WK 5: Reprogramming

What is another name for reprogramming?

Somatic cell reprogramming

What is the definition provided for reprogramming?

Process of converting differentiated adult cells back to a more primitive stem cell state

In order to re-establish the primitive state, what is required?

Reprogramming involves erasing cellular memory (epigenetics) which is typically marked by specific gene expression profiles and re-establishing the zygote nuclei niche thereby allowing ‘reprogrammed’ cells to regain the capacity to differentiate (induced pluripotent stem cells; iPSCs into any cell type within the organism)

What was the story of Dolly

Enucleated oocyte donor (providing the zygote niche) was a blackface + nuclear donor of udder cells (differentiated diploid cell) was provided by a Finn-Dorset

• When fused with electric shock → embryo was cultured

Was Dolly fertile?

Yes, she gave birth to Bonnie

What did the birth of Dolly illustrate?

That the niche of a cell can override epigenetic marks

What did John B. Gordon discover? What was he trying to answer?

→ By the time cells have differentiated, have they lost the genes required for early development? (they had not yet sequenced the genome)

In 1962, Gordon took an enucleated frog cell, and placed an intestinal cell nuclei from a tadpole within it. This produced a new tadpole

What did Yamanaka discover? What was he trying to answer?

→ Minimum # of transcription factors required to override differentiation?

Transfected a mouse fibroblast with four specific TFs. This fibroblast then went on to produce teratomas and a new mouse

• Zygote niche (4 TFs) → affects signaling pathways → TFs are the drivers → specific gene expression

What did Gordon and Yamanaka show?

Differentiated cells can become reprogrammed to a pluripotent state by the expression of four defined factors (all of which are transcription factors)

What are the four required transcription factors (the Yamanaka factors)? How did we use to produce iPSCs?

Oct3/4, c-Myc, Sox2, Klf4

• Mouse fibroblast transfected by viruses expression the 4 TFs

• Selection for antibiotic resistance restricts growth of only viral-infected cells

• Resulting culture only containing iPSCs → ability to create a variety of tissues

What did Gordon’s method require? Did Yamanaka challenge this?

Gordon required oocytes to do the reprogramming (enucleated frog egg)

Yamanaka proved that oocytes are not required for reprogramming, but only the 4 TFs

What is a major medical advantage of iPSC cells?

They can be derived from a patient’s own differentiated cells; therefore there is no danger of rejection if reintroduced into the patient, potentially for therapeutic reasons

What is the protocol for curing sickle cell anemia in a mouse using iPSC cells plus recombinant genetics?

Humanized sickle cell anemia mouse (HbS/HbS where we only need one copy to be normal to have normal phenotype)

→ harvest tail fibroblasts and culture

→ Infect with the Yamanaka factors: Oct3/4, Sox2, Klf4, c-Myc viruses

→ Mouse-derived iPSC clones

→ Correct sickle-cell mutation in iPSC cells by specific gene targeting

→ HbA/HbS iPSC clones

→ Differentiate into embryoid bodies

→ Transplant corrected hematopoietic progenitors back into irradiated mice

Can adult cells be reprogramed in vivo?

Yes, through fetal reversion