Introduction: The secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds. Methods: A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively. Results: UCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM2D-treated wounds in vivo. Although CM2D proved to be beneficial, CM3D-treated wounds revealed a completely regenerated tissue by day 14 after excisions, with a more mature vascular system already showing glands and hair follicles. Conclusions: This work unravels an important alternative to the use of cells in the final formulation of advanced therapy medicinal products by providing a proof of concept that a reproducible system for the production of UCX®-conditioned medium can be used to prime a secretome for eventual clinical applications.

The Escherichia coli morphogene bolA causes round morphology when overexpressed. The expression of bolA is mainly regulated by a σ^s‐dependent gearbox promoter bolA1p. Such regulation results in increased relative levels of expression at slow growth rates, as seen with those attained at the onset of stationary phase. We demonstrate that bolA1p is also induced during early logarithmic growth in response to several forms of stress, and that this induction can be partially σ^s independent. Sudden carbon starvation results in a 17‐fold increase in mRNA levels derived from bolA1p 1 h after stress imposition. Increased osmolarity results in a more than 20‐fold increase after the same period. Considerable increases in bolA1p mRNA levels were also detected as a result of heat shock, acidic stress and oxidative stress, which has been shown to inhibit σ^s translation. The orders of magnitude of bolA1p induction in log phase due to sudden starvation, osmotic shock and oxidative stress surpass the levels reached in stationary phase. Under sudden carbon starvation and osmotic shock, the cells changed their morphology, resembling those cells in which bolA is overexpressed in stationary phase. Increased expression and morphological changes due to sudden carbon starvation and osmotic shock still occur when σ^S is not present in a rpoS⁻ background. The results show that expression of bolA is not confined to stationary phase, but it can also play an important role in general stress response. We propose that bolA1p stress induction overrides the normal regulation imposed by growth rate, which is strictly the result of σ^S‐directed transcription.

Introduction: Among the plethora of cells under investigation to restore a functional myocardium, mesenchymal stromal cells (MSCs) have been granted considerable interest. However, whereas the beneficial effects of bone marrow MSCs (BM-MSCs) in the context of the diseased heart are widely reported, data are still scarce on MSCs from the umbilical cord matrix (UCM-MSCs). Herein we report on the effect of UCM-MSC transplantation to the infarcted murine heart, seconded by the dissection of the molecular mechanisms at play. Methods: Human umbilical cord tissue-derived MSCs (UCX®), obtained by using a proprietary technology developed by ECBio, were delivered via intramyocardial injection to C57BL/6 females subjected to permanent ligation of the left descending coronary artery. Moreover, medium produced by cultured UCX® preconditioned under normoxia (CM) or hypoxia (CMH) was collected for subsequent in vitro assays. Results: Evaluation of the effects upon intramyocardial transplantation shows that UCX® preserved cardiac function and attenuated cardiac remodeling subsequent to myocardial infarction (MI). UCX® further led to increased capillary density and decreased apoptosis in the injured tissue. In vitro, UCX®-conditioned medium displayed (a) proangiogenic activity by promoting the formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs), and (b) antiapoptotic activity in HL-1 cardiomyocytes subjected to hypoxia. Moreover, in adult murine cardiac Sca-1⁺ progenitor cells (CPCs), conditioned medium enhanced mitogenic activity while activating a gene program characteristic of cardiomyogenic differentiation. Conclusions: UCX® preserve cardiac function after intramyocardial transplantation in a MI murine model. The cardioprotective effects of UCX® were attributed to paracrine mechanisms that appear to enhance angiogenesis, limit the extent of the apoptosis, augment proliferation, and activate a pool of resident CPCs. Overall, these results suggest that UCX® should be considered an alternative cell source when designing new therapeutic approaches to treat MI.

Summary: The gene bolA has been shown to trigger the formation of osmotically stable round cells when overexpressed in stationary phase. We show that in poor growth conditions bolA is essential for normal cell morphology in stationary phase and under conditions of starvation. During exponential growth bolA promotes round morphology through a mechanism that is strictly dependent on the two main Escherichia colid,d‐carboxypeptidases, PBP5 and PBP6. The results show that bolA controls the levels of transcription of dacA (PBP5), dacC (PBP6) and ampC (AmpC), a class C β‐lactamase, thus connecting for the first time penicillin binding proteins (PBPs) and β‐lactamases at the level of gene regulation. Furthermore, PBP5 and PBP6 are shown to be independently regulated and to have distinct effects on the peptidoglycan layer. The evidence presented demonstrates that bolA is a regulator of cell wall biosynthetic enzymes with different roles in cell morphology and cell division.

MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroboratedin vivoin a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression ofHLA-DRA,HO-1,IGFBP1, 4 and 6,ILR1,IL6RandPTGESand increased expression ofCD200,CD273,CD274,IL1B,IL-8,LIFandTGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1β, IL-8, LIF and TGFβ2.

Rheumatoid arthritis (RA) is an autoimmune disorder whose treatment is mostly restricted to pain and symptom management and to the delay of joint destruction. Mesenchymal stem/stromal cells from the umbilical cord tissue (UC-MSCs) have previously been proven to be immunomodulatory and more efficient than bone marrow-derived MSCs in causing remission of local and systemic arthritic manifestations in vivo. Given the paracrine nature of UC-MSC activity, their application as active substances can be replaced by their secretome, thus avoiding allogeneic rejection and safety issues related to unwanted grafting. In this work, we aimed at demonstrating the viability of applying the 3D-primed UC-MSC secretome for the amelioration of arthritic signs. A proteomic analysis was performed to both, media conditioned by UC-MSC monolayer (CM2D) and 3D cultures (CM3D). The analysis of relevant trophic factors confirmed secretome profiles with very significant differences in terms of therapeutic potential. Whereas, CM3D was characterised by a prevailing expression of anti-inflammatory cytokines such as IL-10 and LIF, along with trophic factors involved in different mechanisms leading to tissue regeneration, such as PDGF-BB, FGF-2, I-309, SCF, and GM-CSF; CM2D presented relatively higher levels of IL-6, MCP-1, and IL-21, with recognised pro-inflammatory roles in joint disease and pleiotropic effects in the progression of rheumatoid arthritis (RA). Accordingly, different motogenic effects over mouse chondrocytes and distinct capacities of inducing glycosaminoglycan synthesis in vitro were observed between CM3D and CM2D. Finally, the evaluation of arthritic manifestations in vivo, using an adjuvant-induced model for arthritis (AIA), suggested a significantly higher therapeutic potential of CM3D over CM2D and even UC-MSCs. Histological analysis confirmed a faster remission of local and systemic arthritic manifestations of CM3D-treated animals. Overall, the results show that the use of UC-MSC CM3D is a viable and better strategy than direct UC-MSC administration for counteracting AIA-related signs. This strategy represents a novel MSC-based but nonetheless cell-free treatment for arthritic conditions such as those characterising RA.