Multipotential stromal cells (MSCs) have been touted to provide an alternative to conservative procedures of therapy, be it heart transplants, bone reconstruction, kidney grafts, or skin, neuronal and cartilage repair. A wide gap exists, however, between the number of MSCs that can be obtained from the donor site and the number of MSCs needed for implantation to regenerate tissue. Standard methods of MSC expansion being followed in laboratories are not fully suitable due to time and age-related constraints for autologous therapies, and transplant issues leave questions for allogenic therapies. Beyond these issues of sufficient numbers, there also exists a problem of MSC survival at the graft. Experiments in small animals have shown that MSCs do not persist well in the graft environment. Either there is no incorporation into the host tissue, or, if there is incorporation, most of the cells are lost within a month. The use of growth and other trophic factors may be helpful in counteracting these twin issues of MSC expansion and death. Growth factors are known to influence cell proliferation, motility, survival and morphogenesis. In the case of MSCs, it would be beneficial that the growth factor does not induce differentiation at an early stage since the number of early-differentiating progenitors would be very low. The present review looks at the effect of and downstream signaling of various growth factors on proliferation and survival in MSCs.

There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.wound healing | diabetes | drug delivery | small molecule | angiogenesis D iabetes mellitus affects over 25 million people in the United States (1, 2) and costs nearly $250 billion per year (3). Chronic diabetic wounds and decubiti are important long-term sequalae of both diabetes mellitus types 1 and 2 (4). There is a high mortality in diabetic patients who develop decubiti (5-7), and owing to prolonged disability and the high rates of recurrence these wounds represent an especially severe complication of diabetes (8). This is further underscored by the fact that diabetic nonhealing wounds are the leading cause of nontraumatic amputations in the United States (3, 9-11). As such, there is a clear need for new approaches to effectively manage and treat diabetic ulcers.The propensity for wound development in diabetes is associated with a reduced capacity for ischemia-driven neovascularization (12, 13). Hypoxia inducible factor-1 (HIF-1), which consists of a highly regulated α-subunit and a constitutively expressed β-subunit, is a critical transcriptional regulator of the normal cellular response to hypoxia, promoting progenitor cell recruitment, proliferation, survival, and neovascularization (14, 15). In nondiabetics, hypoxia causes stabilization of HIF-1α protein by preventing the normal rapid proteasomal degradation of HIF-1α. It does this by inhibiting the prolyl hydroxylases (PHDs), which hydroxylate specific prolyl residues on HIF-1α. Without proline hydroxylation HIF-1α is not ...

Stem cells have shown substantial promise for various diseases in preclinical and clinical trials. However, low cell engraftment rates significantly limit the clinical translation of stem cell therapeutics. Numerous injectable hydrogels have been developed to enhance cell retention. Yet, the design of an ideal material with tunable properties that can mimic different tissue niches and regulate stem cell behaviors remains an unfulfilled promise. Here, an injectable poly(ethylene glycol) (PEG)-gelatin hydrogel is designed with highly tunable properties, from a multifunctional PEG-based hyperbranched polymer and a commercially available thiolated gelatin. Spontaneous gelation occurs within about 2 min under the physiological condition. Murine adiposederived stem cells (ASCs) can be easily encapsulated into the hydrogel, which supports ASC growth and maintains their stemness. The hydrogel mechanical properties, biodegradability, and cellular responses can be finely controlled by changing hydrogel formulation and cell seeding densities. An animal study shows that the in situ formed hydrogel significantly improves cell retention, enhances angiogenesis, and accelerates wound closure using a murine wound healing model. These data suggest that injectable PEG-gelatin hydrogel can be used for regulating stem cell behaviors in 3D culture, delivering cells for wound healing and other tissue regeneration applications.

Scarring and tissue fibrosis represent a significant source of morbidity in the United States. Despite considerable research focused on elucidating the mechanisms underlying cutaneous scar formation, effective clinical therapies are still in the early stages of development. A thorough understanding of the various signaling pathways involved is essential to formulate strategies to combat fibrosis and scarring. While initial efforts focused primarily on the biochemical mechanisms involved in scar formation, more recent research has revealed a central role for mechanical forces in modulating these pathways. Mechanotransduction, which refers to the mechanisms by which mechanical forces are converted to biochemical stimuli, has been closely linked to inflammation and fibrosis and is believed to play a critical role in scarring. This review provides an overview of our current understanding of the mechanisms underlying scar formation, with an emphasis on the relationship between mechanotransduction pathways and their therapeutic implications.

