Disclaimer: this is a mock NIH pre-doctoral fellowship proposal written to satisfy a molecular biology project assignment that required that I write as if I were a member of an existing lab (I chose the Willenbring group) requesting NIH support for ongoing research. I am, however, not in any way affiliated with Silvia Espejel or Garrett Roll et al., or the Department of Surgery, Division of Transplantation, or any other subsection of the University of California, San Francisco. I did not participate in their very fascinating research, and I am not claiming credit for any of the discoveries herein described. This is only a mock proposal.
The recent development of pluripotence induction in somatic cell lines raised the possibility of organ regeneration from a patient’s own cells. But until our lab’s research, it wasn’t known whether these induced pluripotent stem cells (iPSC) could fully restore vital organ function. We demonstrated that iPSC-derived differentiated hepatocytes possess the functional and proliferative capacities required for complete liver regeneration in a murine model of fumarylacetoacetate hydrolase (FAH) deficiency 1. However, because we transplanted fibroblast-derived iPSCs into strain-matched blastocysts, our model relied exclusively on iPSC differentiation in vivo during embryonic development, where exposure to developmental signaling may have contributed significantly to their functional capacities; it therefore remains undetermined whether iPSC-derived, in vitro-differentiated hepatocytes would display the same functional and proliferative capacities – an uncertainty with explicit clinical relevance, as any patient-derived iPSCs used in human stem therapies would need to undergo differentiation in vitro prior to transplantation 2. We here propose an experimental model to answer this critical question.
The Liver and Constituent Cells
Liver structure and function.
As the body’s largest internal organ and gland, the liver is vital to basic functions as the primary seat for a wide spectrum of physiological processes such as amino acid synthesis, carbohydrate, protein and lipid metabolisms, insulin breakdown, detoxification of xenobiotic compounds, and glycogen, vitamin and mineral storage. The hepatic lobule is the primary functional unit of the liver and is bordered by the portal triad (portal vein, bile duct, hepatic artery), through which blood enters the liver, flowing through the sinusoids toward the central vein. Sinusoidal vessels formed from fenestrated endothelium at the basolateral/liver-cell surface facilitate molecular exchanges between liver tissue and the bloodstream.
The liver’s largest constituent cell type, hepatocytes – which contribute 70-80% of the liver’s cellular mass – are uniquely able to re-enter the cell cycle to partially regenerate damaged tissue. They are also a heterogeneous group, displaying functional divergence among cells occupying different anatomical positions, either periportal or pericentral within the hepatic lobule, known as metabolic zonation, resulting from nuanced microenvironmental cues that trigger differential gene expression 3. The liver can also uniquely adopt and integrate transplanted hepatocytes 4, making it a promising focus of cell therapy technology research such as that herein proposed.
Yamanaka et al., in an unexpected 2006 paper, demonstrated successful generation of pluripotent embryonic stem-like cells from reprogrammed, fully differentiated terminal-line murine fibroblasts via retroviral transduction of four transcription factors: octamer-binding transcription factor-3/4 (OCT3/4), SRY-related high-mobility-group box protein-2 (SOX2), and MYC and Kruppel-like factor-4 (KLF4) 5. These induced pluripotent stem cells (iPSCs) were successfully derived from human somatic cells the following year 6 and have been shown to resemble embryonic stem cells (ESC) in morphology, capacity for unlimited self-renewal, and pluripotency. Since, the protocol has been applied to other types of murine and human cells; the genes used for pluripotency induction have been varied; and viral vector-free non-genetic techniques have been developed, all with widely replicated success 7.
iPSC vs. ESC.
We now have two viable sources for pluripotent stem cells, iPSCs and ESCs, and both are important complementary research avenues that deserve exhaustive exploration. However, a number of defining limitations specific to ESCs, including safety, immune-rejection and bioethics, have primed the biomedical community for considerable enthusiasm regarding iPSCs. But despite their circumnavigation of those limitations inherent to ESCs, iPSCs as a successful substrate for the development of rational clinical therapies initially faced a number of hurdles of their own. Multiple viral insertions, the technique originally pioneered for pluripotence induction, can cause permanent genetic alterations; differentiation of iPSCs produced via this method could result in the reactivation of these transgenes. Further, the exogenous transcription factors used to induce pluripotency could cause unforeseen cellular alterations. These concerns have driven the development of non-viral vector, non-genetic methods for somatic pluripotency induction 8.
