The generation of induced pluripotent stem cells (iPSCs) represents one of the most important scientific breakthroughs of our times, bringing us to the brink of patient-specific stem cell-based therapies. The “simple” generation process involves the forced expression of transcription factor genes in differentiated cells taken from the patient (normally skin cells) to “kick-start” the pluripotency-associated gene expression network. Once this network begins to rev-up, the differentiated cells slowly convert into cells that resemble embryonic stem cells (ESCs). From here, researchers can differentiate iPSCs into most cell types in the body and this, coupled with the foreseen lack of immune rejection due to their patient-specific nature, makes them an important therapeutic option.
But here is the question – do we need iPSCs in the first place? Their production is costly and time-consuming, their epigenetic memory of their original cell origin can mess up differentiation towards the cell that you may need, and the efficiency is generally low (but improving). Combine this with the worry over producing a final cell product containing potentially tumor-forming remainder pluripotent cells and we may be right to ask ourselves – Is there another option?
Enhancing Direct Reprogramming to Avoid iPSCs
One other viable option is a process known as Direct Reprogramming or transdifferentiation. This entails the reprogramming of one differentiated cell directly into another differentiated cell via the forced expression of cell-specific transcription factors. This forgoes the iPSC stage but still suffers from similar problems of low efficiency.
But now, a new study  published in Nature Communications from the laboratory of Jian Feng has described a new strategy to aid direct reprogramming efforts. Their attempts to convert human fibroblasts into dopaminergic neurons via the expression of Ascl1, Nurr1, Lmx1a, and miR124 were beset by low-efficiency. However, they discovered that reducing levels of the p53 tumor suppressor gene and arresting the cells in the G1 phase of the cell cycle significantly boosted the production of functional dopaminergic neurons. Interestingly, they found that this increase was dependent on the expression of the TET1 DNA demethylase enzyme, suggesting that chromatin modulation was a key factor.
Chromatin features again in another related study which sought to increase the efficiency of both iPSC production of direct reprogramming . Using RNAi screens to look for inhibitors of the reprogramming process, researchers from the laboratories of Johannes Zuber and Konrad Hochedlinger found several epigenetic factors whose expression blocked iPSC production. The two most promising hits (Chaf1a and Chaf1b) are the two subunits of the chromatin assembly factor-1 (CAF-1) complex, and the new study found that inhibition of these two factors increased iPSC production efficiency. Likewise, suppression of CAF-1 also increased the efficiency of B cell conversion into macrophages and fibroblast conversion into neurons, suggesting that CAF-1 knockdown could help in other direct reprogramming strategies.
Look Mum! – No viruses or Tumors
But even with direct reprogramming, the use of integrating viruses may promote a tumorigenic phenotype and/or introduce unwanted mutations. So we got rid of the need for iPSCs, can we get rid of the viruses now?!
Xi-Yong Yu and Jianjun Wang think so. Their new study, published in Stem Cell Translational Medicine , has outlined an improved strategy for the production of cardiac progenitor cells (CPCs) from fibroblasts using direct protein transduction. So no viruses, no RNA, no DNA, and just 4 proteins (Gata4, Hand2, Mef2c, and Tbx5) modified using their recently developed QQ transduction reagent (Polyethylenimine 2000, DOTAP, and DOPE) grown in the presence of an optimized combination of cytokines. This very simple process mediated high protein transduction and high cell conversion to CPCs which had the ability to improve heart function following an induced heart attack in a rat model.
Small Molecules – Big Potential?
Another option is to transdifferentiate cells using small molecule drugs, which may represent an easier and cheaper method. A recent study in Cell brought us up to date with the enhanced conversion of somatic cells into iPSCs (up to 1,000-fold greater than previously reported protocols!) using small molecule drugs only . Some studies have begun to investigate chemically-induced direct reprogramming of human skin fibroblasts into neuronal cells  and this promises to be an exciting area of intense study in the coming years.
Where do Embryonic Stem Cells Fit?
While ESCs and iPSCs are now seen as being functionally equivalent, the main obstacle to their widespread use in therapies is the potential problem of immune-mismatch. As ESCs are not patient-specific, they may be rejected after transplantation, negating their usefulness. While creating individual ESC lines via human somatic cell nuclear transfer (SCNT)  is possible, the moral and cost implications are almost insurmountable. However, one recent study has reduced the immunogenicity of ESCs by genetically interfering with the primary mediators of immune rejection in humans – leukocyte antigen class I (HLA-I) molecules . This is a small first step towards the production pf a single non-immunogenic parental ESC line for the generation of therapeutics relevant to most, if not all, patients.
Final Thoughts on Direct Reprogramming
In conclusion, we have some fantastic strategies to produce the endless numbers of cells required for therapies, drug screens, and disease modeling. However, while direct reprogramming has many advantages, there is at least one potentially troubling complication. One study has found that while iPSC-derived cells resemble (at the molecular level) young and healthy cells independent of the age of the donor cells, any cells derived from direct reprogramming retain the signs of normal aging which may be present. As a consequence, the functionality of these cells may suffer and this may affect those more likely to need cell-based interventions: the elderly. While this has yet to be definitely proved, it could represent an important barrier to the widespread application of direct reprogramming.
- Jiang H, Xu Z, Zhong P, et al. Cell cycle and p53 gate the direct conversion of human fibroblasts to dopaminergic neurons. Nat Commun 2015;6:10100.
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