Functional Life-Long Maintenance of Engineered Liver Tissue in Mice Following Transplantation Under The Kidney Capsule
Functional Life-Long Maintenance of Engineered Liver Tissue in Mice Following Transplantation Under The Kidney Capsule
Authors: Kazuo Ohashi, Fumikazu Koyama, Kohei Tatsumi, Midori Shima, Frank Park, Yoshiyuki Nakajima, Teruo Okano
Introduction
An area of active research in the treatment of liver diseases is the use of hepatocytes in cell-based therapies. Hepatocyte transplantation has been shown to be effective but is limited by the number of cells that can be transplanted at one time. An alternative approach is to change an individual hepatocyte into biologically active tissue capable of carrying out liver function in an ectopic site. The current challenge is to increase the lifespan of these functional hepatocytes. In this study, functional hepatocytes were engineered in a mouse kidney capsule. This liver system was monitored for roughly the life-span of a normal mouse (about 450 days). Alongside stability, liver-specific functions, drug metabolism and regenerative potential were also analyzed.
Materials/Methods
Hepatocytes were isolated from transgenic mice expressing human α-1 antitrypsin (hA1AT) under hepatocyte-specific promoter (hA1AT-FVB/N). Cells were isolated using a modified two-step collagenase perfusion method, as described in Ohashi et al. Cells were then filtered through a nylon mesh membrane and purified by centrifugation. Cells were resuspended in equal amounts of serum-free DMEM media and EHS-gel to a final ratio of 1.5 x 106 cells/100 μL. Cell viability was assessed using Trypan blue exclusion. Experiments were only conducted when the viability exceeded 90%. 1.5 x 106 cells were transplanted under the left kidney capsule space in female wild-type FVB/N mice. hA1AT serum samples were collected periodically and assayed through ELISA . Serum analysis showed hA1AT levels in the range of 9000-30,000 ng/mL, which suggested the engineered liver was viable throughout the 450 days (Figure 1).
After 450 days, histological analyses were run on collected samples to test for liver regeneration stimulus and cytochrome P induction. In each case, specimens were collected from the naive liver, engineered liver and duodenum (as a positive control).
Some collected samples came from rats receiving intraperitoneal injections of Phenobarbital (PB) or 3-methylcholantree (3-MC) for 3 consecutive days before having their native and engineered liver tissues processed for histological analysis. The drugs were given to test the liver’s ability to uptake compounds and induce production of drug-metabolizing enzymes, which in this case was cytochrome P (CYP). PB is a CYP2B inducer, while 3-MC is a CYP1A inducer. Immunostaining for both showed strong responses in the engineered and naive liver samples. (Figure 2)
Fig 2. Immunohistochemical staining for CYP2B (A-C) and CYP1A (D-F).A & D show naïve mouse liver before respective treatments, B & E show them after treatment, and C & F show the engineered liver after treatment.
A 70% partial hepatectomy (PH) was performed on some naive rat livers to stimulate liver regeneration. Post surgery, 1 mg BrdU was administered to the naïve and engineered liver tissue per day for 14 days to measure hepatocyte proliferation. After 14 days, the naive liver, engineered liver and duodenum were removed for histological analysis. Analysis showed a 23±37% increase in serum hA1AT levels in PH mice relative to non-PH mice (Figure 3A). There was a significant increase in the amount of BrdU-labelling index (LI) in the PH mice than the sham-operated control, consistent with the values obtained from the naïve livers (Figure 3B). hA1AT staining showed a strong positive signal in the engineered tissue (Figure 3E, H) but not anywhere else.
Fig 3. Results from testing the regenerative capability of the engineered liver tissues. (A) hA1AT levels were measured by ELISA between day 450 and day 464. Triangles represent the control group while circles represent the PH group. # indicates p <> C-F are the controls, while F-H are PH. C & F are from duodenum, D & G from naïve liver and E & H from engineered liver tissue.
