In Vitro Organogenesis of the Liver: Current Progress and Future Applications

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1 JUST, Vol. V, No. 1, 2017 Trent University In Vitro Organogenesis of the Liver: Current Progress and Future Applications Eliza McColl Abstract Tissue engineering, the process of growing cells on a 3D scaffold to form organ-like structures, represents a growing field of in vitro developmental biology. Since receiving its name in 1987, the field of tissue engineering has evolved considerably from being used to grow simple tissues such as skin and cartilage to generating 3D, semi-functional precursors to organs such as the pancreas, kidney, heart, and liver. However, the generation of these more complex organs requires the ability to generate extensive vascularization and condense complex cell mixtures, both of which have thus far provided a major barrier in culturing full-sized, functional organs. In order to advance to culturing full-sized, functional livers in vitro, current research is striving to optimize the cell compositions, culturing conditions, and 3D scaffolds used for in vitro liver organogenesis. This review outlines recent studies that have made important advances regarding protocols used to generate 3D-culture liver organoids as well as their potential applications in health care. Keywords Tissue Engineering Developmental Biology Health Sciences Champlain College 1. Introduction 1.1 What is Tissue Engineering? The term tissue engineering refers to the use of living cells, biocompatible materials, and other factors to create tissue-like structures in vitro (Berthiaume et al. 2011). The process of tissue engineering typically involves using 3D scaffolds as structural support to encourage embryonic stem cells, adult stem cells, or mature cells to condense and develop based on natural cell affinities to form new tissue (Cortesini 2005; Stamatialis et al. 2008). The 3D scaffolds are often biological in origin, such as protein-based materials and polysaccharide-based materials that have been cross-linked with synthetic agents to prevent the scaffold from degrading over time (Berthiaume et al. 2011). Solely synthetic scaffolds are often avoided due to their low biocompatibility. These matrices are seeded with stem cells that are then induced to differentiate using cell line-specific chemicals that mimic endogenous signals required for proliferation. This allows the cells to proliferate into different cell types to generate 3D tissue and organ-like structures in vitro. The ultimate goal of in vitro tissue generation is to generate tissue or organs that can be implanted into the human body or used to repair failing organs (Berthiaume et al. 2011). The first successes in the field of tissue engineering were with regards to skin and cartilage generation; generating these tissues was relatively simple (compared to engineering other organs) because it only required combining cells and 3D matrices, as these tissues don t require extensive vascularization to perform their endogenous functions (Berthiaume et al. 2011). The true challenge in tissue engineering arises when this technology is applied in an attempt to generate more complex organs that require vasculature and diverse cell compositions to function properly. For this reason, the only whole tissueengineered organ to have been successfully grown in vitro at this point is a trachea. However, substantial advances have been made with regards to other organs such as the pancreas, kidney, heart, spinal cord, and the liver. 1.2 The Liver: An Important Target for Culturing The main targets of modern tissue engineering are organs that are commonly prone to injury, disease, and degeneration (Berthiaume et al. 2011). For this reason, the liver is a prime candidate for in vitro organogenesis. Currently, the only successful treatment for patients with end stage liver disease is liver transplantation; however, 20% of end stage liver failure patients die on the waiting list for a transplant due to a shortage of donors, and many patients are not eligible for transplantation in the first place due to the severity of their illness (Berthiaume et al. 2011; Mazza et al. 2015). Additionally, the total number of deaths worldwide caused by cirrhosis and liver cancer has been increasing by 50 million per year since 1990 (Mazza et al. 2015). Due to the increasing number of deaths related to liver cirrhosis, cancer, and the lack of viable transplantation options it is necessary for novel treatments to be assessed. Using tissue engineering to grow livers in vitro for the purpose of transplantation represents only one of many applications of in vitro liver organogenesis. The ability to engineer livers in vitro could also have applications such as providing

