RESUMO
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.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Peptídeos/metabolismo , Sequência de Bases , Linhagem Celular , Técnicas de Visualização da Superfície Celular , Biologia Computacional , Primers do DNA/genética , Citometria de Fluxo , Humanos , Ligantes , Microscopia de Fluorescência , Dados de Sequência Molecular , Pontos Quânticos , Análise de Sequência de DNA , EspectrofotometriaRESUMO
AIM: The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs). MATERIALS & METHODS: The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR. RESULTS: In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-ß3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation. CONCLUSION: The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.
Assuntos
Linhagem da Célula , Condrogênese , Células-Tronco Embrionárias/citologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Células Clonais , Colágeno Tipo II/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Coloração e Rotulagem , Transplante de Células-Tronco , Engenharia Tecidual , Fator de Crescimento Transformador beta/farmacologiaRESUMO
AIM: The identification of molecular markers that are upregulated in multiple tumor types could lead to novel diagnostic and therapeutic strategies. The authors screened a panel of RNAs prepared from diverse tumors and tumor cell lines, and compared them with normal tissues and cultured somatic cell types, in order to identify candidate genes expressed in a broad spectrum of tumor types. MATERIALS & METHODS: Gene expression microarray analysis was carried out on 128 individual tumor samples representing over 20 tumor types, 85 samples representing 31 diverse normal tissue types, 68 tumor cell lines and 97 diverse normal primary cell cultures. Genes were ranked for elevated expression across a large number and variety of tumors relative to normal tissues. RESULTS & CONCLUSION: COL10A1 was identified as a gene with restricted expression in most normal tissues and elevated expression in many diverse tumor types. By contrast, COL10A1 expression was undetectable in the 68 tumor cell lines surveyed in this study. Immunofluorescence studies localized collagen, type X, α-1 (collagen X) staining to tumor vasculature in breast tumors, whereas the vasculature of normal breast tissue was either collagen X-negative or had markedly lower levels. The tumor microenvironment-specific expression of collagen X, together with its localization in the vasculature, may facilitate its use as a novel target for the diagnosis and treatment of diverse solid tumor types.
Assuntos
Colágeno Tipo XI/genética , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neovascularização Patológica/genética , Linhagem Celular Tumoral , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Colágeno Tipo XI/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Reprodutibilidade dos TestesRESUMO
AIMS: We screened 100 diverse human embryonic stem-derived progenitor cell lines to identify novel lines with chondrogenic potential. MATERIALS & METHODS: The 4D20.8 cell line was compared with mesenchymal stem cells and dental pulp stem cells by assessing osteochondral markers using immunohistochemical methods, gene expression microarrays, quantitative real-time PCR and in vivo repair of rat articular condyles. RESULTS: 4D20.8 expressed the site-specific gene markers LHX8 and BARX1 and robustly upregulated chondrocyte markers upon differentiation. Differentiated 4D20.8 cells expressed relatively low levels of COL10A1 and lacked IHH and CD74 expression. Transplantation of 4D20.8 cells into experimentally induced defects in the femoral condyle of athymic rats resulted in cartilage and bone differentiation approximating that of the original tissue architecture. Relatively high COL2A1 and minimal COL10A1 expression occurred during differentiation in HyStem-C hydrogel with TGF-ß3 and GDF-5. CONCLUSION: Human embryonic stem cell-derived embryonic progenitor cell lines may provide a novel means of generating purified site-specific osteochondral progenitor cell lines that are useful in research and therapy.
Assuntos
Condrogênese , Células-Tronco Embrionárias/citologia , Face/embriologia , Mesoderma/metabolismo , Crânio/embriologia , Animais , Antraquinonas , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Células Clonais , Colágeno/genética , Colágeno/metabolismo , Polpa Dentária/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sus scrofa , Engenharia Tecidual , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count, and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation, using both microarray and sequence-based analyses, provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines, the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly, genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes, such as tumor suppressors and genetic disease genes, do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens, such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents.
Assuntos
Células-Tronco Embrionárias/metabolismo , Genoma Humano/genética , Sistema ABO de Grupos Sanguíneos/genética , Alelos , Apolipoproteínas E/genética , Sequência de Bases , Linhagem Celular , Variações do Número de Cópias de DNA/genética , Células-Tronco Embrionárias/citologia , Éxons/genética , Antígenos HLA/genética , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único/genética , Telômero/genéticaRESUMO
The R-spondin (Rspo) family of proteins consists of secreted cysteine-rich proteins that can activate beta-catenin signaling via the Frizzled/LRP5/6 receptor complex. Here, we report that targeted inactivation of the mouse Rspo2 gene causes developmental limb defects, especially in the hindlimb. Although the initiation of the expression of apical ectodermal ridge (AER)-specific genes, including fibroblast growth factor 8 (FGF8) and FGF4 occurred normally, the maintenance of these marker expressions was significantly defective in the hindlimb of Rspo2(-/-) mice. Consistent with the ligand role of R-spondins in the Wnt/beta-catenin signaling pathway, expression of Axin2 and Sp8, targets for beta-catenin signaling, within AER was greatly reduced in Rspo2(-/-) embryos. Furthermore, sonic hedgehog (Shh) signaling within the hindlimbs of Rspo2(-/-) mice was also significantly decreased. Rspo2 is expressed in the AER of all limb buds, however the stunted phenotype is significantly more severe in the hindlimbs than the forelimbs and strongly biased to the left side. Our findings strongly suggest that Rspo2 expression in the AER is required for AER maintenance likely by regulating Wnt/beta-catenin signaling.
