Author + information
- Ju Zhihai and
- Feilong Hei
Exosomes are tiny lipid bilayer vesicles as a result of intraluminal vesicles (ILVs) releasing that were generated by inward budding of endosomal multivesicular bodies (MVBs). Induced pluripotent stem cells generated from adult somatic cells to avoid immune rejection and ethic issues are regarded as candidates for cell therapy and autologous transplantation. Meanwhile, its exosomes may represent a nontoxic, efficient and non-immunogenic alternative to transferring endogenous or exogenous bioactive substances such as microRNA and siRNA to target cells for changing the recipient cells' fate and behavior. However, the isolation, characterization of exosomes derived from human induced pluripotent stem cells and whether they can be incepted by the recipient cells have not been investigated.
Induced pluripotent stem cells reprogrammed from human urine exfoliated renal epithelial cells (huiPSCs) were generated using established protocols described in our previous study and the pluripotent properties of huiPSCs were analyzed in both vitro and vivo. HuiPSC-derived exosomes (huiPSC-exo) were isolated from culture supernatants by ultracentrifugation. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), resistive pulse sensing (qNaNo), Western Blot were used to characterize huiPSC-exo and real-time RT-PCR were used to characterize iPSC-specific makers. Human pulmonary microvascular endothelial cells (HPMVECs) were exposed to huiPSC-exo in culture to evaluate intake of huiPSC-exo by recipient cells.
Alkaline phosphatase staining, immunofluorescence staining of pluripotency markers, and teratoma experiment confirmed that our huiPSCs were pluripotent. TEM, NTA and qNaNo showed that huiPSC-exo are homogenous, round and cup-shaped appearance with a diameter range from 46nm to 166nm. Immunoblotting confirmed the presence of several typical exosomal markers (CD63, CD81, TSG 101 and Alix) on huiPSC-exo. Real-time RT-PCR showed that huiPSC-exo contained iPSC-originated transcript-related mRNA including Oct-4, Nanog, and Klf-4. Moreover, laser scanning confocal microscopy exhibited a fusion of these huiPSC-exo with recipient HPMVECs in coculture.
Here, firstly, we, generated human induced pluripotent stem cells using noninvasive methods through reprogramming urine exfoliated renal epithelial cells. Experiments both in vitro and vivo confirmed they possess pluripotent properties. Meanwhile, our data, for the first time, provide a purification and characterization protocol for huiPSC-exo that as a natural nanocarriers may be vital for cellular communication. Further, the uptake of huiPSC-exo by HPMVECs could be consider as a natural carrier for transferring endogenous and exogenous bioactive contents to recipient cells affecting target cell function and makes a further study about their biological function possible.