Abstract
BackgroundMesenchymal stromal cells (MSCs) are an attractive therapeutic agent in regenerative medicine. Recently, there has been a paradigm shift from differentiation of MSCs to their paracrine effects at the injury site. Several reports elucidate the role of trophic factors secreted by MSCs toward the repair of injured tissues. We hypothesize that fractionating the MSC secretome will enrich exosomes containing soluble bioactive molecules, improving its therapeutic potential for liver failure.MethodsRat bone marrow MSCs were isolated and the conditioned media filtered, concentrated and ultracentrifuged to generate fractionated secretome. This secretome was characterized for the presence of exosomes and recovery from liver injury assessed in in-vitro liver injury models. The results were further validated in vivo.ResultsStudies on in-vitro liver injury models using acetaminophen and hydrogen peroxide show better cell recovery and reduced cytotoxicity in the presence of fractionated as opposed to unfractionated secretome. Further, the cells showed reduced oxidative stress in the presence of fractionated secretome, suggesting a potential antioxidative effect. These results were further validated in vivo in liver failure models, wherein improved liver regeneration in the presence of fractionated secretome (0.819 ± 0.035) was observed as compared to unfractionated secretome (0.718 ± 0.042).ConclusionsThe work presented is a proof of concept that fractionating the secretome enriches certain bioactive molecules involved in the repair and recovery of injured liver tissue.Graphical abstractExosome enriched mesenchymal stromal cell-derived fractionated secretome potentiates recovery upon injection in injured liver
Highlights
Mesenchymal stromal cells (MSCs) are an attractive therapeutic agent in regenerative medicine
The liver has a remarkable capacity to self-heal or regenerate—a feature presumably evolved over the years to protect the liver from the catastrophic consequences of liver loss caused by food toxins [1]
Several studies have hinted at the role of chemokines and trophic factors released by MSCs in reducing tissue inflammation, cellular apoptosis and liver fibrosis, contributing to the overall improvement of liver function [20, 21]
Summary
All materials used were procured from Sigma Aldrich, USA. Animals Male Wistar rats (250–350 g) were used for isolation of bone marrow-derived MSCs and development of ALF models as per approval from the Institute Animal Ethics Committee (IITK/IAEC/2014/1023 and IITK/IAEC/ 2014/1022, respectively) of IIT Kanpur, under the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Collection and characterization of exosome-rich fractionated secretome Once the MSCs in passage 3 reached 70–80% confluency, complete medium was replaced with serum-free medium supplemented with 1% antibiotic and cells cultured for 48 h. Effect of exosome-rich fractionated MSC secretome on viability of liver cells To check for cytotoxicity of the EFS, different concentrations of the secretome were incubated with HepG2 cells. Effect of EFS on liver regeneration rate and recovery of liver functions in rodent models of liver failure In the ischemic/reperfusion injury model, before removing the clamp, 50 μg of EFS in 100 μl phosphate buffered saline (PBS) was injected via the HPV in one group of animals (n = 5) and 50 μg of unfractionated secretome (reconstituted in PBS after lyophilizing) was injected in another group of animals (n = 5). The differences between two groups were analyzed using Student’s t test
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