Finally, the study also assessed the ability of the engineered liver tissues to synthesize glycogen. PAS staining was done on sections from sham-operated and PH mice at day 464. Similar intensity levels were shown for both naïve and engineered livers, though the naïve liver images were not shown. Pretreatment with salivary amylase to remove the glycogen diminished the PAS staining, as expected (Figure 4)
Fig 4. PAS staining of the engineered liver tissues to assess glycogen synthesis. A & B were from non-PH mice, while B & D were from PH mice. A & C were not treated with salivary amylase, while B & D were.
Critique
The paper was very interesting, as the use of engineered organs for therapy is a hot area of research. However, this paper did have some drawbacks. First, they sometimes referred to data that was not presented in the paper. For example, they claimed that hA1AT staining was done on all organs when testing regeneration potential of the engineered liver tissue. They say staining showed that hA1AT was only being produced from the engineered liver tissues, but it would have been nice to see the justification. Figure labeling was also an issue for me. Sometimes they did not label their figures properly, such as in Figure 3B. It can be inferred that white represents naïve liver tissue and black represents engineered, but that should be clearly stated on the figure itself. Also, they only tested a few aspects of the liver’s abilities; much more testing is needed before this method can be used for therapy. Along with this, the hepatocytes will also need to be tested in other ectopic areas, ideally those closer to the conditions a normal liver would experience. The renal capsule is fine, but most likely it is a different environment than what the liver experiences sitting in the abdominal cavity. Finally, they only used female mice as the recipients for the engineered hepatocytes. Ideally, the tests would be run on equal numbers of both sexes.
6 comments:
I like the paper and your critique but a deeper analysis of their results and experimental data would be nice. It would help clarify why they performed those experiments and how it contributes to their overall study.
Did they discuss any ideas on how well this method would transfer to a real therapy? Since the goal seems to be an actual clinical treatment, some higher throughput system seems like it would be necessary to treat real patients, rather than the impractical smaller scale methods used here as a proof of concept.
While this paper is interesting as a first move towards liver therapies, I wonder why they used wild-type mice in the study. In theory, evidence towards the creation and maintenance of an artificial liver under the kidney capsule is good data, but wouldn't this therapy be used for patients with little to no liver function? It seems that the researchers should have tested whether the ectopic liver could, in essence, rescue an animal suffering from a deficient liver. It would be interesting to see whether ectopic liver-gene expression would be sufficient to rescue an animal.
Sorry if I missed it, but the paper doesn't seem to address the hepatocyte function. Usually tests are run on urea and albumin secretion to determine how well functionally the cells are holding up. The paper really needs to further investigate this sort of functionality to ensure that the transplant is actually working well. A further test with antibody staining would probably clarify the functionality question
I think that although this experiment is very helpful for liver therapy procedures, that was not their main goal. They wanted to show that an engineered tissue can maintain itself inside a body, and survive the life of its host, instead of being replaced by the native tissue.
And I think they clearly demonstrated that this happens through their measurement of hA1AT. As an added bonus, they also showed that the liver tissue still could metabolize chemicals. I feel since it wasn't their main focus, they did not need to conduct more thorough examinations on functionality.
Sharp- Apologies. Did you have any specific questions about the paper that I didn't illuminate well enough in my write-up?
Luke- Their discussion of potential therapeutic uses of this method was brief. The paper is obviously just a first step; much more work is needed before this can be an effective aid in therapy. However, they did say that the fact that they could induce cytochrome P450 gene expression was suggestive that this system could be developed into a supportive liver-assisting device for patients suffering liver failure. As expected, they seem confident that given time, this method can be useful for therapy.
Jeni- My guess is that mice suffering from liver failure would have a lot of variables in their overall health and physiology. Since the study just aimed to see if these transplanted livers could survive, regenerate and carry out some basic liver functions, they probably used wild-type to ensure the success of this system. That's purely guesswork though. What do you think?
David- My understanding is that this paper is building on previous results, so I would guess that they did those tests in a previous paper.
Amir- While they were interested in showing the long-term viability of these cells, they did want to show that functionality is preserved. After all, what use are the cells if they lose their ability to function properly?
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