Accessing stem cells via proteomics

Stem cells have two important characteristics that distinguish them from other cells: the ability to self-renew through cell division for a prolonged period, and to differentiate into multiple cells with specialised functions. The power of stem cells for tissue development, regeneration and renewal has been well known to embryologists for many years. The recent concept of adult tissue stem cells as pluripotent progenitors for various tissues has led to the rapid expansion of stem cell research.

The demonstration that both embryonic and adult tissue stem cells have the ability to produce progenitor cells for tissue renewal has opened up vast possibilities for the treatment of congenital deficiencies as well as for regeneration of damaged tissues that cannot be cured today, particularly in organs where the capacity for repair is inherently low, such as the heart, nervous system and pancreas.

Embryonic stem cells which are derived from mammalian pre-implantation embryos have the ability to differentiate into most tissues, however, their clinical application is limited because they are allogeneic to the recipient and as such they have large potential to evoke immune response and rejection. Hence new technologies involving somatic cell nuclear transfer and reprogramming have to be improved to overcome this restriction. Compared to embryonic stem cells, adult stem cells tend to be tissue specific cells which can differentiate into cell types associated with the organ of their origin. Adult stem cells are quite rare but have the advantage that they can be used in autologous transplantation without risk of immune rejection1.

Hematopoietic stem cells are capable of forming different types of blood cells in the body and clinical results achieved by transplantation of hematopoietic precursor cells to treat many haematological malignancies, represent one successful example of cell-based therapy. Additionally, there are expectations that stem cells will be used to treat many other diseases, however, preclinical and clinical use of stem cells is progressing slower than optimistic forecasts supposed. Stem cells directed to differentiate into specific cell types offer the possibility of a renewable source of replacement cells and tissues. One such example is where stem cells differentiated into neural cells could be used to treat spinal cord injury, stroke and neurodegenerative conditions including Parkinson’s and Alzheimer’s diseases. Another example is when specialised cell types derived from stem cells could be used for cell-based therapies for burns, heart diseases, diabetes, osteoarthritis and rheumatoid arthritis. Apart from these clinical applications, stem cells from individuals with various diseases can be utilised for drug screening.

There are however many technical hurdles between the promise of stem cells and the realisation of their applications, which will only be overcome by continued intensive stem cell research. It is essential to realise that each clinical trial to include stem cells, or better, differentiated cells resulting from either embryonic or organ specific stem cells will require a complex approach with the biomedical models to exclude absolutely any possible harm for future patients. Additionally, this effort has to combine standardised stem cell culture conditions, directed and reproducible differentiation into specific cell lineages, and the development of proper implantation protocols to deliver cells into animals or humans including the use of biomaterials such as scaffolds that are used to seed the cells. Rapid progress in basic biomedical research that has generated a huge amount of experimental data was mostly conducted on small laboratory rodents. Before this newly gained knowledge can find its way into designs of new therapies, we need to validate it on animal models closer related to humans. Compared to non-human primates, the primary candidate species for bridging this gap appears to be the pig. Miniature pigs are cheap and easy to maintain in controlled conditions. Their human-like physiology assures a high relevance of the data obtained in this species for human-related therapeutic research. Moreover, the recent advances in methods of transgenic animal production will allow a certain ‘humanisation’ of pigs for use in xenotransplantation, and also the creation of transgenic disease models.

All stem cells could be propagated and differentiated extensively in vitro to generate sufficient quantities of cells for transplantation experiments. The cells could be transplanted safely and effectively to an autologous or allogeneic host and their functionality as well as their long lasting fate after transplantation has to be monitored via their efficient labelling (nano-particles of iron, surface fluorochromes). The functional status of cells after transplantation, especially their ability to cooperate with neighbour cells and to contribute to physiological functions of repaired organs reflects the cellular changes at the molecular level. It means that global characterisation of transcriptomes, epigenomes, proteomes and interaction networks of cellular processes underlying stem cell biology will be essential on the way to improving our approaches for cell-based therapies.

Data gained with the spectrum of today’s proteomic technologies should provide indispensable information. It is evident that Federal and Drug Administration will consider well-characterised proteomes of cells destined for cell therapies as proof of their safe behaviour in the host tissue. Therefore, all stem cell experiments focused on future clinical use may have to include a complex proteomic study in their itinerary. This information will substantially help to optimise differentiation protocols and will provide unequivocal proof about the safety of the application of stem cell therapy.