2 In Vitro Organogenesis of the Liver: Current Progress and Future Applications 2/6 better models for studying human disease and testing novel therapeutics. Since the liver plays a prominent role in metabolizing xenobiotics, livers cultured in vitro could not only be used to test the response of liver tissue to novel therapeutics, but could also give an indication of the metabolism of a given drug which would aid in determining proper therapeutic doses during preclinical trials (Lancaster & Knoblich 2014). However, only small, 3D liver precursors, termed organoids, have successfully been cultured in vitro to date, with the first liver organoid being successfully cultured only six years ago (Lancaster & Knoblich 2014). This review will outline some of the challenges currently preventing full-sized liver culturing, how current research is striving to overcome these challenges, and the potential applications that in vitro liver organogenesis could offer. Research aiming to improve current liver organoid culturing methods, inspire progression towards fully cultured livers in vitro, and implicate the applications of these endeavours are novel fields of developmental biology and thus represent an exciting current topic in biochemistry. 2. Current Research: Striving to Overcome the Hurdles of Growing Full Livers While a full-sized liver has not yet been grown in vitro, many studies have had success growing liver organoids or organ buds, which are small-scale precursors of the liver grown in vitro (Lancaster & Knoblich 2014). A major barrier faced when attempting to progress from engineering liver organoids to full-sized organs is the ability to expand 3D liver progenitors for long periods of time in vitro (Broutier et al. 2016). For example, a previous study by Michalopolous et al. (2001) found that when trying to culture hepatocytes, apoptosis occurred in 50% of the cultured cells after only five days. Even more recently, 3D hepatocyte cultures have only been sustained for approximately one week which only allowed for a 10-fold expansion of the culture (Shan et al. 2013). Without the capability to culture organ progenitors for long periods of time, cells composing liver organoids do not have the opportunity to self-organize into functional components of the liver, such as the hepatic ductal compartment, or the vascularization required to support the liver s growth and function (Broutier et al. 2016). Therefore, culturing methods must be improved in order to progress towards culturing entire functional livers in vitro. In an attempt to overcome the barrier of long-term liver organoid culturing, Broutier et al. (2016) have developed a novel culturing method for 3D liver organoids. This method has increased the length of time that liver progenitors can be cultured in vitro from a couple weeks at maximum to as long as three months. This new culturing method involves combining a scaffold called Matrigel, which has properties similar to the extracellular matrix, with hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and R-spondin-1 (Rspo1). HGF, FGF, EGF, and Rspo1 are factors responsible for embryonic development, progenitor proliferation, and migration of liver cells. It had previously been established that EGF, HGF, and dexamethasone aided in the maintenance of primary liver cells in culture. The combination of these factors with FGF and Rspo1 resulted in the improved culture medium proposed by Broutier et al. (2016). When combined with the Matrigel matrix, these additional factors contributed to the survival and genetic stability of liver organoids over longer periods of time (months) in vitro. This survival and stability allowed single liver progenitors to give rise to up to 10 6 cells, allowing sufficient time for the cells to self-organize into an epithelial structure that resembled the hepatic ductal compartment as well as compartments that partially resembled liver buds of an early embryo. Shifting liver organoid cultures to a differentiation medium that promotes the expression of hepatocyte markers allowed the cells composing the liver organoids to develop functional hepatocyte characteristics (Broutier et al. 2016). The differentiation medium contained dexamethasone and bone morphogenetic protein (BMP), both of which promote hepatocyte differentiation, as well as components that blocked Notch, a factor that promotes ductal development. Blocking ductal development was an important aspect as it promoted differentiation of the organoids cells into functional hepatocytes as opposed to just ductal cells. The resulting differentiated cells produced albumin or bile acid in vitro which indicated hepatocyte functionality. This advancement in the ability to culture liver organoids for months as opposed to days offers great potential for growing full, functional livers in vitro. However, this procedure for long-term culturing can so far only be done using the epithelial components of the liver tissue and has not yet shown success with liver organoids that contain both mesenchymal (multipotent cells) and epithelial cells, which would be required to achieve the complexity of full livers. The challenge faced by Broutier et al. (2016) when attempting to culture multiple cell types in unison is another common barrier faced by in vitro liver engineering. For this reason, many of the current examples of successful tissue engineering are derived from only epithelial structures and lack more complex