(193d) Investigation of the Variation in Exosome Release By Human Pluripotent Stem Cells in Static and Stirred Suspension Cultures | AIChE

(193d) Investigation of the Variation in Exosome Release By Human Pluripotent Stem Cells in Static and Stirred Suspension Cultures


Tzanakakis, E. - Presenter, Tufts University
Ashok, P., Oklahoma State University
Human pluripotent stem cells (hPSCs) have the capacity to differentiate into multiple cell types of great significant to regenerative medicine. Exosomes, which are nanovesicles released from cells and with size ranging between 50-130 nm, carry proteins, mRNAs, and microRNAs (miRNAs) [1]. As vehicles for information transfer among cells, exosomes can be used as gene/protein delivery therapeutics with advantages over conventional technologies. We hypothesize that exosomes released from hPSCs and their differentiated products influence the process of stem cell fate specification.

The isolation of exosomes from hPSCs has not been reported to date. To that end, we successfully obtained exosomes from hPSCs and identified morphological attributes and relevant protein markers. Messenger RNAs and miRNAs linked to the hPSC pluripotent state were detected in these exosomes. Specifically, we discovered miR302A and miR302C, which induce and maintain pluripotency in hPSCs in a POU5F1 (OCT4)-dependent manner [2], in the hPSC-derived exosome cargo. Exposure of human embryonic kidney 293 (HEK293) cells to the exosomes from hPSCs led to upregulation of the aforementioned miRNAs thereby confirming exosomal capacity to transfer information between cells.

While investigation of the role of exosomes on hPSC differentiation is ongoing, we also noted that hPSCs secrete minute quantities of exosomes in dishes when compared to stirred-suspension cultures. Human PSCs cultured in stirred suspension release as much as 10 times higher amounts of exosomes than the same number of hPSCs cultured in dishes. Based on our findings, this difference in exosome production could not be attributed to potentially increased apoptosis or aberrant differentiation. We have investigated the TP53 (p53) downstream products STEAP3 and CHMP4C, which are known to regulate vesicle secretion [3], as causal agents for increased exosome secretion. TP53 may be activated due to increased shear stress experienced by cells in stirred suspension. Based on qPCR and Western blot analyses, the expression of STEAP3 is higher in stirred-suspension hPSC cultures compared to dishes, based on both mRNA (three-fold; qPCR) and protein levels (Western blot).

Our study has ramifications for the use of stirred-suspension culture platforms to obtain large yields of exosomes from self-renewing and differentiating hPSCs and for identifying markers which may facilitate hPSC specification. Moreover, the modulation of STEAP3 expression can conceivably provide a handle on the amount of exosomes released from hPSCs in culture.


  1. Kourembanas, S., Exosomes: Vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Ann. Rev. Physiol. 77(7): 7.1-7.15,2014.
  2. Li, H. L., et al., miR-302 regulates pluripotency, teratoma formation and differentiation in stem cells via an AKT1/OCT4-dependent manner. Cell Death Dis. 7: e2078,2016.
  3. Yu, X., et al., The regulation of the endosomal compartment by p53 the tumor suppressor gene. FEBS J. 276(8): 2201-2212,2009.