Potential iPSC applications.
The potential for this technology is far-reaching: cell transplantation, gene therapy, patient-specific drug toxicity testing and disease modeling, and even organ regeneration are all conceivable applications. Further, iPSC capacity for tri-germ layer functional differentiation suggests their suitability for a wide spectrum of developmental and genetic research.
iPSC-derived cell transplantation
Our research is most relevant to transplantation therapies, which are increasingly being used to treat several congenital metabolic disorders as an alternative to orthotopic liver transplantation. However, success in hepatocyte transplantations, so far conducted using allogeneic cells, has been limited because initial metabolic improvements cannot be sustained without repeated hepatocyte infusions 9. Further, transplantable allogeneic hepatocytes are scarce, their engraftment rates and low, and their rejection is difficult to monitor 10. There is therefore a demand for isogeneic cell sources for hepatocyte transplantation therapies, and iPSCs are currently the most promising. Differentiated iPS-derived cell transplantations have been successful in a number of non-human models, but successful iPSC-derived hepatocyte transplantations were, until ours 1, unreported 10.
The research herein proposed continues from that of our principal investigators, Silvia Espejel and Garrett Roll, of the Department of Surgery, Division of Transplantation, University of California, San Francisco. Our previous findings, published in The Journal of Clinical Investigation as “Induced Pluripotent Stem Cell–Derived Hepatocytes Have the Functional and Proliferative Capabilities Needed for Liver Regeneration in Mice” earlier this year 1, represented a significant advancement toward harnessing the potential of iPSCs for functional organ regeneration.
We conducted three complementary experiments: we first used iPSCs to successfully regenerate liver tissue in fumarylacetoacetate hydrolase (FAH) -deficient mice. By adulthood, the fully regenerated liver was composed exclusively of iPSC-derived cells, allowing, for the first time, liver function analysis of fully differentiated iPSC-derived tissue in the absence of endogenous hepatocytes 1.
Further, we demonstrated iPSC proliferative capacity in two ways: 1) through iPSC-derived tissue regeneration after partial hepatectomy 1, and 2) through a competitive transplant model of both iPSC-derived and wild-type hepatocytes in FAH-deficient mice, each of which contributed equally to liver regeneration 1. In each experiment, genetic marker assays confirmed the iPSC-lineage identity of regenerated tissue 1,2.
We implanted wild-type iPCS-derived hepatocytes into (FAH)-deficient, Rosa26 reporter-heterozygous blastocysts to induce progressive postnatal liver repopulation 1. FAH, a tyrosine-degrading enzyme, is specifically expressed in liver hepatocytes; its absence is associated with tyrosinemia type I, a fatal condition causing neonatal liver failure. However, drug treatment with 2-(2-nitro-4-fluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC) can ameliorate symptoms, preventing the accumulation of hepatotoxic metabolites by blocking an enzymatic step upstream of FAH 1,2. FAH-deficient hepatocytes are disadvantaged and are outcompeted by wild-type transplanted hepatocytes with intact FAH production, allowing a relatively small number of wild-type cells to repopulate FAH-deficient livers 11. However, current in vitro differentiation protocols for producing ESC-derived hepatocytes have so far failed to produce cells capable of full restoration of liver function in other regenerative models 12, making our success with iPSC-derived hepatocytes especially exciting.
To avoid complications associated with viral vector-iPSCs 7,13-15, we obtained the iPSC line 440A-3 1 generated by repeated Oct4, Sox2, Klf4, and c-Myc –expressing plasmid transfection of murine embryonic fibroblasts 15. Another hurdle was to ensure stochastic liver chimerism, as it was important to restrain the iPSCs’ proliferative advantage until after birth. This was achieved by matching mouse strains between the iPSCs and FAH-deficient blastocysts; to restrain FAH-competent iPSC advantage during gestation, iPSC-injected blastocyst-carrying foster mothers were treated with NTBC. After birth, NTBC supplementation (delivered to foster mothers via drinking water) was slowly reduced and finally removed. The chimeral neonates were unaffected by this manipulation, but their non-chimeral (FAH-deficient) littermates were weakened by liver failure such that NTBC treatment was required to prevent death.
