Combined pharmacokinetic monitoring of MSCs and MSC-secreted IL-6.

<p>(A) Bioluminescent images of C57Bl/6 mice over a period of three days after IV cell administration of one million luciferase-engineered human MSCs. (B) Photon flux of bioluminescent signal over time after IV cell administration. Durable BLI signals were detected up to 24 hours in mice that were injected IV with MSCs. (C) Serum ELISA measurements of human IL-6 released by IV cell transplants over time. Time points for serum and imaging analyses were 0.5, 8, 24, and 72 hours after cell injection. Pooled mouse serum was serially analyzed as batches of N = 5.</p

Enhanced delivery of IL-6 by MSC transplants compared to MSC conditioned medium.

<p>(A) Serum profiles of human IL-6 after IV administration of concentrated conditioned medium into C57Bl/6 mice. The plot was normalized to the dose of conditioned medium that was contributed by 1×10⁶ cells. Pharmacokinetic parameters (B) Cmax, (C) AUC, (D) Tmax, (E) Half-life, and (F) Elimination constant were calculated for IL-6 exposure by cell transplants compared to CM administration. Significant differences between cell transplants compared to CM whereby higher levels of IL-6 and longer artificial duration was observed in plasma after cell transplantation. Time points for serum analyses were 0.5, 8, and 24 hours after cell or media injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.</p

Golgi-dependent secretion mechanism of MSC-derived IL-6 in vivo.

<p>Brefeldin A pre-treatment of MSCs was used to evaluate blockade of IL-6 release in vitro and in vivo. (A) MTT assay of MSCs treated at different concentrations of brefeldin. A non-toxic dose of 5 ug/ml was used for functional studies. (B) Human IL-6 levels in vitro after brefeldin pre-treatment. Significant reduction in 24 hour release of IL-6 was observed across all doses. (C) Alteration in serum IL-6 delivery by MSCs pretreated with a Golgi-apparatus inhibitor, Brefeldin A. MSCs were incubated with 5 µg/ml of BFA for one day and then injected into C57Bl/6 mice and compared to untreated MSCs in terms of serum IL-6 delivery. Brefeldin treatment of MSCs led to diminished release of human IL-6 in vitro and in vivo. (D) Area-under-curve analysis of human IL-6 after MSC pre-treatment with brefeldin A and transplantation. Exposure to IL-6 was significantly reduced by inhibition of the Golgi apparatus. Time points for serum analyses were 0.5, 8, and 24 hours after cell injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.</p

Blood Monitoring of Engineered Human MSCs with the Secreted Gaussia Luciferase Reporter.

<p>A lentivirus vector expressing GLuc and GFP was transduced into human MSCs at a confluence of 70% and multiplicity of infection of 4∶1 in complex with 8 ug/ml of polybrene. (A) GFP micrograph and (B) flow cytometry showing high expression level and therefore transduction efficiency of construct. (C) The activity of GLuc was successfully measured in MSCs conditioned medium using a luminometer. (D) Five different engineered cell lines were infused into NOD-SCID mice and serum was individually collected at 0.5, 8, 24, 72, and 168 hours after cell injection in batches of N = 5 per study. MSCs constitutively expressing GLuc were detected in many cases over a week in duration.</p