Transdifferentiation and Cellular Reprogramming Lecture Flashcards

A set of flashcards covering the concepts of transdifferentiation, metaplasia, Barrett's metaplasia, criteria for cellular reprogramming, and experimental models using pancreatic and liver cells.

What is the definition of transdifferentiation?

The direct conversion of one differentiated, mature cell type into another entirely different cell type (e.g., converting a pancreatic cell into a liver cell).

What clinical term is used by pathologists to describe ectopic tissue found in a location where it normally should not be?

Metaplasia.

Why is understanding the molecular basis of metaplasia important for cancer research?

In some cases, transdifferentiation predisposes to neoplasia (cancer), so understanding it can help in early diagnosis and provide targets for new therapies.

Who first described Barrett’s metaplasia, and in what decade?

Surgeon Norman Barrett in the $1950s$.

What is the underlying cause of Barrett’s metaplasia?

Chronic acid reflux.

How does the tissue of the lower oesophagus change in patients with Barrett’s metaplasia?

It changes from its normal stratified squamous epithelium to a columnar-like intestinal epithelium.

Why is Barrett's metaplasia considered highly important to study?

It is the only known precursor to oesophageal adenocarcinoma.

What is the increased chance of developing adenocarcinoma for patients with Barrett's metaplasia?

A $40-100$ fold increased chance.

What is the $5$-year survival rate for oesophageal adenocarcinoma?

Just $10-14\%$.

What is the visual difference between a normal oesophagus and Barrett's tissue during a gastroscopy?

A normal oesophagus is bright pink, while Barrett's tissue is visibly slightly reddish.

What characteristic shape does Barrett's tissue form when viewed by pathologists?

It forms distinct "tongues" of reddish tissue extending upwards from the gastroesophageal junction.

What physical complication occurs if Barrett's progresses to adenocarcinoma?

The cancer mass grows into the lumen of the oesophagus, making it difficult to swallow and physically blocking food from the stomach.

How can identifying "master" transcription factors benefit regenerative therapies?

Scientists can use these factors to artificially reprogram stem cells into specific, differentiated cell types.

What is the developmental link between cell types that can interconvert, such as the pancreas and liver?

They often originate from the exact same region of the developing embryo.

What role do morphogen gradients play in determine cell type during development?

Depending on the concentration of a morphogen a cell receives, it switches on a specific "master transcription factor" that determines its genotype.

What happens if a mutation switches on a master transcription factor induced by a morphogen post-development?

The cell would become the cell type of the adjacent tissue, resulting in an ectopic cell found in a tissue where it is not normally located.

Who defined the two strict criteria for proving a cell has transdifferentiated?

Eguchi and Kodama.

What are the two criteria for transdifferentiation according to Eguchi and Kodama?

$1.$ Describe the phenotype before and after the switch (loss of original and gain of new) and $2.$ Demonstrate a direct lineage relationship between the ancestor and descendant.

What four distinct levels must be analyzed to prove a change in phenotype?

Morphology, Gene Expression ($RNA$), Protein Expression, and Functional Characterisation.

How does morphology change when pancreatic cells convert to liver cells?

Control pancreatic cells are small and round, while the resulting liver cells are larger and flatter.

What is considered the "Gold Standard" for testing if a cell has truly transdifferentiated?

Functional Characterisation (testing if the new cell actually performs the target cell's job).

What are three functional tests for a lab-created hepatocyte?

Testing if it can synthesise lipids, secrete albumin, and metabolise drugs.

In terms of lineage, what does direct transdifferentiation exclude?

It excludes shifting backward through an intervening stem cell stage.

What is the purpose of using a reporter protein like Green Fluorescent Protein ($GFP$) in lineage analysis?

To permanently "label" the starting cell so that if it converts, the new cell still possesses the label, proving its exact origin.

What are the two classic examples of transdifferentiation described in the lecture?

The conversion of pancreatic cells to hepatocytes and the conversion of hepatocytes to pancreatic cells.

From which embryonic layer do both the liver and pancreas arise?

The endoderm.

Which master transcription factor is shared by both liver and pancreatic cells?

Hepatocyte Nuclear Factor $4$ alpha ($HNF4\alpha$).

Which enzyme that phosphorylates glucose is shared by both liver and pancreatic cells?

Glucokinase.

Which glucose transporter is naturally expressed by both the liver and the pancreas?

Glucose transporter type $2$ ($GLUT2$).

What are the two broad functional and histological categories of the pancreas?

The endocrine compartment and the exocrine compartment.

What is the name of the cell clusters in the pancreas that perform endocrine functions?

Islets of Langerhans.

What do Alpha ($\alpha$) cells in the pancreas express?

Glucagon.

What do Beta ($\beta$) cells in the pancreas express?

Insulin.

What is expressed by Pancreatic polypeptide ($PP$) cells?

Pancreatic polypeptide.

What hormone is expressed by Delta ($\delta$) cells?

Somatostatin.

What hormone is expressed by Epsilon ($\epsilon$) cells?

Ghrelin.

What type of cells make up the majority of pancreatic tissue and synthesise digestive enzymes?

Acinar cells (exocrine tissue).

How do pancreatic enzymes reach the intestine?

Through a branching pancreatic ductal tree that empties into the main pancreatic duct and then into the duodenum.

What two specific markers can be used in the lab to identify exocrine cell types?

Amylase (for acinar cells) or ductal cell markers.

How does the liver perform its detoxification function regarding protein metabolism?

It takes toxic by-products like ammonia and detoxifies them.

What plasma proteins are synthesized by the liver?

Albumin and blood clotting factors.

What substance is produced exclusively by the liver tissue?

Bile.

What is the current trend of liver disease incidence and death rates in the UK?

They are increasing year-on-year.

What are the main drivers of rising liver disease rates?

Viral hepatitis and non-alcoholic fatty liver disease ($NAFLD$), which is linked to obesity and diabetes.

What is the name of the pancreatic cell line model derived from azaserine-treated rats?

$AR42J-B13$ (or $B13$ cells).

What is the effect of treating $B13$ cells with the synthetic glucocorticoid Dexamethasone ($Dex$)?

Initial treatment increases amylase production, but prolonged treatment forces transdifferentiation into hepatocyte-like cells.

What morphological change occurs in $B13$ cells following $Dex$ treatment?

They change from small and round to bigger, flatter, and less compact.

What hallmark of mature hepatocytes is observed in $Dex$-treated $B13$ cells?

Polyploidy (specifically the appearance of binucleate cells).

What specific protein expression changes occur when $B13$ cells transdifferentiate?

The cells stop expressing amylase and begin expressing albumin and Glucose-$6$-phosphatase.

Which drug-metabolising enzymes are induced in transdifferentiated $B13$ cells according to microarray analysis?

Phase I and Phase II enzymes, such as $CYP3A1$ and $UGT$.

How did researchers prove $B13$ cells acquired liver-specific functionality?

By demonstrating they could support Hepatitis B Virus ($HBV$) replication.

What was required to allow $B13$ cells to be tested for $HBV$ replication?

The $HBV$ $cDNA$ had to be transfected directly into the cells because they lack the natural receptor for infection.

Which molecules were used alongside $Dex$ to achieve mature liver functionality in $B13$ cells for the $HBV$ test?

Oncostatin M ($OSM$).

What is the "GFP Perdurance Experiment" intended to prove?

The direct lineage relationship between pancreatic cells and liver cells without a stem cell intermediate.

In the lineage tracing experiment, which promoter was used to drive $GFP$ expression in starting acinar cells?

The pancreatic elastase promoter.

Why does $GFP$ allow researchers to track cells even after the pancreatic promoter shuts off?

Because of "perdurance," where the stable $GFP$ protein lingers in the nucleus for days after the gene is deactivated.

What co-localization of markers proved the direct lineage switch in the $GFP$ experiment?

Individual cells possessed both lingering green pancreatic nuclei ($GFP$) and newly synthesized red liver enzyme (Glucose-$6$-phosphatase) in their cytoplasm.

Identify the transcription factor that serves as the molecular driver of the pancreas-to-liver switch.

$C/EBP\beta$.

Is $C/EBP\beta$ normally present in pancreatic cells?

No, it is highly induced only following $Dex$ treatment.

What happens when $C/EBP\beta$ is experimentally overexpressed in control pancreatic cells?

It actively inhibits the pancreatic phenotype and turns on liver proteins.

What is LIP (Liver Inhibitory Protein)?

A dominant-negative form of $C/EBP\beta$ that completely blocks transdifferentiation.

Why are embryonic pancreatic buds used as a model instead of cell lines like $B13$?

Cell lines can be poor models for complex physiology; embryonic buds better represent normal development.

At what embryonic stage were the mouse pancreatic buds cultured?

$E11.5$.

What master transcription factor is essential for pancreas formation and tracked in the embryonic bud model?

$PDX1$.

What liver markers are expressed when $E11.5$ mouse embryonic pancreatic buds are treated with $Dex$?

Albumin, Glucose-$6$-phosphatase, and alpha-$1$-antitrypsin.