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1.
Biomaterials ; 230: 119638, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31810728

RESUMO

Human induced pluripotent stem cells (hiPSCs) were generated on several biomaterials from human amniotic fluid in completely xeno-free and feeder-free conditions via the transfection of pluripotent genes using a nonintegrating RNA Sendai virus vector. The effect of xeno-free culture medium on the efficiency of the establishment of human amniotic fluid stem cells from amniotic fluid was evaluated. Subsequently, the effect of cell culture biomaterials on the reprogramming efficiency was investigated during the reprogramming of human amniotic fluid stem cells into hiPSCs. Cells cultured in laminin-511, laminin-521, and Synthemax II-coated dishes and hydrogels having optimal elasticity that were engrafted with specific oligopeptides derived from vitronectin could be reprogrammed into hiPSCs with high efficiency. The reprogrammed cells expressed pluripotency proteins and had the capability to differentiate into cells derived from all three germ layers in vitro and in vivo. Human iPSCs could be generated successfully and at high efficiency (0.15-0.25%) in completely xeno-free conditions from the selection of optimal cell culture biomaterials.

2.
Biomater Sci ; 7(12): 5467-5481, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31656967

RESUMO

Current xeno-free and chemically defined methods for the differentiation of hPSCs (human pluripotent stem cells) into cardiomyocytes are not efficient and are sometimes not reproducible. Therefore, it is necessary to develop reliable and efficient methods for the differentiation of hPSCs into cardiomyocytes for future use in cardiovascular research related to drug discovery, cardiotoxicity screening, and disease modeling. We evaluated two representative differentiation methods that were reported previously, and we further developed original, more efficient methods for the differentiation of hPSCs into cardiomyocytes under xeno-free, chemically defined conditions. The developed protocol successively differentiated hPSCs into cardiomyocytes, approximately 90-97% of which expressed the cardiac marker cTnT, with beating speeds and sarcomere lengths that were similar to those of a healthy adult human heart. The optimal cell culture biomaterials for the cardiac differentiation of hPSCs were also evaluated using extracellular matrix-mimetic material-coated dishes. Synthemax II-coated and Laminin-521-coated dishes were found to be the most effective and efficient biomaterials for the cardiac differentiation of hPSCs according to the observation of hPSC-derived cardiomyocytes with high survival ratios, high beating colony numbers, a similar beating frequency to that of a healthy adult human heart, high purity levels (high cTnT expression) and longer sarcomere lengths similar to those of a healthy adult human heart.

3.
J Mater Chem B ; 7(45): 7110-7119, 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31513217

RESUMO

Human mesenchymal stem cells (hMSCs), such as human adipose-derived stem cells (hADSCs), present heterogeneous characteristics, including varying differentiation abilities and genotypes. hADSCs isolated under different conditions exhibit differences in stemness. We isolated hADSCs from human fat tissues via culture on different cell culture biomaterials including tissue culture polystyrene (TCPS) dishes and extracellular matrix protein (ECM)-coated dishes in medium supplemented with 5% or 10% serum-converted human platelet lysate (hPL) or 10% fetal bovine serum (FBS) as a control. Currently, it is not clear whether xeno-free hPL in the cell culture medium promotes the ability of hMSCs such as hADSCs to differentiate into several cell lineages compared to the xenomaterial FBS. We investigated whether a synchronized effect of ECM (Matrigel, fibronectin, and recombinant vitronectin) coatings on TCPS dishes for efficient hADSC differentiation could be observed when hADSCs were cultured in hPL medium. We found that Matrigel-coated dishes promoted hADSC differentiation into osteoblasts and suppressed differentiation into chondrocytes in 10% hPL medium. Recombinant vitronectin- and fibronectin-coated dishes greatly promoted hADSC differentiation into osteoblasts and chondrocytes in 5% and 10% hPL media. hPL promoted hADSC differentiation into osteoblasts and chondrocytes compared to FBS on the fibronectin-coated surface and recombinant vitronectin-coated surface.

