Adipose-tissue derived hepatic stem cells for gene therapy: Fat goes to liver’s rescue?
Authors: Nicolas Ferry
Journal: Journal of Hepatology (2010)

Adipose tissue-derived mesenchymal stem cell-based liver gene deliveryJournal of HepatologyVol. 54Issue 5PreviewThe adipose tissue represents an accessible, abundant, and replenishable source of adult stem cells for potential applications in regenerative medicine. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) resemble bone marrow-derived mesenchymal stem cells (BM-MSCs) with respect to morphology, immune-phenotype, and multiple differentiation capability. In the present study, we investigated the feasibility of AT-MSC-based liver gene delivery for the treatment of alpha 1-antitrypsin deficiency. Full-Text PDF The question of whether, and to what extent, stem cells are involved in liver regeneration has been debated for several decades. It has long been known, as highlighted by the ancient myth of Prometheus, that the normal liver has an amazing capacity to regenerate after partial resection. In 1962, a convincing demonstration was published showing that mature differentiated hepatocytes alone were involved in this process [[4]Grisham J. W. A morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver; autoradiography with thymidine-3H.Cancer Res. 1962; 22: 842-849PubMed Google Scholar]. However, when hepatocyte division is impaired, the involvement of stem cells is mandatory for correct liver regeneration. More than 50 years ago, the group of Farber revealed the existence of a new type of epithelial cells, the oval cells, in the liver of rats treated with hepatocarcinogens blocking hepatocyte division [[3]Farber E. Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylaminofluorene and 3′-methyl-4-dimethylaminoazobenzene.Cancer Res. 1956; 16: 142-148PubMed Google Scholar]. These oval cells were bipotent progenitors able to give rise to both hepatocytes and biliary cells [[2]Evarts R.P. Nagy P. Marsden E. Thorgeirsson S.S. A precursor-product relationship exists between oval cells and hepatocytes in rat liver.Carcinogenesis. 1987; 8: 1737-1740Crossref PubMed Scopus (398) Google Scholar]. Although the fate of these new hepatic stem cells was intensively studied, the exact location of the histological niche where they remain in a quiescent state is still not clearly defined. In 1999, a provocative study by Petersen et al. unveiled the ability of bone marrow to serve as a potential source of oval cells and subsequently, the transdifferentiation of bone marrow stem cells to the hepatic lineage was reported in a number of studies [[9]Petersen B.E. Bowen W.C. Patrene K.D. Mars W.M. Sullivan A.K. Murase N. et al.Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Crossref PubMed Scopus (2204) Google Scholar]. Mesenchymal stem cells from bone marrow were also identified as a potential source of hepatic progenitors and, more recently, convincing evidence that adipose tissue-derived mesenchymal stem cells (AT-MSCs) were also a source of hepatocytes was reported [1Banas A. Teratani T. Yamamoto Y. Tokuhara M. Takeshita F. Quinn G. et al.Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes.Hepatology. 2007; 46: 219-228Crossref PubMed Scopus (472) Google Scholar, 10Seo M.J. Suh S.Y. Bae Y.C. Jung J.S. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo.Biochem Biophys Res Commun. 2005; 328: 258-264Crossref PubMed Scopus (489) Google Scholar]. These results have given rise to much controversy and some investigators have claimed that transdifferentiation of bone marrow stem cells into hepatocytes was actually the result of a cell fusion process [12Vassilopoulos G. Wang P.R. Russell D.W. Transplanted bone marrow regenerates liver by cell fusion.Nature. 2003; 422: 901-904Crossref PubMed Scopus (1191) Google Scholar, 13Wang X. Willenbring H. Akkari Y. Torimaru Y. Foster M. Al-Dhalimy M. et al.Cell fusion is the principal source of bone-marrow-derived hepatocytes.Nature. 2003; 422: 897-901Crossref PubMed Scopus (1467) Google Scholar]. Nonetheless, and whatever the mechanism involved, it seems now well documented that mesenchymal stem cells may undergo hepatic differentiation whether in vitro or in vivo. Because such higly replicative stem cells are readily isolated from adipose tissue, which is easily harvested in most patients, it becomes theoretically possible to cultivate and amplify large amounts of autologous cells that can be used for cell therapy of liver failure. In their present study, Li et al. have built up on these premises and they evaluated whether AT-MSCs may be used as a cell therapy platform for ex vivo gene therapy of liver inherited disorders as well [[6]Li H. Zhang B. Lu Y. Jorgensen M. Petersen B. Song S. Adipose tissue-derived mesenchymal stem cell-based liver gene delivery.J Hepatol. 2011; 54: 930-938Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar]. They prepared AT-MSCs from peritoneal tissue of adult male mice. They verified that they were able to differentiate these cells along adipogenic or osteogenic lineages. They then comparatively evaluated the efficiency of different recombinant adeno-associated virus (AAV) serotypes with a single stranded or a double stranded genome to transduce AT-MSCs using the human alpha 1-antitrypsin (AAT) cDNA as a reporter gene. Using a high multiplicity of infection, AAV from serotype 1 was by far the most potent serotype. However, at odds with previous studies of AAV-mediated gene transfer in vivo [[7]McCarty D.M. Fu H. Monahan P.E. Toulson C.E. Naik P. Samulski R.J. Adeno-associated virus terminal repeat [TR] mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.Gene Ther. 2003; 10: 2112-2118Crossref PubMed Scopus (423) Google Scholar], single strand AAV vectors were more efficient than their double strand counterpart. This may be explained by the fact that AT-MSCs were transduced in vitro. Also, a CMV promoter was used in double strand vectors whereas the CB promoter drove expression of human AAT in single strand vectors. After transduction, AT-MSCs were re-implanted in female recipients immediately after a two third partial hepatectomy. Furthermore, the