Induced pluripotent stem cell (iPSCs) research burst on to the scene in 2006 with a paper which has since spawned thousands of studies and has proved to be a quantum leap for regenerative medicine. This reached a crescendo this year when a Japanese patient became the first recipient of retinal cells generated from iPSCs made from her very own cells.
One problem does however hover over the field; no one actually fully understands the processes that occur during the reprogramming of a somatic cell to a pluripotent stem cell-like state!
A Grand Project to Understand Cellular Reprogramming
This prompted stem cell biologist Andras Nagy to embark on massive multivariate analysis on how cellular reprogramming proceeds – a project he courageously and unapologetically calls “Project Grandiose”.
The publication of 5 papers marks the culmination of this epic mission [1-5], and came with an unexpected, but not unwelcomed finding – the description of a new pluripotent stem cell type. The newly describe F-class cell may represent a rich new vein of stem cell research, and brings with it the exciting prospect of even more distinct and stable pluripotent states.
Tonge et al,  first set out to analyze iPSC production from a totally unprejudiced perspective, so to fully appreciate any and all findings. This was established through the forced expression of high levels of the OSKM (Oct4, Sox2, Klf4, c-Myc) transcription factors in mouse embryonic fibroblasts (MEFs) using an inducible PiggyBac transposon system, coupled with an unbiased colony selection strategy.
‘Fuzzy’ New Class of Induced Pluripotent Stem Cells
Interestingly, this strategy led to the emergence of traditional “C-class” pluripotent cells (Compact) which resemble mESCs, and a brand new class of pluripotent cells, the so called “F-class” cells (Fuzzy), which do not resemble traditional mESC colonies and display a fuzzy appearance because of the low adherence of cells.
Thorough analysis found that F-class cells:
- Displayed low adhesion and high proliferation.
- Required the forced expression of the reprogramming factors to maintain the F-class state, as loss of transgene expression led to rapid differentiation.
- Expressed a distinct gene expression pattern that was stable over time and similar between clones, suggesting a bone fide new pluripotent state.
- Underwent specific epigenetic changes different from both fibroblasts and mESCs, further promoting the existence of a new pluripotent state.
- Formed teratomas and displayed ectodermal, mesodermal and endodermal differentiation capacity similar to mESCs, but did not contribute to chimera formation.
- Converted to an ESC-like state after treatment with histone deacetylase inhibitors, while iPSCs converted to the F-class state following forced expression of reprogramming factors.
Clinical Implications of Alternative Stem Cells States
This new class of pluripotent cells may be suited for large scale production due to the low adhesiveness and fast proliferation, making them also desirable for cell-based therapies which demand large quantities of specific differentiated cell types. However, the requirement of transgenes, due to insertional mutagenesis or incomplete reactivation following differentiation, may make these cells unsafe for clinical applications.
The research does however clear the way for a new understanding of the pluripotent state; multiple interconvertible acquired states may arise in vitro and in vivo, and the discovery and stabilization of said states may prove highly utile to the future of regenerative medicine.
Reprogramming Roadmap Reveals Unique Epigenetics of ‘Fuzzy’ Stem Cells
In the second study Hussein et al,  began with the aim of elaborating the first thorough roadmap of iPSC reprogramming, and whilst doing so, also provided an explanation for the emergence of the new F-class pluripotent state. Using the reprogramming system from Tonge et al , the group studied the emergence of C- and F-class cells with regards to:
- Transcriptomic analysis – Long (mRNA, lncRNA) and short (miRNA, snoRNA, piRNA, etc.) transcripts.
- Proteomic analysis – Global and cell surface proteins.
- Epigenomic analysis – H3K4me3, H3K36me3 and H3K27me3, and DNA methylation.
Transcriptomics proved that F-class cells were distinct from other pluripotent cell types at every point during their reprogramming, and this extensive coverage of the entire reprogramming time period uncovered key determinants surrounding the generation of F-class cells.
- During the initial “deterministic” phase of reprogramming, H3K4me3 and H3K27me3 dictate transcriptomic and cell fate changes:
- Loss of H3K27me3 leads to massive chromatin structure opening in both C-class and F-class destined cells.
- High transgene expression during the following “stochastic” period leads to the acquisition of specific H3K27me3 and DNA methylation patterns which lock in the F-class state:
- This involves the repression of traditional ESC-associated genes, and the retention of some somatic epigenetic patterns.
- This may allow for the production of large amounts of fast cycling cells with a predestined, and understood differentiation bias.
- Lower transgene expression leads to the acquisition of H3K4me3 and somatic-origin DNA demethylation and reprogramming to an ESC-like state in both epigenetic and transcriptomic terms.
These alterations are discussed in more detail in three Nature Communications studies [3-5] that make up the remainder of the Project Grandiose findings. These studies concentrate on the in-depth analyses of epigenetic changes (Lee et al.), small RNA changes (Clancy et al.), and protein expression changes (Benevento et al.). Encouragingly for everyone in the field, the large datasets generated during these studies are conveniently and freely available at www.stemformatics.org.
It’s certain that these studies will open the flood doors, and the deluge of papers to come will hopefully provide even more insight and lead to another quantum leap forward for stem cell research and regenerative medicine.
- Tonge PD, Corso AJ, Monetti C, et al. Divergent reprogramming routes lead to alternative stem-cell states. Nature 2014;516:192-197.
- Hussein SM, Puri MC, Tonge PD, et al. Genome-wide characterization of the routes to pluripotency. Nature 2014;516:198-206.
- Lee DS, Shin JY, Tonge PD, et al. An epigenomic roadmap to induced pluripotency reveals DNA methylation as a reprogramming modulator. Nat Commun 2014;5:5619.
- Clancy JL, Patel HR, Hussein SM, et al. Small RNA changes en route to distinct cellular states of induced pluripotency. Nat Commun 2014;5:5522.
- Benevento M, Tonge PD, Puri MC, et al. Proteome adaptation in cell reprogramming proceeds via distinct transcriptional networks. Nat Commun 2014;5:5613.