L16: Stem cells part 2 Flashcards
Lgr5+ Organoids Technology?
Harvesting Biopsies:
Intestinal biopsies are obtained endoscopically.
Isolation of Crypts:
Intestinal crypts are isolated from the biopsy.
Establishing Culture Systems:
Specialized culture systems are set up to support growth.
Organoid Formation:
Organoids form from the isolated crypts in the culture.
Enrichment of Stem Cells:
The culture enriches intestinal stem cells as organoids grow.
Stem Cell Transplantation:
Stem cell transplantation may be performed, using the enriched organoids for further study or therapy.
professional vs faculative adult stem cells?
Professional Stem Cells:
Satellite cells in muscle – These are active and constantly renewing in tissues like blood or intestine.
Muscle regeneration: Satellite cells stay quiescent (non-dividing) under normal conditions but can re-enter the cell cycle upon injury or insult to the muscle. They then amplify and differentiate to form myofibers and regenerate the damaged tissue.
Facultative Stem Cells:
Liver and pancreas do not have a constant stem cell compartment like blood or intestine. They rely on facultative stem cells that activate only when needed.
Liver regeneration:
Hepatocytes (liver cells) and biliary duct cells are responsible for most of the liver’s regenerative capacity.
The liver typically only needs to renew hepatocytes once a year, so there is no need for an active, professional stem cell compartment under normal conditions.
In the case of massive liver injury, facultative stem cells (either hepatocyte or biliary duct cells) are activated. These cells enter a progenitor state, expand, and then differentiate into both hepatocytes and biliary duct cells to regenerate the liver tissue.
applications of pluripotent stem cells?
Applications of pluripotent stem cells
Open up field of regenerative medicine including possibility of transplantation- stem cell therapy.
D9sease modelling.
Enable drug screening.
Need to learn how to do direct differentiation to do these things. Push pluripotency out of pluripotency and induce diff along lineage and cell type of interest. Directed differentiation.
progress in therapies based on ipscs?
Since ips derived from any somatic cell types in our body they can be derived from patients. Biopsy from patient. These cells will have exact genetic background or possible polmorphism of this individual so good for personalised medicine either for gene correction for example or transplantation or drug screening.
Can use pluripotent stem cells but ipsc has genetic background of the individual.
Autologous iPSC therapy: Cells from the same individual (e.g., human patient) are reprogrammed into iPSCs for therapeutic purposes.
intestine paradigm vs liver and pancreas and methods of studying stem cells
er and Pancreas Regeneration:
Liver: The liver has remarkable regenerative abilities due to its ability to activate facultative stem cells when needed, particularly during injury or after significant tissue loss. However, under normal conditions, it doesn’t require a constant adult stem cell compartment for everyday maintenance, as hepatocytes and bile duct cells can re-enter the cell cycle to maintain tissue mass.
Pancreas: The pancreas, on the other hand, has limited regenerative capacity. While there is some regenerative ability, it is much less robust compared to the liver. Diseases like diabetes, pancreatitis, or pancreatic cancer represent major challenges, and research into using pluripotent stem cells (iPSCs) to regenerate pancreatic cells, particularly insulin-producing beta cells, is an area of significant interest.
Stem Cell and Progenitor Cell Definitions:
Stem Cells: These are long-term self-renewing cells with multipotency (ability to differentiate into multiple cell types). In tissues like the liver, facultative stem cells can be activated when needed to regenerate tissue.
Progenitor Cells: These cells also have multipotency, but their renewal capacity is limited compared to stem cells. They can divide and differentiate, but they do not have the ability for long-term self-renewal.
Differentiated Cells: These are fully specialized cells with limited or no ability to renew. Once differentiated, they typically cannot divide or regenerate the tissue from which they came.
Techniques for Studying Stem Cells:
(a) In Vivo Lineage Tracing: This technique is used to track stem cell differentiation over time. It helps researchers understand how stem cells contribute to tissue regeneration and maintenance in vivo. This method can also be used to trace the fate of transplanted pluripotent stem cells in animal models.
(b) Clonogenic Assays: These assays measure the ability of stem cells or progenitor cells to proliferate and form colonies. It helps assess the self-renewal and multipotency of a given cell population, including how efficiently they can differentiate into functional cells in culture.
(c) In Vitro Culture vs In Vivo Transplantation:
In Vitro Culture: Stem cells can be cultured in controlled laboratory conditions to study their behavior, such as self-renewal, differentiation, and their ability to form organoids or specialized tissues. This allows researchers to investigate how stem cells respond to different signals and factors in a controlled environment.
In Vivo Transplantation: This method involves transplanting stem cells or progenitor cells into an animal model (typically mice) to observe how they behave in a living organism. In vivo transplantation helps assess how well stem cells integrate into tissues, their potential to regenerate damaged tissue, and their ability to function in a complex, living environment. It’s a critical technique for evaluating the therapeutic potential of stem cells before clinical application.
liver and pancreas development?
Common Origin: The liver and pancreas both arise from a common bipotent endoderm progenitor cell during development. In just a few hours, this progenitor decides whether to become liver or pancreas, and two distinct transcriptional programs are activated to guide the development of each tissue.
Distinct Pathways:
The liver and pancreas develop into different tissue architectures and have distinct metabolic properties, but they are related in the adult body as part of the digestive system and are connected through a network of ducts.
Liver:
Hepatocytes: Liver cells, called hepatocytes, play a central role in various metabolic processes such as coagulation, detoxification of nutrients, and absorption of nutrients.
Biliary Epithelial Cells: These cells are involved in bile production and secretion, essential for digestion.
Pancreas:
The pancreas functions as two organs in one:
Exocrine Compartment: The majority of the pancreas is made up of exocrine tissue, including pancreatic duct cells and acinar cells. These cells are responsible for producing digestive enzymes to aid in food digestion.
Endocrine Compartment: A smaller portion of the pancreas is made up of endocrine tissue, which is organized into islets of Langerhans. These clusters contain several cell types:
Beta cells: Produce insulin, which regulates blood glucose levels.
Alpha cells: Produce glucagon, which also plays a role in glucose metabolism.
Islet Dysfunction: When the islets lose function, such as in diabetes, it results in a disruption of blood glucose regulation.
Challenges and Clinical Interest:
Pancreatic Cancer: Both the exocrine and endocrine compartments of the pancreas are vulnerable to cancer, with pancreatic cancer being one of the most aggressive and least treatable cancers.
Liver Regeneration: The liver is known for its regenerative ability, whereas the pancreas has limited regenerative properties and lacks a defined adult stem cell compartment.
Islet Transplantation: For patients with diabetes, islet transplantation has become a therapeutic option to restore insulin production. However, this approach is still limited by availability and long-term success.
Stem Cell Research: There’s active research on using stem cells to create surrogate pancreatic or islet cells as a potential cure for diabetes. Clinical trials are exploring the development of stem cell-derived islets to replace dysfunctional or lost pancreatic cell
liver disease?
Biliary Tract Disease:
Affects bile ducts, can lead to obstruction or inflammation.
Causes: Genetic, infections, autoimmune, or bile duct stones.
Liver Cancer (Hepatocellular Carcinoma):
Often develops from chronic liver diseases like cirrhosis or hepatitis.
Causes: Hepatitis B/C, alcohol abuse, aflatoxins, NAFLD.
Viral Hepatitis (Hepatitis B & C):
Viral infections causing liver inflammation.
Causes: Transmitted through blood, sex, or mother-to-child.
Chronic: Can lead to cirrhosis or liver cancer.
Non-Alcoholic Fatty Liver Disease (NAFLD):
Fat buildup in the liver, not caused by alcohol. The accumulation of fat and the resulting liver damage trigger the activation of resident macrophages (Kupffer cells) and the recruitment of additional macrophages from the bloodstream.
Inflammation: Activated macrophages release pro-inflammatory cytokines, such as TNF-α and IL-6, which contribute to liver inflammation.
Causes: Obesity, insulin resistance, type 2 diabetes, dyslipidemia.
Chronic: Can progress to NASH, cirrhosis, and liver cancer.
Liver Injury:
Resulting from toxins, drugs, infections, or trauma.
Causes: Alcohol, medications, viruses, physical injury.
Chronic: Can lead to liver failure, cirrhosis.
Alcoholic Liver Disease:
Caused by excessive alcohol consumption.
Causes: Chronic alcohol abuse.
Chronic: Can lead to cirrhosis and liver cancer.
how is celll identity establushed?
Transcriptional and chromatin program
Extrinsic signals
Morphogenesis and cell interactions
Need constant interplay between transcription factor cascade, epigenetic reg, inextrinsic factors, this knowledge is needed to define directed differentiation
Unraveling control mechanisms of cell identity has direct clinical and translational releva
making pancreatic cells?
pancreatic lineage fate decision: Pancreatic progenitor cells give rise to all the cell types in the pancreas.
Transcription factors like Pdx1 and Neurogenin3 (Ngn3) are important for beta-cell development.
Intrinsic factors play a role in acquiring pancreatic cell identity:
FGF10 (Fibroblast Growth Factor 10)
RA (Retinoic Acid)
Notch signaling
approaches for making pancreatic cells:
Lineage reprogramming refers to the process of converting one differentiated cell type into another, either within the same organ (intra-organ) or across different organs (inter-organ). This can be achieved by manipulating transcription factors and signaling pathways that regulate cellular fate.
Intra-Organ Lineage Reprogramming:
Intra-organ reprogramming involves converting cells within the same organ to another cell type. For example, converting pancreatic duct cells into beta cells within the pancreas.
This approach typically involves reprogramming pancreatic progenitors or ductal cells into insulin-producing beta cells, which can be useful in treating conditions like Type 1 diabetes where beta cells are lost.
Key transcription factors for reprogramming include:
Pdx1, Ngn3, and MafA for driving beta cell differentiation in the pancreas.
Inter-Organ Lineage Reprogramming:
Inter-organ lineage reprogramming refers to the conversion of cells from one organ into a cell type from another organ. For example, reprogramming liver cells to produce beta cells for diabetes treatment.
This type of reprogramming typically involves greater challenges as it requires cross-tissue reprogramming, which may need extensive manipulation of signaling pathways and epigenetic remodeling to recreate the cellular environment necessary for functional beta cell formation.
Differentiation from Pluripotent Stem Cells (PSCs):
PSCs, including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), are a powerful tool for generating various cell types, including beta cells.
Differentiation of beta cells from PSCs typically follows a multi-step protocol that mimics embryonic development.
The differentiation process is driven by the application of specific growth factors and signaling molecules that guide PSCs to differentiate into pancreatic progenitors and, eventually, mature insulin-producing beta cells.
Key stages in the differentiation of beta cells from PSCs include:
Endodermal differentiation – First, PSCs are directed to differentiate into endodermal cells, which are the precursors of pancreas cells.
Pancreatic progenitor stage – Endodermal cells are then directed towards pancreatic progenitors using factors like Pdx1, Ngn3, and FGF10.
Beta-cell maturation – These progenitors are then induced to become mature beta cells, which produce insulin. Factors like MafA and Neurogenin3 (Ngn3) are crucial at this stage.
Beta Cell Differentiation in the Context of Diabetes Therapy:
One of the main therapeutic strategies for Type 1 diabetes is to generate functional beta cells from iPSCs derived from the patient’s own cells. This avoids issues of immune rejection.
Current research focuses on improving the efficiency and functionality of beta cells generated from iPSCs, ensuring they can produce insulin in response to blood glucose levels.
how has the field used developmentl biology knowledge?
The field of stem cell differentiation has applied insights from developmental biology to create direct differentiation protocols. This has allowed the differentiation of pluripotent stem cells (such as human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs)) into various pancreatic cell types, including endodermal cells, pancreatic progenitors, and beta cells.
Key Signaling Pathways for Pancreatic Development:
Retinoic Acid (RA): Known to play a crucial role in the early stages of pancreatic development by regulating gene expression.
FGF (Fibroblast Growth Factor): FGF signaling has been shown to promote the differentiation of progenitors into specific pancreatic cell types.
Notch Signaling: Regulates the development of pancreatic progenitors and helps in the determination of cell fate during embryonic development.
These pathways were originally discovered in embryonic development to be essential for pancreatic formation, and researchers have leveraged this knowledge to direct the differentiation of stem cells into functional pancreatic cell types, including insulin-producing beta cells.
