Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure

Abstract

Modulation of the immune system may be a viable alternative in the treatment of fulminant hepatic failure (FHF) and can potentially eliminate the need for donor hepatocytes for cellular therapies. Multipotent bone marrow-derived mesenchymal stem cells (MSCs) have been shown to inhibit the function of various immune cells by undefined paracrine mediators in vitro. Yet, the therapeutic potential of MSC-derived molecules has not been tested in immunological conditions in vivo. Herein, we report that the administration of MSC-derived molecules in two clinically relevant forms-intravenous bolus of conditioned medium (MSC-CM) or extracorporeal perfusion with a bioreactor containing MSCs (MSC-EB)-can provide a significant survival benefit in rats undergoing FHF. We observed a cell mass-dependent reduction in mortality that was abolished at high cell numbers indicating a therapeutic window. Histopathological analysis of liver tissue after MSC-CM treatment showed dramatic reduction of panlobular leukocytic infiltrates, hepatocellular death and bile duct duplication. Furthermore, we demonstrate using computed tomography of adoptively transferred leukocytes that MSC-CM functionally diverts immune cells from the injured organ indicating that altered leukocyte migration by MSC-CM therapy may account for the absence of immune cells in liver tissue. Preliminary analysis of the MSC secretome using a protein array screen revealed a large fraction of chemotactic cytokines, or chemokines. When MSC-CM was fractionated based on heparin binding affinity, a known ligand for all chemokines, only the heparin-bound eluent reversed FHF indicating that the active components of MSC-CM reside in this fraction. These data provide the first experimental evidence of the medicinal use of MSC-derived molecules in the treatment of an inflammatory condition and support the role of chemokines and altered leukocyte migration as a novel therapeutic modality for FHF.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Infusion of MSC lysate provides a survival trend in an animal model of FHF.

Sprague-Dawley rats were administered lethal intraperitoneal injections of a hepatotoxin. Animals were treated with i.v. injections of MSCs, or MSC lysates from the same cell mass (2×106 cells). Controls received vehicle or NIH 3T3-J2 fibroblast components. Kaplan-Meier survival analysis of Gal-N administered rats treated with cell transplants or lysates. Time points of interventions are stated above survival plots. Results are cumulative data of two independent experiments (N = 8 per each group) using different batches of MSCs. P-value determined by Log Rank Test.

Figure 2. MSC-CM reverses FHF in a cell mass-dependent manner.

(A) Kaplan-Meier survival analysis of Gal-N administered rats treated with concentrated MSC-CM. (B) Dose response of animal survival 72 hours after liver failure induction as a function of MSC mass from which MSC-CM was derived. Controls received vehicle or fibroblast conditioned medium (fibroblast-CM). Time points of interventions are stated above survival plots. Results for both panels are cumulative data of two independent experiments using different batches of MSC-CM (N = 8 per each group). P-value determined by Log Rank Test.

Figure 3. MSC-EB support reduces liver injury biomarkers and increases survival.

Animals were treated with an MSC-EB, using a 3T3 fibroblast-based bioreactor (fibroblast-EB) and an acellular bioreactor (acellular-EB) as controls. (A) Serum biomarkers of liver injury, aspartate aminotransferase and alanine aminotransferase preceding and 24 hours after treatment with a MSC-EB (n = 5) or an acellular-EB (n = 3). Due to mortality, n = 1 in the acellular group after treatment. (B) Kaplan-Meier survival analysis of Gal-N administered rats treated with EBs. Time points of interventions are stated above survival plots. Each result for (B) was an independent experiment using different batches of MSCs. P-value determined by student's t-test analysis for panel (A). P-value determined by Log Rank Test for panel (B).

Figure 4. MSC-CM treatment inhibits immune cell infiltration and hepatobiliary cell change in Gal-N injured liver tissue.

Representative H&E histology sections of liver tissue from Gal-N injured rats 36 hours post-treatment with vehicle (A,C) or MSC-CM (B, D). Scale bars are indicated on the micrographs. Images (A, B) and (C,D) are captured 10× and 20× magnification, respectively.

Figure 5. Alteration in leukocyte migration after MSC-CM treatment.

(A) Experimental design of adoptive transfer study. Gal-N injured rats were treated with vehicle or MSC-CM followed by infusion of In111-labeled leukocytes. SPECT images were acquired at t = 0, 3, and 24 hr. for MSC-CM (B–D) and vehicle (E–G) treated rats, respectively.

Figure 6. MSC-CM is composed of high levels of chemokines that correlate with survival benefit seen in FHF.

Serum-free MSC-CM was analyzed using an antibody array for 174 specified proteins. (A) Densiometry of spotted antibody array results. Data are presented as spot intensity relative to the negative control and normalized to positive control. (B) Pie chart showing cluster analysis of MSC secreted proteins based on reported function. MSC-CM was fractionated over a heparin-agarose column into heparin bound and unbound fractions. (C) Kaplan-Meier survival analysis of Gal-N administered rats treated with the (+) heparin MSC-CM and (−) heparin MSC-CM. Time points of interventions are stated above survival plots. Results for (C) are cumulative data of two independent experiments using different batches of MSC-CM (N = 8 per each group). P-value determined by Log Rank Test.