4.
Biomater Sci ; 7(10): 4345-4362, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31411209

RESUMO

Recombinant vitronectin-grafted hydrogels were developed by adjusting surface charge of the hydrogels with grafting of poly-l-lysine for optimal culture of human embryonic stem cells (hESCs) under xeno- and feeder-free culture conditions, with elasticity regulated by crosslinking time (10-30 kPa), in contrast to conventional recombinant vitronectin coating dishes, which have a fixed stiff surface (3 GPa). hESCs proliferated on the hydrogels for over 10 passages and differentiated into the cells derived from three germ layers indicating the maintenance of pluripotency. hESCs on the hydrogels differentiated into cardiomyocytes under xeno-free culture conditions with much higher efficiency (80% of cTnT+ cells) than those on conventional recombinant vitronectin or Matrigel-coating dishes just only after 12 days of induction. It is important to have an optimal design of cell culture biomaterials where biological cues (recombinant vitronectin) and physical cues (optimal elasticity) are combined for high differentiation of hESCs into specific cell lineages, such as cardiomyocytes, under xeno-free and feeder-free culture conditions.

5.
Biomaterials ; 221: 119411, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31419657

RESUMO

Commonly, stem cell culture is based on batch-type culture, which is laborious and expensive. We continuously cultured human pluripotent stem cells (hPSCs) on thermoresponsive dish surfaces, where hPSCs were partially detached on the same thermoresponsive dish by decreasing the temperature of the thermoresponsive dish to be below the lower critical solution temperature for only 30 min. Then, the remaining cells were continuously cultured in fresh culture medium, and the detached stem cells were harvested in the exchanged culture medium. hPSCs were continuously cultured for ten cycles on the thermoresponsive dish surface, which was prepared by coating the surface with poly(N-isopropylacrylamide-co-styrene) and oligovitronectin-grafted poly(acrylic acid-co-styrene) or recombinant vitronectin for hPSC binding sites to maintain hPSC pluripotency. After ten cycles of continuous culture on the thermoresponsive dish surface, the detached cells expressed pluripotency proteins and had the ability to differentiate into cells derived from the three germ layers in vitro and in vivo. Furthermore, the detached cells differentiated into specific cell lineages, such as cardiomyocytes, with high efficiency.

6.
Taiwan J Obstet Gynecol ; 57(4): 507-516, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30122569

RESUMO

OBJECTIVE: Defects in L-selectin ligand (LSL) expression have been reported to cause implantation failure, but little is known about LSL expression in adenomyosis. This study evaluates LSL expression throughout the menstrual cycle in women with adenomyosis. MATERIALS AND METHODS: Endometrial samples were obtained from reproductive-aged women with adenomyosis who underwent hysterectomy. A total of 42 endometrial biopsies were included. There were 12 women in proliferative phase, 10 in early-secretory phase, 9 in mid-secretory phase, and 11 in late-secretory phase. Immunohistochemistry, western blotting, and RT-PCR were performed to evaluate LSL expression. A non-parametric Kruskal-Wallis one-way analysis of variance with multiple comparisons was performed to examine differences among menstrual phases. RESULTS: Immunohistochemistry analysis with MECA-79 shows that LSL is expressed with weak intensity in the endometrium in all phases. In the luminal epithelium, MECA-79 reactivity increased from the proliferative to the late-secretory phase but decreased in the mid-secretory phase. There were significant differences in the mean histological scores (HSCOREs) among the proliferative, early-secretory, and late-secretory phases (p < 0.05). Five LSL genes were detected in the adenomyotic endometria: PODXL, EMCN, CD300LG, GLYCAM1, and CD34. The mRNA expression of LSL genes occurred differentially among phases. Moreover, PODXL differed significantly among phases (p < 0.05). CONCLUSIONS: LSL expressions were downregulated in the luminal epithelium of adenomyotic endometria in the mid-secretory phase. The mRNA expressions of LSL genes also had differential expression patterns throughout the menstrual cycle, especially for PODXL. Our study showed that adenomyosis may cause abnormalities of LSL production in the mid-secretory phase, which may contribute to impaired endometrial receptivity and implantation failure.


