Characterization of Induced Pluripotent Stem Cell Microvesicle Genesis, Morphology and Pluripotent Content

Abstract

Microvesicles (MVs) are lipid bilayer-covered cell fragments that range in diameter from 30 nm–1uM and are released from all cell types. An increasing number of studies reveal that MVs contain microRNA, mRNA and protein that can be detected in the extracellular space. In this study, we characterized induced pluripotent stem cell (iPSC) MV genesis, content and fusion to retinal progenitor cells (RPCs) in vitro. Nanoparticle tracking revealed that iPSCs released approximately 2200 MVs cell/hour in the first 12 hrs with an average diameter of 122 nm. Electron and light microscopic analysis of iPSCs showed MV release via lipid bilayer budding. The mRNA content of iPSC MVs was characterized and revealed the presence of the transcription factors Oct-3/4, Nanog, Klf4, and C-Myc. The protein content of iPSCs MVs, detected by immunogold electron microscopy, revealed the presence of the Oct-3/4 and Nanog. Isolated iPSC MVs were shown to fuse with RPCs in vitro at multiple points along the plasma membrane. These findings demonstrate that the mRNA and protein cargo in iPSC MVs have established roles in maintenance of pluripotency. Building on this work, iPSC derived MVs may be shown to be involved in maintaining cellular pluripotency and may have application in regenerative strategies for neural tissue.

Herpes Simplex Virus Capsid Localization to ESCRT-VPS4 Complexes in the Presence and Absence of the Large Tegument Protein UL36p

“…Regions of VP26-mRFP1 red fluorescence aligned with spherical structures of ϳ140 nm in diameter; we previously saw identical structures emitting green fluorescence in the presence of VP26-GFP (48); this shape and size correspond well to the expected 125-nm diameter of the HSV capsid plus the approximately 16-nm-thick chromium sputter coating deposited during SEM preparation. Capsids were frequently in close association with pleomorphic vesiculotubular structures similar to those we earlier showed to be stained with lipophilic dyes (48) and that resemble isolated organelles and vesicles imaged by SEM (91,92). CLEM analysis revealed that particles with high levels of red/green fluorescence colocalization correspond to capsids in intimate association with organellar envelopment sites.…”

Human Pluripotent Stem Cell-Derived Extracellular Vesicles: Characteristics and Applications

“…Undifferentiated iPSC-derived EVs/exosomes Undifferentiated iPSCs were reported to release about 2200 EVs/cell/hour in the first 12 h (with an average diameter of 122 nm) in culture, producing 16-fold more EVs than various types of MSCs in a chemically defined medium. 61,62 mRNAs in iPSC-EVs were found to contain reprogramming factors Oct3/4, Nanog, Klf4, and c-Myc. Glycome of EVs derived from hiPSCs was analyzed using high-density lectin microarray, 63 which found that the characteristic glycan signature of hiPSCs was captured in the derived EVs.…”

Exosomes Generated From iPSC-Derivatives

“…In addition, the microvesicles from the hiPSCs exhibit enhanced cardiac and endothelial differentiation potential of cMSCs 94 . Microvesicles derived from mouse iPSC were shown to contain a group of iPSC-specific pluripotent transcription factors critical in maintaining iPSC pluripotency 95 . The promising results encourage more investigation on iPSC-derived exosomes although information inside the iPSC-exosomes are not completely identified yet.…”