Pluripotent cells are stem cells which can give rise to all cell types in the body. Pluripotent stem cells have been isolated from a variety of human sources as models for studying early human development as well as for in vitro
differentiation into cardiocytes, motor neurons, hematopoetic cells and others for the purpose of transplantation therapy [1
]. Two of the most well-studied cell types include embryonic stem cells (ESCs) derived from the inner cell mass of blastocyst-staged embryos and embryonal carcinoma cells (ECCs), the stem cells of teratocarcinomas (mixed germ cell tumors) derived from progenitors of the germline [3
]. Both of these cell types share the general properties of pluripotent stem cells in that they exhibit unlimited self-renewal and can give rise to derivatives of all three embryonic germ layers as demonstrated by embryoid bodies in cell culture and in the development of tumors after injection into adult mice. Thus, given these attributes, pluripotent stem cells can potentially provide sufficient numbers of differentiated cells to treat a wide variety of human conditions, including heart disease, diabetes, and many neurological disorders.
However, several major hurdles remain to be overcome if such cells are to be used clinically. Most importantly, these cells must be easily and reproducibly cultured and manipulated so that they possess the necessary characteristics for successful differentiation, transplantation and engraftment. For this purpose, identifying the factors involved in stem cell survival, proliferation and pluripotency is critical. Another critical factor lies with their chromosomal stability. For instance, most ECC lines are heteroploid, and those that are diploid exhibit alterations in their genomes as revealed through comparative genomic hybridization [4
]. Nonetheless, to date the only clinical trial reported on the use of a pluripotent stem cell-derived source in humans are human ECC-derived postmitotic neurons implanted in regions of the brain damaged by stroke [5
]. Although the outcome has been promising, the safety concerns regarding the use of a karyotypically unstable cell line will require further monitoring. In contrast, ESC lines are routinely maintained as normal diploids, except over long extended cultures which some lines have shown chromosomal abnormalities similar to those seen in ECCs [8
]. Like karyotypic instability, the expression of factors associated with oncogenesis inherent in embryonic stem cells also raises concerns for their use in transplantation. Altered expression of many factors has now been associated with some cancers even though their role is unknown or is a secondary effect downstream of the cause of the tumorigenicity. Therefore, it is essential to determine those factors which turn on the oncogenic state versus those that enhance proliferation and self-renewal without inferring aberrant cell cycles and genomic instability. These factors can then be controlled and screened in cells before transplantation to minimize the risk of potential carcinogenic outcomes. The need for this information is highlighted by the recent approval by the FDA for the first human clinical trial utilizing human embryonic stem cells. This trial involves treating patients with spinal cord injury with hESC-derived oligodendrocyte neural progenitors [9
]. The significance of identifying factors associated with pluripotency while avoiding those associated with tumorigenesis has also been highlighted by a series of studies that have shown the conversion of adult fibroblast cells into pluripotent-like stem cells by inserting four genes [10
]. The resulting cells designated as induced pluripotent stem (iPS) cells express two pluripotent genes, OCT4
, and two genes c-Myc and KLF4
which are frequently upregulated in tumors. Although this combination of genes successfully produced ES-like colonies that could generate chimeric animals including germline transmission, nearly 20% of the iPS-derived chimeric offspring developed tumors [2
]. In addition, Maherali et al. [10
] demonstrated that the expression of OCT4
was no longer required for iPS cell survival. Thus, while these types of studies provide hope for reprogramming adult cells for therapeutic uses, it further reiterates the necessity of finding genes associated with pluripotency while avoiding those associated with oncogenesis.
To define such genes, many attempts have been made to study the global stem cell genome as well as its chromatin state[14
]. While these studies provide critical information for finding the factors associated with pluripotency, investigation into the protein levels in these cells are also required as levels of protein expression do not always directly correlate to transcriptomic changes. Indeed, with current developments in proteome-wide approaches, the characterization of the proteome of these cells has just begun. Some of the these proteomic studies which include analysis of mouse ESCs [15
], human ESCs [16
] and human embryonal carcinoma cells (ECCs) [17
] involve non-quantitative analyses, which while useful, do not allow for differential analyses among these populations.
To date, a few proteomics studies and several transcriptomics studies have been reported comparing ESCs and ECCs [18
]. Membrane proteomic approaches have also been reported recently using a label-free method of quantitation after extensive membrane fractionation [17
]. More recent developments in quantitative methods to study proteomics have been employed to study ESCs in mice [21
] but have not yet been applied to study human pluripotent stem cells. For this purpose, isobaric tagged for relative and absolute quantitation labeling (iTRAQ) is an effective method for comparing the expression level of even low abundance proteins. Alternatively, stable isotope labeling with amino acids in cell culture (SILAC) is another straightforward and simple approach for labeling proteins for mass spectrometry based analysis. This approach has been recently used for quantitative comparison of the membrane proteomes in human embryonic stem cells and their differentiation after their adaptation to SILAC media [22
Here, we report the use of an iTRAQ coupled to two-dimensional liquid chromatography and tandem mass spectrometry to compare the protein expression between two distinct, but phenotypically related, pluripotent populations - human ESCs and human ECCs. Our goal was to study the proteomic differences between ESCs and ECCs to identify potential candidates that might explain regulation of pluripotency and malignancy. This approach generated an initial high quality reference proteins of ~1,800 proteins, which include low abundance protein classes such as transcription factors and kinases that were not previously described in stem cells as well as previously documented stem cell markers. We also examined compartmental distribution of nuclear, cytoplasmic, and membrane proteins. Bioinformatics analysis of ESCs and ECCs revealed shared features of their pluripotent nature as well as distinguish the expression of key factors which may be related to the oncogenetic nature of ECCs.