Proteomic dataset of Sca-1+ progenitor cells

Embryonic stem cells (ES cells) can differentiate into endothelial cells and smooth muscle cells (SMCs), which participate in vascular angiogenesis. In this study, we differentiated mouse ES cells into Sca-1(+) cells, which have the potential to serve as vascular progenitor cells, and mapped their proteome by 2-DE using a pH 3-10 non-linear gradient and 12% SDS-polyacrylamide gels. A subset of 300 protein spots was analysed and mapped, with 241 protein spots being identified by their PMF using MALDI-TOF MS or by partial amino acid sequencing using MS/MS. Our protein map is the first of Sca-1(+) progenitor cells and will facilitate the identification of proteins differentially expressed during stem cell differentiation. The proteome of adult arterial SMCs is described in an accompanying paper (in this issue, DOI 10.1002/pmic.200402045). All data are made accessible on our website http://www.vascular-proteomics.com.     

Four stage liquid chromatographic selection of methionyl peptides for peptide-centric proteome analysis: the proteome of human multipotent adult progenitor cells

Serial application of strong cation-exchange and diagonal reversed-phase chromatography selecting methionyl peptides by stepwise shifting them from their reduced to their sulfoxide and sulfone forms generates a four-stage fractionation system, allowing high coverage analysis of complex proteome digests by LC-MALDI-MS/MS. Application to the proteome of a human multipotent adult progenitor cell line (MAPC) identified 2151 proteins with high confidence as on average four MS/MS-spectra were linked to each protein. Our dataset contains several novel, potential marker proteins that may be evaluated as affinity-anchors for isolating different adult stem cells in further studies. Furthermore, at least 2 tyrosine kinases that were previously linked to the self-renewal potential of stem cells were identified, validating the stemness of the analyzed cells. We also present data hinting at possible involvement of the ubiquitin/proteasome machinery in steering proliferation and/or differentiation of MAPC. Finally, following comparison of the MAPC proteome with proteomes of four human differentiated cell lines reveals differential usage of chromosomal information: compared to differentiated cells, MAPC do not appear to hold any preference for expressing genes located on specific chromosomes.     

2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM)

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.     

Proteomic profiling of distinct clonal populations of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi‐lineage differentiation. The molecular mechanisms governing MSC self‐renewal and differentiation remain largely unknown. The development of sophisticated techniques, in particular clinical proteomics, has enabled researchers in various fields to identify and characterize cell specific biomarkers for therapeutic purposes. This study seeks to understand the cellular and sub‐cellular processes responsible for the existence of stem cell populations in bone marrow samples by revealing the whole cell proteome of the clonal cultures of bone marrow‐derived MSCs (BMSCs). Protein profiling of the MSC clonal populations was conducted by Two‐Dimensional Liquid Chromatography/Matrix‐Assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry (MS). A total of 83 proteins were identified with high confidence of which 11 showed differential expression between subpopulations, which included cytoskeletal and structural proteins, calcium binding proteins, cytokinetic proteins, and members of the intermediate filament family. This study generated a proteome reference map of BMSCs from the clonal populations, which will be valuable to better understand the underlying mechanism of BMSC self‐renewal and differentiation. J. Cell. Biochem. 106: 776–786, 2009. © 2009 Wiley‐Liss, Inc.     

Integrated analysis of the cerebrospinal fluid peptidome and proteome

Cerebrospinal fluid (CSF) is the only body fluid in direct contact with the brain and thus is a potential source of biomarkers. Furthermore, CSF serves as a medium of endocrine signaling and contains a multitude of regulatory peptides. A combined study of the peptidome and proteome of CSF or any other body fluid has not been reported previously. We report confident identification in CSF of 563 peptide products derived from 91 precursor proteins as well as a high confidence CSF proteome of 798 proteins. For the CSF peptidome, we use high accuracy mass spectrometry (MS) for MS and MS/MS modes, allowing unambiguous identification of neuropeptides. Combination of the peptidome and proteome data suggests that enzymatic processing of membrane proteins causes release of their extracellular parts into CSF. The CSF proteome has only partial overlap with the plasma proteome, thus it is produced locally rather than deriving from plasma. Our work offers insights into CSF composition and origin.     