structures such as vasculature (Takebe et al. 2015). In vivo, the liver develops from a combination of mesenchymal stem cells, undifferentiated vascular endothelial cells, and endoderm cells. In order to generate livers in vitro that closely resemble and function like livers in vivo, a protocol must be developed that allows liver organoids to be composed from cultures containing multiple cell types. Takebe et al. (2015) have developed a protocol, depicted in Figure 1, in which mesenchymal stem cells are used to initiate condensation of heterotypic cell mixtures which allowed them to generate vascularized and complex liver organoids. This protocol eventually led to the first ever generation of human-like liver organoids, as previous successful liver organoids had only been grown from animal cells lines (Lancaster &

3 In Vitro Organogenesis of the Liver: Current Progress and Future Applications 3/6 Figure 1. Schematic showing the mesenchymal-directed condensation of multiple cell types to generate vascularized organ buds of multiple organ types. Image courtesy of Takebe et al. (2015). Knoblich 2014). Previous research had established that mesenchymal stem cells can enhance the contraction force of the self-organization of cells (Sondergaard et al. 2010). Takebe et al. (2015) implemented the use of the mesenchymal stem cells to apply this knowledge to growing liver organoids. In their initial research, they labelled human induced pluripotent stem cell (ipsc)-derived hepatic endoderm cells and umbilical cord-derived endothelial cells with two different fluorescent markers, co-cultured them on a 3D matrix, and then used fluorescent live imaging to observe any cell migration or condensation. It was observed that in the absence of mesenchymal stem cells, the co-cultures of the two different cell types failed to condense into liver organ buds. Conversely, when mesenchymal stem cells were added to the co-culture of the endoderm and endothelial cells, the mesenchymal stem cells appeared to increase the contraction force to result in condensation of the two cell lines. The live image cell tracking was also used to observe that the condensed cells eventually developed into vascularized liver organoids, as opposed to epithelialbased organoids. This phenomenon was observed in culture medium similar to that designed by Broutier et al (2016) in that it contained factors for hepatocyte development such as dexamethasone and hepatocyte growth factor (HGF). This protocol was also successful in developing complex organ buds for the intestine, lung, kidney, heart, and brain, all of which are pictured in Figure 2. The success of Takebe et al. (2015) in generating complex, vascularized liver organoids demonstrates the potential role of mesenchymal stem cells in guiding the condensation of multiple cell types for complex organoid generation of not only the liver, but multiple other organs. A third ongoing challenge in the field of in vitro liver engineering is the optimization of an ideal 3D scaffold. In order for a liver to be grown to full size in vitro and maintain functionality, the 3D matrix on which it is grown must support the growth and differentiation of the cells composing the organoid (Mazza et al. 2015). Additionally, as previously mentioned, growing livers with sufficient vascularization for proper functioning and survival has also been a challenge. This vascularization would be required in order to supply oxygen and nutrients to the cultured tissue for long-term survival (Cortesini 2005). Thus, if the matrix used to grow the liver could somehow already exhibit this vascularization, it would greatly enhance the ease with which functioning livers could be grown in vitro. In an attempt to achieve an ideal 3D scaffold, Mazza et al. (2015) proposed using decellularized liver tissue as a 3D scaffold. This technique had previously been shown to promote successful growth of multiple liver cells types while retaining their functionality. For example, Uygun et al. (2010) were the first to successfully decellularize rat livers and use them to create tissue-engineered liver grafts with functioning hepatocytes. However, all previous research using this technique had been done using animal liver tissue and until recently it was not known whether this technique could be applied to human liver tissue. Mazza et al. (2015) used healthy human livers deemed ineligible for transplantation and decellularized them using a retrograde perfusion protocol. The purpose of this protocol was to remove immunogenic cellular materials from the livers while maintaining the 3D shape, essential matrix proteins, and vascularization of the organ so that it could be used to generate fresh tissue in vitro. The perfusion protocol involved flushing the livers (either a single lobe or a whole liver) with solutions containing varying concentrations of detergents such as sodium dodecyl sulfate (SDS) and Triton X100. Throughout the perfusion, the flow rate of the decellularization solutions was varied; a slow flow rate was used to begin the decellularization and then was rapidly increased before being