Novel tools to discern the pluripotency gene network establishment

Not only does the reprogramming of somatic cells to induced
pluripotent stem cells (iPSCs) by exogenous transcription
factors remain highly elusive, but there is also characteristic
discrepancy among the discoveries of different labs.
In order to address this, first we developed a system for a
robust and reproducible systematic mechanistic analysis of
reprogramming. Briefly, we generated a single targeting vector
carrying both the reverse tetracycline-controlled transactivator
(rtTA) and the doxycycline (Dox)-inducible reprogramming
factors (2A-peptide-linked c-Myc-Klf4-Oct4-Sox2 = MKOS)
for a novel genomic locus that assured optimal transgene
expression. One round of targeting with this vector makes any
ES cell (ESC) line a source of Dox-inducible reprogrammable
cells/mouse.
Intriguingly, there is a variety of reprogramming cassettes
used by different labs to perform mechanistic analysis; thus, we
compared the existing cassettes, and to our surprise, they
generated different number of both fully reprogrammed and
partially reprogrammed colonies. Keeping that in mind, we
used our novel reprogramming system to generate Nanog-GFP
ESC lines with the reprogramming factors in a different order
(TNG MKOS/OKMS ESCs). TNG OKMS reprogramming
gave rise to *10 times more colonies upon the addition of
Dox, but with poor activation of the Nanog-GFP reporter,
compared to the TNG MKOS one. CD44 and ICAM1
expression, which we recently utilized to dissect the reprogramming
process, along with E-cadherin staining, enabled us
to identify a clear roadblock in OKMS reprogramming. Finally,
RNA sequencing of these reprogramming intermediate subpopulations
provided us with many candidate genes which
potentially affect the pluripotency acquisition.
The tools described here enable the precise analysis of the
reprogramming intermediates and will make a large contribution
to reveal the molecular mechanisms for the establishment
of the pluripotency gene network and to shed light to the
existing conflict.