First steps to define murine amniotic fluid stem cell microenvironment

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells

Decellularized diaphragmatic muscle drives a constructive angiogenic response in vivo

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1\u3b1. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31+, \u3b1SMA+, and vWF+ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF+ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration

Containment of a genetically modified microorganism by an activated sludge system

Abstract The effectiveness of physical, chemical and biological barriers to the diffusion of genetically modified microorganisms (GMMs) to prevent their release into the environment is currently under scrutiny worldwide because of the associated potential ecological impacts. An industrial discharge of a non-sterilized fermentation broth containing GMM biomass into a conventional municipal wastewater treatment plant would deliver the GMMs into the activated sludge system process (ASSP). The present work aimed to model and evaluate the containment capability of a small ASSP (part of a 20,000 people equivalent municipal plant) in the event of receiving GMM biomass from a medium-small biotechnological plant dedicated to the production of polyhydroxyalkanoates (3000 t/year of biopolymer). An actual GMM (Pseudomonas putida KTOY06) was injected into a bench-scale ASSP (ASSPLab) in a quantity proportional to the relative dimensions of the plants mentioned. The experimental and model results indicated that the ASSP of the target municipal treatment plant would not be capable of holding back such a sudden input of GMM; 6 h after the discharge, 11–15 % of injected GMM cells were released through the clarified stream of the ASSPLab, with the rest being gradually released over time. Since the GMM employed did not exhibit any growth in the ASSPLab, its concentration in the clarified water stream would not represent a substantial risk of release into the environment if appropriate tertiary treatments were integrated. This study confirmed the necessity of a thorough risk assessment of biotechnological processes prior to their implementation