Self-Organization Provides Cell Fate Commitment in MSC Sheet Condensed Areas via ROCK-Dependent Mechanism

Abstract

Multipotent mesenchymal stem/stromal cells (MSC) are one of the crucial regulators of regeneration and tissue repair and possess an intrinsic program from self-organization mediated by condensation, migration and self-patterning. The ability to self-organize has been successfully exploited in tissue engineering approaches using cell sheets (CS) and their modifications. In this study, we used CS as a model of human MSC spontaneous self-organization to demonstrate its structural, transcriptomic impact and multipotent stromal cell commitment. We used CS formation to visualize MSC self-organization and evaluated the role of the Rho-GTPase pathway in spontaneous condensation, resulting in a significant anisotropy of the cell density within the construct. Differentiation assays were carried out using conventional protocols, and microdissection and RNA-sequencing were applied to establish putative targets behind the observed phenomena. The differentiation of MSC to bone and cartilage, but not to adipocytes in CS, occurred more effectively than in the monolayer. RNA-sequencing indicated transcriptional shifts involving the activation of the Rho-GTPase pathway and repression of SREBP, which was concordant with the lack of adipogenesis in CS. Eventually, we used an inhibitory analysis to validate our findings and suggested a model where the self-organization of MSC defined their commitment and cell fate via ROCK1/2 and SREBP as major effectors under the putative switching control of AMP kinase.

Extracellular Matrix Deposition Defines the Duration of Cell Sheet Assembly from Human Adipose-Derived MSC

“…Previously, we demonstrated the activation of MSC proliferation at later terms of CS assembly (days 9-12) [9]. The relation between MSC proliferation and rapid CS formation seemed obvious, but our data revealed no significant correlation between the CS assembly duration and PDT (Figure 1C).…”

“…In non-permeabilized monolayers, fibronectin was visualized as a network of thin extracellular filaments (Figure 3A, "Extracellular ECM"), while in CSs, the strength of the signal was vividly higher, covering the field of view. This might also have resulted from the CS multilayered structure we have previously shown using confocal microscopy [9][10][11]. The use of permeabilization had minimal impact on the visualized patterns in the monolayers and CSs (Figure 3A, "Total ECM"), suggesting an even distribution of fibronectin between the intra-and extracellular compartments.…”

Expression, Purification, and Characterization of Plasmodium vivax Lactate Dehydrogenase from Bacteria without Codon Optimization

“…This fluorescence-based LDH assay is based on the fact that LDH converts lactate to pyruvate and NAD + to NADH, and the formed NADH acts on the PicoProbe TM to generate a fluorescence signal (Ex/Em = 535/587 nm) from a fluorogenic substrate ( Figure 8 a). Several studies have reported successful monitoring LDH activity by this assay method [ 49 , 50 , 51 ]. The concentration of PvLDH was serially diluted by one-half from 10 nM.…”

Insight Into Rho Kinase Isoforms in Obesity and Energy Homeostasis

“…Moreover, a reciprocal enhancement of cell adhesion mediated by focal adhesion kinase activity and actin dynamics mediated by RhoA/ROCK signaling is involved in the commitment towards osteogenic versus adipogenic lineages of human adipose stem cells ( 163 ). In addition, RhoA/ROCK/AMPK/SREBP axis is involved in inhibiting adipogenesis and promoting osteogenesis during cell sheet formation of human adipose tissue-derived stem cells, which represents a cell culture model of spontaneous self-organization of multipotent mesenchymal stem/stromal cells ( 164 ). These studies support a negative role of RhoA/ROCK/F-actin/MRTF-A/SRF, RhoA/ROCK/F-actin/YAP/TAZ, or RhoA/ROCK/AMPK/SREBP pathway in the commitment of mesenchymal stem cells to the adipogenic lineage.…”