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Reprogramming the methods that induce pluripotency

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By Carla Mellough

The clinical applicability of induced pluripotent stem cells (iPSC) has been limited by the inefficiency of reprogramming methods, modification of the genome using most reprogramming protocols, and the lack of failsafe differentiation protocols to then generate functional differentiated phenotypes from iPSCs to provide patient-specific cell types for autologous transplantation. Various non-integrating and thus safer methods of reprogramming have been achieved but these too come with their own limitations. For example, repeat administration of transient vectors demonstrate low reprogramming efficiency1-3, it is challenging to generate and purify the quantities of recombinant proteins required in protein based strategies4 and methods based on DNA transfection come with the risk of genetic recombination or insertional mutagenesis. To this end, a recent paper in Cell Stem Cell5 from the laboratory of Derrick Rossi at Harvard Medical School describes how they achieve reprogramming of multiple human somatic cell types using a simple non-integrating method which achieves highly efficient reprogramming to levels much higher than what has previously been achieved.

The authors synthesised RNA by generating in vitro transcription templates (IVT) for each of the genes of interest. Cellular entry was facilitated by making a complex between the RNA and a cationic vehicle to allow endocytotic uptake but initial experiments utilising synthetic RNA encoding GFP yielded high dose-dependent cytotoxicity in both fibroblasts and keratinocytes. A modest reduction in cytotoxicity was achieved by treating RNAs with a phosphatase capable of removing a fraction of the uncapped IVT products bearing 5’ triphosphates that are known to activate antiviral responses (through interferon and NF-ĸB pathways) and repress protein translation. However cell viability was vastly improved when the authors generated synthesised mRNAs with modified ribonucleoside bases, particularly when two bases were completely substituted at the same time (e.g. 5-methylcytidine replacing cytidine and pseudouridine for uridine). Although a small residual activation of the interferon pathway could still be detected, media supplementation with an interferon inhibitor further increased cell viability following RNA transfection.

Hence ‘modified’ RNAs were produced by treatment of synthetic RNAs with phosphatase alongside complete substitution of specific ribonucleoside bases. When used in media supplemented with an interferon inhibitor Warren et al.5 demonstrated that repeat administration of synthetic modified RNA encoding GFP alone or together with mCherry had highly penetrant expression across 6 human cell lines (50-90% positive cells) and that co-expression of multiple proteins could be achieved. They go on to demonstrate the applicability of modified RNAs to direct cell fate; murine C3H10T1/2 cells transfected with synthesised modified RNA encoding the myogenic transcription factor MYOD yielded multinucleated myotubes that expressed myogenic markers.

The authors then address whether modified RNAs can induce pluripotency. Daily transfection of somatic human cells (fetal fibroblasts, postnatal fibroblasts and adult cystic fibrosis patient-derived fibroblasts from a skin biopsy) with modified RNAs encoding for the four Yamanaka factors KLF4, c-MYC, OCT4 and SOX2 alongside an additional modified RNA encoding for LIN28 (KMOSL) generated RNA-derived iPSCs (RiPSCs) with high conversion efficiency (in the best case 4.4%) in all cell types. Additional experiments showed no enhancement of efficiency using valproic acid, implicated in increased reprogramming efficiency6, however low oxygen (5%) conditions increased efficiency levels by 1.5 fold. Rapid reprogramming kinetics were also observed using modified RNA, with colonies appearing almost twice as fast (present by 17 days) compared to other methods which typically require 4 weeks. More than 10 RiPSC clones were expanded for each somatic line with almost all establishing proliferative cultures. RiPSC lines were demonstrated to express pluripotency markers and transcripts with extensive demethylation at the Oct4 locus and a molecular signature closely mimicking that of human embryonic stem cells (hESCs). Cluster analysis of transcriptional profiles clustered RiPSCs with hESCs more closely than virally-transduced iPSCs. Embryoid body formation then confirmed in vitro differentiation of RiPSCs into cells from all germ layers, as did teratoma formation in vivo. Further, transfection of an RiPSC line with modified RNA encoding for MYOD during differentiation generated enriched cultures of multinucleated myotubes (approximately 35% of the culture using high dosage modified RNA).

In conclusion, synthetic RNA modified to bypass the innate antiviral response can reprogram multiple human somatic cell types to pluripotency with great efficiency and rapid conversion kinetics. This method is transient, comes with no risk of genomic integration or insertional mutagenesis and the resulting RiPSCs are to date the closest equivalent of hESCs. The demonstrated efficient direction of cell fate utilising synthetic modified RNAs provides an efficient means by which to enhance the generation of useful patient-specific cell types without compromising genomic integrity and which offers an additional level of phenotypic orchestration over current differentiation protocols that are largely regulated by extracellular cytokine cocktails. Further, this technology potentially lends itself to multiple other clinical applications, such as the safe and transient expression of antigens for cancer therapy.

References

  1. Jia et al. (2010) A nonviral minicircle vector for deriving human iPS cells. Nat. Methods 7, 197-199.
  2. Kim et al. (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472-476.
  3. Yu et al. (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801.
  4. Zhou et al. (2009) Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381-384.
  5. Warren et al. (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, doi:10.1016/j.stem.2010.08.012
  6. HuangFu et al. (2008) Induction of pluripotent stem cells by defined factors is greatly improved by small molecule compounds. Nat. Biotechnol. 26, 795-797.