Enginerring liver tissue Flashcards
Describe the structure of a healthy liver and explain how this structure is affected by liver disease
A healthy liver is comprised of four lobes, each with an organized lobule structure crucial for its multifunctionality and regenerative capacity. Liver disease causes a gradual breakdown of this lobule structure, leading to loss of function and regenerative ability.
What are the limitations of current approaches to modeling human liver disease in vitro?
- Adult hepatocytes are scarce and expensive, and lose functionality over time in culture
- hepatic cell lines lose functionality during expansion, even tho inexpensive and easy to expand
- animal hepatocytes exhibit interspecies differences with humans, particularly in the cytochrome P450 system, less accurate models .
Compare and contrast embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as sources for liver tissue engineering.
Both embryonic stem cells (derived from blastocysts) and induced pluripotent stem cells (reprogrammed somatic cells) can differentiate into any cell type in the body. However, ethical concerns and potential immune rejection are considerations for embryonic stem cells, while iPSCs offer patient-specific cell sources.
Outline the key steps involved in the chemically defined differentiation of pluripotent stem cells into hepatocytes.
The chemically defined differentiation involves
- priming pluripotent stem cells with Activin A and Wnt3a, followed by
- hepatic differentiation induced by DMSO, and finally,
- hepatocyte maturation using HGF and Hepatozyme™.
How did researchers evaluate the metabolic competence and functionality of the stem cell-derived hepatocytes?
Functionality was assessed through
- gene expression profiling of drug metabolism and transporter genes,
- biochemical assays for metabolic activity (e.g., cytochrome P450 activity), and
- pathway analysis using drugs like paracetamol to evaluate drug metabolism and toxicity.
What advantages did using laminin as a substrate offer compared to Matrigel in hepatocyte differentiation?
Laminin as a substrate led to
- improved cell organization,
- enhanced function (demonstrated by increased dye clearance),
- better metabolic activity (compared to cryopreserved primary hepatocytes)
- improved gene expression with a reduction in contaminating markers.
Explain the rationale for developing a 3D liver tissue model and describe the specific approach utilized in the study
The 3D model was developed to address rapid dedifferentiation observed in 2D cultures. Researchers employed microwell differentiation, allowing the formation of liver spheres with a defined size suitable for in vitro and in vivo applications.
What key observations supported the long-term stability and functionality of the 3D liver spheres?
The 3D liver spheres exhibited long-term stability with over a year in culture, maintained key structural markers like HNF4α and albumin, displayed organized polarity, and retained drug-inducible metabolic activity over the culture period.
Describe the animal model used to assess the therapeutic potential of the engineered liver tissue and the main findings of this study
The study used a Tyrosinemia type 1 mouse model with a FAH enzyme mutation leading to liver damage. Transplantation of the engineered liver tissue showed improved host liver histology, human albumin production, decreased liver damage markers, and reduced circulating toxins.
How did the researchers investigate the potential for immune modulation by the stem cell-derived hepatocytes?
Researchers co-cultured stem cell-derived hepatocytes with quiescent or activated T cells. They observed reduced T cell proliferation in the presence of hepatocytes, indicating potential immune modulation. Further investigation revealed high ID1 expression in hepatocytes, leading to tryptophan starvation and subsequent downregulation of T cell activation.
What key findings emerged from the collaboration with Bristol Myers and Squibb regarding the functionality of stem cell-derived hepatocytes?
The study showed that stem cell-derived hepatocytes were comparable to primary human hepatocytes in predicting drug toxicity at day 7. However, the study also revealed areas where the model needed improvement, such as longevity and inclusion of other cell types.
What evidence supports the claim that the stem cell-derived liver implants have immune-modulatory capabilities?
The stem cell-derived liver implants persisted in immune competent animals for 14 days, suggesting immune-modulatory properties. Further studies revealed that these hepatocytes express high levels of ID1, leading to tryptophan starvation in T-cells. This tryptophan depletion reduces T-cell activation and proliferation, contributing to implant tolerance.
Describe the structure and key characteristics of the 3D liver spheres created using microwell technology.
3D liver spheres consist of hepatocytes arranged on the outer layer, with mesenchymal cells in the core. They exhibit proper morphology, structural markers like zona occludens 1 and E-cadherin, and maintain expression of HNF4 Alpha, a key transcription factor for liver identity.
What is the primary challenge associated with two-dimensional culture of stem cell-derived hepatocytes, and how did the researchers address this issue?
The main challenge is the rapid dedifferentiation of hepatocytes in 2D culture, leading to loss of function and cell death within 10-15 days. To address this, researchers transitioned to 3D culture systems, specifically microwell technology, which allowed for the creation of stable liver tissue for over a year.
Explain how the automated system was used to study fatty liver disease and what insights were gained.
Researchers fat-loaded stem cell-derived hepatocytes to mimic fatty liver disease. Using the automated system, they identified mitochondrial dysfunction and rewiring of the TCA cycle, leading to the accumulation of large fat globules (macrovesicular steatosis). This highlighted potential therapeutic targets for metabolic intervention.