Microvesicles Released By Human Embryonic Stem Cell Derived-mesenchymal Stem Cells Inhibit Proliferation Of Leukemia Cells In Vitro

Background Recently,several studies have suggested that human umbilical cord and bone marrow mesenchymal stem cells(MSCs)could obviously inhibit the proliferation of leukemia cells after direct coculture.Our laboratory focused on the culture and oriented differentiation of human embryonic stem cells(h ESCs)before and found h ESCs could differentiate into mesenchymal stem cells under certain conditions,which was also called ‘human embryonic stem cell derived mesenchymal stem cells(h ESC-MSCs)’.We expected that this kind of MSCs might also inhibit the proliferation of leukemia cells.To deal with this problem,following studies were conducted and results showed h ESC-MSCs could actually inhibit leukemia cells through indirect actions or paracrine.Microvesicles were active component in conditioned medium and played an important role in the exchange of information and material.Corresponding to this,we hypothesized that microvesicles released by human embryonic stem cell derived-mesenchymal stem cells(h ESC-MSC-MVs)could also inhibit the proliferation of leukemia cells in vitro and conducted further studies.Objectives We aimed to investigate the inhibition effect of h ESC-MSC-MVs on leukemia cells with two different human leukemia cell lines--K562 and HL60 and preliminarily explore the molecular mechanisms involved.Methods(1)Serum-free medium or conditioned medium of h ESC-MSCs was collected and h ESC-MSC-MVs were isolated through ultracentrifugation.Then h ESC-MSC-MVs were identified under a scanning electron microscope and transmission electron microscope separately.(2)After 48 h cocultured with h ESC-MSCs or h ESC-MSC-MVs,the number of leukemia cells was counted under microscope and viability of K562 and HL60 was measured by CCK-8 assay.(3)Transmission electron microscope was used to count the amount of autophagosomes and Western blot was adopted to evaluate the expression of autophagy related proteins such as LC3 and Beclin-1.(4)Flow cytometry was used to analyze the apoptosis rate of tumor cells and the expression level of proteins Bax and Bcl-2 related to apoptosis were estimated by Western blot analysis.Results(1)Both h ESC-MSCs and h ESC-MSC-MVs could inhibit proliferation of leukemia cells in a concentration-dependent manner in vitro.(2)After 48 h stimulation with h ESC-MSC-MVs,the treatment group showed a remarkable autophagosome accumulation in leukemia cells.Western blot suggested an increase of expression level of Beclin-1 and LC3-II conversion in stimulation group.(3)Flow cytometry analysis showed the apoptosis rate was increased in stimulation group and the ratio of Bcl-2/Bax was reduced,which was reflected by Western blot.Conclusions(1)h ESC-MSC-MVs could inhibit proliferation of human leukemia cell lines K562 and HL60 in vitro.(2)h ESC-MSC-MVs could up-regulate the level of autophagy in leukemia cells.(3)Excessive autophagy induced by h ESC-MSC-MVs might promote apoptosis of tumor cells further.