Assuntos
Adenomiose/metabolismo , Endométrio/metabolismo , Glicoproteínas/genética , Selectina L/metabolismo , Ligantes , Ciclo Menstrual/metabolismo , Adulto , Regulação para Baixo , Endométrio/química , Feminino , Glicoproteínas/análise , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , RNA Mensageiro/análise , Sialoglicoproteínas/análise , Sialoglicoproteínas/genética
7.
J Vis Exp ; (132)2018 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-29443075

RESUMO

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.


Assuntos
Células-Tronco Pluripotentes/metabolismo , Álcool de Polivinil/uso terapêutico , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/citologia , Álcool de Polivinil/farmacologia
8.
Sci Rep ; 8(1): 1443, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29362381

RESUMO

This study investigates peptide components of L-selectin ligand (LSL) and their gene expressions in human endometrium during the natural menstrual cycle. We recruited 41 endometrial samples from reproductive-aged women with leiomyoma and undergoing hysterectomy and 11 endometrial samples from menopausal women as controls. Immunohistochemistry revealed strong MECA-79 expression from the early through the mid-secretory phase and low expression in menopausal endometrium. Five peptide components of LSL were detected in reproductive and menopausal endometrium by one-step quantitative RT-PCR: podocalyxin, endomucin, nepmucin, GlyCAM-1, and CD34. Endomucin differed significantly between the proliferative and early-secretory phases. CHST2 and CHST4 genes (which are involved in the generation of LSL epitopes) were expressed without significant differences among phases. The gene expression of progesterone receptor decreased from the proliferative to the late-secretory phase, and the difference was significant. However, estrogen receptor α expression showed stability among phases. The significant expression of endomucin between the proliferative and early-secretory phases might play a vital role in endometrial receptivity. Further studies are needed to investigate the factors that regulate the expression of endomucin and other LSL peptide components in different phases of the menstrual cycle.


Assuntos
Antígenos de Superfície/metabolismo , Endométrio/metabolismo , Expressão Gênica , Leiomioma/cirurgia , Proteínas de Membrana/metabolismo , Menopausa/metabolismo , Ciclo Menstrual/metabolismo , Adulto , Antígenos CD/genética , Antígenos CD34/genética , Antígenos de Superfície/genética , Feminino , Humanos , Histerectomia , Leiomioma/genética , Leiomioma/metabolismo , Proteínas de Membrana/genética , Menopausa/genética , Ciclo Menstrual/genética , Pessoa de Meia-Idade , Mucinas/genética , Receptores Imunológicos/genética , Receptores de Progesterona/genética , Sialoglicoproteínas/genética , Sulfotransferases/genética
9.
Lab Invest ; 97(10): 1167-1179, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28869589

RESUMO

Cardiovascular disease remains the leading cause of death and disability in advanced countries. Stem cell transplantation has emerged as a promising therapeutic strategy for acute and chronic ischemic cardiomyopathy. The current status of stem cell therapies for patients with myocardial infarction is discussed from a bioengineering and biomaterial perspective in this review. We describe (a) the current status of clinical trials of human pluripotent stem cells (hPSCs) compared with clinical trials of human adult or fetal stem cells, (b) the gap between fundamental research and application of human stem cells, (c) the use of biomaterials in clinical and pre-clinical studies of stem cells, and finally (d) trends in bioengineering to promote stem cell therapies for patients with myocardial infarction. We explain why the number of clinical trials using hPSCs is so limited compared with clinical trials using human adult and fetal stem cells such as bone marrow-derived stem cells.


Assuntos
Bioengenharia , Ensaios Clínicos como Assunto , Infarto do Miocárdio/terapia , Transplante de Células-Tronco , Animais , Materiais Biocompatíveis , Bioengenharia/métodos , Bioengenharia/tendências , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/transplante , Pesquisa com Células-Tronco
10.
Sci Rep ; 7: 45146, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28332572

RESUMO

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.


Assuntos
Hidrogéis/química , Oligopeptídeos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
11.
Data Brief ; 6: 603-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26909373

RESUMO

This data article contains two figures and one table supporting the research article entitled: "Continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surface" [1]. The table shows coating conditions of three copolymers, poly(styrene-co-acrylic acid) grafted with oligovitronectin, poly(styrene-co-N-isopropylacrylamide) and poly(styrene-co-polyethylene glycol methacrylate) to prepare thermoresponsive surface. XPS spectra show the nitrogen peak of the polystyrene surface coated with poly(styrene-co-acrylic acid) grafted with oligovitronectin. The surface coating density analyzed from sorption of poly(styrene-co-acrylic acid) grafted with oligovitronectin by UV-vis spectroscopy is also presented.

