Gap junctional intercellular communication in adipose‐derived stromal/stem cells is cell density‐dependent and positively impacts adipogenic differentiation

Abstract

Adipose-derived stromal/stem cells (ASCs) represent a widely used cell source with multi-lineage differentiation capacity in approaches for tissue engineering and regenerative medicine. Despite the multitude of literature on their differentiation capacity, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction-forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in human ASCs, and its significance for adipogenic differentiation of these cells. Cx43 expression in ASCs was demonstrated histologically and on the gene and protein expression level, and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of ASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α-glycyrrhetinic acid (AGA) significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process. Altogether, this study demonstrates the impact of direct cell-cell communication via gap junctions on the adipogenic differentiation process of ASCs, and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches.

Human MuStem Cell Grafting into Infarcted Rat Heart Attenuates Adverse Tissue Remodeling and Preserves Cardiac Function

“…Other classical markers, including voltage-gated sodium channel α subunit 5 ( SCN5A ) and troponin T2 ( TNNT2 ), were expressed at low levels. Only the gap junction protein α1 ( GJA1 ) gene encoding connexin 43 (Cx43), which is required to establish intercellular connections between CMs and is also expressed by other progenitor cells, such as MSCs, 38 , 39 , 40 was detected in hMuStem cells. In addition, a large amount of the phosphorylated isoform of Cx43, which is involved in the electrical cell-to-cell coupling through its action on the gap junction function, were detected by western blotting on hMuStem cells compared to those obtained in proliferating myoblasts known to express Cx43 ( Figure 1 C).…”

Connexin43 is Dispensable for Early Stage Human Mesenchymal Stem Cell Adipogenic Differentiation But is Protective against Cell Senescence

“…Undocked connexin hemichannels have also been shown to play some role in mediating autocrine and paracrine signaling, although their role beyond pathological cell processes remains poorly understood in vivo [12]. MSCs, adipocyte-derived stromal cells, and bone marrow-derived stromal cells all express the prototypical connexin, Cx43 [13,14,15,16,17,18]. Cx43 is widely found in the human anatomy, and more than 80 different mutations in the gene encoding Cx43 ( GJA1 ) are associated with the pleiotropic developmental disorder known as oculodentodigital dysplasia (ODDD).…”

“…However, this does not appear to be the case in fat, as adipocytes only express Cx43 and there is no evidence in ODDD patients that white or brown fat formation is abnormal. Yet, Cx43 has recently been proposed to play a role in adipose-derived stromal cell differentiation into adipocytes, as well as adipose beiging, in rodents and humans [15,24,25,26,27,28].…”

“Synapse-like” Connections between Adipocytes and Stem Cells: Morphological and Molecular Features of Human Adipose Tissue

“…Confocal image stacks allowed us to affirm that "Cx43+ gap junctions" are widespread on adipocyte surfaces and in promoting contact between adipocytes and AdSCs. This observation, together with the finding by TEM of the presence of small vesicles near the membranes of the adipocytes, suggests the existence of a "synapse-like" communication link where stem cells may be receiving signals from adipocytes to continue in the pluripotency state [35]. This type of connection could also play a role in cellular latency, or for activation toward a proliferative state when exposed to different stress conditions.…”