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Clinical implications of induced pluripotent stem (iPS) cell technology are enormous for personalized medicine. However, extensive use of viral approach for ectopic expression of reprogramming factors is a major hurdle in realization of its true potential. Non-viral methods for making iPS cells, although plausible, are impractical because of high cost. MicroRNAs are important cellular modulators that have been shown to rival transcription factors and are important players in embryonic development. We have generated distinct ‘microRNA-omes’ signature of iPS cells that remain in a near embryonic stem (ES) cell state and distinct from differentiated cells. Recent advances in the microRNA field and experimentally validated microRNAs warrant a review in experimental protocols for microRNA expression profile.
One of the most clinically relevant finding of the past decade is the ability to reprogram a terminally differentiated cell to an ES cell-like state. Yamanaka et al utilized ectopic expression of transcription factors Oct4, Sox2, Klf4 and cMyc in terminally differentiated somatic cells to achieve pluripotency . Later, Thompson and colleagues published NANOG and LIN28 along with OCT4 and SOX2 to make iPS cell lines . Despite rapid advancements in the field, two major hurdles in clinical application of iPS cells remaining are the inability to avoid stable genomic integration of viral vectors used to overexpress ‘pluripotency factors’ and the residual epigenetic signature from the cells used to derive iPS cells . Efforts have been made to address these issues with limited success, and all of these approaches have their shortcomings. For instance, one promising approach is to use full-length mRNAs for ‘pluripotency factors’ . However, this approach is still too expensive at present. Another approach is to use piggyback vectors in reprograming, but this also has its limitations in being cumbersome . Efforts are therefore underway to device a new techniques to generate iPS cells without confounding factors to realize their true clinical potential. However, more effective strategies of cellular reprograming will require a better understanding of underlying changes that take place during the process of reprograming .
Along with transcription factors, microRNAs have emerged as a novel class of regulators of gene expression . These small RNAs are increasingly being recognized as important regulators of various biological pathways that control gene expression by either mediating target mRNA degradation or stalling protein synthesis [8, 9]. Interestingly, the involvement of various microRNA families have long been known in various developmental processes . Therefore it is not surprising that microRNAs play a key role in the generation and maintenance of iPS cell-like state. We have previously shown a distinct microRNA expression profile in ES cells compared to terminally differentiated somatic cells and the similarity of ‘microRNA-omes’ of iPS cells to ES cells . We have also assessed dynamic microRNA expression programs during cardiac differentiation of human ES cells . Others have analyzed common changes in the expression of microRNAs (miRNAs) and mRNAs in different human ES cell lines during early commitment and examined the expression of key ES cell-enriched miRNAs in earlier developmental states . Several microRNAs clusters such as miR-17-92, miR-106a-363, miR-302-367, and miR-200 showed a similar expression signature in ES and iPS cell lines. Recent advances in techniques for microRNA expression profiling and the ever growing list of validated microRNAs make it would be worthwhile to establish the protocols for genome-wide ‘microRNA-omes’ expression profiling, which will help us understand molecular events that culminate in cellular dedifferentiation.
DMEM containing 10% FBS, and 50 units and 50 mg/ml penicillin and streptomycin. To prepare 450 ml of DMEM medium, mix 50 ml of FBS and 2.5 ml of penicillin/streptomycin, and then fill up to 500 ml with DMEM. Store at 4°C up to a week.
To prepare thaw 5x supplement at 4°C overnight and mix with MteSR1 media. Store at 4°C.
The miRNeasy Mini Kit combines phenol/guanidine-based lysis of samples and silica membrane–based purification of total RNA. The upper, aqueous phase is extracted, and ethanol is added to provide appropriate binding conditions for all RNA molecules from 18 nucleotides (nt) upwards. A specialized protocol is provided for enrichment of miRNAs and other small RNAs (less than ~200 nt) in a separate fraction. The manufacturer’s instructions should be followed to efficiently isolate the small RNA from fibroblasts, ES cells, and iPS cells.
Microarray assay can be performed by outsourcing to service provider, or by using following protocol.
In order to determine functionally relevant differences in microRNA expression, normalization needs to be performed to reduce system-related variations, including variations such as difference in sample amount, efficiency of labeling dyes, and signal gain that is subject to the scanner used to acquire the image.
We recommend these or any other commercial kits for the extraction of the total RNA including small RNA fraction. Do not use the size fractionation or small RNA enrichment protocol. You must use the same total RNA extraction methods to obtain consistent results for comparative experiments. Different total RNA extraction methods may result in slightly different miRNA profiles. Extraction methods that use organic solvents such as TRIZOL may result in inaccurate quantification because organic solvent contamination from carry-over during the RNA extraction may compress the 260/230 ratio. The affected 260 measurements may result in inaccurate quantification of the total RNA.
We thank funding support from Burroughs Welcome Foundation, NIH DP2OD004437, and NIH RC1G036142 (JCW).