The deeper understanding of molecular mechanisms underlying ‘stemness’ of stem cells and their differentiation is essential to move towards promising the utilisation of stem cells and certainly the application of proteomics can help to reach this goal. Proteomics is aimed at studying protein properties on a large-scale to obtain global view of cellular processes at the protein level (proteomes). In this sense it is complementing genomic and transcriptomic analyses. Genome, the entire complement of genetic material in a chromosome set, is a static entity. On the other hand, its corresponding transcriptome and proteome are dynamic units, representing a set of cellular mRNAs and proteins respectively at defined time-points. The cell transcriptome can be analysed with the use of microarray technology and global gene expression profiles of different stem cell populations have been generated. To be able to really understand processes of stem cell self-renewal and differentiation, it is essential to analyse proteins which are responsible for ultimate function of expressed genes and to know protein post-translational modifications, interactions and cellular localisations. The two-dimensional gel electrophoresis still remains the core technology for separation and comparison of thousands of proteins in complex mixtures. Typical two-dimensional gel electrophoresis studies require relatively large amounts of protein samples and they suffer from various shortcomings, mainly problems of solubilisation and separation of hydrophobic and membrane proteins and limited accuracy of quantification due to dynamic range of color stains and the possible comigration of proteins. Therefore, many improvements and recent developments in technologies have been remarkable, including separation techniques such as differential in gel electrophoresis (DIGE) or nano-flow high-pressure liquid chromatography (nano-HPLC). The second generation of mass spectrometry techniques allowing gel-independent relative quantification such as isotope coded affinity tag (ICAT), stable isotope labelling by amino acids in cell culture (SILAC), isobaric tag for relative and absolute quantitation (iTRAQ) is a vast improvement in obtaining better data. With relatively low protein loadings direct 2-D liquid chromatography separation of peptides coupled online to tandem mass spectrometer (high throughput) multidimensional protein identification technology (MudPIT) approach represents example of very powerful techniques, which can provide detection of thousands of proteins in one analysis. Due to the large amount of data obtained, the system requires sophisticated software associated with mass spectrometer to analyse mass spectra and to avoid false positive identifications.

The initial approach of proteomic analysis made on stem cell populations has concentrated mostly on profiling, for example global proteome description used to obtain protein fingerprints of a given cell population and to characterise its typical behavior and properties. Such studies focused mainly on murine embryonic stem cells, neural, hematopoietic and mesenchymal stem cells, and stem cell environment, including secretome. Because the secretome strictly depends on the cell type and cellular state, it has the potential to reveal the biomarkers involved in cellular processes, including growth and differentiation. With advances in proteomic techniques, the functional differential studies comparing proteome of undifferentiated stem cells with their differentiated progenies have been initiated, and they currently appear to prevail over simple characterisation of static stem cell populations. The representative example of studies on the basis of maintenance and regulation of differentiation of embryonic stem cells support the idea that epigenetic regulation mediated by chromatin dynamics plays an important role in the regulation of stem cell differentiation and early lineage decisions during embryogenesis2.

The studies on neural stem cell differentiation (Figure 1) identified proteins constitutive for the cellular cytoskeleton and also rearranging cytoskeleton, the proteins involved in transcription and RNA or other nucleic acid metabolism and transport (nuclear proteins), protein synthesis and metabolism, energy, chaperones and stress response proteins and signalling proteins3. Together, these studies of neural stem cells suggest massive rearrangement of stem cell proteome during differentiation to terminally differentiated neuronal and glial cells and indicate potential targets for further characterisation of mechanisms driving differentiation of neural stem cells. Using the antibody microarray analysis revealed several signalling proteins and phosphoproteins such as GRK2 and enhanced phosphorylations of αB-crystallin (S45), PKCγ (T655), PKD (PKCμ;S738+S742), and eIF2α (S51) associated with differentiation of neural stem cells and raised intriguing questions with regards to their potential functionality within stem cells4 (see Figure 2).

