Reversible photoregulation of cell-cell adhesions with opto-E-cadherin.

E-cadherin-based cell-cell adhesions are dynamically and locally regulated in many essential processes, including embryogenesis, wound healing and tissue organization, with dysregulation manifesting as tumorigenesis and metastasis. However, the lack of tools that would provide control of the high spatiotemporal precision observed with E-cadherin adhesions hampers investigation of the underlying mechanisms. Here, we present an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light. With opto-E-cadherin, functionally essential calcium binding is photoregulated such that cells expressing opto-E-cadherin at their surface adhere to each other in the dark but not upon illumination. Consequently, opto-E-cadherin provides remote control over multicellular aggregation, E-cadherin-associated intracellular signalling and F-actin organization in 2D and 3D cell cultures. Opto-E-cadherin also allows switching of multicellular behaviour between single and collective cell migration, as well as of cell invasiveness in vitro and in vivo. Overall, opto-E-cadherin is a powerful optogenetic tool capable of controlling cell-cell adhesions at the molecular, cellular and behavioural level that opens up perspectives for the study of dynamics and spatiotemporal control of E-cadherin in biological processes.

Spatiotemporal Control Over Multicellular Migration Using Green Light Reversible Cell-Cell Interactions.

The regulation of cell-cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell-cell interactions with high precision are of great interest to a better understanding of their roles and building tissue-like structures. Herein, the green light-responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion receptor, so that upon addition of its cofactor vitamin B12 specific cell-cell interactions form and lead to cell clustering in a concentration-dependent manner. Upon green light illumination, the CarH based cell-cell interactions disassemble and allow for their reversion with high spatiotemporal control. Moreover, these artificial cell-cell interactions impact cell migration, as observed in a wound-healing assay. When the cells interact with each other in the presence of vitamin B12 in the dark, the cells form on a solid front and migrate collectively; however, under green light illumination, individual cells migrate randomly out of the monolayer. Overall, the possibility of precisely controlling cell-cell interactions and regulating multicellular behavior is a potential pathway to gaining more insight into cell-cell interactions in biological processes.

NAD+/NADH redox alterations reconfigure metabolism and rejuvenate senescent human mesenchymal stem cells in vitro.

Human mesenchymal stem cells (hMSCs) promote endogenous tissue regeneration and have become a promising candidate for cell therapy. However, in vitro culture expansion of hMSCs induces a rapid decline of stem cell properties through replicative senescence. Here, we characterize metabolic profiles of hMSCs during expansion. We show that alterations of cellular nicotinamide adenine dinucleotide (NAD + /NADH) redox balance and activity of the Sirtuin (Sirt) family enzymes regulate cellular senescence of hMSCs. Treatment with NAD + precursor nicotinamide increases the intracellular NAD + level and re-balances the NAD + /NADH ratio, with enhanced Sirt-1 activity in hMSCs at high passage, partially restores mitochondrial fitness and rejuvenates senescent hMSCs. By contrast, human fibroblasts exhibit limited senescence as their cellular NAD + /NADH balance is comparatively stable during expansion. These results indicate a potential metabolic and redox connection to replicative senescence in adult stem cells and identify NAD + as a metabolic regulator that distinguishes stem cells from mature cells. This study also suggests potential strategies to maintain cellular homeostasis of hMSCs in clinical applications.

Cyclical aggregation extends in vitro expansion potential of human mesenchymal stem cells.

Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why aggregation was responsible for this phenomenon, the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging. This method holds the potential to increase the clinical relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression.

Aggregation-induced integrated stress response rejuvenates culture-expanded human mesenchymal stem cells.

Protein homeostasis is critical for cellular function, as loss of homeostasis is attributed to aging and the accumulation of unwanted proteins. Human mesenchymal stem cells (MSCs) have shown promising therapeutic potential due to their impressive abilities to secrete inflammatory modulators, angiogenic, and regenerative cytokines. However, there exists the problem of human MSC expansion with compromised therapeutic quality. Duringin vitro expansion, human MSCs are plated on stiff plastics and undergo culture adaptation, which results in aberrant proliferation, shifts in metabolism, and decreased autophagic activity. It has previously been shown that three-dimensional (3D) aggregation can reverse some of these alterations by heightening autophagy and recovering the metabolic state back to a naïve phenotype. To further understand the proteostasis in human MSC culture, this study investigated the effects of 3D aggregation on the human MSC proteome to determine the specific pathways altered by aggregation. The 3D aggregates and 2D cultures of human MSCs derived from bone marrow (bMSC) and adipose tissue (ASC) were analyzed along with differentiated human dermal fibroblasts (FB). The proteomics analysis showed the elevated eukaryotic initiation factor 2 pathway and the upregulated activity of the integrated stress response (ISR) in 3D aggregates. Specific protein quantification further determined that bMSC and ASC responded to ISR, while FB did not. 3D aggregation significantly increased the ischemic survival of bMSCs and ASCs. Perturbation of ISR with small molecules salubrinal and GSK2606414 resulted in differential responses of bMSC, ASC, and FB. This study indicates that aggregation-based preconditioning culture holds the potential for improving the therapeutic efficacy of expanded human MSCs via the establishment of ISR and homeostasis.

Size-Dependent Cortical Compaction Induces Metabolic Adaptation in Mesenchymal Stem Cell Aggregates.

IMPACT STATEMENT: This study reveals that multicellular aggregation induces metabolic reprogramming via mechanical compaction in lieu of formation of a hypoxic core. Utilizing biomechanical knowledge gained from planar culture, we set forth a novel three-dimensional (3D) model of size-dependent cortical compaction and demonstrated its role in metabolic reconfiguration. Ultimately, this study establishes mechanical compaction and its spatial gradients as key regulatory factors and design parameters in the development of 3D human adipose-derived mesenchymal stem cell aggregates.

Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization.

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (ΔP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ΔP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.

Bioreactor design for perfusion-based, highly-vascularized organ regeneration.

Bioartificial or laboratory-grown organs is a growing field centered on developing replacement organs and tissues to restore body function and providing a potential solution to the shortage of donor organs for transplantation. With the entry of engineered planar tissues, such as bladder and trachea, into clinical studies, an increasing focus is being given to designing complex, three-dimensional solid organs. As tissues become larger, thicker and more complex, the vascular network becomes crucial for supplying nutrients and maintaining viability and growth of the neo-organ. Perfusion decellularization, the process of removing cells from an entire organ, leaves the matrix of the vascular network intact. Organ engineering requires a delicate process of decellularization, sterilization, reseeding with appropriate cells, and organ maturation and stimulation to ensure optimal development. The design of bioreactors to facilitate this sequence of events has been refined to the extent that some bioartificial organs grown in these systems have been transplanted into recipient animals with sustained, though limited, function. This review focuses on the state-of-art in bioreactor development for perfusion-based bioartificial organs and highlights specific design components in need of further refinement.