stabilized for the remainder of the decellularization. The decellularized livers (shown in Figure 3) were then examined using immunostaining analysis, as well as techniques that quantified DNA, collagen, and elastin present in the matrices to determine the success of the perfusion. Mazza et al. (2015) were then able to successfully re-engraft three different human liver cell types (LX2, HepG2, and Sk-Hep-1 cells) onto cubes of the decellularized matrix by using a syringe to deposit the cells onto the cubes. Using tissue staining techniques, successful migration and cellular proliferation of each of the cell types on the matrix was observed. This success represents the potential for decellularized tissue to provide an optimal 3D basis for new full-sized, vascularized, functional liver formation, specifically in humans. The donation of the livers to be decellularized could come from organs that are deemed unfit for transplantation, which thus would not deprive a transplant

4 In Vitro Organogenesis of the Liver: Current Progress and Future Applications 4/6 Figure 2. The protocol derived by Takebe et al. (2015) in which mesenchymal-directed condensation of multiple cell types is used for organoid formation resulted in successful generation of organoids of multiple tissue types. Image courtesy of Takebe et al. (2015). Figure 3. Decellularized left lobe of a human liver. The perfusion process results in the tissue taking on a translucent appearance as the liver becomes increasingly decellularized. When lit up from behind, the conserved vasculature of the decellularized matrix is visible. Image courtesy of Mazza et al. (2015). recipient of a viable organ. However, questions still remain about optimal ways to recellularize the tissue to regenerate the more complex areas of the liver including the hepatic sinusoids and the portal triad vasculature. 3. Applications of In Vitro Organogenesis of the Liver The contributions of the research discussed above will hopefully aid in the ability to move toward growing full-sized, functional livers in vitro, which would have multiple applications in the medical field. Three main applications will be focused on: the generation of livers to be used for modelling human disease, using these models to test the response of liver tissue to drugs, and the potential for engineered livers to be used for transplantation purposes. To begin, 3D in vitro models of functional, viable livers have the potential to significantly improve our knowledge of human disease development and the response of tissue to novel therapeutics. The models currently used to study these processes include 2D cell culture methods, animal models, or cadaver tissues are used to study these processes; however, all three of these methods have significant shortcomings. For example, 2D cultures fail to take into account the microenvironment of 3D tissues in vivo and thus have limited applicability to in vivo conditions (Skardal et al. 2015). Similarly, animal models don t always resemble human tissue types and can yield results that don t translate to human tissue (Elliott & Yuan 2010). While human cadaver tissues may offer the most accurate depiction of live human tissue, problems occur due to a lack of donor tissue and difficulty associated with maintaining the viability of excised tissues (Elliott & Yuan 2010). Therefore, there is a great need for improved models to more effectively study human disease and drug response. The generation of viable, functional human liver tissue in vitro could offer an invaluable model for studying liver disease and drug response. Skardal et al. (2015) demonstrated the ability to engineer a 3D liver-tumor hybrid organoid system that could be used to study both tumor development and the response of the tissue to chemotherapeutic treatment. In order to achieve this, they generated 3D liver organoids composed of both healthy liver cells and fluorescently labelled carcinoma cells by co-culturing them in the presence of a 3D matrix in a rotating wall vessel (RWV) bioreactor. The use of the RWV allowed the cell cultures to be suspended in microgravity which caused them to condense based on natural cell affinities into liver organoids containing tumor foci representative of metastatic colon carcinoma. The researchers were able to study the development of the tumor foci within the healthy liver tissue over time by imaging the developing liver-tumor hybrid organoids with both light and fluorescence microscopy. Figure 4 depicts the result of merging images of the growing organoids taken using these two visualization techniques. Imaging the organoids using these techniques gave the researchers insight into how the tumors (composed of fluorescent carcinoma cells) formed within the healthy liver tissue over time. Upon confirming the viability of the resulting organoids by measuring increases in their size and metabolism

