The importance of knowing your identity: sources of confusion in stem cell biology.

Recent progress in stem cell biology has created much enthusiasm and hope about the therapeutic potential of such cells. While this resurgence in interest applies to multiple tissues, some of the most promising advances pertain to our favorite organ, the liver. Human clinical trials have demonstrated some clinical benefits of hepatocyte transplantation.1 Furthermore, the liver is similar to the hematopoietic system in that extensive tissue replacement (repopulation) can be achieved when the transplanted cells have a selective growth advantage.2 Spontaneous liver repopulation occurs in humans and multiple preclinical animal models of therapeutic liver reconstitution exist.3–9 The procurement of human hepatocytes for transplantation is difficult,10 and, for this reason, much effort has been placed into the isolation and characterization of liver stem cells. Recently, another compelling reason for the interest in hepatic stem cells has emerged: liver stem cells may be useful for the treatment of type 1 diabetes. Many observations suggest that there is a tight relationship between pancreatic progenitors/stem cells and hepatic progenitors. During embryonic development, both organs originate from the same region of the ventral foregut, suggesting the existence of a common hepatopancreatic precursor cell in this location. In addition, multiple examples of pancreatichepatic cell fate switches in adult mammals exist.11 Now, several studies have indicated that hepatic progenitors can give rise to insulin-producing cells in vitro12 and in vivo.13 Liver-derived insulin-producing cells have even been effective in reducing experimental diabetes.13,14 These hopeful preclinical results have already resulted in company spin-offs. Type 1 diabetes is an important medical problem, and currently, most patients are treated by lifelong insulin replacement therapy. Cell therapy, which restores the missing -cell mass, can replace insulin15 and, therefore, a very large (and lucrative) commercial market exists for -cell transplantation. There are 2 basic techniques to demonstrate the differentiation of putative progenitors/stem cells into their differentiated progeny. The first is in vitro differentiation in a tissue culture system. Progenitor cells (preferably at single cell density) are exposed to conditions that promote their conversion to the cell type of interest, which is then ascertained by the presence of markers, such as insulin production. In many cases, the in vitro conditions involve co-culture with other cells or culture on feeder layers. The second method is transplantation of the stem cell in vivo. The donor derived cells are tracked in the organ of interest to determine whether they have differentiated into the desired functional progeny. The purpose of this editorial is to highlight and discuss the potential pitfalls that exist when these techniques are used to study stem cells. The lessons to be learned do not only apply to liver and pancreatic stem cells, but to stem cell biology in general. The first artifact to be cognizant of is cell fusion in tissue culture. Co-culture or culture on feeder-layers is a frequently used technique to provide stem cells with growth factors and other signals that influence their cellular phenotype. For example, embryonic stem cells are usually cultured on feeder layers of embryonic fibroblasts.16 Some groups have attempted to differentiate embryonic stem (ES) cells into desired cell fates by co-culture with other cell types. During the course of such experiments, 2 groups independently discovered that ES cells could actually fuse with the cells with whom they are being co-cultured and thereby become “differentiated.”17,18 The “differentiated” ES cells contained genetic markers from both the ES cells and the cells with which they had been mixed. Without this careful analysis, the authors of these studies could have concluded that the ES had actually differentiated and acquired a phenotype similar to that of the co-culture population. The second artifact is cell fusion in vivo. Several studies have demonstrated the unexpected plasticity of adult bone marrow derived stem cells to become epithelial cell types in vivo.19–22 In general, these studies interpreted From the Department of Molecular and Medical Genetics, Oregon Health & Sciences University, Portland, OR. Received September 9, 2003; accepted October 15, 2003 . Address reprint requests to:Markus Grompe,M.D., Professor, Dept. ofMolecular andMedical Genetics, L103, Oregon Health & Sciences University, 3181 SW Sam Jackson Pk Rd., Portland, OR 97239. E-mail: grompem@ohsu.edu; fax: 503-494-6886. Copyright © 2004 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.20020