Undifferentiated human ES cells were cultured according to the protocol provided by the University of California, San Francisco in human ES culture medium [DMEM supplemented with 20% KnockOut Serum Replacement (Invitrogen) and 5 ng/mL of bFGF (R&D Systems)]. commit to further maturation along a specific lineage. While stem cells can be best defined functionally, a good number of molecular markers have been used to prospectively identify numerous stem cell populations. Although the functional importance of many of these antigens remains unknown, their unique expression pattern Ro 48-8071 fumarate and timing of expression provide a useful tool for scientists to identify as well as isolate stem cells. Embryonic stem cells (ESC), derived from the inner cell mass of pre-implantation embryos, have been recognized as the earliest stem cell populace [1,2]. This pluripotent populace can differentiate into all somatic tissue including germ cells. In the case of human ESC, they can differentiate into some extra-embryonic derivatives as well. Like mouse ESC, human ES cells can be managed and propagated on mouse fibroblast feeders for extended periods in media containing basic fibroblast growth factor (bFGF) [3]. Gene expression Ro 48-8071 fumarate of undifferentiated human ES cells has been investigated among several ES cell lines by a variety of techniques. They include comparison with databases, reverse transcriptase-polymerase chain reaction, focused cDNA microarrays, and immunocytochemistry. A list of molecules comprised of known ES-specific or -highly expressed genes and candidates that can serve as markers for human ESCs and may also contribute to the “stemness” phenotype has been established [3-11]. For example, pluripotent ESC can be characterized by high level expression of Oct3/4 (POU domain name, class 5, transcription factor 1, Pou5f1) and Nanog, which are a member of POU domain name and homeobox transcription factors respectively. A critical amount of Oct3/4 and Nanog expression is required to sustain stem-cell pluripotency and both of these markers are downregulated as cells differentiate in vitro and in vivo [4-9]. Antibodies to Oct3/4 which cross react with human Oct 3/4 have been widely used to monitor the presence of undifferentiated ESC. No single marker however is sufficient or unique for identifying ESCs. Oct3/4 for example is expressed by germ cells and may be expressed by specific populations later in development. Similarly, Nanog has been shown to express in other tissues. We and other have noted however, that while no single marker is sufficient a constellation of positive and negative markers can in concert unambiguously allow one to define the state of ESC cultures and that surface markers in combination can be used to prospectively sort for ESC. Based on Ro 48-8071 fumarate published data at the level of gene expression, we have cloned a number of candidate marker genes. We have also expressed the recombinant protein and generated a panel of monoclonal or polyclonal antibodies to these proteins. Using these antibodies we have confirmed the specificity and selectivity of these antibodies on several ESC lines and established their power as stem cells markers. Our results confirm the expression pattern and timing of these cell markers at the protein level, whereas previous data reported at the level of gene expression. Ro 48-8071 fumarate Results and conversation Characterization of undifferentiated human ES cells and differentiated EBs by antibodies All monoclonal antibodies were initially selected for their abilities to recognize recombinant proteins in direct ELISAs. A subset were also tested by Western Blot analysis using recombinant proteins and cell lysate to confirm binding to a single epitope. Klf2 The best clone was later screened for its applications for immunocytochemistry and circulation cytometry using numerous cell lines. Human peripheral blood platelets were used for screening mouse anti-human CD9 antibody. MCF-7 cells were used for screening mouse anti-human E-Cadherin and PODXL (podocalyxin-like) antibodies. MG-63 cells were used for screening mouse anti-human GATA1 (GATA binding protein 1) antibody. Beta-TC6 cells were used for screening for mouse anti-human/mouse PDX-1 (pancreatic duodenal homeobox-1) antibody. NTERA-2 cells were used for screening mouse anti-human Oct3/4 and SOX2 (sex-determining region Y-box 2) antibodies. All polyclonal antibodies were affinity-purified using recombinant proteins and validated by direct ELISAs and Western. Caco-2 cells were used for validation of goat anti-human GATA6 antibody and NTERA-2 cells were used for validation of goat anti-human Nanog and anti-human Oct3/4 antibodies (Summarized in Table ?Table11). Table 1 Summary list of antibody verification by western blot.
AntibodySample used for analysisMol. Wt. (KD)Gt hBrachyurymouse ES-derived EB lysate48Ms hDPPA5N/AN/AGt hGATA6Caco2 cell lysate65Gt hNanogNTERA-2 cell lysate33Gt hOct 3/4NTERA-2 cell lysate39Gt hPDX1beta-TC 6 cell lysate32Gt hSOX17mouse ES-derived EB lysate45Ms hCD9PBMC25Rt hGATA-1N/AN/AMs hE-CadherinMCF-7 cell lysate97Ms hPODXLMCF-7 cell lysate57Ms hSOX2NTERA-2 cell lysate36 Open Ro 48-8071 fumarate in a separate windows N/A: 1. DPPA5.