5 In Vitro Organogenesis of the Liver: Current Progress and Future Applications 5/6 Figure 4. Merged light microscopy and fluorescence microscopy images that show the development of tumor foci (red) within the liver organoids over time during culturing. The transparent circles in the images are hyaluronic acid-coated microcarriers, which were used as the 3D matrix for culturing. Image courtesy of Skardal et al. (2015). during development, the researchers suggested that the model could be used as a method for real-time tumor tracking. They were also able to use immunohistochemical staining of multiple cell markers to determine that the phenotype of the cells composing the organoids more closely resembled that of liver cancer in vivo than those of typical 2D cultures. These results support the potential for liver organoids to be used as more representative model of human disease development. In addition to exemplifying the potential of in vitro liver engineering to provide a better model for disease development, Skardal et al. (2015) were able to simultaneously demonstrate the potential for this technology to be advantageous in testing therapeutics. Skardal et al. (2015) used their liver-tumor organoids to test the specificity of a common chemotherapeutic agent, 5-fluorouracil (5-FU), in targeting the cancer cells versus the surrounding healthy liver cells. By incubating the organoids in 5-FU and counting how many of the cells that underwent apoptosis were cancer cells versus normal liver cells, they were able to establish at what concentration 5-FU began non-selectively targeting cancer cells and imposed damage on healthy cells. Considering that the phenotype of the cells composing the organoids was more representative of that of an in vivo liver cancer system, these results are likely more applicable to human disease and drug response in vivo. This would therefore supply researchers with more accurate data related to drug dosage for chemotherapies and how tissue may react to new therapeutics. Perhaps the most compelling application of in vitro liver organogenesis is the potential to generate transplant organs. As previously mentioned, the only current treatment option for end stage liver disease is a liver transplant, but a shortage of donors causes 20% of patients on the transplant wait list to die before receiving a liver (Mazza et al. 2015). Thus, the ability to transplant livers without the need for a donor could alleviate the number of patients that die waiting for a transplant. Current research is showing the potential of this application by generating liver organoids and transplanting them into animal models to see how they function and are accepted in vivo. For example, Saito et al. (2011) successfully grew liver organoids and transplanted them into mice. The liver organoids were grown from a combination of immortalized mouse hepatocytes, hepatic stellate cells, and sinusoidal endothelial cells on a 3D matrix in a radial flow bioreactor (RFB). Within the RFB, culture medium containing the three cell types was perfused over the 3D scaffold (AFS scaffold) to produce viable liver organoids. The resulting organoids were then transplanted into either a pocket under the renal capsule of the kidney or the porta-hepatis region of the omentum in mice; both locations were chosen based on the ease of the operation required to perform the transplant. At either 4 or 8 weeks post-transplant, the mice were euthanized and the transplanted organoids were examined. Histological staining and scanning electron microscopy (SEM) of the excised organoids were used to determine that the transplanted organoids were able to proliferate and survive in vivo post-transplantation. Additionally, Saito et al. (2011) observed that the expression of genes commonly transcribed in the liver (albumin, HNF-4α and G6Pase) was higher in the organoids after the transplant than during culturing. The results of this research indicate the potential for engineered livers to be successfully transplanted into live patients to continue to develop and maintain functionality. Not only would this reduce the wait time of patients requiring a liver transplant, but it could also reduce the risk of transplant rejection if the livers could be grown using a patient s own stem cells. Overall, this would lower the number of patients that die from liver disease; however, this technology would first need to be tested in human patients as opposed to animal models to assess its true application in health care. 4. Conclusion In conclusion, while the current state of in vitro liver organogenesis represents significant advances in the field of tissue engineering since the formation of more simple structures such as skin or cartilage, various challenges still remain that have thus far prevented the ability to culture a full-sized, functional liver in vitro. Current research in this field is attempting to optimize the cell types, culturing parameters, and 3D matrices required to overcome these challenges. This is a worthwhile pursuit, as the ability to generate a functioning liver from a patient s own stem cells in vitro could not only be useful for transplantation purposes, but also for gaining a better understanding of liver diseases and drug response of tissues in vivo.

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