12.
Biomaterials ; 76: 76-86, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26519650

RESUMO

Stem cell culture is typically based on batch-type culture, which is laborious and expensive. Here, we propose a continuous harvest method for stem cells cultured on thermoresponsive nanobrush surfaces. In this method, stem cells are partially detached from the nanobrush surface by reducing the temperature of the culture medium below the critical solution temperature needed for thermoresponse. The detached stem cells are harvested by exchange into fresh culture medium. Following this, the remaining cells are continuously cultured by expansion in fresh culture medium at 37 °C. Thermoresponsive nanobrush surfaces were prepared by coating block copolymers containing polystyrene (for hydrophobic anchoring onto culture dishes) with three types of polymers: (a) polyacrylic acid with cell-binding oligopeptides, (b) thermoresponsive poly-N-isopropylacrylamide, and (c) hydrophilic poly(ethyleneglycol)methacrylate. The optimal coating durations and compositions for these copolymers to facilitate adequate attachment and detachment of human adipose-derived stem cells (hADSCs) and embryonic stem cells (hESCs) were determined. hADSCs and hESCs were continuously harvested for 5 and 3 cycles, respectively, via the partial detachment of cells from thermoresponsive nanobrush surfaces.


Assuntos
Adesão Celular , Nanotecnologia , Células-Tronco/citologia , Tecido Adiposo/citologia , Meios de Cultura , Humanos
13.
Mol Biosyst ; 12(1): 283-94, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26595144

RESUMO

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a system level. Through high throughput experiments and system biology analysis, we investigated the genes and pathways associated with NGF induced neuronal differentiation. We performed microarray experiments and used the KEGG database, system biology analysis, and annotation of pathway functions to study NGF-induced differentiation in PC12 cells. We identified 2020 NGF-induced genes with altered expressions over time. Cross-matching with the KEGG database revealed 830 genes; among which, 395 altered genes were found to have a 2-fold increase in gene expression over a two-hour period. We then identified 191 associated biologic pathways in the KEGG database; the top 15 pathways showed correlation with neural differentiation. These included the neurotrophin pathways, mitogen-activated protein kinase (MAPK) pathways, genes associated with axonal guidance and the Wnt pathways. The activation of these pathways synchronized with nerve growth factor (NGF)-induced differentiation in PC12 cells. In summary, we have established a model system that allows one to systematically characterize the functional pathway changes in a group of neuronal population after an external stimulus.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Genômica , Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular/efeitos dos fármacos , Análise por Conglomerados , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Genômica/métodos , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Células PC12 , Ratos , Transdução de Sinais/efeitos dos fármacos , Transcriptoma
14.
Sci Rep ; 5: 18136, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-26656754

RESUMO

The tentative clinical application of human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human induced pluripotent stem cells, is restricted by the possibility of xenogenic contamination resulting from the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. Therefore, we investigated hPSC cultures on biomaterials with different elasticities that were grafted with different nanosegments. We prepared dishes coated with polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture, whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions. These results indicate that cell culture matrices with optimal elasticity can maintain the pluripotency of hPSCs in culture.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células , Hidrogéis/química , Células-Tronco Pluripotentes/citologia , Sequência de Aminoácidos , Linhagem Celular , Meios de Cultura/química , Módulo de Elasticidade , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Álcool de Polivinil/química , Reprodutibilidade dos Testes , Fatores de Transcrição SOXB1/metabolismo , Succinatos/química , Fatores de Tempo , Vitronectina/química , Xenobióticos/química
15.
Sci Rep ; 5: 10217, 2015 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-25970301

RESUMO

Human adipose-derived stem cells (hADSCs) exhibit heterogeneous characteristics, indicating various genotypes and differentiation abilities. The isolated hADSCs can possess different purity levels and divergent properties depending on the purification methods used. We developed a hybrid-membrane migration method that purifies hADSCs from a fat tissue solution with extremely high purity and pluripotency. A primary fat-tissue solution was permeated through the porous membranes with a pore size from 8 to 25 µm, and the membranes were incubated in cell culture medium for 15-18 days. The hADSCs that migrated from the membranes contained an extremely high percentage (e.g., >98%) of cells positive for mesenchymal stem cell markers and showed almost one order of magnitude higher expression of some pluripotency genes (Oct4, Sox2, Klf4 and Nanog) compared with cells isolated using the conventional culture method.


