Titanium Oxide Nanotube Platforms for Bioartificial Livers and for Transplantation of Hepatocytes Derived from Human Embryonic Stem Cells
The liver is the largest organ in the human body. It is essential for life. The production of blood proteins involved in coagulation and the detoxification of poisons that enter the body are among the most important functions of the liver. Serious health consequences occur when the liver fails to perform these functions. For example, human beings born with defective coagulation proteins acquire Hemophilia (a genetic disease). Or humans with livers destroyed by incurable infections (hepatitis) or work-related chemicals often contract liver failure and fibrosis. When Hemophilia, liver failure and liver fibrosis are uncontrollable, these people often die of ‘end-stage liver disease’ (ESLD). ESLDs such as chronic hepatitis and liver cancer are rampant in California and clinical treatments are becoming increasingly strained. A recent study (2005) found that chronic liver disease ranked as one of the leading causes of death in California, resulting in 3,725 deaths in 2002; it was also found that minorities suffered disproportionately from ESLD. Liver organ transplantation is the current therapy for ESLD. However, it is very costly and complex, it depends upon the availablity of donor livers, and there are many associated problems. Donor livers are rare, transplant waiting lists are long, and transplant waiting times are long (so long that some patients die before a donor liver is available). The major problem -- unless the donor comes from an identical twin – is that patients reject the donor liver. To prevent rejection, patients are currently treated lifelong with drugs, but often these drugs fail or are themselves dangerously toxic and life-threatening. The research in this proposal will lay the groundwork for the development of a device that can replace a failing liver (without drugs), much as dialysis machines can save the lives of people with kidney failure. This device is called a BAL (bioartificial liver). A prototype BAL will be made by a team of biologists, physicians and bioengineers. Federally approved human embryonic stem cells (hESCs), which can be converted into liver cells, will be placed on ‘computer-like’ chips made from titanium (a metal harmless to the body) designed to simulate small livers. To see if hESC-derived liver cells-on-chips (LCOCs) maintain liver functions and survive transplantation, LCOCs will be put into special mice (which do not reject human cells) for up to a month. During this time, the LCOCs will be removed and tests for liver cell functions (e.g. production of blood proteins) will be made. If these experiments work, future research will be geared to (a) designing LCOCs that cure liver disease in animals, and (b) producing hESCs that resist rejection. If these problems are solved, studies will move into human trials. If human trials work, we hope to build universal, inexpensive, LCOCs to cure ESLD in California and worldwide, without resorting to liver transplantation and drugs.
Statement of Benefit to California:
End-stage liver diseases (ESLDs) such as chronic hepatitis and liver cancer are rampant in California and therapeutic modalities are becoming increasingly strained. Many afflicted die of these conditions including those associated with alcoholic liver disease. According to the California DHS and Center for Health Statistics (Data Summary No. DS05-05000, May, 2005, pp. 1-11), in a study entitled “End Stage Liver Disease (ESLD): Morbidity, Mortality, and Transplantation California, 1999-2003”, chronic liver disease and cirrhosis ranked as one of the leading causes of death in California, resulting in 3,725 deaths statewide in 2002. Not surprisingly, minorities suffered disproportionately: American Indians, Alaska Natives, Hispanics and Latinos had significantly higher ESLD death and hospitalization rates, but lower liver transplant rates, despite many Adult Liver Transplant centers in the State (11 of 91 throughout the US, as determined 5/30/06 [https://www.cms.hhs.gov/ApprovedTransplantCenters/downloads/liver_list.pdf]). More surprisingly, the incidence of ESLD in California was higher than the incidence of newly diagnosed Parkinson’s disease cases as judged from a 1994–1995 study using information from Kaiser Permanente of Northern California (Van Den Eeden SK et al. Amer. J. Epidemiol. 2003;157:1015-1022). Current cures for ESLD depend mainly upon liver transplantation. However, liver donor organs are limited, matched organs rarely exist, and the medical costs for transplantation and post-operative care are prohibitive. Transplantation of suspensions of committed liver stem cells is one future option; but scientific controversy and technical issues plague isolation, culture, directed and stable differentiation of these cells, as well as the universal problems of (a) transplantation without rejection, and (b) provision of sufficient liver function to sustain normal life. State-of-the art materials science and nanotechnology, coupled with recent advances in the hepatocyte-directed differentiation of human embryonic stem cells (hESCs) in vitro, may provide tissue and biomedical engineering approaches that can lead to breakthroughs towards curing ESLDs without resorting to organ transplantation. These breakthroughs may well come from functional extracorporeal and transplantable hESC-based bioartificial livers (BALs), constructed from inexpensive TiO2 chips carrying liver acinar-like stacks of hESC-drived hepatocytes, to assist or cure human beings suffering from ESLDs of infectious (hepatitis), genetic (Hemophilia A and B) or chemical origin (alcohol abuse). Apart from therapeutic transplantation devices, significant benefits from these novel BALs would be quickly evident, as they would provide normal, homogeneous cell sources for robotic screening of potential specificities, metabolism, polymorphisms and toxicities of new or experimental drugs, chemicals, and therapeutics developed by pharmaceutical and chemical industries.
