ROJW - cellular reprogramming

Question 1

Cellular programming by nuclear transplantation

Answer 1

• First performed by John Gurdon on a frog
• Replace the nucleus of an egg cell with the nucleus from a differentiated somatic cell (ex. intestinal cell)
• Can still result in development of a whole animal (information from the nucleus programs the development of the egg cell)
• Proves that the nuclei of all cells harbor all the genetic materials required for the development of a whole animal

Question 2

General principles:

Answer 2

• All fully differentiated somatic cells contain a complete genome that is the same as the genome found in embryonic cells
• But fully differentiated cells just express a subset of genes specific for a particular cell type that is different from another cell type (ex: a neuron vs. a hepatocyte – express different subsets of genes)
• The different gene expression in different somatic cells are created and maintained by the expression of gene-specific transcription factors to establish and maintain tissue identity
• Artificial nuclear reprogramming methods work by changing the expression of gene-specific transcription factors (thus change the subset of expressed genes) to change the cell type, or reset it to less differentiated stem cell states

Question 3

1. Totipotent stem cell

Answer 3

• The early stage of fertilized egg cell
• The most versatile stem cell type, because they are formed shortly after fertilization of an egg cell by a sperm cell
• Can become all of the cells of the human body, as well as the cells of the embryo and developing fetus.

Question 4

2. Pluripotent stem cell

Answer 4

• Can give rise to all of the cell types that form the human body, but are not as versatile as totipotent cells.
• Can’t give rise to extra embryonic tissue that are required for embryogenesis
• Differentiate into cells from any of the 3 germ layers

Question 5

3. Multipotent stem cell

Answer 5

• Differentiate into a limited range of cell types which are more specialized/ specific
• Involved in producing cells that are continuously produced in the body (ex. Blood cells)

Question 6

4. Final/ Fully differentiated cells

Answer 6

• Committed to the function of the cell type that it has become to ensure tissue integrity
• So usually, differentiation is not reversible
• BUT: Somatic cells can be experimentally induced to change their identity by expressing one or more specific gene-specific transcription factors (artificially induced nuclear reprogramming)

Question 7

Artificially induced nuclear reprogramming

Answer 7

• Discovered in 2006 by Shinya Yamanaka
• Expression of a combination of 4 gene-specific transcription factors: OCT4, KLF4, SOX2, and MYC (Yamanaka factors) - in any differentiated cells will revert it back to a pluripotent stem cell (forming induced pluripotent stem cells – iPSCs) that can differentiate into a variety of any other cell type – complete reprogramming
  o All these transcription factors, especially MYC, are also oncoproteins! overexpression and/or mutations in these proteins can be found in many types of human cancer
• Possible therapeutic advantage to correct a problematic cell type (ex. neurodegenerative disease)

Question 8

Anti-aging research can also use:

Answer 8

• Partial reprogramming involves applying Yamanaka factors to cells for long enough to roll back cellular aging and repair tissues but without returning to pluripotency
• Partial reprogramming can dramatically reverse age-related phenotypes in the eye, muscle and other tissues in cultured mammalian cells and rodent models by countering epigenetic changes associated with aging

Question 9

Ex of Yamanaka factors:

Answer 9

OCT4

• a moderately sized gene-specific transcription factor of (360 amino acids)
• Has a “POU” DNA-binding domain is located centrally
  o consists of two helix-turn-helix motifs linked to each other
• Transcriptional activation domains (ADs) located in the flanking N- & C-terminal portions
• needs to be expressed in pluripotent cells to maintain their undifferentiated state
• OCT4 acts with SOX2 to recognise and bind in a sequence-specific manner to DNA target sites located next to regulatory elements, driving the transcription of embryonic stem cell-specific transcription factors
• Overexpression of OCT4 & SOX2 enforces aberrant the transcription of embryonic stem cell-specific transcription factors in differentiated cells, transforming them into iPSC

Question 10

iPSC Characteristics

Answer 10

• Immortal - no limit to number of cellular divisions (propagate indefinitely in cell culture)
• Can be differentiated into any cell type found in the human body
• Have a specific morphology
  o large prominent nuclei
  o narrow cytoplasm
  o defined border
  o tight cellular packaging
• Express various specific proteins that are not expressed in differentiated cells (serve as pluripotency markers)
  o gene-specific transcription factors, such as OCT4
  o cell surface proteins, such as SSEA3, SSEA4,TRA-1-60, TRA-1-81

Question 11

Applications of iPSC

Answer 11

• Therapeutic cure of degenerative diseases (Stem cell therapy)
• Genome edited iPSCs to cure genetic diseases
• Experimental purposes

Question 12

Therapeutic cure of degenerative diseases (Stem cell therapy)

