L16: Stem cells part 3 Flashcards
regenerative cell therapies for diabetes?
pluripotent cell lines hesc or ipsc—> differentiation—> pancreatic progenitors or beta cells—-> transplantation. micro or macro encapsulation—> reversal of hyperglycemia.
Although stem cell derived products are like cell type: referred to as beta-like cells as they are not fully mature, not perfectly functional. Problems due to rejection. Immune system. Diabetes (type1) autoimmune. Cells can be attacked again by immune system. Different challenges.
studying human pancreas development?
Studying Human Pancreas Development Using Stem Cells
Stem cell-derived pancreatic cells are now being used to study human pancreatic development, a task that was previously difficult due to limited access to human fetal tissue. These stem cell models also allow for disease modeling.
Applications of iPSCs and CRISPR/Cas9 in Pancreatic Research:
Induced Pluripotent Stem Cells (iPSCs) and CRISPR/Cas9 are used to create and manipulate pancreatic progenitor cells.
Researchers can knock down the expression of various transcription factors involved in pancreatic development to generate pancreatic progenitor cells or beta cells.
For example, when the expression of Neurogenin-3 (Ngn3), a key transcription factor for endocrine cell differentiation, was switched off in iPSC-derived pancreatic progenitors, the cells were unable to differentiate into insulin-producing beta cells. This experiment demonstrated that the mechanisms of pancreatic development in humans are similar to those in mice.
sequential stages of liver formation?
Liver formation involves the crosstalk between intrinsic and extrinsic factors:
Extrinsic factors include signaling molecules like BMP (Bone Morphogenetic Protein) and FGF (Fibroblast Growth Factor).
Intrinsic factors are key transcription factors and epigenetic regulators that guide cell fate decisions. Liver Development Process (From Liver Diverticulum to Differentiation)
Liver Diverticulum:
The liver diverticulum is the initial bud-like structure that forms from the endodermal layer during early embryonic development. It eventually develops into the liver.
Cell Expansion (Getting “Fatter”):
As the diverticulum grows, proliferation (cell division) occurs to expand the structure.
Cell “Bursting” Out:
This refers to the migration of cells from the liver diverticulum. Cells leave the initial site and spread to form the basic structure of the liver, involving key processes like migration, adhesion loss, and differentiation.
Key Biological Processes in Liver Development (as shown by arrows):
Proliferation: Cells divide to increase the number of liver progenitor cells.
Migration: Liver precursor cells move to specific locations during development.
Loss of Adhesion: Cells detach from each other to enable migration and further differentiation.
Interkinetic Nuclear Migration: This refers to the movement of the nucleus during cell cycle progression in proliferating cells, important for cell division and differentiation.
Differentiation: Liver progenitor cells differentiate into hepatocytes, biliary cells, and other liver cell types.
Developmental biology knowledge, especially regarding how liver cells are formed, is being used to generate hepatocytes from pluripotent stem cells (PSCs) for potential clinical use, such as for liver disease treatment or regenerative medicine.
key transcription factors in image
cell-based regenerative therapy for liver disease?
hPSCs to Primitive Streak:
Extrinsic signaling factors (e.g., FGF, BMP) promote the transition of hPSCs into the primitive streak (a key step in mesoderm and endoderm formation). This also blocks the differentiation of ectoderm cells that would give rise to skin, neural tissues, etc.
Primitive Streak to Definitive Endoderm:
The signals from FGF and BMP direct hPSCs into the definitive endoderm, which restricts the potential for differentiation into mesoderm or ectoderm cell types (like muscle, bone, or nervous system cells). This ensures the focus remains on endodermal fates, which include the liver, pancreas, lungs, and intestines.
Definitive Endoderm to Posterior Foregut:
The posterior foregut cells are specified from the definitive endoderm. Signaling pathways such as FGF and BMP suppress the pancreatic progenitor fate and other foregut derivatives like the biliary cells, ensuring that these cells remain directed towards liver development. This process helps to block the pancreatic progenitors and biliary cells from diverging into other lineages.
Posterior Foregut to Liver Bud:
At this stage, the liver bud forms from the posterior foregut and extrinsic factors like FGF and BMP help the cells differentiate into hepatic progenitors and hepatocyte-like cells, while simultaneously blocking the differentiation into pancreatic or biliary progenitors.
Liver Bud to Hepatic Progenitors:
Transcription factors like Hex, Prox-1, and Gata-6 drive the progenitors towards the liver lineage while blocking the development of other potential fates such as pancreatic progenitors or biliary epithelial cells.
Hepatic Progenitors to Hepatocyte-like Cells:
As the progenitors differentiate into hepatocyte-like cells, this is a highly specific process that blocks the alternative fates of pancreatic cells and biliary epithelial cells. The cells are primed to become hepatocytes, and this is regulated by the appropriate transcription factors like HNF4α, HNF-6, and C/EBPα, ensuring no loss of liver identity.
Generation of Pure Liver Progenitors and Hepatocyte-like Cells:
Using extrinsic factors (FGF, BMP, RA), pure liver progenitors and hepatocyte-like cells are generated while suppressing unwanted differentiation into pancreatic, biliary, or other endodermal cell types. These factors are critical in blocking off-lineages.
Liver-Specific Cell Surface Markers:
To verify that the cells have properly differentiated into hepatocytes or biliary cells, liver-specific markers are used. This confirms that the pancreatic or biliary cell fates have been blocked effectively.
Engraftment of PSC-Derived Liver Cells into Fah-/- Mice:
After differentiation into liver progenitors or hepatocyte-like cells, these cells are engrafted into Fah-/- mice to assess liver functionality and determine the cells’ potential for restoration of liver function. The engraftment is successful because of the differentiation protocol that blocks other cell fates.
generating ipsc-derived liver organoids?
To generate iPSC-derived liver organoids, the cells are cultured in 3D, allowing them to better mimic the structure and function of the liver. In 3D, cells can interact with each other, which helps them mature and become functional.
For organoids to be more mature, they need to vascularize (form blood vessels) and interact with supporting cells like mesenchymal cells. These interactions help the liver cells develop properly.
This system is useful for toxicology and drug screening since it allows researchers to test drug responses in a more realistic, human-like model compared to 2D cultures or animal testing
