Hematopoietic stem cells and liver regeneration: Differentially acting hematopoietic stem cell mobilization agents reverse induced chronic liver injury Eleftheria Tsolaki a,b , Evangelia Athanasiou a , Eleni Gounari a,b , Nikolaos Zogas a,b , Eleni Siotou a , Minas Yiangou b , Achilles Anagnostopoulos a , Evangelia Yannaki a, ⁎ a Gene and Cell Therapy Center, Hematology Department—BMT Unit, George Papanicolaou Hospital, Thessaloniki, Greece b Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece abstract article info Article history: Submitted 9 January 2014 Revised 2 February 2014 Accepted 3 February 2014 Available online 10 June 2014 (Communicated by G. Stamatoyannopoulos, M.D., Dr. Sci., 03 February 2014) Keywords: Liver regeneration Chronic liver injury Granulocyte-Colony Stimulating Factor Plerixafor Stem cells Bone marrow (BM) could serve as a source of cells facilitating liver repopulation in case of hepatic damage. Currently available hematopoietic stem cell (HSC) mobilizing agents, were comparatively tested for healing potential in liver fibrosis. Carbon tetrachloride (CCl 4 )-injured mice previously reconstituted with Green Fluorescent Protein BM were mobilized with Granulocyte-Colony Stimulating Factor (G-CSF), Plerixafor or G-CSF + Plerixafor. Hepatic fibrosis, stellate cell activation and oval stem cell frequency were measured by Gomori and by immunohistochemistry for a-Smooth Muscle Actin and Cytokeratin-19, respectively. Angiogene- sis was evaluated by ELISA and immunohistochemistry. Quantitative real-time PCR was used to determine the mRNA levels of liver Peroxisome Proliferator-Activated Receptor gamma (PPAR-γ), Interleukin-6 (IL-6) and Tumor Necrosis-alpha (TNFα). BM-derived cells were tracked by double immunofluorescence. The spontaneous migration of mobilized HSCs towards injured liver and its cytokine secretion profile was determined in transwell culture systems. Either single-agent mobilization or the combination of agents significantly ameliorated hepatic damage by decreasing fibrosis and restoring the abnormal vascular network in the liver of mobilized mice compared to CCl 4 -only mice. The degree of fibrosis reduction was similar among all mobilized mice despite that G-CSF + Plerixafor yielded significantly higher numbers of circulating HSCs over other agents. The liver homing potential of variously mobilized HSCs differed among the agents. An extended G-CSF treatment provided the highest anti-fibrotic effect over all tested modalities, induced by the proliferation of hepatic stem cells and decreased hepatic inflammation. Plerixafor-mobilized HSCs, despite their reduced liver homing potential, reversed fibrosis mainly by increasing hepatic PPAR-γ and VEGF expression. In all groups, BM-derived mature hepatocytes as well as liver-committed BM stem cells were detected only at low frequencies, further supporting the concept that alternative mechanisms rather than direct HSC effects regulate liver recovery. Overall, our data suggest that G-CSF, Plerixafor and G-CSF + Plerixafor act differentially during the wound healing process, ulti- mately providing a potent anti-fibrotic effect. © 2014 Elsevier Inc. All rights reserved. Introduction Liver transplantation is the only effective treatment for end-stage liver cirrhosis, but it is limited by donor shortage, postoperative morbid- ity and mortality, immune rejection, high costs and long-term side effects. In order to fulfill the unmet medical needs in the field, alterna- tive, cell-based therapies for the treatment of end-stage hepatic diseases are under investigation. Mature hepatocytes have been traditionally recognized as the major contributors to liver repair [1,2]. However, recent evidence suggests that intrahepatic stem cell populations, the so-called “hepatic progenitor/ stem cells” or oval cells (OCs) become activated, expand and actively con- tribute to the regenerative responses by giving rise to hepatocytes and biliary epithelial cells when hepatocyte proliferation is overwhelmed Blood Cells, Molecules and Diseases 53 (2014) 124–132 Abbreviations: a-SMA, a-Smooth Muscle Actin; BM, bone marrow; CCl 4 , carbon tetrachloride; CK19, Cytokeratin-19; FCM, flow cytometry; FVIII, Factor VIII; G-CSF, Granulocyte-Colony Stimulating Factor; GFP, Green Fluorescent Protein; HSCs, hema- topoietic stem cells; IL-6, Interleukin-6; LK, Lin - /c-Kit + ; OCs, oval cells; panCK, pan Cytokeratin; PPAR-γ, Peroxisome Proliferator-Activated Receptor gamma; SDF-1, stromal cell-derived factor-1; SGPT, serum glutamic–pyruvic transaminase; TNFα, Tumor Necrosis Factor alpha; VEGF, Vascular Endothelial Growth Factor. ⁎ Corresponding author at: George Papanicolaou Hospital, Gene and Cell Therapy Center, Hematology Department—BMT Unit, Thessaloniki 57010, Greece. Fax: +30 2313307521. E-mail address: eyannaki@u.washington.edu (E. Yannaki). http://dx.doi.org/10.1016/j.bcmd.2014.05.003 1079-9796/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd