David A. Hess

Type 2 diabetes mellitus (T2DM) disrupts the quantity, phenotype, and function of circulating vascular regenerative (VR) progenitor cells that are critical to vessel repair. These impairments include reduced progenitor clonogenicity, mobilization and homing to areas of ischemia, and imbalances in secretory function, collectively contributing to poor vascular healing and complications such as peripheral artery disease, critical limb ischemia, and cardiovascular disease. Despite growing recognition of VR cell exhaustion, regenerative strategies remain hindered by imprecise cell phenotyping, underused functional assays, and insufficient integration of disease-relevant in vivo models. This article examines the evolving landscape for the integrated assessment of function, encompassing multipotent colony-forming cell assays, single-cell RNA sequencing, advanced proteomic and metabolomic profiling, and novel models to evaluate vessel regeneration through human cell transplantation into immune-deficient mice with T2DM-associated comorbidities. We also highlight promising regenerative strategies using glucose-reducing agents, alongside rigorous omics-driven precision profiling to quantify the recovery of VR cell function during T2DM, underscoring an urgent need to progress beyond phenotypic studies toward functionally integrated models that predict translational efficacy. Article Highlights: Vascular regenerative cells, including endothelial precursor cells and hematopoietic progenitor cells, exhibit altered regenerative function in type 2 diabetes. Traditional definitions of endothelial precursor cells lack phenotypic specificity, limiting interpretation of regenerative capacity across studies. Functional assays, including colony formation, migration, and in vivo ischemic models for type 2 diabetes, have revealed defects in angiogenesis, proliferation, and repair. Emerging omics approaches provide mechanistic insight into vascular regenerative cell exhaustion across metabolic and inflammatory states. Standardized phenotyping and functional assays are needed to improve the translational success of regenerative therapies for cardiometabolic diseases.

Cellular therapies harnessing the potential of endothelial colony-forming cells (ECFCs) for therapeutic revascularization in patients with critical limb ischemia (CLI) have garnered much interest. However, limited cell persistence following intramuscular injection into ischemic tissues has prompted the development of biomaterials as cell-delivery platforms to support cell survival and enhance therapeutic outcomes. This study explored the use of hydrogels derived from human decellularized adipose tissue (DAT) as a cell-instructive platform for ECFC delivery. In vitro studies confirmed high initial ECFC viability at 1 day following encapsulation in the DAT hydrogels but revealed that cell viability and proliferation were reduced over time in culture as compared to ECFCs cultured on tissue culture polystyrene (TCP). However, the ECFCs cultured within the DAT hydrogels showed similar ECFC cell surface marker expression patterns compared to ECFCs cultured on TCP after 6 days in culture, supporting that their phenotype was maintained. Subsequent testing focused on comparing a low (2.4 × 10⁵ cells) and high (1 × 10⁶ cells) dose of ECFCs delivered in either saline or within DAT hydrogels in a femoral artery ligation-induced CLI (FAL-CLI) model in NOD/SCID mice over 35 days. Bioluminescence imaging results showed that the low dose of ECFCs delivered in DAT hydrogels demonstrated enhanced retention on days 14, 21, and 28 compared to delivery in saline. Despite this finding, there were no observed improvements in hindlimb perfusion between delivery strategies, which may be related to the robust collateral vessel formation observed in this model with a µCT-based angiography method. Overall, this work supports that DAT hydrogels can enhance localized ECFC retention following intramuscular injection in mice with femoral artery ligation but emphasizes that enhanced retention may be insufficient for improving functional vascular regeneration.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have revolutionized the management of type 2 diabetes and obesity. Due to class-wide reduction in major adverse cardiovascular events in cardiovascular outcome trials and pleiotropic actions in multiple tissues, the use of GLP-1RAs has expanded beyond metabolic diseases. Recent studies have reported GLP-1RA efficacy for the treatment of atherosclerosis, heart failure and peripheral artery disease, alongside evolving potential in chronic kidney disease. The recent discovery that GLP-1RAs can improve vascular regenerative progenitor cell flux during type 2 diabetes has uncovered a novel mechanism implicating 3 classical hallmarks of vascular aging: (1) stem cell exhaustion, (2) altered intercellular communication, and (3) chronic systemic inflammation. In this review we discuss recent evidence demonstrating that imbalances in hematopoiesis during cardiometabolic diseases intersect with the senescence-associated secretory phenotype to elevate chronic inflammation and accelerate vascular aging. With a focus on stem cells as the master regulators of regenerative processes, we integrate the activities of GLP-1RAs that shift the balance from damage accumulation to repair competence in blood vessels prematurely aged by cardiometabolic syndrome.

Introduction: Diabetes affects >500 million people worldwide. Despite insulin therapy, most patients develop devastating complications, emphasizing the need for curative strategies. Human multipotent stromal/stem cells (MSC) secrete factors that promote islet regeneration. We previously showed that intrapancreatic (iPan) delivery of Wnt-activated MSC-conditioned media (Wnt+ CM) stimulates islet regeneration without cell transfer. This study aimed to identify and functionally validate specific MSC-secreted proteins with islet regenerative potential. Methods: Comprehensive mass spectrometry-based, quantitative proteomic analyses comparing Wnt-pathway activated versus untreated MSC secretomes were cross-referenced with a prior dataset distinguishing regenerative from nonregenerative MSC CM. Proteins enriched in both conditions were iPan-injected individually or in combination into streptozotocin-treated NOD/SCID mice. Nonfasting glucose, glucose tolerance, beta cell mass, islet morphology, and islet cell proliferation were assessed at 4- and 32-days post-treatment. Results: Cross-referenced secretome analyses identified eight proteins implicated in islet regeneration: CALU, CTSB, FAM3C, GAL1, PPIA, PSAP, SOD1, and TGM2. A single iPan-injection of the 8-protein combination significantly lowered hyperglycemia, improved glucose tolerance, and increased beta cell mass, comparable to Wnt+ CM. Regenerative effects such as increased beta cell proliferation appeared as early as day 4. Single-protein testing identified CALU and SOD1 as leading candidates, improving glucose tolerance and reducing nonfasting glucose. Conclusion: This study defines a set of MSC-secreted proteins that promote islet regeneration in vivo, supporting the development of protein-based biologics to preserve or restore beta cell function during diabetes, with potential applications alongside islet replacement therapies to enhance graft survival and function.

Diabetes mellitus is a global healthcare issue of epidemic proportions. At the root of these disorders, characterized by poor glucose regulation and insulin deficiencies, is the pancreatic beta cell and insufficient insulin signal transduction in peripheral tissues. Residual c-peptide secretion and persisting beta cells have been found in patients who have been living with type 1 diabetes for over 50 years. Thus, beta cell regeneration has been vastly studied in rodents, and many agents to expand beta cell mass are under rigorous investigation for the treatment of diabetes. Multipotent stromal cells (MSC), isolated from human bone marrow, have an immunomodulatory and pro-regenerative secretome that can aid in repairing damaged tissues, including pancreatic islets. MSC transplantation has been shown to reduce hyperglycemia and orchestrate islet repair in experimental diabetes models and is currently being assessed in clinical trials. While the immunomodulatory mechanisms of MSC are well-studied, the beta-cell-regenerative mechanisms are unknown. MSC likely play a regenerative role by signaling to resident progenitor or precursor cells in the pancreas; however, the decades-long controversy surrounding the origin of regenerated adult beta cells remains unresolved. Herein, we take a deep dive into the role of MSC in the treatment of diabetes and the potential cellular mechanisms behind the MSC stimulation of beta cell regeneration.

Background and Aims: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) reduce major atherosclerotic cardiovascular events in individuals living with either diabetes or obesity. Since the turnover of vascular regenerative (VR) stem and progenitor cells has been demonstrated to modulate vessel repair and atherothrombotic risk, this study aimed to determine the effect of the GLP-1RA semaglutide on the levels of circulating VR cells. Methods: SEMA-VR CardioLink-15 was a randomized translational trial of usual care vs semaglutide for 6 months in 46 participants with either type 2 diabetes and/or obesity plus atherosclerotic cardiovascular disease (ASCVD) or ASCVD risk factors. Vascular regenerative cells were enumerated using multi-parametric flow cytometry for high aldehyde dehydrogenase activity (ALDHhi) and lineage-specific cell surface marker expression. The primary endpoint was the 6-month change in VR cell content. Results: Compared with usual care (n = 24), semaglutide (n = 22) led to a greater increase in the number of VR cells [high aldehyde dehydrogenase 1A1 activity and low side scatter (ALDHhiSSClow): +0.8% vs +34.8%; P = .036], pan-haematopoietic myeloid progenitors (ALDHhiSSClowCD45+: +2.8% vs +40.1%; P = .017), and endothelial precursors (ALDHhiSSClowCD34+ CD133+ CD45−: −2.3% vs +66.2%; P = .037) from baseline. Semaglutide also decreased granulocyte precursors (ALDHhiSSChi: +0.3% vs −50.8%; P = .002), particularly those expressing the neutrophil activation marker CD66b and chemokine receptor CXCR2. Semaglutide down-regulated serum proteins over-represented in pro-inflammatory tumour necrosis factor and interleukin signalling pathways. Conclusions: In people living with either type 2 diabetes or obesity plus ASCVD risk, semaglutide increased circulating VR cell content while reducing pro-inflammatory granulocyte precursors and cytokine production. Collectively, these findings suggest that semaglutide may improve endogenous progenitor cell–mediated blood vessel repair processes.

