Single Cell Imaging to Probe Mesenchymal Stem Cell N-Cadherin Mediated Signaling within Hydrogels

Abstract

N-cadherin (Ncad) mediates cell-cell interactions, regulates β-catenin (βcat) signaling, and promotes the chondrogenic differentiation of mesenchymal lineage cells. Here, we utilized confocal imaging to investigate the influence of Ncad interactions on single mesenchymal stem cell (MSC) behavior within 3-dimensional hydrogel environments under conditions that promote chondrogenic differentiation. Human MSCs were photoencapsulated in hyaluronic acid (HA) hydrogels functionalized with Ncad mimetic peptides and compared to cells in environments with control non-active peptides (Ctrl). Using single-cell imaging, we observed a significant increase in membrane βcat, nuclear βcat, and cell roundness after 3 days in Ncad hydrogels compared to Ctrl hydrogels. The extent of membrane and nuclear βcat localization and MSC roundness decreased to Ctrl hydrogel levels via pre-treatment with Ncad-specific antibodies prior to encapsulation in the Ncad hydrogels, confirming the activity of the peptide. Interestingly, there was a pronounced (>80% increase) in βcat nuclear localization in two-cell clusters within the Ctrl hydrogels, which was much greater than the increase (~30%) in βcat nuclear localization in two-cell clusters within the Ncad hydrogels. In summary, we utilized fluorescent imaging to demonstrate Ncad-mediated single cell responses to developmental cues within hydrogels towards chondrogenesis.

Recent advances in hydrogels for cartilage tissue engineering

“…This outcome was even observed in vivo and it was shown recently that the presence of the N-cadherin signal from the hydrogel also influenced β-catenin signaling in human MSCs (Fig. 6 c-d ) (Vega et al ., 2016). …”

“…( c ) Cross-sectional images of MSCs stained for β-catenin (green) show that N-cadherin mimics recruit β-catenin to the cell membrane. Additionally, N-cadherin mimics induced an increase in nuclear β-catenin, as confirmed by ( d ) representative maximum (top) and average (bottom) projections of single MSCs stained for actin (red), nucleus (blue) and β-catenin (green) (Vega et al ., 2016). Scale bars: b = 50 μm; c,d = 25 μm.…”

Dose and Timing of N‐Cadherin Mimetic Peptides Regulate MSC Chondrogenesis within Hydrogels

“…Prior work has established that tethered NCad mimetic HAV peptides in HA hydrogels exhibit NCad agonistic activity due to one end being conjugated to the HA backbone, resulting in the expected nuclear β-catenin localization in MSCs when encapsulated within this material. [17, 18] Furthermore, blocking studies using antibody treatment have confirmed that the observed enhancement of chondrogenic gene expression in this system was indeed due to NCad interactions with these peptides. [17] …”

“…[17] Furthermore, incorporation of this peptide into hydrogels resulted in increased β-catenin recruitment to the membrane and subsequent translocation to the nucleus, as is observed with NCad signaling. [18] …”

3D material cytometry (3DMaC): a very high-replicate, high-throughput analytical method using microfabricated, shape-specific, cell-material niches

“…This includes work from the Burdick group, where single cell or multi-cell units were imaged and masked to analyze the impact of N-cadherin-mimetic peptide on hMSCs 24 , and work from the Gartner group using DNA-programmed assembly of cells to build specific cellular microenvironments which were analyzed in a 3D bulk hydrogel array 25 . Neither of the systems was suspension-based which limited their replicate number, a major advantage of 3DMaC; however, these demonstrate that there is a need for a high-throughput way to analyze biomaterial-cell interactions in the context of the encapsulating material for both single cells and multi-cellular constructs.…”