Comparative proteomics of human embryonic stem cells and embryonal carcinoma cells

Abstract

Pluripotent human embryonic stem cells (ESCs) can be differentiated in vitro into a variety of cells which hold promise for transplantation therapy. Human embryonal carcinoma cells (ECCs), stem cells of human teratocarcinomas, are considered a close but malignant counterpart to human ESCs. In this study, a comprehensive quantitative proteomic analysis of ESCs and ECCs was carried out using the iTRAQ method. Using two-dimensional LC and MS/MS analyses, we identified and quantitated approximately 1800 proteins. Among these are proteins associated with pluripotency and development as well as tight junction signaling and TGFbeta receptor pathway. Nearly approximately 200 proteins exhibit more than twofold difference in abundance between ESCs and ECCs. Examples of early developmental markers high in ESCs include beta-galactoside-binding lectin, undifferentiated embryonic cell transcription factor-1, DNA cytosine methyltransferase 3beta isoform-B, melanoma antigen family-A4, and interferon-induced transmembrane protein-1. In contrast, CD99-antigen (CD99), growth differentiation factor-3, cellular retinoic acid binding protein-2, and developmental pluripotency associated-4 were among the highly expressed proteins in ECCs. Several proteins that were highly expressed in ECCs such as heat shock 27 kDa protein-1, mitogen-activated protein kinase kinase-1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor like-2, and S100 calcium-binding protein-A4 have also been attributed to malignancy in other systems. Importantly, immunocytochemistry was used to validate the proteomic analyses for a subset of the proteins. In summary, this is the first large-scale quantitative proteomic study of human ESCs and ECCs, which provides critical information about the regulators of these two closely related, but developmentally distinct, stem cells.

Fig. 1. Outline of the quantitative proteomic strategy using 4-plex iTRAQ reagents

iTRAQ labeling was carried out separately using whole cell lysate, cytosolic or non-cytosolic fractions. Samples were digested using trypsin in duplicate and labeled using iTRAQ reagents. Peptides from ECCs were labeled with iTRAQ reagent having 114 and 115 reporters and peptides from ESCs were labeled with iTRAQ reagent having 116 and 117 reporters. After labeling, peptides from all four samples were combined and fractionated by strong cation exchange (SCX) chromatography. Each fraction was then analyzed by LC-MS/MS on a quadrupole time of flight mass spectrometer.

Fig. 2. MS/MS spectra of iTRAQ labeled peptides from selected proteins

Panels A to F show the MS/MS spectra of peptides from undifferentiated embryonic cell transcription factor 1 (UTF1) and DNA cytosine-5 methyltransferase 3 beta isoform 1 (DNMT3B), heat shock 27kDa protein 1 (HSPB1), CD99 antigen (CD99), podocalyxin-like isoform 1 (PODXL) and LIN28 homolog (LIN28), respectively. The reporter ions in the inset show the examples of high, low and equal expression of proteins in ESCs and ECCs.

Fig. 3. Localization and functional annotation of proteins identified from ESCs and ECCs

Panel A shows the distribution of iTRAQ fold changes (proteins expression levels) observed between ESCs and ECCs. Panel B shows the gene ontology analysis for cellular localization of all the proteins identified. Primary and alternate localization data was downloaded from human protein reference database (www.hprd.org) [25] and Panel C shows functional classification of all the proteins quantitated in this study. Using Ingenuity pathway analysis tool, proteins justifying specific biological function significantly (p <0.05) are listed.

Fig. 4. Immunocytochemical analysis of proteins expressed at high levels in ESCs

Indirect immunofluorescence labeling of different cell types was carried out using Alexa Fluor 594 or Alexa Fluor 488 conjugated secondary antibodies. DAPI (blue) was used to stain nuclei. Panels A to H show proteins found to be expressed at higher levels in ESCs. Panel A to G includes immunocytochemical staining for proteins encoded by DNMT3B (blue-green nuclei), DNMT3A (blue-green nuclei), GSN (red cytoplasm), UTF1 (blue-green nuclei), BGN (red secretory), LGALS1 (green cell surface), CTNNB1 (green cell surface) and HELLS (blue-green nuclei), respectively.

Fig. 4. Immunocytochemical analysis of proteins expressed at high levels in ESCs

Indirect immunofluorescence labeling of different cell types was carried out using Alexa Fluor 594 or Alexa Fluor 488 conjugated secondary antibodies. DAPI (blue) was used to stain nuclei. Panels A to H show proteins found to be expressed at higher levels in ESCs. Panel A to G includes immunocytochemical staining for proteins encoded by DNMT3B (blue-green nuclei), DNMT3A (blue-green nuclei), GSN (red cytoplasm), UTF1 (blue-green nuclei), BGN (red secretory), LGALS1 (green cell surface), CTNNB1 (green cell surface) and HELLS (blue-green nuclei), respectively.

Fig. 5. Immunocytochemical analysis of proteins expressed at high levels in ECCs

Indirect immunofluorescence labeling of different cell types was carried out using Alexa Fluor 594 or Alexa Fluor 488 conjugated secondary antibodies. DAPI (blue) was used to stain nuclei. Panels A to D show proteins found to be expressed at high levels in ECCs. Panel includes immunocytochemical staining for proteins encoded by DPPA4 (blue-green nuclei), MFGE8 (red cytoplasm), GDF3 (blue-green nuclei) and HSPB1 (also known as HSP27, green cytoplasm), respectively. Panel E shows similar expression level of TLN1 (red cytoplasm) in ESCs and ECCs.

Fig. 5. Immunocytochemical analysis of proteins expressed at high levels in ECCs

Indirect immunofluorescence labeling of different cell types was carried out using Alexa Fluor 594 or Alexa Fluor 488 conjugated secondary antibodies. DAPI (blue) was used to stain nuclei. Panels A to D show proteins found to be expressed at high levels in ECCs. Panel includes immunocytochemical staining for proteins encoded by DPPA4 (blue-green nuclei), MFGE8 (red cytoplasm), GDF3 (blue-green nuclei) and HSPB1 (also known as HSP27, green cytoplasm), respectively. Panel E shows similar expression level of TLN1 (red cytoplasm) in ESCs and ECCs.