Cell Transplantation to Replace Whole Liver Transplantation
Article Outline
See “Differentiation and transplantation of human embryonic stem cell-derived hepatocytes,” by Basma H, Soto-Gutierrez A, Yannam GR, et al, on page 990.
Liver transplantation is the only established, successful treatment for end-stage liver diseases; however, the number of donor livers is inadequate. As alternatives, extracorporeal bioartificial liver support devices and hepatocyte transplantation offer the possibility of effective treatment for many inherited and acquired hepatic disorders and other liver-based congenital metabolic diseases. Unfortunately, the lack of donor livers makes it difficult to obtain enough viable human hepatocytes for the further advancement of hepatocyte-based therapies. A technology that might address these issues is the development of a human liver cell line that can be employed for liver cell transplantation or in a bioartificial liver. Developing such a cell line from human embryonic stem cells (hESC) or from other human stem cell sources would provide a valuable tool for pharmacology studies, as well as for use in cell-based therapeutics.
Bone marrow-derived stem cells, liver stem cells/oval cells, cord blood cells, amnionic stem cells, fetal hepatocytes, and hESC, among others, are candidate cell types that display potential to develop into viable hepatocytes. However, conditions for directing stem cells to differentiate into hepatocytes are not yet fully defined; moreover, controversies exist as to the function, replication potential, and plasticity of all the different stem cell populations,1 and it is not at all clear which stem cell type will ultimately be most effective in developing clinically relevant cell lines (Figure).
Figure.
Potential human stem cell sources.
These concerns have not stopped the use of bone marrow-derived stem cells for the treatment of patients with advanced liver disease, paralleling a similar approach in cardiovascular medicine. Although the engraftment rate and effectiveness of bone marrow-derived cell transplantation into the liver in animals and in humans remains controversial, small pilot studies have been conducted successfully employing bone marrow-derived cells to treat patients with advanced liver disease. This field has been reviewed recently in another issue of Gastroenterolgy.2 The obvious advantage of the bone marrow-derived cells is that they can be harvested from the patient, then reinfused after a variety of manipulations, and the risks of tumorigenicity and need for immunosuppression are obviated.
It is postulated by many that hESC may be the most effective cells in regenerative medicine because they have the potential to provide a limitless supply of differentiated cells. Recently, there have been a host of studies demonstrating new approaches to differentiate hESC along a hepatocyte lineage,3, 4 and there has been marked improvement in the ability of the variety of differentiation approaches to develop cells with many of the characteristics of primary human hepatocytes. The present study in this issue of Gastroenterology is the latest to improve on the findings of previous reports by generating a more homogeneous population of cells that can provide levels of function comparable in some ways to primary liver cells.5 Fox and co-workers have employed the multistep approach initiated by D'Amour et al,6 which begins with directing the hESC toward the definitive endoderm with activin A and low serum. With this as a starting point, they have been successful in developing a differentiation approach that derives a reasonably high percentage of the cells expressing a mature hepatocyte phenotype. They have accomplished this by fluorescence-activated cell sorting for the asialoglycoprotein receptor. The resulting cells secrete albumin and α-1-antitrypsin into the culture media, demonstrate functional levels of cytochrome p450 metabolic activity in vitro, and most impressively, express significant levels of human albumin and 1-antitrypsin in the serum of immunosuppressed rodents for more than 2 months.
Thus, their work has moved the field forward. However, as with any field still in its infancy, there are many issues that must be addressed and many questions to be answered. Although the in vivo results are encouraging, even their best results provide serum albumin levels that are orders of magnitude less than therapeutic. Obtaining good in vivo data employing human cells in rodent livers has been a persistent problem for stem cell studies. The possible explanations are immune rejection even in immunocompromised rodents, poor quality cells, or most likely, the presence of a relatively hostile environment for the transplanted human cells in the rodent host. Human hepatic cells are not exactly happy with the extracellular matrix and growth factor niche provided by rodent livers. Perhaps studies in nonhuman primates, where the environment should be more conducive to human hepatocyte engraftment, differentiation, and proliferation, may yield better results.
When evaluating even an extremely good preclinical study, we must be cognizant of the difficulties in moving the research to become clinically relevant. An immediate issue is the feasibility of scaling up the author's approach to treat an animal that is 3000 times the size of a mouse. One concern that must be addressed is the inverse relationship that often exists between cell proliferation and its differentiated state. Simply stated, as cells become terminally differentiated, they usually have a limited proliferative capacity, and hepatocytes are certainly no exception. The authors do not comment about the ability of their cells to proliferate as they move from undifferentiated hESC to become hepatocyte-like cells with abundant liver-specific gene expression. Were they proliferating extensively at day 18 of culture when they were already differentiated, then sorted and ready for transplantation? How much of a problem will this be when scaling up is required?