Correlation of survival in the absence of NTBC with Fah expression in liver tissue was demonstrated with quantitative Real-Time Polymerase Chain Reaction (RT-PCR); Fah expression approached wild-type levels in chimeric mice, while FAH-deficient mice did not express Fah, as expected. Upon sacrifice and dissection, livers of chimeric mice were found to be composed of 50% at P28 and 100% at P70 FAH-positive cells, suggesting that differentiated iPSC–derived hepatocytes responded to FAH deficiency by dividing until all FAH–deficient hepatocytes were replaced 11. Further, liver function in chimeric mice reached normal levels within 22 days of NTBC removal, though iPSC-derived liver repopulation by this point was less than 50%. Liver function continued normally through P300, more than 10 months after full NTBC removal.
Post-transplantation proliferation of iPSC-derived hepatocytes was a critical measure of our model’s clinical relevance. To measure their proliferation capacity, 1 x 106 iPSC-derived chimera-isolated hepatocytes were transplanted into FAH-deficient adult mice. The iPSC-derived hepatocytes rapidly repopulated the livers in these subjects after NTBC removal, leading to NTBC independence. We then subjected the FAH-deficient, iPSC-derived hepatocyte-treated mice to two-thirds partial hepatectomies. These two-thirds hepatectomized livers, constituted exclusively by iPSC-derived FAH-producing transplanted hepatocytes, fully regenerated, matching the proliferative and regenerative capacities measured among wild-type and chimeric mice.
But in all of these experiments, iPSC-derived hepatocytes had a competitive advantage over FAH-deficient cells. We wanted to definitively test proliferative capacity in the absence of this advantage. To do so, we transplanted equal numbers of iPSC-derived, chimera-isolated hepatocytes and hepatocytes procured from age-matched β-galactosidase marker gene-expressing mice 16 into FAH-deficient, immunodeficient mice. After NTBC withdrawal, iPSC-derived hepatocytes and β-galactosidase-marked hepatocytes constituted the repopulated tissue in equal parts, demonstrating the normal proliferative abilities of these iPSC-derived cells.
In conclusion, we observed, in vivo, the complete differentiation of iPSCs to fully functional hepatocytes capable of normal tissue growth and even organ regeneration. These cells performed as well as both ESC-derived and normal hepatocytes, demonstrating that neither their fibroblast origin nor the non-genetic reprogramming process diminish these capacities. Since our results were obtained with iPSCs produced without viral genomic integration, they suggest that faithful in vitro non-viral iPSC hepatocyte-lineage differentiation may be possible and should be a near-term goal of iPSC research; if successful, such cells could begin a new era of effective therapies for human liver disease.
We previously demonstrated the normal functional capacity of iPSC-derived hepatocytes in a novel murine in vivo differentiation model 1. The next step in our research will be to generate iPSC-derived hepatocytes with normal functional capacity – including proliferative and regenerative capacities – differentiated in vitro, outside the signaling environment of the developing organism. Clinical treatment strategies for human liver disease utilizing stem cell therapies will need to induce pluripotency in patient-derived somatic cells and then differentiate them in vitro before transplantation to avoid teratoma-like tumor formation 17. Therefore, not only is there a need to demonstrate that stable, fully differentiated hepatocytes derived from iPSCs can be produced in vitro, but also that these cells, once transplanted into the subject, can successfully compete with disease-state endogenous cells, proliferate, and ultimately regenerate normally functioning organ tissue. Similar experiments have very recently been reported using embryonic stem cell (ESC) -derived, in vitro-differentiated hepatocyte-like cells to regenerate liver tissue in a murine model 18; however, these successes are limited by the ethical and practical limitations of ESCs – precisely the strengths of the iPSC model – reminding us of the exciting potential for translational applications inherent in our model herein proposed.
To determine the functional, proliferative, and regenerative capacity of in vitro-differentiated, iPSC-derived hepatocytes in our established murine model, a number of complementary experiments will be required: first, using iPSC line 440A-3 (generated by repeated transfection of murine embryonic fibroblasts with Oct4, Sox2, Klf4 and c-Myc –expressing plasmids 15 to avoid complications associated with viral vector-iPSCs 7,13-15) as our iPSC source, in vitro hepatocyte differentiation will be induced using a slight modification of the highly efficient and consistent (80-85% hepatocyte formation) protocol recently developed by Si-Tayeb, Noto, and Duncan et al. 19.