Aims: Lipoprotein(a) [Lp(a)] is a causal risk factor for atherosclerotic cardiovascular disease (ASCVD); however, the relationship between Lp(a) and the capacity for vascular repair remains unclear. Depletion of vascular regenerative (VR) progenitor cells has been shown to be a novel indicator of compromised vascular repair in people living with cardiometabolic disorders. The purpose of this study was to determine if elevated levels of Lp(a) modify VR cell content properties. Methods and results: The cross-sectional, multi-site Lipoprotein(a) and Vascular Regenerative Cell Content CardioLink-16 [Lp(a)-VRCE] study enrolled 40 individuals—20 with Lp(a) ≥100 nmol/L and 20 with Lp(a) <100 nmol/L. Isolated peripheral blood mononuclear cells were analysed by multi-parameter flow cytometry. VR progenitor cells were identified based on high aldehyde dehydrogenase (ALDH) activity, in combination with primitive vs. mature lineage-specific cell surface markers. The Lp(a) ≥100 nmol/L group exhibited baseline characteristic differences compared with the Lp(a) <100 nmol/L group such as lower estimated glomerular filtration rate (86.9 vs. 100.1 mL/min/1.73 m2), lower total cholesterol (4.0 vs. 4.8 mmol/L), greater statin use (90 vs. 60%), and a higher prevalence of ASCVD (60 vs. 25%). The Lp(a) ≥100 nmol/L group had lower frequencies of pro-angiogenic ALDHhiSSClowCD133+ (P = 0.0008) and ALDHhiSSClowCD34+CD133+ (P = 0.005) progenitor cells with pro-angiogenic secretory function. Compared with those in the Lp(a) <100 nmol/L group, individuals in the Lp(a) ≥100 nmol/L group demonstrated a higher frequency of M1-polarized pro-inflammatory monocytes (ALDHhiSSCmidCD86+CD163−; P = 0.007) and a lower frequency of ALDHhiSSChiCD49d+ granulocyte precursor cells (P = 0.04) that are involved in vessel repair. Conclusion: In this translational study, people with an Lp(a) ≥100 nmol/L had fewer VR cells and more pro-inflammatory polarized monocyte precursor cells than those with Lp(a) levels <100 nmol/L. These findings suggest that vessel repair activities may be compromised in individuals with elevated Lp(a) levels. Registration: Lp(a)-VRCE ClinicalTrials.gov: NCT06626659

Residual beta cell function has been documented in “medalist” patients who have lived with Type 1 diabetes (T1D) for >50 years. In addition, endocrine cell neogenesis first occurs in the developing human embryo from progenitor cells derived from pancreatic ductal epithelial structure. Thus, beta cell conversion from a dormant epithelial precursor remains a promising approach to regenerate islets during T1D. We have previously shown that intra-pancreatic (iPan) injection of Wnt pathway-stimulated conditioned media (Wnt+ CdM) generated from human bone marrow-derived multipotent stromal cells (MSC) contained islet regenerative factors that reduced hyperglycemia and recovered beta cell mass in streptozotocin-treated mice. However, the endogenous source of regenerated beta cells remains unknown. Herein, we employed cytokeratin 19 (CK19)-CreERT Rosa26-mTomato lineage-tracing mice to assess the endocrine conversion of CK19+ cells during MSC CdM-induced islet regeneration. Mice iPan-injected with Wnt+ CdM demonstrated reduced blood glucose levels and improved glucose tolerance compared to mice injected with unconditioned basal media. CdM-injected mice also showed increased islet number and beta cell mass, as well as CK19+ cells within regenerating islets. The frequency of insulin + cells that co-expressed tdTomato within dissociated pancreas samples observed via flow cytometry was 5-fold higher in Wnt+ CdM-injected mice (~5%) compared to basal media-injected controls (~1%). Collectively, in vivo lineage tracing revealed conversion of CK19+ cells to functional beta cells partially contributed to islet regeneration induced by Wnt-activated MSC CdM. Future studies are required to delineate alternate cell types and mechanisms participating in islet regeneration induced by direct delivery of MSC-CdM.