Matricellular protein Tenascin-C enhances mesenchymal stem cell angiogenic and wound healing efficacy under ischemic conditions.

Human mesenchymal stem cells/multipotent stromal cells (MSCs) hold great promise in aiding wound healing through their ability to modulate all phases of repair and regeneration, most notably their secretion of pro-regenerative paracrine factors. However, MSC clinical utility is hindered by poor survival rates post-transplantation due to the harsh microenvironment in injured tissue. Previous work has shown that the matricellular protein Tenascin-C (TNC) provides survival signaling to MSCs via the epidermal growth factor receptor by restricting its activation at the plasma membrane, resulting in enhanced prosurvival signals. Herein, we investigate how TNC influences MSC survival and MSC-mediated promotion of the wound healing process. This study examined the survival and angiogenic potential of MSCs cultured on TNC-coated surfaces under ischemic duress in vitro. We also assessed the angiogenic and wound healing outcomes of MSC + TNC in vivo using a CXCR3-/- mouse model that exhibits a delayed healing phenotype within the tissue replacement phase of repair. We found that MSCs in the presence of TNC exhibit higher levels of angiogenic-promoting processes, collagen maturation, and an overall better wound healing outcome than MSCs administered alone. This was seen in vitro in terms of enhanced tube formation. In vivo, the MSCs in the presence of TNC stabilized with a coacervate delivery system resulted in more regenerative wounds with accelerated maturation of the dermis. These findings suggest the coupling of TNC to MSCs as a promising tool for future MSC-ECM combinatorial therapies for wound healing applications.

The matricellular protein decorin delivered intradermally with coacervate improves wound resolution in the CXCR3-deficient mouse model of hypertrophic scarring.

Cutaneous wound healing is an intricate orchestration of three overlapping phases of repair that encompass numerous cell types, signalling cascades, and microenvironment modifications to reach a successful resolution. Disruption of any of these steps will create an abnormal healing response resulting in either ulceration or excessive scarring. It has become evident that the extracellular matrix and its associated components are key orchestrators during this process. One of these essential matrix proteins is decorin, a small leucine-rich proteoglycan (SLRP) that acts as a regulator of collagen fibrillogenesis and a non-competitive inhibitor of multiple growth factors signalling cascades. Decorin is a necessary shut-off switch for the pro-reparative mechanism of the tissue replacement phase and limits the occurrence of hypertrophic scarring by preventing excessive repair. We investigated the use of decorin as a therapeutic by administering the matrix protein anchored in a slow-release coacervate in a hypertrophic scarring mouse model. The results show that early wound healing phase measurements exhibit little difference in performance compared to our coacervate-only baseline or HB-EGF-treated control mice. However, during the resolution phase of wound healing, the decorin-treatment significantly reduces cutaneous thickness, enhances collagen alignment, and improves overall wound scoring in the mice. Thus, mice treated with decorin display better healing outcomes and could limit the hypertrophic scarring phenotype in the coacervate only, and HB-EGF controls. These results suggest that decorin may be a promising tool and alternative therapy for patients who suffer from over-exuberant matrix deposition during wound healing.

ECM-regulation of autophagy: The yin and the yang of autophagy during wound healing.

Wound healing is a complex sequence of tissue protection, replacement, and reorganization leading to regenerated tissue. Disruption of any of these steps results in the process being incomplete as an ulcer or over-exuberant as a hypertrophic scar. Over the past decade, it has become evident that the extracellular matrix and associated components orchestrate this process. However, the cellular events that are induced by the extracellular matrix to accomplish wound healing remain to be defined. Herein we propose that matrix-regulated cellular macro-autophagy is key to both the tissue replacement and resolution stages of healing by directing cellular function or apoptosis. Further, disruptions in matrix turnover alter autophagic function leading to chronic wounds or scarring. While the literature that directly investigates autophagy during wound healing is sparse, the emerging picture supports our proposing a model of the centrality of the matrix-autophagy modulation as central to physiologic and pathologic healing.

Mesenchymal Stem Cell/Multipotent Stromal Cell Augmentation of Wound Healing: Lessons from the Physiology of Matrix and Hypoxia Support.

Cutaneous wounds requiring tissue replacement are often challenging to treat and result in substantial economic burden. Many of the challenges inherent to therapy-mediated healing are due to comorbidities of disease and aging that render many wounds as chronic or nonhealing. Repeated failure to resolve chronic wounds compromises the reserve or functioning of localized reparative cells. Transplantation of mesenchymal stem cells/multipotent stromal cells (MSCs) has been proposed to augment the reparative capacity of resident cells within the wound bed to overcome stalled wound healing. However, MSCs face a variety of challenges within the wound micro-environment that curtail their survival after transplantation. MSCs are naturally pro-angiogenic and proreparative, and thus numerous techniques have been attempted to improve their survival and efficacy after transplantation, many with little impact. These setbacks have prompted researchers to re-examine the normal wound bed physiology, resulting in new approaches to MSC transplantation using extracellular matrix proteins and hypoxia preconditioning. These studies have also led to new insights on associated intracellular mechanisms, particularly autophagy, which play key roles in further regulating MSC survival and paracrine signaling. This review provides a brief overview of cutaneous wound healing with discussion on how extracellular matrix proteins and hypoxia can be utilized to improve MSC retention and therapeutic outcome.