Hepatocyte transplantation and proliferation.
Differentiated hepatocytes will then be sorted using fluorescence-activated cell sorting techniques (to reduce the possibility of teratoma formation) and immediately transplanted into our strain-matched FAH-deficient immunocompromised mice by direct intrasplenic injection before population expansion, as the long-term cultivation required to expand the culture in vitro may result in reduced hepatocyte metabolism potential 2. Upon successful transplantation, NTBC supplementation will be removed to confer a proliferative advantage to the iPSC-derived hepatocytes. Those mice that survive NTBC withdrawal will be treated as transplant positive subjects throughout the remaining experiments. At measured intervals, liver function will be defined using plasma assays to determine total bilirubin, albumin, alkaline phosphatase, and alanine aminotransferase levels. Mice will be sacrificed at P28 and P70 and dissected to determine the level of FAH-positive cell proliferation in liver tissue using a Real-Time PCR System to simultaneously amplify the wild-type (iPSC-derived) and knockout Fah allele in genomic DNA 20. In addition, established R26R 21 and GFP 22 PCR protocols will be used to distinguish between endogenous (R26R, FAH-deficient) and transplanted (GFP gene-carrying 15, iPSC-derived) cells, as previously described 1.
Hepatocyte regenerative capacity.
Finally, two-thirds partial hepatectomies will be performed on mice displaying sustained normal liver function 23 after iPSC-derived, in vitro-differentiated hepatocyte transplantation to determine the regenerative capacity of the experimental cells. Tissue taken during and periodically after hepatectomy will be characterized using immunostaining techniques (anti-FAH, anti-BrdU, anti-Ki67 and anti-phosphorylated histone H3 antibodies). Following hepatectomy, liver function will be monitored as before until subjects are sacrificed and dissected (post-hepatectomy 28 and 70) to determine the degree of iPSC-derived hepatocyte-driven tissue regeneration as compared to controls. All subjects not sacrificed at 28 or 70 days post-hepatectomy will be monitored and their liver function measured until natural death or sacrifice at P300, at which time subjects will be dissected and their liver tissue characterized by previously described assays.
Line 440A-3 iPSCs, generated from repeated plasmid transfection rather than retroviral transduction 15, will be cultured in monolayers in Roswell Park Memorial Institute (RPMI) media with B27 supplements and 100mg/mL activin A 19 for 5 days in 5% CO2 and ambient oxygen. Cultures will then be placed in a 4% O2 + 5% CO2 environment with RPMI / B27 media supplemented with 20ng/mL bone morphogenic protein 4 (BMP4) and 10 ng/mL fibroblast growth factor 2 (FGF2) – both critical for hepatic specification 24 – for an additional 5 days. Following, hepatic-lineage cells will be identified, isolated, and cultured in RPMI / B27 with 20ng/mL hepatocyte growth factor under 4% O2 + 5% CO2 conditions. Finally, cultures will be placed in hepatocyte culture medium with Oncostatin M (20ng/mL) in 5% CO2 + ambient O2 conditions for the remaining 5 days of the protocol. Success at each step described will be confirmed by multiple immunohistochemical assays using appropriate antibodies as described by Si-Tayeb, Noto, and Duncan et al. 19. Further, the fully differentiated iPSC-derived hepatocytes will be characterized by several assays: periodic acid-Schiff staining to confirm glycogen synthesis; oil red O staining to identify lipid droplets; fluoresceinated low-density lipoprotein-incubation to confirm low-density lipoprotein accumulation. Experimental cell-line morphology will be compared to that of primary hepatocytes and hepatocytes derived from embryonic stem cells using the same methods.
All animal procedures will first gain approval from the Institutional Animal Care Committee at the University of California, San Francisco. All FAH-deficient mice to be used in these experiments have been extensively describe by us previously and others elsewhere 1,20. All control mice to be used are from the 129S4 strain background, while immunocompromised FAH-deficient mice lacking B, T, and natural killer cells are from a mixed 129S4 and C57BL/6 background 25. Perioperative antibiotic treatments will be applied to all transplant and hepatectomized subjects as described by Azuma et al. 25.
