The vasculature is an essential, physiological element in virtually all human tissues. Formation of perfusable vasculature is therefore crucial for reliable tissue modeling. Three-dimensional vascular networks can be formed through the co-culture of endothelial cells (ECs) with stromal cells embedded in hydrogel. Mesenchymal stem/stromal cells (MSCs) derived from bone marrow (BMSCs) and adipose tissue (ASCs) are an attractive choice as stromal cells due to their natural perivascular localization and ability to support formation of mature and stable microvessels in vitro. So far, BMSCs and ASCs have been compared as vasculature-supporting cells in static cultures. In this study, BMSCs and ASCs were co-cultured with endothelial cells in a fibrin hydrogel in a perfusable microfluidic chip. We demonstrated that using MSCs of different origin resulted in vascular networks with distinct phenotypes. Both types of MSCs supported formation of mature and interconnected microvascular networks-on-a-chip. However, BMSCs induced formation of fully perfusable microvasculature with larger vessel area and length whereas ASCs resulted in partially perfusable microvascular networks. Immunostainings revealed that BMSCs outperformed ASCs in pericytic characteristics. Moreover, co-culture with BMSCs resulted in significantly higher expression levels of endothelial and pericyte-specific genes, as well as genes involved in vasculature maturation. Overall, our study provides valuable knowledge on the properties of MSCs as vasculature-supporting cells and highlights the importance of choosing the application-specific stromal cell source for vascularized organotypic models.

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein.