Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery.

Advances in biomedical research have generated an unprecedented number of potential targets for therapeutic intervention to treat disease or delay disease progression. Unfortunately, many of these targets are not druggable as they are intracellular, present in many cell types, poorly soluble or rapidly inactivated. Although synthetic drug vehicles have successfully circumvented many of these problems, natural particulates such as exosomes that intrinsically possess many attributes of a drug delivery vehicle are highly attractive as potentially better alternatives. Of the cell types known to produce exosomes, the readily available proliferative, immunosuppressive and clinically tested human mesenchymal stem cell (MSC) is the most prolific producer. Its exosomes are therapeutic in animal model of disease and exhibit immunosuppressive activity. The quality and quantity of exosome production is not compromised by immortalization to create a permanent MSC cell line. Therefore, MSC is well suited for mass production of exosomes that are ideal for drug delivery.

Proteolytic Potential of the MSC Exosome Proteome: Implications for an Exosome-Mediated Delivery of Therapeutic Proteasome.

Mesenchymal stem cells (MSCs) are used in many of the current stem cell-based clinical trials and their therapeutic efficacy has increasingly been attributed to secretion of paracrine factors. We have previously demonstrated that a therapeutic constituent of this secretion is exosome, a secreted bilipid membrane vesicle of ~50-100 nm with a complex cargo that is readily internalized by H9C2 cardiomyocytes. It reduces infarct size in a mouse model of myocardial ischemia/reperfusion (MI/R) injury. We postulate that this therapeutic efficacy is derived from the synergy of a select permutation of individual exosome components. To identify protein candidates in this permutation, the proteome was profiled and here we identified 20S proteasome as a protein candidate. Mass spectrometry analysis detected all seven α and seven ß chains of the 20S proteasome, and also the three beta subunits of "immunoproteasome" with a very high confidence level. We demonstrated that a functional proteasome copurified with MSC exosomes with a density of 1.10-1.18 g/mL, and its presence correlated with a modest but significant reduction in oligomerized protein in a mouse model of myocardial infarction. Circulating proteasomes in human blood also copurified with exosomes. Therefore, 20S proteasome is a candidate exosome protein that could synergize with other constituents to ameliorate tissue damage.

Delineating biological pathways unique to embryonic stem cell-derived insulin-producing cell lines from their noninsulin-producing progenitor cell lines.

To identify unique biochemical pathways in embryonic stem cell-derived insulin-producing cells as potential therapeutic targets to prevent or delay beta-cell dysfunction or death in diabetic patients, comparative genome-wide gene expression studies of recently derived mouse insulin-producing cell lines and their progenitor cell lines were performed using microarray technology. Differentially expressed genes were functionally clustered to identify important biochemical pathways in these insulin-producing cell lines. Biochemical or cellular assays were then performed to assess the relevance of these pathways to the biology of these cells. A total of 185 genes were highly expressed in the insulin-producing cell lines, and computational analysis predicted the pentose phosphate pathway (PPP), clathrin-mediated endocytosis, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway as important pathways in these cell lines. Insulin-producing ERoSHK cells were more resistant to hydrogen peroxide (H(2)O(2))-induced oxidative stress. Inhibition of PPP by dehydroepiandrosterone and 6-aminonicotinamide abrogated this H(2)O(2) resistance with a concomitant decrease in PPP activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Clathrin-mediated endocytosis, which is essential in maintaining membrane homeostasis in secreting cells, was up-regulated by glucose in ERoSHK but not in their progenitor ERoSH cells. Its inhibition by chlorpromazine at high glucose concentration was toxic to the cells. Troglitazone, a PPARG agonist, up-regulated expression of Ins1 and Ins2 but not Glut2. Gene expression analysis has identified the PPP, clathrin-mediated endocytosis, and the PPAR signaling pathway as the major delineating pathways in these insulin-producing cell lines, and their biological relevance was confirmed by biochemical and cellular assays.

Derivation and characterization of human fetal MSCs: an alternative cell source for large-scale production of cardioprotective microparticles.

The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 10(16-19) cells or 10(7-10) doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r(2)>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50-65 nm. This purified population of microparticles was cardioprotective at approximately 1/10 dosage of the crude secretion.