Safety concerns are also major issues and the focus of considerable attention in the stem cell field. When the differentiated hESC in this report were transplanted before sorting, teratomas quickly developed. Even after the sorting process, adenocarcinomas were found in the peritoneum. How certain must we be that similar results will not occur in humans before hESC transplantation can be attempted? Some recent, unpublished reports have employed neutralizing antibodies or suicide gene regimens to eliminate undifferentiated cells. But the question remains: How does one guarantee a lack of tumor formation, especially when immune suppressive regimens will likely be required for clinical use? Where previously there was optimism that immunosuppression will not be required for cell transplantation using differentiated hESC, it now seems that immunosuppression regimens will be required because hESC cells are immunogenic. This could limit the usefulness of hESC transplantation if the risks of the immunosuppression regimen outweigh the potential benefit of the cell transplantation that is required. Thus, the search for novel and less toxic immunosuppression approaches continues in the stem cell field.
This also raises the issue of which types of liver disease should/can be treated with cell transplantation. Chronic genetic diseases are the easiest choices. In many cases, only a percentage of the normal gene expression would be required to effectively replace a loss-of-function defect. Acute liver failure would also seem to be a likely etiology for eventual liver cell transplantation employing differentiated hESC. On the positive side, transplanted cells could be engineered with an “off switch” that could lead to transplanted cell removal if the endogenous liver eventually regained sufficient function. By contrast, the question arises if sufficient liver cell function can be rapidly developed by cell transplantation of hESC, especially given the results of the Fox study, which showed that it required several weeks after cell transplantation for the optimal levels of exogenous cell function to occur. The hope is that this delay in function might well be the result of host/donor species differences, and that nonhuman primate studies might demonstrate a rapid and improved rate of engraftment, proliferation, and function of hESC in vivo. The potential benefit of cell transplantation for end-stage liver disease from cirrhosis is controversial. It might be counterintuitive to expect stem cell transplantation to improve advanced liver disease; however, the evidence for such an improvement comes from several sources. It is known to all liver clinicians that improvement in liver function and even some of the sequelae of portal hypertension occurs with the successful treatment of autoimmune liver disease, sobriety in alcohol-induced cirrhosis, and even viral eradication in hepatitis C or B virus infections. Moreover, there are preclinical studies demonstrating that liver cell transplantation is effective in improving cirrhosis in animal models.7 Finally, although no controlled trial has been done, there is an expanding list of case reports demonstrating improvement in liver function, even suggestions of improved mortality, with autologous bone marrow-derived stem cell transplantation in patients with end-stage liver disease.2 It is certainly possible that some “contaminating cells,” such as hematopoietic or mesenchymal cells, may also play a role in improving liver function after the transplantation of differentiated hESC. These cells may act in a paracrine manner to enhance the function and proliferation of endogenous hepatocytes and the transplanted differentiated hepatic cells. A series of preclinical studies have been done recently, which suggest that bone marrow-derived cells can enhance liver function by increasing hepatocyte proliferation and by inhibiting fibrosis through increasing metalloproteinase production and/or decreasing the synthesis of tissue inhibitor of metalloproteinase.8
The present paper employs a federally approved hESC cell line that has been grown on mouse feeder layers and has been exposed to a series of animal products. Clearly, the use of newly developed cell lines without these impediments to human use will be advantageous if differentiated hESC are to be employed. Moreover, the hESC field is being completely transformed by the use of induced pluripotent stem cells. The ability to reprogram terminally differentiated somatic cells, including hepatocytes, to function in many ways similar to hESC has led to a vast number of studies employing these cells.9 The advantages and disadvantages of using these cells for clinical applications are now a major area of stem cell research, and studies employing them for hepatocyte differentiation are being undertaken.
A final question that is implicitly raised by the Fox paper is which cell type will most likely be clinically employed in human liver cell transplantation studies to be done in the future. All the stem cell populations are candidates. Each cell type has its advantages and disadvantages, with bone marrow-derived cells already being employed in clinical studies and by for-profit companies around the world. Critical to the ultimate decision as to which will be the best cell type is the open scientific analysis of all of them. If stem cell research is ever to meet the very high expectations placed on it to develop therapeutic applications, hard questions should be asked and answered objectively. The present manuscript meets this criterion in that it provides a careful, reasoned analysis of an improved approach to differentiate hESC along a hepatocyte lineage.
References
- Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 2002;297:2256–2259
- . Critical review of clinical trials of bone marrow stem cells in liver disease. Gastroenterology. 2008;135:438–450
- Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells. 2007;25:3058–3068
- Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proc Natl Acad Sci U S A. 2008;105:12301–12306
- Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology. 2009;136:990–999
- Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005;23:1534–1541
- Treatment of cirrhosis and liver failure in rats by hepatocyte xenotransplantation. Gastroenterology. 2003;124:422–431
- Bone marrow-derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice. Hepatology. 2007;45:213–222
- Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872
Conflicts of interest The author discloses no conflicts.
PII: S0016-5085(09)00049-3
doi:10.1053/j.gastro.2009.01.019
© 2009 AGA Institute. Published by Elsevier Inc. All rights reserved.
Refers to article:
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Differentiation and Transplantation of Human Embryonic Stem Cell–Derived Hepatocytes
, 30 October 2008