Comparative proteomic analysis of primary mouse liver c-Kit-(CD45/TER119)- stem/progenitor cells

Liver stem/progenitor cells play a key role in liver development and maybe also in liver cancer development. In our previous study a population of c-Kit-(CD45/TER119)- liver stem/progenitor cells in mouse fetal liver, was successfully sorted with large amount (10(6)-10(7)) by using immuno-magnetic microbeads. In this study, the sorted liver stem/progenitor cells were used for proteomic study. Proteins of the sorted liver stem/progenitor cells and unsorted fetal liver cells were investigated using two-dimensional electrophoresis. A two-dimensional proteome map of liver stem/progenitor cells was obtained for the first time. Proteins that exhibited significantly upregulation in liver stem/progenitor cells were identified by peptide mass fingerprinting and peptide sequencing. Nineteen protein spots corresponding to 12 different proteins were identified as showing significant upregulation in liver stem/progenitor cells and seem to play important roles in such cells in cell metabolism, cell cycle regulation, and stress. An interesting finding is that most of the upregulated proteins were overexpressed in various cancers (11 of 12, including 6 in human hepatocellular carcinoma (HCC)) and involved in cancer development as reported in previous studies. Some of the identified proteins were validated by real-time PCR, Western blotting, and immunostaining. Taken together, the data presented provide a significant new protein-level insight into the biology of liver stem/progenitor cells, a key population of cells that might be also involved in liver cancer development.     

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.     

Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.     

Stem cell-derived neural stem/progenitor cell supporting factor is an autocrine/paracrine survival factor for adult neural stem/progenitor cells

Recent evidence suggests that adult neural stem/progenitor cells (ANSCs) secrete autocrine/paracrine factors and that these intrinsic factors are involved in the maintenance of adult neurogenesis. We identified a novel secretory molecule, stem cell-derived neural stem/progenitor cell supporting factor (SDNSF), from adult hippocampal neural stem/progenitor cells by using the signal sequence trap method. The expression of SDNSF in adult central nervous system was localized to hippocampus including dentate gyrus, where the neurogenesis persists throughout life. In induced neurogenesis status seen in ischemically treated hippocampus, the expression of SDNSF was up-regulated. As functional aspects, SDNSF protein provided a dose-dependent survival effect for ANSC following basic fibroblast growth factor 2 (FGF-2) withdrawal. ANSCs treated by SDNSF also retain self-renewal potential and multipotency in the absence of FGF-2. However, SDNSF did not have mitogenic activity, nor was it a cofactor that promoted the mitogenic effects of FGF-2. These data suggested an important role of SDNSF as an autocrine/paracrine factor in maintaining stem cell potential and lifelong neurogenesis in adult central nervous system.     

Proteomic analysis of neural differentiation of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) can differentiate into different types of cells, and serve as a good model system to study human embryonic stem cells (hESCs). We showed that mESCs differentiated into two types of neurons with different time courses. To determine the global protein expression changes after neural differentiation, we employed a proteomic strategy to analyze the differences between the proteomes of ES cells (E14) and neurons. Using 2-DE plus LC/MS/MS, we have generated proteome reference maps of E14 cells and derived dopaminergic neurons. Around 23 proteins with an increase or decrease in expression or phosphorylation after differentiation have been identified. We confirmed the downregulation of translationally controlled tumor protein (TCTP) and upregulation of alpha-tubulin by Western blotting. We also showed that TCTP was further downregulated in derived motor neurons than in dopaminergic neurons, and its expression level was independent of extracellular Ca(2+) concentration during neural differentiation. Potential roles of TCTP in modulating neural differentiation through binding to Ca(2+), tubulin and Na,K-ATPase, as well as the functional significance of regulation of other proteins such as actin-related protein 3 (Arp3) and Ran GTPase are discussed. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.     

