Reprogramming of murine and human somatic cells using a single polycistronic vector

Bryce W. Carey, Styliani Markoulaki, Jacob Hanna, Kris Saha, Qing Gao, Maisam Mitalipova

PNAS, January 6, 2009, Vol. 106, no. 1, 157-162


Summary

Typically, generation of induced pluripotent stem cells (iPS) requires multiple proviral integrations to acheive viral-based delivery of the reprogramming factor; multiple proviral integrations pose the danger of insertional mutagenesis. This paper suggests a new approach to reprogram human somatic cells into patient-specific stem cells using just a single vector. Authors used 2A "self-cleaving" peptides which support efficeint polycistronic expression from a single promotor to deliver reprogramming factors in a single virus. They found four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus to generate iPS cells in both embryonic and adult somatic mouse cells. Additionally, human induced pluripotent stem cell (hiPS) lines from human keratinocyts were generated using the same approach.


Key Concept

• 2A "self-cleaving" peptides - 2A peptides originally identified and characterized in apthovirus foot-and-mouth disase virus. 2A oligopeptides contain a higly conserved c-terminal D(V/I)EXNPGP motif that mediates "ribosomal skipping" at the terminal 2A proline and subsequent amino acid. They used F2A, T2A and E2A peptides derived from different viruses.


• Single polycistronic vector - This vector can translate several reprogramming vectors from one promoter using 2A peptides. In this study they made 4F2A vector which contains Oct4, Sox2, Klf4 and c-Myc. Three different 2A peptides seperate those four factors when they are translated.
• Doxycycline (DOX)-inducible lentivirus - A tetracycline inducible lentivirus vector was constructed where expression of the genes was controlled from the tetracycline operator minimal promoter (tetOP) They, first, added DOX to generate four reprogramming factors. Once the somatic cells were transformed into iPS cells, they stopped adding DOX to check that iPS cells show 'stemness'; if iPS cells contain 'stemness', cells will renew themselves automatically even without external expression of reprogramming factors.


• Nanog GFP - The expression of Nanog is used as a key limestone to ensure the stemness of iPS cells. Green flourescent protein was attaced to nanog and used as a marker.


• Three germ layer - Endoderm, Ectoderm, Mesoderm were used as key markers for stemness of iPS cell line.

Significance

• Multiple technologies may converge to allow the generation of therapeutically acceptable iPS cells without viral integrations or lingering reprogramming factors
• Because the four factors are expressed from a defined location, the polycistronic vector system should simplify the study of reprogramming mechanism.