animals were pre-treated with monocrotaline. The partial hepatectomy was aimed at stimulating liver regeneration and monocrotaline is a pyrrolizidine alkaloid that blocks hepatocyte division. In this setting, mature hepatocytes are unable to divide and there is a strong selective growth advantage for injected cells that have not been intoxicated by monocrotaline. After 8 weeks, recipient mice were sacrificed. Immunohistochemical detection of human AAT revealed that 20% of the cells in the liver parenchyma were from donor origin. The human AAT positive cells also expressed mouse albumin indicating that they were differentiated hepatocytes. Unfortunately, the authors were not able to quantitate the amount of Y positive cells in the liver. Therefore, it is not possible to discriminate whether co-expression of human AAT cDNA and mouse albumin resulted from transdifferentiation of AT-MSCs in hepatocytes or from cell fusion between stem cells and hepatocytes. The human AAT serum level was in the range of 100–250 ng/ml. This value corresponds to less than 0.1% of the normal value and is surprisingly lower than the value expected from the number of human AAT-transduced hepatocytes present in the liver (20%). This may be explained by a poor activity of the CB promoter in hepatocytes in vivo. Also, it cannot be excluded that when transduced AT-MScs differentiated into hepatocytes, they synthesized both human and mouse AAT and that a competition of both forms in the secretory pathway resulted in lower amounts of the human protein in the serum. A very encouraging finding for future clinical applications was the absence of immune response to the transgene product. Indeed, many gene therapy strategies are hampered by the induction of humoral or cytotoxic immune responses that occur after gene-transfer mediated expression of non-self proteins. Such an absence of immune reaction may be related to the defective priming of antigen presenting cells after implantation of ex vivo transduced cells or to the known tolerogenic capacity of mesenchymal cells [[11]Uccelli A. Moretta L. Pistoia V. Mesenchymal stem cells in health and disease.Nat Rev Immunol. 2008 Sep; 8: 726-736Crossref PubMed Scopus (2667) Google Scholar]. It may also result from direct tolerance after selective AAV-mediated gene transfer to hepatocytes as already observed [[8]Mingozzi F. Liu Y.L. Dobrzynski E. Kaufhold A. Liu J.H. Wang Y. et al.Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer.J Clin Invest. 2003; 111: 1347-1356Crossref PubMed Scopus (379) Google Scholar]. Finally, using GFP as a marker gene, the authors analyzed the dissemination of injected cells in the whole body. By using immunostaining, GFP positive cells were detected only in spleen, lung, and bone marrow. These GFP positive cells were not further characterized. Although immunostaining is less sensitive than other methods such as PCR, the absence of engraftment in a large number of organs is promising for future cell therapy protocols. These data open new ways that deserve consideration for future clinical application for the treatment of liver inherited diseases. Indeed, a major bottleneck for liver directed cell therapy resides in the need for a huge amount of cells that should be infused to reach a therapeutic threshold. The shortage of liver graft is a strong limitation in the preparation of large batches of allogenic hepatocytes. On the other hand, the use of embryonic stem cells or induced pluripotent stem cells is still limited by considerations related to ethics or biosafety. Therefore, the demonstration that autologous mesenchymal cells obtained from fat and transduced ex vivo may result in long term expression of the transgene is promising. However, some issues remain to be addressed in future studies. First, these results were obtained in animals that were treated with monocrotaline to stop hepatocyte division and the clinical application of this strategy is not conspicuous. With the exception of inherited diseases in which a strong growth advantage for corrected cells is present, such as type 1 tyrosinemia, other strategies should be evaluated to harness AT-MSCs with some selective advantage to efficiently repopulate the diseased liver. Second, the authors used recombinant AAV vectors that are not integrative vectors. Although transgene was still expressed after 8 weeks, permanent expression cannot be guaranteed unless integration of the recombinant genome in the corrected cell is achieved. This is particularly true when transduced cells are committed to further division. Therefore, comparison of the efficacy of a panel of gene transfer vectors, whether integrative [such as lentiviral vectors] or not, to achieve permanent correction in a regenerating liver should be considered. The first clinical trial for gene therapy of a liver inherited disorder was carried out 20 years ago by the group of Jim Wilson in Philadelphia [[5]Grossman M. Rader D.J. Muler D.W.M. Kolansky D.M. Kozarsky K. Clark III, B.J. et al.A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolemia.Nature Med. 1995; 1: 1148-1154Crossref PubMed Scopus (469) Google Scholar]. It involved the harvest and culture of billions of hepatocytes from LDL receptor deficient patients. The cells were transduced with MoMULV retroviral vectors and infused back to patients via the portal vein. Although the procedure was safe it did not yield therapeutic results and the trial demonstrated that the efficiency of the vector as well as the handling of large amounts of cells both were major limitations to widespread clinical use. Twenty years later, the quality of the vectors has greatly improved. Similarly, the availability of diverse sources of hepatic stem cells obviates the need to perform a partial hepatectomy to get the cells to be corrected. It is not overly optimistic to think that liver gene therapy, either using an ex vivo or an in vivo strategy, is on the verge of a new era, provided that the biosafety of the procedure is fully validated to reach the high standards required by regulatory agencies. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.