old (Jackson Laboratories, Bar Harbor, ME, USA) by intraperitoneal injection of streptozotocin (35 mg/kg/day) for 5 consecutive days, as previously described 41 . On Day 10, hyperglycemic (15-25 mmol/l) mice were transplanted by intrapancreatic injection 41 of basal AmnioMAX C-100 or 8µg total protein of concentrated CM. Non-fasted systemic blood glycemic levels were monitored in a blinded-fashion weekly for up to 42 days.…”

“…Due to the pancreatic source of Panc-MSC and associated with islets of Langerhans, we sought to assess the islet regenerative properties of EV-or EV+ CM in a more complex murine model in which endogenous regeneration has reported to be extremely limited or absent. STZ-induced βcell ablation generates a model of chronic hyperglycemia, which demonstrates extremely limited regeneration of ablated endogenous β-cells 41,56 . Similar to use the LDPI in the FAL model, temporal monitoring of resting blood glucoses can be utilized as a surrogate to assess the regeneration of insulin-producing β-cells ( Figure 6A).…”

“…Alternatively, we have recently demonstrated bone marrow (BM) MSC may be utilized as 'biofactories' to generate a potent serum-free cocktail capable of stimulating pancreatic islet regeneration in hyperglycemic NOD/SCID mice following intrapancreatic injection 12,13 . The use of a cell-free biotherapeutic approach mitigated deficiencies with direct cell transplantation 14 ; yet additional investigations remain necessary to establish the safety and to maximize the efficacy of this approach.…”