Enginerring liver tissue Flashcards

1
Q

Describe the structure of a healthy liver and explain how this structure is affected by liver disease

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are the limitations of current approaches to modeling human liver disease in vitro?

A
  • 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 .
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Compare and contrast embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as sources for liver tissue engineering.

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Outline the key steps involved in the chemically defined differentiation of pluripotent stem cells into hepatocytes.

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How did researchers evaluate the metabolic competence and functionality of the stem cell-derived hepatocytes?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What advantages did using laminin as a substrate offer compared to Matrigel in hepatocyte differentiation?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Explain the rationale for developing a 3D liver tissue model and describe the specific approach utilized in the study

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What key observations supported the long-term stability and functionality of the 3D liver spheres?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Describe the animal model used to assess the therapeutic potential of the engineered liver tissue and the main findings of this study

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

How did the researchers investigate the potential for immune modulation by the stem cell-derived hepatocytes?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What key findings emerged from the collaboration with Bristol Myers and Squibb regarding the functionality of stem cell-derived hepatocytes?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What evidence supports the claim that the stem cell-derived liver implants have immune-modulatory capabilities?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Describe the structure and key characteristics of the 3D liver spheres created using microwell technology.

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is the primary challenge associated with two-dimensional culture of stem cell-derived hepatocytes, and how did the researchers address this issue?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Explain how the automated system was used to study fatty liver disease and what insights were gained.

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are the advantages of using pluripotent stem cells (PSCs) for liver tissue engineering?

A

Renewable: Derived from embryos or induced from somatic cells, eliminating reliance on organ donation.
Defined genetics: Offer control over genetic background, enabling disease modeling and drug screening.
Maturation potential: Can differentiate into mature liver cells, providing more accurate models of liver function.

17
Q

How are implantable liver tissues being engineered from PSCs?

A

Chemically defined differentiation: PSCs are guided through specific stages of differentiation using defined media and growth factors, resulting in hepatocyte-like cells.
3D culture: Cells are cultured in 3D systems like microwells to promote self-assembly into liver spheres, improving tissue organization, function, and longevity.
Vascularization: Endothelial cells are incorporated to form vascularized liver spheres, enhancing nutrient supply and waste removal.
Scaffold integration: Liver spheres are seeded onto biocompatible scaffolds to create implantable constructs for transplantation.

18
Q

What are the potential applications of engineered liver tissue?

A

In vitro modeling: Drug metabolism and toxicity testing, disease modeling, and virology studies.
In vivo transplantation: Providing support for failing liver function in patients with liver disease.

19
Q

How do engineered liver tissues show promise for treating liver failure?

A

Preclinical studies using a mouse model of Tyrosinemia type 1 (a progressive liver disease) have shown encouraging results:

Improved liver function: Transplanted liver tissue supported host liver function, leading to reduced liver damage markers and circulating toxins.
Immune modulation: Implanted liver tissues exhibited immune-modulatory properties, enabling tolerance in immune-competent animals.

20
Q

What are the advantages of using microwell technology for 3D liver tissue engineering?

A

Controlled size: Enables the creation of uniform liver spheres with defined size and cellular composition.
Improved stability: 3D structure promotes cell-cell interactions and enhances long-term stability, overcoming limitations of 2D cultures.
Scalability: Amenable to automation, facilitating large-scale production of liver tissues for research and therapeutic applications.

21
Q

What are the future directions for research in this field?

A

Refining tissue engineering processes: Optimizing vascularization, cell composition, and maturation of engineered tissues.
Developing new biomaterials: Identifying ideal scaffold materials for implantation and integration with host tissues.
Conducting preclinical studies: Evaluating the safety and efficacy of engineered liver tissues in animal models over longer time periods.
Translating to clinical trials: Ultimately, progressing toward clinical trials to test the therapeutic potential of implantable liver tissues in humans.

22
Q

What is the significance of the immune-modulatory properties of these stem cell-derived liver tissues?

A

Studies show that stem cell-derived hepatocytes can modulate T-cell proliferation and activation, leading to immune tolerance. This is crucial for the success of liver tissue implants, as it suggests they may evade rejection by the host immune system, even in immune-competent animals. This finding opens up possibilities for using these tissues in a wider range of patients without the need for strong immunosuppression.