Assuntos
Tecido Adiposo/citologia , Células-Tronco Adultas/citologia , Separação Celular/métodos , Células-Tronco Adultas/metabolismo , Antígenos de Superfície/metabolismo , Biomarcadores , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem
16.
Lab Invest ; 95(1): 26-42, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25365202

RESUMO

Induced pluripotent stem cells (iPSCs) provide a platform to obtain patient-specific cells for use as a cell source in regenerative medicine. Although iPSCs do not have the ethical concerns of embryonic stem cells, iPSCs have not been widely used in clinical applications, as they are generated by gene transduction. Recently, iPSCs have been generated without the use of genetic material. For example, protein-induced PSCs and chemically induced PSCs have been generated by the use of small and large (protein) molecules. Several epigenetic characteristics are important for cell differentiation; therefore, several small-molecule inhibitors of epigenetic-modifying enzymes, such as DNA methyltransferases, histone deacetylases, histone methyltransferases, and histone demethylases, are potential candidates for the reprogramming of somatic cells into iPSCs. In this review, we discuss what types of small chemical or large (protein) molecules could be used to replace the viral transduction of genes and/or genetic reprogramming to obtain human iPSCs.


Assuntos
Células-Tronco Pluripotentes/citologia , Animais , Técnicas Genéticas , Humanos , Camundongos
17.
Biomaterials ; 35(14): 4278-87, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24565521

RESUMO

The purification of human adipose-derived stem cells (hADSCs) from human adipose tissue cells (stromal vascular fraction) was investigated using membrane filtration through poly(lactide-co-glycolic acid)/silk screen hybrid membranes. Membrane filtration methods are attractive in regenerative medicine because they reduce the time required to purify hADSCs (i.e., less than 30 min) compared with conventional culture methods, which require 5-12 days. hADSCs expressing the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the permeation solution from the hybrid membranes. Expression of the surface markers CD44, CD73, and CD99 on the cells in the permeation solution from the hybrid membranes, which were obtained using 18 mL of feed solution containing 50 × 104 cells, was statistically significantly higher than that of the primary adipose tissue cells, indicating that the hADSCs can be purified in the permeation solution by the membrane filtration method. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the permeation solution, whereas CD34⁺ cells could not be purified by the conventional culture method. The hADSCs in the permeation solution demonstrated a superior capacity for osteogenic differentiation based on their alkali phosphatase activity, their osterix gene expression, and the results of mineralization analysis by Alizarin Red S and von Kossa staining compared with the cells from the suspension of human adipose tissue. These results suggest that the hADSCs capable of osteogenic differentiation preferentially permeate through the hybrid membranes.


Assuntos
Tecido Adiposo/citologia , Ácido Láctico/farmacologia , Membranas Artificiais , Ácido Poliglicólico/farmacologia , Seda/farmacologia , Células-Tronco/citologia , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Separação Celular , Filtração , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Soluções , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo
18.
J Biomed Mater Res B Appl Biomater ; 102(3): 463-76, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24039170

RESUMO

Cancer-initiating cells [cancer stem cells (CSCs)] in colon cancer cells can be selectively suppressed when they are cultured on Pluronic (nanosegment)-grafted dishes, whereas CSCs are maintained on conventional tissue culture dishes and extracellular matrix-coated dishes. CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumorigenic clones. The purification or depletion (suppression) of CSCs should be useful for analyzing CSC characteristics and for clinical application. CSCs can be selectively suppressed from colon cancer cells containing adipose-derived stem cells (ADSCs) on Pluronic-grafted dishes, while ADSCs remain on the dishes. ADSCs on Pluronic-grafted dishes after the suppression of the CSCs can differentiate into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, and neuronal cells. The CSCs and ADSCs exhibited different characteristics. The selection of ADSCs was possible on Pluronic-grafted dishes that suppressed the CSCs from the fat tissues of cancer patients (i.e., cell-sorting dishes), which was explained by specific biomedical characteristics of Pluronic.


