Alexandra M. Smink¹, Erika Pinheiro-Machado¹, Timara Kuiper¹, Romy H. Huurman¹, Luis Henrique Corrêa², Arjen H. Petersen¹, Bart J. de Haan¹, Martin C. Harmsen¹.

¹University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology. ²Institute of Physiological Chemistry and Pathobiochemistry, Cells-in-Motion Interfaculty Center, University of Münster. E-mail: a.m.smink@umcg.nl.

Background: Subcutaneous polymeric scaffolds for insulin-producing cell transplantation require a microenvironment that includes supportive extracellular matrix (ECM) alongside a robust vascular network to ensure adequate oxygen, nutrient delivery, and insulin transport. This study evaluated adipose-derived stromal cell (ASC) secretome for fostering such a scaffold microenvironment.

Methods: Rat ASCs were exposed to various culture conditions to determine the alterations in the composition and functional properties of their secretomes. Subsequently, secretome-containing scaffolds were subcutaneously implanted in a non-diabetic immunocompromised rat model and on days 7, 14, 21, and 28 the microenvironment was investigated by lectin infusion, gene expression analysis, and histology.

Results: Exposure of ASCs to normoxia, hypoxia, or a combination of hypoxia + high glucose resulted in enrichment of secretome proteins associated with angiogenesis and ECM organization. These conditions also stimulated in vitro endothelial tube formation (number of branching points 1.3-2.4 times higher than the control) and collagen deposition by fibroblasts (1.7-2.2 times increase in collagen staining than the control). In vivo experiments demonstrated that all three secretomes enhanced the formation of a vascular network and ECM deposition within the scaffold. Notably, the most significant improvement was observed with the hypoxia + high glucose-derived secretome. Starting from day 14, this secretome significantly increased pro-angiogenic genes like VEGF (1.7-2.5 fold change), Angiopoietin1 (1.3-1.6 fold change), and Ve-Cadherin (1.4-1.6 fold change), and the presence of perfused blood vessels (1.4-5 fold change). Furthermore, the gene expression of several matrix metalloproteinases and the protein expression of Collagen IV and Laminin were improved.

Discussion/conclusion: By modifying the ASC secretome, it becomes highly suitable for engineering a sustainable microenvironment within a subcutaneous scaffold which might enhance Type 1 Diabetes transplantation outcomes.