While many across the universe have been pondering over the decision between the light and the dark side of the Force, a more pressing decision confronts CRISPR-based gene editing in stem cells: the primed or the naïve side of pluripotency!?
Pluripotent Stem Cells (PSCs), such as human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs) and mouse epiblast stem cells (mEpiSCs), exist in a “primed” pluripotent state. This state actually confers a lower self-renewal ability and multi-differentiation capacity than the “naïve” state of pluripotency (mouse ESCs and iPSCs) (Click here for some extra background from EpiGenie).
In a recent study from researchers from Tongji University, Shanghai, People’s Republic of China directly produced naïve hiPSCs from β-thalassemia patient-derived fibroblasts and then compared the efficiency of CRISPR-based gene editing between their new naïve hiPSCs and primed hiPSCs.
Their new strategy involved:
- Reprogramming of β-thalassemia patient fibroblasts with non-integrating episomal vectors expressing the mother lode of reprogramming factors
- OCT4, sh-p53, SOX2, KLF4, MYC, LIN28, and NANOG
- Culture of emerging colonies in a combination of inhibitors and growth factors (5i/L/FA medium) which can convert primed human PSCs into naïve PSCs in culture.
- Medium includes inhibitors of MEK, GSK3, BRAF, ROCK, and SRC, human leukemia inhibitory factor (LIF), and FGF and Activin A
The resultant dome shaped hiPSC colonies exhibited the morphological, transcriptional, and epigenetic traits of the naïve pluripotent state and, importantly, these naïve iPSCs were able to form interspecies chimeras with widespread integration, an ability that primed human PSCs do not have.
The next stage was to assess β-thalassemia mutation correction in primed vs naïve iPSCs using CRISPR/Cas9 and a specific single-guide RNA.
- At the single allele level, the correction efficiency in naïve hiPSCs (57%) was almost double that of primed hiPSCs (23%), while one naïve hiPSC colony contained cells with both alleles corrected.
- Naïve hiPSCs contained no off-target events while primed hiPSCs did display some unwanted off-target events.
- Importantly, corrected naïve hiPSCs maintained their hematopoietic differentiation potential.
So the way forward for all you gene-editing Jedi’s seems to be the naïve side of pluripotency! May the reprogramming and gene editing force be with you!
Believe the hype; see all the details at Stem Cells Translational Medicine, Dec 2015.