IntroductionMesenchymal stem cell-conditioned medium (MSC-CM) has been shown to have protective effects against various cellular-injury models. This mechanism of protection, however, has yet to be elucidated. Recently, exosomes were identified as the active component in MSC-CM. The aim of this study is to investigate the effect of MSC-derived exosomes in an established carbon tetrachloride (CCl4)-induced liver injury mouse model. This potential effect is then validated by using in vitro xenobiotic-induced liver-injury assays: (1) acetaminophen (APAP)- and (2) hydrogen peroxide (H2O2)-induced liver injury.MethodsThe exosomes were introduced concurrent with CCl4 into a mouse model through different routes of administration. Biochemical analysis was performed based on the blood and liver tissues. Subsequently the exosomes were treated in APAP and H2O2-toxicants with in vitro models. Cell viability was measured, and biomarkers indicative of regenerative and oxidative biochemical responses were determined to probe the mechanism of any hepatoprotective activity observed.ResultsIn contrast to mice treated with phosphate-buffered saline, CCl4 injury in mice was attenuated by concurrent-treatment exosomes, and characterized by an increase in hepatocyte proliferation, as demonstrated with proliferating cell nuclear antigen (PCNA) elevation. Significantly higher cell viability was demonstrated in the exosomes-treated group compared with the non-exosome-treated group in both injury models. The higher survival rate was associated with upregulation of the priming-phase genes during liver regeneration, which subsequently led to higher expression of proliferation proteins (PCNA and cyclin D1) in the exosomes-treated group. Exosomes also inhibited the APAP- and H2O2-induced hepatocytes apoptosis through upregulation of Bcl-xL protein expression. However, exosomes do not mitigate hepatocyte injury via modulation of oxidative stress.ConclusionsIn summary, these results suggest that MSC-derived exosomes can elicit hepatoprotective effects against toxicants-induced injury, mainly through activation of proliferative and regenerative responses.

Adult tissue-derived mesenchymal stem cells (MSCs) have demonstrated therapeutic efficacy in treating diseases or repairing damaged tissues through mechanisms thought to be mediated by either cell replacement or secretion of paracrine factors. Characterized, self-renewing human ESCs could potentially be an invariable source of consistently uniform MSCs for therapeutic applications. Here we describe a clinically relevant and reproducible manner of generating identical batches of hESC-derived MSC (hESC-MSC) cultures that circumvents exposure to virus, mouse cells, or serum. Trypsinization and propagation of HuES9 or H1 hESCs in feeder- and serum-free selection media generated three polyclonal, karyotypically stable, and phenotypically MSC-like cultures that do not express pluripotency-associated markers but displayed MSC-like surface antigens and gene expression profile. They differentiate into adipocytes, osteocytes, and chondrocytes in vitro. Gene expression and fluorescence-activated cell sorter analysis identified CD105 and CD24 as highly expressed antigens on hESC-MSCs and hESCs, respectively. CD105+, CD24- monoclonal isolates have a typical MSC gene expression profiles and were identical to each other with a highly correlated gene expression profile (r(2) > .90). We have developed a protocol to reproducibly generate clinically compliant and identical hESC-MSC cultures.

Although the hormone erythropoietin (Epo) and its receptor (EpoR) are known to play important roles in the regulation of erythropoiesis, several questions remain concerning the developmental role of Epo/EpoR signaling. As the functions of Epo have been defined primarily through studies of definitive erythroid cells, its importance for primitive, embryonic erythropoiesis remains uncertain, as does the significance of EpoR expression in several nonerythroid cell types. To address these questions, mouse embryonic stem cells and embryos lacking a functional EpoR gene were produced by gene targeting. The effects of the mutation were examined in embryos developing in vivo, in chimeric adult mice produced with homozygous mutant embryonic stem cells, and in hemopoietic cells cultured in vitro. No defects were apparent in nonerythroid cell lineages in which the EpoR normally is expressed, including megakaryocytes and endothelial cells. In the mutant yolk sac, primitive erythrocytes were produced in normal numbers, they underwent terminal differentiation, and expressed near normal levels of embryonic globins, although they were reduced in size and their proliferation was severely retarded after E9.5. In contrast, in the fetal liver, definitive erythropoiesis beyond the late progenitor (CFU-E) stage was drastically inhibited by the EpoR mutation, and virtually no definitive erythrocytes were produced in vivo, leading to embryonic death by E13.5. Thus, our results suggest a fundamental difference in the molecular mechanisms stimulating primitive and definitive erythropoiesis. It was also observed that a few mutant definitive erythroid cells could terminally differentiate when cultured with additional cytokines, demonstrating that although Epo/EpoR signaling is important for definitive erythroid cell survival and proliferation, it is not an obligatory step in differentiation.