RESUMO
INTRODUCTION: Tumourigenesis attributed to residual undifferentiated cells in a graft is considered to be a significant issue in cell therapy using human pluripotent stem cells. To ensure the safety of regenerative medicine derived from pluripotent stem cells, residual undifferentiated cells must be eliminated in the manufacturing process. We previously described the lectin probe rBC2LCN, which binds harmlessly and specifically to the cell surface of human pluripotent stem cells. We report here a technique using rBC2LCN to remove pluripotent cells from a heterogenous population to reduce the chance of teratoma formation. METHODS: We demonstrate a method for separating residual tumourigenic cells using rBC2LCN-bound magnetic beads. This technology is a novel use of their previous discovery that rBC2LCN is a lectin that selectively binds to pluripotent cells. We optimize and validate a method to remove hPSCs from a mixture with human fibroblasts using rBC2LCN-conjugated magnetic beads. RESULTS: Cells with the potential to form teratoma could be effectively eliminated from a heterogeneous cell population with biotin-labelled rBC2LCN and streptavidin-bound magnetic beads. The efficiency was measured by FACS, ddPCR, and animal transplantation, suggesting that magnetic cell separation using rBC2LCN is quite efficient for eliminating hPSCs from mixed cell populations. CONCLUSIONS: The removal of residual tumourigenic cells based on rBC2LCN could be a practical option for laboratory use and industrialisation of regenerative medicine using human pluripotent stem cells.
RESUMO
The recombinant N-terminal domain of BC2L-C lectin (rBC2LCN) is useful for detecting not only human pluripotent stem cells but also some cancers. However, the cancer types and stages that can be detected by rBC2LCN remain unclear. In this study, we identified the human breast carcinoma subtypes and stages that can be detected by rBC2LCN. Compared with rBC2LCN-negative breast carcinoma cell lines, the rBC2LCN-positive cells expressed higher levels of human epidermal growth factor receptor 2 (HER2) and epithelial marker genes. Importantly, rBC2LCN histochemical staining of human breast carcinoma tissues demonstrated the utility of rBC2LCN in detecting breast carcinoma types that express HER2 and have not spread much in the early phase of growth. We conclude that rBC2LCN may have potential as a detection probe and a drug delivery vehicle to identify and treat early-stage HER2-positive breast carcinoma.
Assuntos
Proteínas de Bactérias/química , Neoplasias da Mama/diagnóstico , Lectinas/química , Sondas Moleculares/química , Antineoplásicos/administração & dosagem , Proteínas de Bactérias/genética , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Burkholderia cenocepacia , Portadores de Fármacos/química , Estudos de Viabilidade , Feminino , Humanos , Lectinas/genética , Células MCF-7 , Sondas Moleculares/genética , Estadiamento de Neoplasias , Receptor ErbB-2/análise , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise Serial de Tecidos/métodosRESUMO
The recombinant lectin rBC2LCN is a useful marker for discriminating the undifferentiated status of human induced or embryonic stem cells. Recently, rBC2LCN has also been used for detecting some cancers and niche cells. However, the generality of which types of cells are detected by rBC2LCN is unclear. In this study, we demonstrated the potential of rBC2LCN as a probe for detecting and isolating cancer stem-like cells. Interestingly, flow cytometric analysis of various human cell lines indicated that the human prostate cancer cell line PC-3 consisted of rBC2LCN-positive and -negative subpopulations. Compared with the rBC2LCN-negative subpopulation, the rBC2LCN-positive subpopulation possessed representative features of cancer stem cells and malignancy, such as slow proliferation, increased cell motility, anchorage-independent growth, and drug resistance. The comprehensive expression profiles revealed that the rBC2LCN-positive subpopulation expressed higher levels of cancer stem cell markers. These findings indicate that rBC2LCN is useful for detecting not only pluripotent stem cells but also the cancer stem-like subpopulation of PC-3â¯cells. Pluripotent and cancer cells with rBC2LCN positivity would be important for future stem cell research.
Assuntos
Lectinas/metabolismo , Sondas Moleculares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Recombinantes/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lectinas/genética , Células MCF-7 , Masculino , Sondas Moleculares/genética , Células-Tronco Neoplásicas/patologia , Células PC-3 , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
The application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously, we identified a human pluripotent stem-cell-specific lectin probe, called rBC2LCN, by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A, termed rBC2LCN-PE23, which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells, followed by its internalization, allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus, rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies.
Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Lectinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Virulência/metabolismo , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Escherichia coli/metabolismo , Exotoxinas/genética , Humanos , Lectinas/genética , Plasmídeos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
While human pluripotent stem cells are attractive sources for cell-replacement therapies, a major concern remains regarding their tumorigenic potential. Thus, safety assessment of human pluripotent stem cell-based products in terms of tumorigenicity is critical. Previously we have identified a pluripotent stem cell-specific lectin probe rBC2LCN recognizing hyperglycosylated podocalyxin as a cell surface ligand. Here we demonstrate that hyperglycosylated podocalyxin is secreted from human pluripotent stem cells into cell culture supernatants. We establish a sandwich assay system, named the GlycoStem test, targeting the soluble hyperglycosylated podocalyxin using rBC2LCN. The GlycoStem test is sufficiently sensitive and quantitative to detect residual human pluripotent stem cells. This work provides a proof of concept for the noninvasive and quantitative detection of tumorigenic human pluripotent stem cells using cell culture supernatants. The developed method should increase the safety of human pluripotent stem cell-based cell therapies.