Adult Stem Cell Functioning in the Tumor Micro-Environment.

Tumor progression from an expanded cell population in a primary location to disseminated lethal growths subverts attempts at cures. It has become evident that these steps are driven in a large part by cancer cell-extrinsic signaling from the tumor microenvironment (TME), one cellular component of which is becoming more appreciated for potential modulation of the cancer cells directly and the TME globally. That cell is a heterogenous population referred to as adult mesenchymal stem cells/multipotent stromal cells (MSCs). Herein, we review emerging evidence as to how these cells, both from distant sources, mainly the bone marrow, or local resident cells, can impact the progression of solid tumors. These nascent investigations raise more questions than they answer but paint a picture of an orchestrated web of signals and interactions that can be modulated to impact tumor progression.

The Pro-reparative Engine: Stem Cells Aid Healing by Dampening Inflammation.

PURPOSE OF REVIEW: Stem cells have been proposed as sources for tissue replacement when healing does not occur. These cells could contribute directly to skin structures via differentiation, or via producing trophic factors that would 'educate' the micro-environment to encourage tissue repair. Studies in animals have supported both mechanisms, but translation to humans has been challenged by poor cell survival after transplantation. However, the improvement noted with even transient existence suggests another new possibility, that of suppressing the inflammatory response that limits regenerative healing. Herein, we will propose that this immunomodulatory aspect holds promise for promoting skin healing. RECENT FINDINGS: We have found that stem cell transplantation into wounds can dampen both acute and chronic inflammation, leading to more regenerative-like healing and diminished scarring. SUMMARY: Wound healing could be improved by dampening inflammation both initially to allow for tissue replacement to proceed and late to reduce scarring.

Compressed Collagen Enhances Stem Cell Therapy for Corneal Scarring.

Stem cells from human corneal stroma (CSSC) suppress corneal stromal scarring in a mouse wound-healing model and promote regeneration of native transparent tissue (PMID:25504883). This study investigated efficacy of compressed collagen gel (CCG) as a vehicle to deliver CSSC for corneal therapy. CSSC isolated from limbal stroma of human donor corneas were embedded in soluble rat-tendon collagen, gelled at 37°C, and partially dehydrated to a thickness of 100 µm by passive absorption. The CCG disks were dimensionally stable, easy to handle, and could be adhered securely to de-epithelialized mouse cornea with fibrin-based adhesive. CSSC in CCG maintained >80% viability for >1 week in culture media and could be cryopreserved in 20% fetal bovine serum-10%DMSO in liquid nitrogen. CCG containing as few as 500 CSSC effectively prevented visible scarring and suppressed expression of fibrotic Col3a1 mRNA. CSSC in CCG were more effective at blocking scarring on a per-cell basis than CSSC delivered directly in a fibrin gel as previously described. Collagen-embedded cells retained the ability to suppress corneal scarring after conventional cryopreservation. This study demonstrates use of a common biomaterial that can facilitate storage and handling of stem cells in a manner that may provide off-the-shelf delivery of stem cells as a therapy for corneal scarring. Stem Cells Translational Medicine 2018;7:487-494.

Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation.

BACKGROUND: Mesenchymal stem/multipotent stromal cells (MSCs) contribute to tissue repair but are challenged during wound healing when the blood supply is disrupted, thereby limiting nutrient delivery. Survival mechanisms against 'starvation' include autophagy, which we previously found to enhance differentiation efficiency. MSC response to models of in vitro nutrient deprivation are of great interest for improving MSC survival and therapeutic efficacy; however, the rate-limiting nutrients are unknown. METHODS: MSC responses to culture nutrient and/or serum deprivations were assessed through light microscopy, cell survival, and measurements of metabolic levels. Glucose uptake was determined through conditioned media analyses over 3 days of culture. The Seahorse XF24 Flux analysis system was used to determine oxygen consumption and extracellular acidification for glycolytic metabolism. MSC autophagic response to these conditions was assessed via immunoblots for LC3-I and LC3-II, markers of autophagosome turnover. RESULTS: We more closely examined limiting nutritional factors to MSC survival in vitro, finding that glucose is rapidly utilized/depleted whereas amino acids and other required nutrients were used sparingly. This finding concurred with metabolic analyses that showed a primarily glycolytic character to the MSCs at steady state. MSC autophagy, previously linked to MSC function through a unique accumulated autophagosome phenotype, also responded quickly to changes in glucose concentration, with drastic LC3-II changes within 24 h of glucose concentration shifts. CONCLUSIONS: Our results demonstrated a rapid uptake of glucose in MSC cultures that was due to a highly glycolytic phenotype for the cells; MSC starvation with serum or other nutrients appears to have a less notable effect on the cells. These findings highlight the importance of glucose and glucose metabolism on MSC function. The conditions and cellular responses outlined here may be essential in modeling MSC nutrient deprivation.

Wnt signaling regulates hepatobiliary repair following cholestatic liver injury in mice.

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate-tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up-regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low-density lipoprotein-related protein 5/6, transgenic mice expressing stable ß-catenin, and in vitro studies, we show a role of Wnt signaling through ß-catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a ß-catenin-independent manner. CONCLUSION: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both ß-catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666).

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming.

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.