Assuntos
Ectoderma/metabolismo , Membro Posterior/embriologia , Trombospondinas/metabolismo , Animais , Cateninas/metabolismo , Feminino , Proteínas Hedgehog/metabolismo , Masculino , Camundongos , Mutação , Transdução de Sinais , Trombospondinas/genética , Proteínas Wnt/metabolismoRESUMO
BACKGROUND & AIMS: R-spondin 1 (Rspo1) is a novel epithelial mitogen that stimulates the growth of mucosa in both the small and large intestine. METHODS: We investigated the therapeutic potential of Rspo1 in ameliorating experimental colitis induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) as well as nonsteroidal anti-inflammatory drug-induced colitis in interleukin (IL)-10-deficient mice. RESULTS: Therapeutic administration of recombinant Rspo1 protein reduced the loss of body weight, diarrhea, and rectal bleeding in a mouse model of acute or chronic DSS-induced colitis. Histologic evaluation revealed that Rspo1 improved mucosal integrity in both villus and/or crypt compartments in the small intestine and colon by stimulating crypt cell growth and mucosal regeneration in DSS-treated mice. Moreover, Rspo1 significantly reduced DSS-induced myeloperoxidase activity and inhibited the overproduction of proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1alpha, IL-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor, in mouse intestinal tissue, indicating that Rspo1 may reduce DSS-induced inflammation by preserving the mucosal barrier function. Likewise, Rspo1 therapy also alleviated TNBS-induced interstitial inflammation and mucosal erosion in the mouse colon. Furthermore, Rspo1 substantially decreased the histopathologic severity of chronic enterocolitis by repairing crypt epithelium and simultaneously suppressing inflammatory infiltration in piroxicam-exposed IL-10(-/-) mice. Endogenous Rspo1 protein was localized to villus epithelium and crypt Paneth cells in mouse small intestine. CONCLUSIONS: Our results show that Rspo1 may be clinically useful in the therapeutic treatment of inflammatory bowel disease by stimulating crypt cell growth, accelerating mucosal regeneration, and restoring intestinal architecture.
Assuntos
Colite/tratamento farmacológico , Colo/patologia , Mitógenos/uso terapêutico , Trombospondinas/uso terapêutico , Doença Aguda , Animais , Anti-Inflamatórios não Esteroides/toxicidade , Proliferação de Células/efeitos dos fármacos , Colite/induzido quimicamente , Colite/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Piroxicam/toxicidade , Substitutos do Plasma/toxicidade , Proteínas Recombinantes/uso terapêutico , Índice de Gravidade de Doença , Resultado do Tratamento , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
NTB-A is a CD2-related cell surface protein expressed primarily on lymphoid cells including B-lymphocytes from chronic lymphocytic leukaemia (CLL) and lymphoma patients. We have generated a series of monoclonal antibodies (mAbs) against NTB-A and assessed their therapeutic potential for CLL. Selective mAbs to NTB-A were further tested in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicty (ADCC) assays in cell lines and B lymphocytes freshly isolated from CLL patients. While lower levels of NTB-A were detected in T and natural killer (NK) cells, CDC activity was demonstrated primarily in B cells isolated from CLL patients and B lymphoma cell lines. Knockdown of NTB-A by small interfering RNA in target cells results in lower cytotoxicity, demonstrating the specificity of the mAbs. Furthermore, anti NTB-A mAbs demonstrated anti-tumour activity against CA46 human lymphoma xenografts in nude mice and against systemically disseminated Raji human lymphoma cells in severe combined immunodeficient mice. Taken together, these results demonstrate NTB-A as a potential new target for immunotherapy of leukaemia and lymphomas.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Imunização Passiva/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Receptores de Superfície Celular/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Reações Antígeno-Anticorpo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo , Humanos , Hibridomas , Leucemia Linfocítica Crônica de Células B/imunologia , Ativação Linfocitária , Camundongos , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Interferência de RNA , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Transplante HeterólogoRESUMO
We have discovered a family of small secreted proteins in Homo sapiens and Mus musculus. The IGF-like (IGFL) genes encode proteins of approximately 100 amino acids that contain 11 conserved cysteine residues at fixed positions, including two CC motifs. In H. sapiens, the family is composed of four genes and two pseudogenes that are referred as IGFL1 to IGFL4 and IGFL1P1 and IGFL1P2, respectively. Human IGFL genes are clustered together on chromosome 19 within a 35-kb interval. M. musculus has a single IGFL family member that is located on chromosome 7. Further, evolutionary analysis shows a lack of direct orthology between any of the four human members and the mouse gene. This relationship between the mouse and the human family members suggests that the multiple members in the human complement have arisen from recent duplication events that appear limited to the primate lineage. Structural considerations and sequence comparisons would suggest that IGFL proteins are distantly related to the IGF superfamily of growth factors. IGFL mRNAs display specific expression patterns; they are expressed in fetal tissues, breast, and prostate, and in many cancers as well, and this pattern is consistent with that of the IGF family members.
