Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs

Abstract

BackgroundExosomes or secreted bi-lipid vesicles from human ESC-derived mesenchymal stem cells (hESC-MSCs) have been shown to reduce myocardial ischemia/reperfusion injury in animal models. However, as hESC-MSCs are not infinitely expansible, large scale production of these exosomes would require replenishment of hESC-MSC through derivation from hESCs and incur recurring costs for testing and validation of each new batch. Our aim was therefore to investigate if MYC immortalization of hESC-MSC would circumvent this constraint without compromising the production of therapeutically efficacious exosomes.MethodsThe hESC-MSCs were transfected by lentivirus carrying a MYC gene. The transformed cells were analyzed for MYC transgene integration, transcript and protein levels, and surface markers, rate of cell cycling, telomerase activity, karyotype, genome-wide gene expression and differentiation potential. The exosomes were isolated by HPLC fractionation and tested in a mouse model of myocardial ischemia/reperfusion injury, and infarct sizes were further assessed by using Evans' blue dye injection and TTC staining.ResultsMYC-transformed MSCs largely resembled the parental hESC-MSCs with major differences being reduced plastic adherence, faster growth, failure to senesce, increased MYC protein expression, and loss of in vitro adipogenic potential that technically rendered the transformed cells as non-MSCs. Unexpectedly, exosomes from MYC-transformed MSCs were able to reduce relative infarct size in a mouse model of myocardial ischemia/reperfusion injury indicating that the capacity for producing therapeutic exosomes was preserved.ConclusionOur results demonstrated that MYC transformation is a practical strategy in ensuring an infinite supply of cells for the production of exosomes in the milligram range as either therapeutic agents or delivery vehicles. In addition, the increased proliferative rate by MYC transformation reduces the time for cell production and thereby reduces production costs.

Evaluation of electroporation-induced adverse effects on adipose-derived stem cell exosomes

“…These factors included, for example, the correct choice of donor cell, because the exosomes derived for drug delivery should be devoid of any stimulating properties on the immunological system as well as on the disease, in which they are used (Tian et al 2013). The donor cell should also be an efficient producer of exosomes to obtain clinical scale quantities of exosomes for human use (Chen et al 2011;Yeo et al 2013). To secure specific action of the delivered drug, exosomes should be endowed with a targeting ligand to facilitate transport into a particular tissue or area of disease (Alvarez-Erviti et al 2011;Ohno et al 2012;Tian et al 2013).…”

Embryonic Stem Cells for Therapies – Challenges and Possibilities

“…Immortalization of these mesenchymal stem cells did not compromise the production or activity of the exosomes (T.S. Chen et al, 2011). These studies provided for the development of a sustainable scalable manufacturing process to produce potentially therapeutic exosomes for testing in the clinic.…”

Is Immunomodulation a Principal Mechanism Underlying How Cell-Based Therapies Enhance Stroke Recovery?

“…Indeed, the secretomes of cellular therapies have become an active area of research in the hopes of identifying the factors produced by cell therapies that are critical to their effects. Many cell therapies also release microvesicles such as exosomes, which themselves may penetrate and re-engineer the biological properties of target tissues [45][46][47].…”