Proteomic analysis of the hydrophobic fraction of mesenchymal stem cells derived from human umbilical cord blood

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering, but their application has been impeded by lack of knowledge of their core biological properties. In order to identify MSC-specific proteins, the hydrophobic protein fraction was individually prepared from two different umbilical cord blood (UCB)-derived MSC populations; these were then subjected to two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). Although the 2D gel patterns differed somewhat between the two samples, computer-assisted image analysis identified shared protein spots. 35 spots were reliably identified corresponding to 32 different proteins, many of which were chaperones. Based on their primary sub-cellular locations the proteins could be grouped into 6 categories: extracellular, cell surface, endoplasmic reticular, mitochondrial, cytoplasmic and cytoskeletal proteins. This map of the water-insoluble proteome may provide valuable insights into the biology of the cell surface and other compartments of human MSCs.     

Preliminary 2-D chromatographic investigation of the human stem cell proteome

Stem cells represent a promising tool for the treatment of various hematopoietic diseases. In order to identify stem cell-specific proteins, the proteome of human stem cells from umbilical cord blood was explored for the first time. For this purpose, the crude lysate of 4 x 10(5) CD34+ cells was subjected to in solution trypsin digestion. The resulting peptides were then separated via cation exchange followed by reversed phase chromatography and analyzed by nanospray MS/MS. Database search revealed a total of 215 proteins which could be reliably identified. To obtain a more complete picture of the human stell cell proteome and to also access low abundant proteins, pooling of more than one CD34+ preparations seems necessary in order to increase the cell number and thus the protein content.     

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.     

Derivation of myoepithelial progenitor cells from bipotent mammary stem/progenitor cells

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Recent molecular profiling has identified six major subtypes of breast cancer: basal-like, ErbB2-overexpressing, normal breast epithelial-like, luminal A and B, and claudin-low subtypes. To help understand the relationship among mammary stem/progenitor cells and breast cancer subtypes, we have recently derived distinct hTERT-immortalized human mammary stem/progenitor cell lines: a K5(+)/K19(-) type, and a K5(+)/K19(+) type. Under specific culture conditions, bipotent K5(+)/K19(-) stem/progenitor cells differentiated into stable clonal populations that were K5(-)/K19(-) and exhibit self-renewal and unipotent myoepithelial differentiation potential in contrast to the parental K5(+)/K19(-) cells which are bipotent. These K5(-)/K19(-) cells function as myoepithelial progenitor cells and constitutively express markers of an epithelial to mesenchymal transition (EMT) and show high invasive and migratory abilities. In addition, these cells express a microarray signature of claudin-low breast cancers. The EMT characteristics of an un-transformed unipotent mammary myoepithelial progenitor cells together with claudin-low signature suggests that the claudin-low breast cancer subtype may arise from myoepithelial lineage committed progenitors. Availability of immortal MPCs should allow a more definitive analysis of their potential to give rise to claudin-low breast cancer subtype and facilitate biological and molecular/biochemical studies of this disease.     

The remyelination Philosopher's Stone: stem and progenitor cell therapies for multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease that leads to oligodendrocyte loss and subsequent demyelination of the adult central nervous system (CNS). The pathology is characterized by transient phases of recovery during which remyelination can occur as a result of resident oligodendroglial precursor and stem/progenitor cell activation. However, myelin repair efficiency remains low urging the development of new therapeutical approaches that promote remyelination activities. Current MS treatments target primarily the immune system in order to reduce the relapse rate and the formation of inflammatory lesions, whereas no therapies exist in order to regenerate damaged myelin sheaths. During the last few years, several transplantation studies have been conducted with adult neural stem/progenitor cells and glial precursor cells to evaluate their potential to generate mature oligodendrocytes that can remyelinate axons. In parallel, modulation of the endogenous progenitor niche by neural and mesenchymal stem cell transplantation with the aim of promoting CNS progenitor differentiation and myelination has been studied. Here, we summarize these findings and discuss the properties and consequences of the various molecular and cell-mediated remyelination approaches. Moreover, we address age-associated intrinsic cellular changes that might influence the regenerative outcome. We also evaluate the extent to which these experimental treatments might increase the regeneration capacity of the demyelinated human CNS and hence be turned into future therapies.     