Answer 12

• Harvest cells from patients’ easily accessible tissues (ex. blood cells/ skin fibroblasts)
• Grow in tissue culture
• Introduce cloning vectors to overexpress Yamanaka factors – by virus/plasmids/mRNA to obtain iPSC
• Differentiate the iPSCs to different cell types ex. pancreatic cells, cardiomyocytes, motor neurons – the cells that lacking in degenerative disease patients – and re-introduce them back to the right place
• Create cells that are re-introduced into patients to cure/ reduce the severity of the diseases
• Re-introduced iPSCs are genetically identical to the patients so there won’t be problems with immune rejection

Question 13

Genome edited iPSCs to cure genetic diseases

Answer 13

• In case of genetic disease, iPSC genome can be modified and corrected for any defects (ex. Knockout a gene that shouldn’t be on, add gene that is missing in the genome, correct mistake in the sequence of a gene)

Question 14

Experimental purposes

Answer 14

• Differentiate iPSC for desired cell types that are difficult get hold of
• Ex. Cardiomyocytes, Neurons – and use them for drug screening purposes, mobile system for disease detection, especially genetic diseases – ex. Alzheimer’s

Question 15

Differentiation of iPSCs

Answer 15

• After obtaining iPSC from differentiated cells, the iPSCs need to be exposed to different growth factors at different stages to guide them through the differentiation pathway to produce the desired fully differentiated cell

Question 16

Ex: Differentiation of iPSCs into Red Blood Cells

Answer 16

• Laboratory techniques have been developed successfully to generate red blood cells (erythrocytes) from human iPSCs
• could become an alternative treatment option for patients with blood disorders
• sickle cell anaemia
• various forms of a- and b- thalassaemia

Question 17

Pluripotency and Cancer

Answer 17

• Several lines of evidence suggest that reprogramming to pluripotency and oncogenic transformation are related at the molecular and cellular level:
  o both processes induce a switch from an oxidative to a glycolytic metabolic state to generate E from glucose (don’t go through TCA cycle, does not require O2)
   cancer cells: before vasculated, do not have enough access to O2
   embryonic cell: early embryo has not differentiated heart and blood vessels, so low access to O2 too
  o both involve numerous chromatin factors and RNA binding proteins that cooperate with reprogramming factors
  o both lose cellular identity& acquire cell plasticity (rate-limiting steps for both reprogramming towards pluripotency and transformation into cancer cells)

Question 18

Cancer and Pluripotency has the same early steps

Answer 18

• both require specific TFs to transform the cell to an intermediate cell type which characterize a high plasticity state (lose tissue-specific gene expression program)
• cells then decide whether to develop into pluripotent or cancer cells (different paths)

Question 19

Problems with iPSC: Mutations

Answer 19

• Some iPSC lines are heavily mutagenized, with a mutational burden similar to what is typically observed in human cancers
• Mutations can be Due to 2 diff conditions:
  o Mutations present in original somatic cells used for nuclear reprogramming
   Cells that have existed for a long time could have acquired mutations that do not affect the function but no longer a WT genome – if get iPSC, will pass down mutations to iPSC
  o Mutations caused by cell culturing and in vitro reprogramming procedure

Question 20

Mutations Already Present in Somatic Cells

Answer 20

• Usually, fibroblasts from skin cell samples are used for converting them into induced pluripotent cells (main reason: sampling from skin is easy)
• iPSCs derived from such fibroblasts frequently contain a larger number of mutations than those derived from other cell types (due to exposure to sunlight thus UV radiation)
• Any differentiated cells derived from such iPSC cell lines will also contain all these mutations, which increases the cancer risk for therapeutic applications
• Therefore, using easily accessible cells like fibroblast runs a risk of having UV induced mutations ending up in the iPSC and increases the cancer risk for therapeutic applications

Question 21

Cell Culture-Induced Mutations

Answer 21

• Culturing iPSCs in vitro causes additional mutations due to oxidative damage.
• most frequently observed mutation in blood cell-derived iPSCs = single-nucleotide variants in gene-specific transcription factor BCOR (acts as transcriptional repressor)
  o BCOR is BCL6 corepressor, a key transcription factor identified as a potent oncoprotein in the lymphoid lineage
  o BCOR mutations are highly recurrent and oncogenic in mature T-cell lymphoma
  o Therefore, BCOR mutations that arise in iPSCs after reprogramming, can affect the global transcriptomic program of iPSCs and impede differentiation into neural lineages
• BCOR can be statistically classified as a driver gene — a gene in which the prevalence of mutations is higher than would be expected by chance

Question 22

Practical, Social, Ethical and Legal Considerations

Answer 22

• Consent of donor
• Intellectual property and legal considerations (using other ppl’s cells – they might claim to have intellectual property rights)
• Predisposition of recipients to cancer development (mutations already present in the somatic donor cells or caused during the reprogramming procedure may predispose cells to developing cancer when transplanted back into the patient)
  o Therefore, need:
   Informed consent
   High degree of protocols to ensure that reprogramming and cell differentiation is done in a safe environment
   Early detection of damaged cells