SYNOPSIS: The goal of this proposal, in a broad sense, is to bring technology and material from nanotechnology to the study of hESCs and particularly to hESC differentiation into hepatocytes for transplantation. The aims are (i) to determine the sizes, shapes and configuations of TiO2 chips (planar and 3D) that are optimal for hESC maintenance, clonogenicity and directed differentiation to hepatocytes; (ii) to vary culture hESC conditions (growth factors, matrices, and chip geometries) to optimize generation of hepatocytes that have differentiated secretory and metabolic function; and (iii) to follow the survival and function of hESC-generated hepatocytes on TiO2 chips into immunodeficient mice. SIGNIFICANCE AND INNOVATION: The use of nanotechnology in stem cell biology is in its infancy, and so examination of the interface of a TiO2 surface with hESCs represents a logical first step in bringing some of the materials and techniques from nanotechnology into hESC biology. To date, ideal engineered surfaces/substrates for growing cells in general have not been identified, so there is a need for biocompatible materials that can influence cell fate in predictable ways. The investigators will use H1 and H9 cell lines (though they also mention H2) and perhaps some non-federally approved lines. With some preliminary data using H1 and H9 lines, the work would be appropriate for federal funding. It is clear that successful liver tissue engineering/transplant would have major impact on our healthcare system. hESC differentiation into hepatocytes is still somewhat uncertain and successfully accomplishing this task would undoubtedly have important implications for the hESC community, though it would certainly be more significant across the community if the work were to provide molecular insights on the mechanisms by which such differentiation occurred. The materials and material processing are relatively novel but it remains unclear whether they provide a panacea for the problem at hand. STRENGTHS: Although the PI emphasizes that liver disease is an important problem (liver disease in general is poorly studied compared to its importance in medicine), the importance of this application, at least in the short-term, is not going to be a contribution to treatment of liver disease. Especially from the standpoint of the spirit of the RFA, the important part of this application is an evaluation of TiO2 as a substrate for hESC growth and manipulation. The collaboration brings engineers and cell biologists together effectively -- two groups with very distinct specializations focused on a common goal. Bringing engineers into the effort in a meaningful way means the objectives of the RFA have been met. Specific strenghts include: A specific identifiable goal with measurable endpoints; the combination of novel materials technology with stem cells; a proven track record of productivity amongst the investigators; and demonstration of successful use of this material to support adult hepatocytes. WEAKNESSES: The difficulties of the proposed work are not fully acknowledged. There is no one in the group with hESC experience who will assist the investigators, and since they do not cite use of a core facility, there will be a learning curve involved in handling the cells. The remaining expertise and equipment all seem to be in place. There is no reason to believe that stem cell differentiation will be supported by the titanium nanomaterial. It is very likely that the adhesive/niche requirements for active differentiation of cells varies with time and is not equivalent to the requirements for prevention of de-differentiation of adult hepatocytes. There is concern about the validity of the idea that creating an acinus based only on geometries will force cells to act similarly to differentiated hepatocytes in an actual 3D structure. The repeated structures in the liver are the result of signal gradients eminating from the blood supply around which the hepatocytes organize -- so that the biology dictates the structure. Going in the opposite direction may not work if there is a uniform surface of TiO2 through the acinus (why will the central hepatocytes be signaled to be different than those at the periphery as happens in vivo?) In that regard the depot idea is a good one, though very poorly outlined in the application. So again, the combination of the TiO2 and the depots to direct hepatocyte fate seem to be the best part of the proposal and should be the major focus. For example, it is completely unclear why, in the context of the aims, clonogenicity should be important to this work. Furthermore, there is no discussion of adequate detail about the nature of the structures that will be fabricated (e.g., why geometry A and not B, or what the range of parameters specifically are that will be generated). Given the infinite space of structures that could be generated, no clear basis was provided for how investigators will pick what to make and how they will optimize. It looks like a random search for a needle in a haystack of an 'appropriate' material. The actual number of configurations of TiO2 chips is also not made clear. Just how many planar and 3D conformations will be tested and varying which parameters? For example, ranges of thicknesses are depicted in Aim 1 but there is no indication how many of them will be tested with each range. The previous work of the post-doctoral fellow (already funded by CIRM in a training grant, publications cited in the application) should have set the stage for some of these parameters, although the stem cells were not used in this work. Aim 1 involves no hESC work, and on that basis could be funded federally. Finally, it seems very presumptious to begin animal work (Aim 3) at this stage, and depends on the high risk, low probability positive outcome of Aim 2. Reviewers would have liked to see more intellectual resources directed to fleshing out a plan for success in Aim 2. However, one reviewer thought that the transplant studies should just be dropped, given the considerations cited in this review, they are not feasible in the timeframe. DISCUSSION: This proposal brings nanotechnology to hESC, and particularly to hepatocyte differentiation for transplantation. One reviewer commented that aim 1 is entirely dependent on material development, which is disappointing since at this point every aim should be about the cells. Another reviewer pointed out that the proposal is risky and, as such, is responsive to the goals of the SEED grant RFA. The reviewer noted, however, that there are several problems with the proposal. Given that the applicant is adept with materials, there is suprisingly little description of what they propose to make - no sense of strategy or rationale for the structures. There appears to be an assumption that shape change alone will affect differentiation, but reviewers couldn't figure out what the investigators were going to make or how it would make a difference. What is their strategy, and how is it justified? In addition, no details were given on how to recapitulate the in vivo environment, particularly for generating a gradient of expression across the acinus. Aim 2 was therefore considered a long shot with no better chance of succeeding than random chance. Finally, given that the work is at a very early stage, the proposal to perform animal transplants in Aim 3, seems premature. The reviewers felt that the applicants were moving too fast given the little information they currently have.