Molecular dissection of adult liver regeneration to guide the generation of hepatocytes from pluripotent stem cells
The liver is a promising target for cell therapy since it supports and functionally integrates transplanted cells. Human liver contains more than 50 billion cells and more than 10% replacement will be required for most liver diseases. Hence, embryonic stem cells (ESC), which have unlimited growth capacities, represent one of the few cell types with potential for liver cell therapy. However, to be functionally effective and safe, ESC have to be differentiated into hepatocytes, the cells of the liver that provide its typical functions, before transplantation. Unfortunately, current ESC differentiation protocols generate cells that are not fully differentiated or functional. To achieve levels of differentiation that would be therapeutic we propose to identify the mechanisms that establish hepatocyte function in progenitor cells in the adult liver. Adult liver progenitors are typically absent from the normal liver but become apparent in liver disease when hepatocytes are damaged. Remarkably, adult liver progenitors can differentiate into fully functional hepatocytes within a few days. We hope to identify the genes that enable this rapid maturation process in order to apply it to immature cells derived from ESC. If maturation can be induced and these hepatocyte-like cells function to correct a mouse model of a human liver disease we will have provided proof-of-principle for the potential of ESC for liver cell therapy.
Liver transplantation is the only curative option for patients with severe liver diseases. As donor livers are rare, many Californians are currently waiting to receive a transplant. In fact, many patients on the waiting list die before a donor organ becomes available. If successful, the proposed project would help to alleviate the need for donor organs by establishing embryonic stem cells as a source of hepatocytes for transplantation. As hepatocyte transplantation would be less invasive and expensive than orthotopic liver transplantation, funds might become available that could be used to benefit the citizens of California in other areas.
The overall goal of this project is to establish new strategies for liver regeneration. For this purpose, we aim at identifying molecular mechanisms regulating liver regeneration that could be exploited for therapeutic purposes. Along these lines, we have identified small RNAs that either promote or inhibit the proliferation of hepatocytes, the cells in the liver that provide its manifold functions. We are now working on developing tools to modify the levels of these small RNAs in the liver with the goal to enhance liver regeneration. In addition, we aim at developing hepatocyte replacement strategies for liver diseases that cause irreparable hepatocyte damage. We focus on immune-compatible pluripotent stem cells because they can in principle generate the large numbers of hepatocytes required for therapeutically effective cell therapy and would not require life-long immune suppression. We have established proof-of-principle for the therapeutic efficacy of hepatocytes derived from mouse pluripotent stem cells, and are now working towards recapitulating these results in human cells.
In the past year we have made several discoveries that move us closer to our goal to improve the proliferation and function and thus therapeutic efficacy of hepatocytes derived from pluripotent stem cells.
Some of these discoveries have elucidated the role of microRNAs, a class of non-coding small RNAs, in liver regeneration and function. For example, we found that miR-21 acts as a promoter of hepatocyte proliferation during liver regeneration. In addition, we identified several other microRNAs that establish differentiated function in hepatocytes.
Other discoveries of ours have revealed which type of pluripotent stem cell is best for liver cell therapy that does not require chronic immune suppression. Our results show that induced pluripotent stem cells derived from fibroblasts are as effective in reversing liver failure as normal hepatocytes.
In the last year we have made significant progress towards our goal of "Molecular dissection of adult liver regeneration to guide the generation of hepatocytes from pluripotent stem cells". We have identified the mechanism of how microRNA-21 promotes liver regeneration. We are currently working on translating this understanding into a therapeutic strategy for liver failure. We have also gained in-depth insight into the molecular regulation of differentiation of liver progenitor cells into hepatocytes. We have begun to use this insight to direct the differentiation of pluripotent stem cells into hepatocytes that are effective in liver cell therapy.
Being able to generate hepatocytes from human pluripotent stem cells would advance many important research efforts, including studies of the pathobiology of liver diseases and the development of liver cell therapies. Unfortunately, realizing this potential has been hampered by shortcomings of human hepatocyte-like cells (HLCs) generated with current in vitro-differentiation protocols, not only as it pertains to replicating the function of primary human hepatocytes, but also their ability to proliferate in vivo. We have made significant progress toward our goal of identifying regulators of hepatocyte differentiation. In addition, we have established the feasibility of liver repopulation of immune-deficient mice with HLCs generated in vitro, thereby proving their ability to mature and proliferate after transplantation
We have made significant progress toward our goal of generating in the laboratory human liver cells that are therapeutically effective in mouse models of human liver failure. Because these surrogate human liver cells can be derived from readily accessible cell types like skin cells, they have potential for autologous liver cell therapies requiring nor or little immune suppression. Much of this progress was afforded by insight into mouse liver development and regeneration obtained from the investigations performed under this grant.
The overall goal of the research funded by this award was to establish the feasibility of generating therapeutically effective human hepatocytes in the laboratory. Another goal was to generate these hepatocytes so that they would not require immune suppression after transplantation. For this we investigated how hepatocyte differentiation and proliferation are regulated in mice and humans and applied this insight for the directed differentiation of human fibroblasts fully or partially reprogrammed to pluripotency. Our results showed that human fibroblasts can be converted into cells that replicate both function and proliferation of primary human hepatocytes, thereby establishing the feasibility of autologous liver cell therapy not requiring immune suppression.
- Nature (2014) Mouse liver repopulation with hepatocytes generated from human fibroblasts. (PubMed: 24572354)
- Cell Rep (2014) Evidence against a Stem Cell Origin of New Hepatocytes in a Common Mouse Model of Chronic Liver Injury. (PubMed: 25131204)
- J Clin Invest (2012) A microRNA-21 surge facilitates rapid cyclin D1 translation and cell cycle progression in mouse liver regeneration. (PubMed: 22326957)
- J Clin Invest (2012) Cholangiocarcinomas can originate from hepatocytes in mice. (PubMed: 22797301)
- Cell Stem Cell (2011) A simple code for installing hepatocyte function. (PubMed: 21816357)
- Proc Natl Acad Sci U S A (2011) Core promoter recognition complex changes accompany liver development. (PubMed: 21368148)
- J Clin Invest (2011) Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. (PubMed: 22105172)
- Hepatology (2010) MicroRNAs control hepatocyte proliferation during liver regeneration. (PubMed: 20432256)
- Aging (Albany NY) (2010) miRNAs regulate SIRT1 expression during mouse embryonic stem cell differentiation and in adult mouse tissues. (PubMed: 20634564)
- J Clin Invest (2010) Induced pluripotent stem cell-derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice. (PubMed: 20739754)
- Stem Cell Res (2010) Transplanted nonviable human hepatocytes produce appreciable serum albumin levels in mice. (PubMed: 20829142)