Identification of Human Embryonic Progenitor Cell Targeting Peptides Using Phage Display

University of Cincinnati, United States of America
PLoS ONE (Impact Factor: 3.23). 03/2013; 8(3):e58200. DOI: 10.1371/journal.pone.0058200
Source: PubMed


Human pluripotent stem (hPS) cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS) cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES) cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken together these data suggest that selection of phage display libraries against a clonal progenitor stem cell population can be used to identify progenitor stem cell targeting peptides. The peptides may be useful for monitoring hPS cell differentiation and for the development of cell enrichment procedures to improve the efficiency of directed differentiation toward clinically relevant human cell types.

Download full-text


Available from: Paola Bignone,

Click to see the full-text of:

Article: Identification of Human Embryonic Progenitor Cell Targeting Peptides Using Phage Display

5.14 MB

See full-text
  • Source
    • "A representative experiment is shown in Figure 5. The cell line E15, which in other conditions was shown to have chondrogenic potential19 and the line W1027 strongly induced MYH11 in micromass conditions supplemented with 10 ng/mL BMP4, but this induction was essentially ablated in HyStem-4D culture supplemented with BMP4. Instead, in HyStem-4D beads, the line markedly upregulated expression of DCN, a marker of meninges.28 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Human pluripotent stem (hPS) cells provide an attractive opportunity for the manufacture of a wide array of therapeutic cell types. The challenges to commercialization include the thousand-fold diversity of cell types emerging from hPS cells and the associated difficulties in validating processes to reliably generate cells with precise identity and purity. Improved methods of controlling the dosage and migration of hPS-derived cells in solid tissues are also needed. To directly address these issues, we clonally expanded proliferating lineages of cells that were intermediate in regard to their state of differentiation between hPS and terminally differentiated cells. These cells called monoclonal embryonic progenitors (hEP), are expandable mortal lineages with diverse site-specific homeobox gene expression and multipotentiality. In this review, we discuss methods of generating combination products wherein the fate space of precisely identified monoclonal hEP cells is mapped by differentiating the cells in vitro in HyStem-3D bead arrays in the presence of diverse growth factors. This combination of discovery processes has the potential to translate directly into cell-matrix formulations that can be used to generate pre-clinical data leading to human clinical trials and potentially new medical therapies.
    Biomatter 01/2013; 3(1). DOI:10.4161/biom.24496
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Due to the fundamental progress in elucidating the molecular mechanisms of human diseases and the arrival of the post-genomic era, increasing numbers of therapeutic genes and cellular targets are available for gene therapy. Meanwhile, the most important challenge is to develop gene delivery vectors with high efficiency through target cell selectivity, in particular under in situ conditions. The most widely used vector system to transduce cells is based on adenovirus (Ad). Recent endeavors in the development of selective Ad vectors that target cells or tissues of interest and spare the alteration of all others have focused on the modification of the virus broad natural tropism. A popular way of Ad targeting is achieved by directing the vector towards distinct cellular receptors. Redirecting can be accomplished by linking custom-made peptides with specific affinity to cellular surface proteins via genetic integration, chemical coupling or bridging with dual-specific adapter molecules. Ideally, targeted vectors are incapable of entering cells via their native receptors. Such altered vectors offer new opportunities to delineate functional genomics in a natural environment and may enable efficient systemic therapeutic approaches. This review provides a summary of current state-of-the-art techniques to specifically target adenovirus-based gene delivery vectors.
    Viruses 04/2014; 6(4):1540-63. DOI:10.3390/v6041540 · 3.35 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel peptide, BRBP1 (MYPWTEPSYLSN), was identified using an in vitro phage biopanning strategy against human brain-seeking breast carcinoma cells (231-BR cells).The peptide-phage clone, BRBP1-M13 displaying BRBP1 sequence, specifically bound to 231-BR cells and the binding could be competitively abolished by BRBP1. In vivo distribution studies showed that BRBP1-M13 preferentially homed to the 231-BR tumors. Fluorescently-labeled BRBP1, BRBP1-K(5-TAMRA), preferentially bound to 231-BR cells in a dose-dependent and energy-dependent manner and it was efficiently internalized into the cells after 2 h incubation. Near-infrared fluorophores imaging demonstrated the accumulation of Cy5.5-conjugated BRBP1 peptide in the tumors in vivo. Thus, BRBP1 is a promising peptide binding to human brain metastatic breast cancer and it may be applied to targeted delivery of cytotoxic agents to the intended tumor.
    Biotechnology Letters 07/2014; 36(11). DOI:10.1007/s10529-014-1608-0 · 1.59 Impact Factor