Assuntos
Adipócitos/fisiologia , Materiais Biocompatíveis , Células-Tronco Neoplásicas/fisiologia , Células-Tronco/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Poloxâmero , Poliestirenos
19.
Biomaterials ; 34(31): 7632-44, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23876761

RESUMO

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem and progenitor cells (HSPCs) for transplantation. However, the low number of HSPCs from a single UCB donor limits the direct transplantation of UCB to patients. Because little is known about the effects of the physical microenvironment on HSPC expansion, we investigated the ex vivo expansion of HSPCs cultured on biomaterials with different elasticities and grafted with different nanosegments. Polyvinylalcohol-co-itaconic acid (PVA-IA)-coated dishes with different stiffnesses ranging from a 3.7 kPa to 30.4 kPa storage modulus were used. Fibronectin or an oligopeptide (CS1, EILDVPST) was grafted onto the PVA-IA substrates. High ex vivo fold expansion of HSPCs was observed in the PVA-IA dishes grafted with fibronectin or CS1, which displayed an intermediate stiffness ranging from 12.2 kPa to 30.4 kPa. The fold expansion was more than 1.4 times higher than that cultured in tissue culture polystyrene dishes (TCPS, 12 GPa). Furthermore, HSPCs cultured in fibronectin or CS1-grafted PVA-IA-coated dishes with a stiffness of 12.2-30.4 kPa generated more pluripotent colony-forming units (CFU-GM and CFU-GEMM) than those in TCPS dishes. This result indicates that both the physical and biological properties of biomaterials affect the ex vivo expansion of HSPCs.


Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco/citologia , Proliferação de Células , Fibronectinas/metabolismo , Humanos , Oligopeptídeos/metabolismo
20.
Drug Des Devel Ther ; 7: 491-502, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23818760

RESUMO

PURPOSE: We evaluated the higher levels of carcinoembryonic antigen (CEA) secreted by the LoVo human colon carcinoma cells in a medium containing anticancer drugs. Drug-resistant LoVo cells were analyzed by subcutaneously xenotransplanting them into mice. The aim of this study was to evaluate whether the drug-resistant cells isolated in this study were cancer-initiating cells, known also as cancer stem cells (CSCs). METHODS: The production of CEA was investigated in LoVo cells that were cultured with 0-10 mM of anticancer drugs, and we evaluated the increase in CEA production by the LoVo cells that were stimulated by anticancer drug treatment. The expression of several CSC markers in LoVo cells treated with anticancer drugs was also evaluated. Following anticancer drug treatment, LoVo cells were injected subcutaneously into the flanks of severe combined immunodeficiency mice in order to evaluate the CSC fraction. RESULTS: Production of CEA by LoVo cells was stimulated by the addition of anticancer drugs. Drug-resistant LoVo cells expressed lower levels of CSC markers, and LoVo cells treated with any of the anticancer drugs tested did not generate tumors within 8 weeks from when the cells were injected subcutaneously into severe combined immunodeficiency mice. These results suggest that the drug-resistant LoVo cells have a smaller population of CSCs than the untreated LoVo cells. CONCLUSION: Production of CEA by LoVo cells can be stimulated by the addition of anticancer drugs. The drug-resistant subpopulation of LoVo colon cancer cells could stimulate the production of CEA, but these cells did not act as CSCs in in vivo tumor generation experiments.


Assuntos
Antineoplásicos/farmacologia , Antígeno Carcinoembrionário/biossíntese , Neoplasias do Colo/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Antígeno AC133 , Animais , Antígenos CD/análise , Linhagem Celular Tumoral , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos , Glicoproteínas/análise , Humanos , Camundongos , Camundongos SCID , Peptídeos/análise
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