FAH-deficient mice will be kept viable during development via NTBC supplementation provided to the foster mothers (7.5mg/mL drinking water) during pregnancy. NTBC supplementation will be maintained for neonates until NTBC withdrawal (50%, 25%, 12.5%, and 6.25% reduction every two days until complete supplement removal) following iPSC-derived in vitro-differentiated hepatocyte transplantation. Blood plasma will be obtained by retro-orbital bleeding 8 days after complete NTBC withdrawal; body weight will be recorded 0, 13, and 16 days after initiation of withdrawal.
Quantitative reverse transcription PCR.
As previously described 1, samples will be taken from various sections of liver specimens and combined. RNA will be isolated with TRIzol (Molecular Research Center), while DNA will first be treated with DNase1 (Ambion), followed by first-strand cDNA synthesis using TaqMan Reverse Transcription Reagent (Applied Biosystems). PCR will then be performed using a 7300 Real-Time PCR System with SYBR Green (both by Applied Biosystems) running 2 minutes at 50°C, 10 minutes at 90°C, 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C.
Liver samples will be fixed in 4% paraformaldehyde at 4°C for 4 hours, then cryoprotected with 30% sucrose and embedded in optimum cutting temperature compound (Tissue-Tek, Sakura Finetek) as described elsewhere 26. Frozen sections will be stained with several appropriate antibodies, as will cell cultures at each step during the differentiation process (see Appendix, Supplemental Section One for a list of antibodies to be used). Fluorescent microscopy will utilize appropriate secondary antibodies (see Appendix), with nuclear DNA stained using 2μg/mL DAPI (Molecular Probes).
Liver function assays.
Blood plasma extracted using retro-orbital bleeding will be diluted 1:4 in 0.9% sodium chloride. Bilirubin, albumin, alkaline phosphatase, and alanine aminotransferase assays will be conducted using an ADVIA 1800 (Siemens) chemistry analyzer.
Two-thirds partial hepatectomies will be performed using procedural protocol defined by Mitchell and Willenbring 23. Following 40 hours after resection, 100μg/g body weight 5-bromo-2-deoxyuridine (BrdU, Roche) will be injected intraperitoneally. Immunostainings for BrdU incorporation and phosphorylation of histone H3 will be performed on tissue procured 48 hours following the partial hepatectomy procedure.
Estimation of hepatocytes per microscopic field.
Microscopic fields will be measured in pixels using Openlab software (Cellular Imaging). Hepatocytes will be generically calculated at 20 x 20µm; from this, hepatocyte-per-field estimates will be generated.
Calculation of cell numbers and divisions in repopulating nodules.
The number of hepatocytes present in a given 2-dimensional section will be determined using a correction factor described by Wang et al. 27 to estimate the total number comprising the 3-dimensional tissue sample. Cell division cycles required to reach this population estimate will be calculated as log2.
Stem cell therapies and regenerative medicine are still in their infancies, yet radical advancements in our understanding of and control over pluripotent cell differentiation and tissue development have already been achieved. Embryonic stem cells were long thought to be the only pathway to stem technologies until Yamanaka et al. announced their successful induction of pluripotency by reprogramming mature fibroblasts using retroviral transduction of just four transcription factors. As exciting as this discover was, it was not without inbuilt challenges, specifically in the genetic contamination of the iPSCs during their generation. But this technology has evolved rapidly, and by 2009 pluripotency-induction techniques utilizing non-genetic plasmid transfection had been developed 8, allaying those original concerns. Our previous research sought to determine the functional, proliferative, and regenerative capacities of iPSC-derived, in vivo-differentiated hepatocytes. In this model, our experimental cells’ characteristics were indistinguishable from those of primary hepatocytes in each of these three critical categories. But for these findings to translate to clinical treatment, we need to determine whether iPSC-derived in vitro-differentiated hepatocytes, too behave and function as normal cells. We here propose several experiments, all based on our unique, proven murine model, designed to illuminate this critical issue: 1) in vitro hepatocyte-specific differentiation, 2) transplantation of iPSC-derived cells into FAH-deficient mice to determine functional and proliferative capacities, and 3) partial hepatectomy to determine the regenerative capacity of iPSC-derived, in vitro-differentiated hepatocytes in our model.
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