Of special interest is the work using stable isotope labelling by amino acids in cell culture coupled to quantitative mass spectrometry to investigate signalling proteins associated with mesenchymal stem cells differentiation into osteoblasts. The induction of 113 phosphoproteins with 1.5-fold or higher change was detected after treatment with growth factors, while five phosphoprotein levels decreased after activation5. Among other proteomic studies focused on stem cell differentiation, attention has been paid to the analysis of hematopoietic stem cells and muscle cell differentiation. Using cytokine protein array, it has been shown that cytokine induction and signal transduction are important for the differentiation of human mesenchymal stem cells6. In the context of rising incidence of obesity, the proteome changes during adipogenesis were studied at the adipose-derived stem cells and their differentiation into adipocytes7. There are also some more clinically oriented studies done in the field of stem cell biology. Of particular interest is differential and quantitative proteomic analysis of chemically induced ischemia on neural differentiated embryonic stem cells or study using proteomics for follow-up of patients after allogenic hematopoietic stem cells transplantation. This phase 1 diagnostic study used capillary electrophoresis and mass spectrometry for analysis of peptides from patient’s urine samples in an attempt to identify early indicators of graft-versus-host disease. This application yielded the identification of 16 polypeptides that enabled clear distinguishing of patients with graft-versus-host disease from patients without such complications with high specificity (82%) and sensitivity of 100%8.

The availability of approaches to investigate at complex level epigenome and proteome including signal transduction pathways (phosphoproteome) has significantly contributed to basic understanding of stem cell biology and produced data characterising proteins involved in mechanisms that regulate stem cell proliferation and differentiation. Numerous proteomic techniques are continuously being improved and developed for protein or peptide fractionation and identification. Additionally, there are other expectations that mass spectrometry-based quantification approaches and their optimisation as well as protein arrays will soon significantly contribute to the proteome stem cell biology. Another problem is the consistency of the results of which it is difficult to draw relevant conclusions from individual studies using different cell types, cell culture protocols and stimuli. Hence, standardisation of methodologies applied in different laboratories would help to establish unique database organising stem cell proteomic data. Importantly, whenever possible, interesting results should be confirmed by functional studies on relevant models. Possible approaches to data verification include western blotting, immunofluorescence studies of expression and cellular localisation of identified proteins, the use of specific inhibitors that interfere with protein functions or siRNA induced protein knockdown. Thus, only after thorough verification can the proteome studies contribute to our knowledge of stem cells and bring closer their therapeutical use.

Several initiatives were set up to bring the research on stem cell biology to a higher level. International Stem Cell Initiative9 aims to characterise numerous human embryonic stem cell lines using standardised assays to allow stringent comparison of experimental data. This initiative has been established by International Stem Cell Forum (http://www.stemcellforum.org), the NIH Stem Cell Unit (http://www.stemcells.nih.gov/research/nihresearch/scunit) and ATCC (http://stemcells.atcc.org).

More recently, “Proteome Biology of Stem Cells”, a joint initiative shared by the International Society for Stem Cell Research and the Human Proteome Organisation was established10. Considering the many challenges in the field of stem cell biology, this initiative intends to be beneficial for many researchers and to increase the value of proteomics in these endeavours.

References

1. A. Atala: Advances in tissue and organ replacement. Curr Stem Cell Res Ther. 3: pp. 21-31, 2008.
2. A. Kurisaki et al: Chromatin-related proteins in pluripotent mouse embryonic stem cells are downregulated after removal of leukemia inhibitory factor. Biochem. Biophys. Res. Commun. 335: pp. 667-675, 2005.
3. H. Skalnikova et al: A proteomic approach to studying the differentiation of neural stem cells. Proteomics. 7: pp. 1825-1838, 2007.
4. H. Skalnikova et al: Protein signalling pathways in differentiation of neural stem cells. Proteomics, currently in press, 2008.
5. I. Kratchmarova et al: Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation. Science, 308: pp. 1472-1477, 2005.
6. Ch. Liu and S.M. Hwang: Cytokine interaction in mesenchymal stem cells from cord blood. Cytokine, 32: pp. 270-279, 2005.
7. J.P. DeLany et al: Proteomic analysis of primary cultures of human adipose-derived stem cells: modulation by Adipogenesis. Mol. Cell. Proteomics, 4: pp. 731-740, 2005.
8. T. Kaiser et al: Proteomics applied to the clinical follow-up of patients after allogeneic hematopoietic stem cell transplantation. Blood, 104: pp. 340-349, 2004.
9. P.W. Andrews et al: Steering Committee of the International Stem Cell Initiative. The International Stem Cell Initiative: toward benchmarks for human embryonic stem cell research. Nat Biotechnol. 23: pp.795-797, 2005.
10. J. Krijgsveld et al: Proteome biology of stem cells: a new joint HUPO and ISSCR initiative.Mol Cell Proteomics. 7: pp. 204-205, 2008.