Improvements in Maturity and Stability of 3D iPSC-Derived Hepatocyte-like Cell Cultures.

Induced pluripotent stem cell (iPSC) technology enables differentiation of human hepatocytes or hepatocyte-like cells (iPSC-HLCs). Advances in 3D culturing platforms enable the development of more in vivo-like liver models that recapitulate the complex liver architecture and functionality better than traditional 2D monocultures. Moreover, within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation and maintenance of hepatocyte metabolic function. Thus, models combining 3D culture and co-culturing of various cell types potentially create more functional in vitro liver models than 2D monocultures. Here, we report the establishment of 3D cultures of iPSC-HLCs alone and in co-culture with human umbilical vein endothelial cells (HUVECs) and adipose tissue-derived mesenchymal stem/stromal cells (hASCs). The 3D cultures were performed as spheroids or on microfluidic chips utilizing various biomaterials. Our results show that both 3D spheroid and on-chip culture enhance the expression of mature liver marker genes and proteins compared to 2D. Among the spheroid models, we saw the best functionality in iPSC-HLC monoculture spheroids. On the contrary, in the chip system, the multilineage model outperformed the monoculture chip model. Additionally, the optical projection tomography (OPT) and electrical impedance tomography (EIT) system revealed changes in spheroid size and electrical conductivity during spheroid culture, suggesting changes in cell-cell connections. Altogether, the present study demonstrates that iPSC-HLCs can successfully be cultured in 3D as spheroids and on microfluidic chips, and co-culturing iPSC-HLCs with NPCs enhances their functionality. These 3D in vitro liver systems are promising human-derived platforms usable in various liver-related studies, specifically when using patient-specific iPSCs.

Simultaneous induction of vasculature and neuronal network formation on a chip reveals a dynamic interrelationship between cell types.

BACKGROUND: Neuronal networks receive and deliver information to regulate bodily functions while the vascular network provides oxygen, nutrients, and signaling molecules to tissues. Neurovascular interactions are vital for both tissue development and maintaining homeostasis in adulthood; these two network systems align and reciprocally communicate with one another. Although communication between network systems has been acknowledged, the lack of relevant in vitro models has hindered research at the mechanistic level. For example, the current used in vitro neurovascular models are typically established to be short-term (≤ 7 days) culture models, and they miss the supporting vascular mural cells. METHODS: In this study, we utilized human induced pluripotent stem cell (hiPSC) -derived neurons, fluorescence tagged human umbilical vein endothelial cells (HUVECs), and either human bone marrow or adipose stem/stromal cells (BMSCs or ASCs) as the mural cell types to create a novel 3D neurovascular network-on-a-chip model. Collagen 1-fibrin matrix was used to establish long-term (≥ 14 days) 3D cell culture in a perfusable microphysiological environment. RESULTS: Aprotinin-supplemented endothelial cell growth medium-2 (EGM-2) supported the simultaneous formation of neuronal networks, vascular structures, mural cell differentiation, and the stability of the 3D matrix. The formed neuronal and vascular networks were morphologically and functionally characterized. Neuronal networks supported vasculature formation based on direct cell contacts and by dramatically increasing the secretion of angiogenesis-related factors in multicultures in contrast to cocultures without neurons. Both utilized mural cell types supported the formation of neurovascular networks; however, the BMSCs seemed to boost neurovascular networks to greater extent. CONCLUSIONS: Overall, our study provides a novel human neurovascular network model that is applicable for creating in vivo-like tissue models with intrinsic neurovascular interactions. The 3D neurovascular network model on chip forms an initial platform for the development of vascularized and innervated organ-on-chip and further body-on-chip concepts and offers the possibility for mechanistic studies on neurovascular communication both under healthy and in disease conditions. Video Abstract.

Biochemical and Biophysical Characterization of Carbonic Anhydrase VI from Human Milk and Saliva.

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.

Vasculogenic Potency of Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells Results in Differing Vascular Network Phenotypes in a Microfluidic Chip.

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.