Transdifferentiation

• What is transdifferentiation?

  Also known as direct cellular reprogramming - the conversion of one differentiated somatic cell to another without entering an intermediate pluripotent state

  
  

  Also includes interconversion of stem cells
• What is the importance of studying transdifferentiation?

  • Understanding cancer
  • Identifying the responsible transcription factors - reprogramming stem cells for therapeutics
  • Elucidating the normal developmental biology (cellular and molecular differences) of the two cell types that interconvert - what distinguishes them as neighbouring regions of the developing embryo
• What is Barrett's oesophagus (metaplasia)?

  A premalignant condition that predisposes to the development of oesophageal adenocarcinoma
• How is Barrett's metaplasia diagnosed?

  1. Endoscopy - change in appearance of oesophageal lining

  2. Biopsy analysis - histological confirmation of the presence of intestinal metaplasia in the lower oesophagus

  
  

  Expect that the normal squamous epithelium of the oesophagus will be replaced by intestinal-type columnar cells
• What is the criteria Eguchi and Kodama (1993) established to classify "true" transdifferentiation

  
  

  • The differentiated phenotypes before and after the conversion must be clearly defined - normally requires both morphological and molecular/biochemical characterisation
  • The cell lineage relationship between the two cell types must be clearly established - normally requires some form of lineage labelling or direct observation in tissue culture
• What are some examples of transdifferentiation?

  
  

  • Pancreatic cells to hepatocytes
  • Fibroblasts to neurones
  • Fibroblasts to cardiomyocytes
• What are some functions of the liver?

  • Detoxification – metabolises toxic compounds
  • Regulates carbohydrate fat protein metabolism
  • Produces bile
  • Synthesises proteins
• Major symptoms of liver failure


  • Jaundice - breakdown red blood cells that produces bilirubin
  • Oedema - build-up of fluid that causes swelling in legs, ankles, and feet
  • Testicular atrophy - reduced size and potential loss of function (impotence)
  • Caput medusae - cluster of swollen veins in the abdomen
  • Accumulation of oestrogen, e.g. gynecomastia
• Why is there interest in studying Barret's metaplasia?

  Identifying the Barrett’s metaplasia cell of origin could lead to more effective prevention (earlier diagnosis) and treatment strategies (novel targets) for both Barrett’s esophagus and esophageal adenocarcinoma
• How does reprogramming differ from transdifferentiation?

  Reprogramming involves dedifferentiation into an induced pluripotent stage (cells become known as iPSCs), where it can then be redifferentiated into a desired cell type - an indirect approach

  
  

  In contrast, transdifferentiation is more direct - the starting cell directly transforms into the desired cells type

  
• What are morhpogens?

  Signalling factors, often present in a gradient, that act to provide such positional information to cells within developing tissues

  
  

  The gradient in tissues leads to differential gene expression between cells, depending on the local concentrations of morphogen that individual cells encounter
• What are examples of morphogens in vertebrates?

  • Sonic hedgehog (SHH)
  • Bone morphogenetic protein (BMP)
  • Fibroblast growth factor (FGF)
• What is the Edmonton protocol?

  • Method of implanting pancreatic islets to treat type 1 diabetes
  • Typically recieve islets from one to as many as three donos
  • Requires immunosuppresion as the cells are non-self
• What is metaplasia?

  • Describes a wider class of cell type interconversions
  • Recently used to refer to any switch of cell type regardless of pathway
• What is Barrett's metaplasia characterised by?

  Normal esophageal squamous epithelium replaced with metaplastic intestinalised columnar cells due to chronic damage from gastroesophageal reflux

  
• What is an in vitro model for transdifferentiation of pancreatic cells to liver cells?

  AR42J-B13 cells - acinar-like pancreatic cells derived from azaserine-treated rats

  • AR42J-B13 cells are mainly exocrine, but also have some neuroendocrine properties - e.g. amylase, synaptophysin and neurofilament
  • Acinar cells = specialised epithelial cells responsible for producing and secreting substances like digestive enzymes
  • Azaserine = a potent carcinogen used to induce pancreatic cancer
• What experiments generate hepatocytes from AR42J-B13?

  • Amylase expression increased when cultured with synthetic glucocorticoid dexamethasone (Dex)
  • Induce formation of insulin-secreting B-cells with activin and Hepatocyte Growth Factor (HGF)
• How is a lineage relationship demonstrated in AR42J-B13 transdifferentiation to liver cells?

  • Label the AR42J cells with GFP, driven by the pancreatic Elastase (Elas) promoter
  • Incubate the cells with and without Dex
  • Stain and observe GFP fluorescence over time - transdifferentiated pancreatic cells (into mature liver cells) lose activation of the Elastase promoter and thus also GFP expression (fluoresence decreases)
  • Stain with markers of liver proteins, e.g. Glucose-6-phosphatase - co-expression indicates that liver cells arise directly from pancreatic cells
• What tests can be done to prove successful transdifferentiation?

  • Morphological analysis - visual inspection of alterations in cell shape, size, and organisation etc.
  • Immunocytochemistry (ICC) - immunostaining for specific protein markers
  • Gene expression analysis - quantitative PCR (qPCR) or RNA sequencing
  • Functional assays - assess physiological properties of the differentiated cells
  • Transplantation studies - in vivo transplantation
  • Single-cell analysis - reveal cellular heterogeneity and identify rare cell populations
  • Lineage tracing - e.g. fluorescent reporters or Cre-loxP systems,
• How was transdifferention of AR42J-B13 cells into hepatocytes assessed?

  Morphological analysis

  • AR42J-B13 cells showed flattened, epithelial-like morphology - 2 weeks post Dex-treatment
  • Transdifferentiated hepatocytes showed extensive endoplasmic reticulum and structures resembling bile canaliculi

  Gene expression (microarray) analysis

  • Inhibited expression of amylase (pancreas)
  • Strong expression of albumin (liver)

  Immunocytochemistry - liver-specific markers

  • Glucose-6-phosphatase
  • Albumin
• What functions were transdifferentiated hepatocytes able to perform compared to normal liver cells?

  • Synthesise and secrete albumin
  • Synthesis lipid
  • Metabolise drugs
  • Replicate hepatisis B virus
• What is the molecular basis of the switch from pancreas to liver?

  C/EBPβ (CCAAT/enhancer-binding protein beta) is a master TF of this transdifferentiation

  • Not expressed in pancreatic cells typically
  • However, expressed in [amylase-negative (pancreas) + G6Pase-positive (liver)] Dex-treated cells - additionlly these cells show nuclear translocation of HNF-4α and activation of differentiated hepatic products
  • Transfection of C/EBPβ into cells is enough to provoke transdifferentiation - meanwhile while LIP (liver inhibitory protein), its dominant-negative form, inhibits the process