Assuntos
Regulação da Expressão Gênica , Família Multigênica , Somatomedinas/genética , Somatomedinas/metabolismo , Sequência de Aminoácidos , Animais , Cromossomos Humanos Par 19 , Sequência Conservada , Cisteína , Evolução Molecular , Éxons , Feminino , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Pseudogenes , Homologia de Sequência de Aminoácidos , Somatomedinas/químicaRESUMO
The R-spondin (Rspo) protein family is a recently described group of four distinct human secreted proteins. Reported activities for Rspo proteins include essential roles in vertebrate development and their ligand-type activities overlap substantially with those of the canonical Wnt ligands in that both Rspo and canonical Wnt signaling result in the activation of beta-catenin. In a general functional screen for human secreted proteins using transgenic mouse models, we identified human R-spondin1 (hRspo1) protein as a potent and specific mitogen for the gastrointestinal epithelium and demonstrated a potential therapeutic application for the protein in mouse models of cancer therapy-induced mucositis. In contrast to previous studies, our data indicated only partial overlap between Wnt and Rspo ligand activities, suggesting that there may be independent receptor/signaling pathways for Rspo proteins that intersect those of Wnt at the level of beta-catenin. Here we summarize the current reported data on the Rspo family and discuss these results in terms of alternate mechanisms of action. We have extended our observations on the potential therapeutic application of Rspo proteins by showing that all four human Rspo family members are capable of inducing epithelial proliferation and report the first non-vertebrate Rspo family member.
Assuntos
Trombospondinas/metabolismo , beta Catenina/metabolismo , Animais , Humanos , Transdução de Sinais , Trombospondinas/química , Trombospondinas/classificaçãoRESUMO
Several described growth factors influence the proliferation and regeneration of the intestinal epithelium. Using a transgenic mouse model, we identified a human gene, R-spondin1, with potent and specific proliferative effects on intestinal crypt cells. Human R-spondin1 (hRSpo1) is a thrombospondin domain-containing protein expressed in enteroendocrine cells as well as in epithelial cells in various tissues. Upon injection into mice, the protein induced rapid onset of crypt cell proliferation involving beta-catenin stabilization, possibly by a process that is distinct from the canonical Wnt-mediated signaling pathway. The protein also displayed efficacy in a model of chemotherapy-induced intestinal mucositis and may have therapeutic application in gastrointestinal diseases.
Assuntos
Proliferação de Células , Mucosa Intestinal/citologia , Mitógenos , Trombospondinas/fisiologia , Animais , Antineoplásicos/efeitos adversos , Linhagem Celular , Linhagem Celular Tumoral , Quimera , Colo/citologia , Colo/patologia , Proteínas do Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Células Enteroendócrinas/metabolismo , Células Epiteliais/metabolismo , Fator 7 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/farmacologia , Fluoruracila/efeitos adversos , Peptídeos Semelhantes ao Glucagon , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/citologia , Intestino Delgado/patologia , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Peptídeos/farmacologia , Proteínas/farmacologia , Proteínas Recombinantes/farmacologia , Trombospondinas/genética , Trombospondinas/metabolismo , Trombospondinas/farmacologia , Língua/efeitos dos fármacos , Língua/patologia , Transativadores/metabolismo , Proteínas Wnt , Proteína Wnt3 , beta CateninaRESUMO
We have discovered a family of small secreted proteins in Homo sapiens and Mus musculus using a novel database searching strategy. The family is composed of five highly homologous genes referred to as TAFA-1 to -5. The TAFA genes encode proteins of approximately 100 amino acids that contain conserved cysteine residues at fixed positions. TAFA-1 to -4 are more closely related to each other than to TAFA-5, in which a conserved motif including CC in TAFA-1 to -4 is not present. In H. sapiens, TAFA-3 has two isoforms formed by alternative splicing. Sequence homology analyses reveal that TAFA proteins appear distantly related to MIP-1alpha, a member of the CC-chemokine family. TAFA mRNAs are highly expressed in specific brain regions, with little expression seen in other tissues.