IntroductionPathophysiologic changes associated with diabetes impair new blood vessel formation and wound healing. Mesenchymal stem cells derived from adipose tissue (ASCs) have been used clinically to promote healing, although it remains unclear whether diabetes impairs their functional and therapeutic capacity.MethodsIn this study, we examined the impact of diabetes on the murine ASC niche as well as on the potential of isolated cells to promote neovascularization in vitro and in vivo. A novel single-cell analytical approach was used to interrogate ASC heterogeneity and subpopulation dynamics in this pathologic setting.ResultsOur results demonstrate that diabetes alters the ASC niche in situ and that diabetic ASCs are compromised in their ability to establish a vascular network both in vitro and in vivo. Moreover, these diabetic cells were ineffective in promoting soft tissue neovascularization and wound healing. Single-cell transcriptional analysis identified a subpopulation of cells which was diminished in both type 1 and type 2 models of diabetes. These cells were characterized by the high expression of genes known to be important for new blood vessel growth.ConclusionsPerturbations in specific cellular subpopulations, visible only on a single-cell level, represent a previously unreported mechanism for the dysfunction of diabetic ASCs. These data suggest that the utility of autologous ASCs for cell-based therapies in patients with diabetes may be limited and that interventions to improve cell function before application are warranted.

Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and non-hematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein (GFP) and non-hematopoietic cells with red fluorescent protein (RFP). Use of single-cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45− and Cd11b−, representing a previously unidentified population. Both cell types were found to initially peak, then reduce post-healing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time-course of fibrocytes in cutaneous healing.

IntroductionBone marrow mesenchymal stem cells/multipotent stromal cells (MSCs) are recruited to sites of injury and subsequently support regeneration through differentiation or paracrine activity. During periods of stress such as wound site implant or differentiation, MSCs are subjected to a variety of stressors that might activate pathways to improve cell survival and generate energy. In this study, we monitored MSC autophagy in response to the process of differentiation.MethodsMSC autophagosome structures were observed by using transmission electron microscopy and a tandem green fluorescent protein-red fluorescent protein autophagic flux reporter to monitor the mammalian microtubule-associated protein-1 light chain 3 (LC3) turnover in real time. MSCs were differentiated by using standard osteogenic and adipogenic media, and autophagy was examined during short-term and long-term differentiation via immunoblots for LC3I and II. Autophagy was modulated during differentiation by using rapamycin and bafilomycin treatments to disrupt the autophagosome balance during the early stages of the differentiation process, and differentiation was monitored in the long term by using Von Kossa and Oil Red O staining as well as quantitative polymerase chain reaction analysis of typical differentiation markers.ResultsWe found that undifferentiated MSCs showed an accumulation of a large number of undegraded autophagic vacuoles, with little autophagic turnover. Stimulation of autophagy with rapamycin led to rapid degradation of these autophagosomes and greatly increased rough endoplasmic reticulum size. Upon induction of osteogenic differentiation, MSC expression of LC3II, a common autophagosome marker, was lost within 12 hours, consistent with increased turnover. However, during adipogenic differentiation, drug treatment to alter the autophagosome balance during early differentiation led to changes in differentiation efficiency, with inhibited adipocyte formation following rapamycin treatment and accelerated fat accumulation following autophagosome blockade by bafilomycin.ConclusionsOur findings suggest that MSCs exist in a state of arrested autophagy with high autophagosome accumulation and are poised to rapidly undergo autophagic degradation. This phenotype is highly sensitive, and a balance of autophagy appears to be key in efficient MSC differentiation and function, as evidenced by our results implicating autophagic flux in early osteogenesis and adipogenesis.

Multipotential stromal cells/mesenchymal stem cells (MSCs) are attractive candidates for regenerative therapy due to the ability of these cells to differentiate and positively influence neighboring cells. However, on implantation for wound reconstruction, these cells are lost as they are challenged by nonspecific inflammation signals generated in the wound environment and in response to any implanted foreign body. We have previously shown that sustained and surface-restricted epidermal growth factor receptor (EGFR) signaling by a tethered form of its prototypal ligand EGF enhances survival of MSC in the presence of death cytokines such as FasL, serum deprivation, and low oxygen in vitro. This was proposed to be due to the plasma membrane restriction of EGFR signaling. Interestingly, during wound repair, an extracellular matrix (ECM) component Tenascin-C (TNC) containing EGF-like repeats (EGFL) and fibronectin-like repeats (FNL) is upregulated. A few of the 14 EGFL on each of the 6 arms, especially the 14th, bind as low-affinity/high-avidity ligands to EGFR causing sustained surface-restricted EGFR signaling. We queried whether signaling by this physiologically relevant EGFR matrikine also protects MSCs from FasLinduced death. MSCs grown on TNC and Collagen I (as TNC by itself is antiadhesive) displayed a survival advantage in the presence of FasL. TNC neither sequestered nor neutralized FasL; rather, the effects of survival were via cell signaling. This survival was dependent on TNC activating EGFR and downstream pathways of Erk and Akt through EGFL; to a much lesser extent, the FNL of TNC also contributed to survival. Taken together, these results suggest that providing MSCs with a nonimmunogenic naturally occurring ECM moiety such as TNC enhances their survival in the presence of death factors, and this advantage occurs via signaling through EGFR primarily and integrins only to a minor extent. This matrix component is proposed to supplement MSC delivery on the scaffolds to provide a survival advantage against death upon in vivo implantation.