Enhanced detection of CNS cell secretome in plasma protein-depleted cerebrospinal fluid

Human cerebrospinal fluid (CSF) proteome is actively investigated to identify relevant biomarkers and therapeutic targets for neurological disorders. Approximately 80% of CSF proteome originate from plasma, yielding a high dynamic range in CSF protein concentration and precluding identification of potential biomarkers originating from CNS cells. Here, we have adapted the most complete multiaffinity depletion method available to remove 20 abundant plasma proteins from a CSF pool originating from patients with various cognitive disorders. We identified 622 unique CSF proteins in immunodepleted plus retained fractions versus 299 in native CSF, including 22 proteins hitherto not identified in CSF. Parallel analysis of neuronal secretome identified 34 major proteins secreted by cultured cortical neurons (cell adhesion molecules, proteins involved in neurite outgrowth and axonal guidance, modulators of synaptic transmission, proteases and protease inhibitors) of which 76% were detected with a high confidence in immunodepleted CSF versus 50% in native CSF. Moreover, a majority of proteins previously identified as secretory products of choroid plexus cells or astrocytes were detected in immunodepleted CSF. Hence, removal of 20 major plasma proteins from CSF improves detection of brain cell-derived proteins in CSF and should facilitate identification of relevant biomarkers in CSF proteome profiling analyses.     

The proteome of the human neuroblastoma cell line SH-SY5Y: an enlarged proteome

The human neuroblastoma cell line SH-SY5Y (ATCC: CRL-2266) is widely used as a neural cellular model system. The hitherto existing proteome data (115 proteins) are here extended. A total of 1103 unique proteins of this cell line were identified using 2D-LC combined with MALDI-TOF/TOF-MS, SDS-PAGE with nano-LC-MS/MS, N-terminal COFRADIC analysis with nano-LC-MS/MS and 2D-PAGE with MALDI-TOF/TOF-MS peptide mass fingerprinting. The obtained proteome profile of this cell line is discussed.     

Lessons from the stem cell proteome

The proteome of a cell is a molecular fingerprint directly relating to the gene expression snapshot profile at a certain point of time or developmental stage. Monitoring the expansion and the differentiation state of stem cells by proteomic means seems therefore a very attractive method for diagnostic as well as for therapeutic purposes. We have investigated the protein expression patterns of umbilical cord blood-derived CD34+/AC133+ cells in order to obtain a most comprehensive view of the stem cell proteome. For this purpose, we have applied 2-D gel electrophoresis and 2-D chromatography for most efficient protein/peptide separation and characterisation. The proteins were identified after tryptic digestion by nano-HPLC coupled directly to an ion trap mass spectrometer. An extensive bioinformatic analysis of the protein obtained revealed a dynamic stem cell proteome. This means that the heterogeneity of protein expression patterns obtained from different stem cell preparations refers to a limited set of stem cell-specific house keeping proteins as well as to a large number of proteins which depend on (marginal) stimuli from the environment. Since those are difficult to standardise, snapshot views of the stem cell proteome reflect not only stem cell-intrinsic metabolism but also the strong influence of the sample history on protein expression patterns.     

The dynamics of murine mammary stem/progenitor cells

The stem/progenitor cells in the murine mammary gland are a highly dynamic population of cells that are responsible for ductal elongation in puberty, homeostasis maintenance in adult, and lobulo-alveolar genesis during pregnancy. In recent years understanding the epithelial cell hierarchy within the mammary gland is becoming particularly important as these different stem/progenitor cells were perceived to be the cells of origin for various subtypes of breast cancer. Although significant advances have been made in enrichment and isolation of stem/progenitor cells by combinations of antibodies against cell surface proteins together with flow cytometry, and in identification of stem/progenitor cells with multi-lineage differentiation and self-renewal using mammary fat pad reconstitution assay and in vivo genetic labeling technique, a clear understanding of how these different stem/progenitors are orchestrated in the mammary gland is still lacking. Here we discuss the different in vivo and in vitro methods currently available for stem/progenitor identification, their associated caveats, and a possible new hierarchy model to reconcile various putative stem/progenitor cell populations identified by different research groups.