Profiling Study Methods

Gene Expression Analysis of Stem Cells

The establishment of the gene expression profile of stem cells has facilitated defining specific signaling pathways as well as molecular machineries that are associated with the control of pluripotency of stem cells. Gene expression profiling involves the analysis of the proteome of stem cells, including the determination of the amount of each protein secreted, the stability of these products, the localization of each protein within the cell, as well as modifications that occur at the post-translational level. The most commonly used approach for proteome analysis includes two-dimensional gel electrophoresis in relation to the stages of differentiation and pluripotency.

One advantage of using two-dimensional gel electrophoresis of proteins is that thousands of macromolecules could be analyzed at one time. This technique has been utilized since the 1970s, with further improvements integrated in the 1980s. The addition of immobilized pH gradients has significantly enhanced the capacity in separating proteins in a gel, resulting in electropherograms with higher resolution. Furthermore, the gels showed higher reproducibility, as well as enhanced loading capacity. Other essential improvements in two-dimensional gel electrophoresis include the design of highly sensitive protein stains.

A significant innovation in proteomics is the integration of mass spectrometry in two-dimensional gel electrophoresis. Mass spectrometry facilitates the identification of proteins even at the femtomolar levels, thus surpassing the classic protein sequencing methods that are generally manual and of lower sensitivity. Identification [Page 962]of proteins that have been separated using two-dimensional gel electrophoresis by using mass spectrometry includes peptide mapping. In this technique, the proteins are initially digested using trypsin or any other dissociative enzyme, followed by the measurement of proteolytic peptides via mass spectrometry. The instrumentation employed in mass spectrometry allows the determination of each protein’s mass spectra, which are then compared to reference proteolytic peptides of known mass spectral information. This step in the proteomic analysis of stem cells can be automated, although it initially requires the full protein sequence or, alternatively, the coding region of the corresponding gene.

Screening of Epigenetic Modifications in Embryonic Stem Cells and Induced Pluripotent Stem Cells

Another method of studying stem cells is through the investigation of epigenetic factors that are strongly associated with the network of transcription factors influencing pluripotency. By resetting the status of epigenetic markings of somatic cells to those of embryonic stem cells, information on reprogramming factors that play essential roles in the production of induced pluripotent stem cells could be identified. The epigenetic modifications that are required in retaining the pluripotency state of embryonic stem cells must be induced via nuclear reprogramming. Moreover, schemes for controlling the establishment of epigenetic markers should be stronger than those of the natural epigenetic state of somatic cells. Thus, certain epigenetic events might have functioned specifically to delete the epigenetic modifications in somatic cells.

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