RESUMO
BACKGROUND: Suitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa. METHODS: In vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval. RESULTS: Of the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (ß = 0.490), both IMDM (ß = 0.256) and DMEM (ß = 0.198) media and the protein supplements NCS (ß = 0.052) and FBS (ß = 0.022); while for L. loa L3, in addition to feeder cells (ß = 0.259) and both IMDM (ß = 0.401) and DMEM (ß = 0.385) media, the protein supplements BSA (ß = 0.029) were found important in maintaining the worm motility. CONCLUSIONS: The findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.
Assuntos
Loa/crescimento & desenvolvimento , Técnicas Microbiológicas/métodos , Microfilárias/crescimento & desenvolvimento , Parasitologia/métodos , Animais , Meios de Cultura/química , Avaliação Pré-Clínica de Medicamentos/métodos , Células Epiteliais/fisiologia , Células Alimentadoras/fisiologia , Filaricidas/isolamento & purificação , Haplorrinos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Loa/fisiologia , Locomoção , Microfilárias/fisiologia , Muda , Análise de SobrevidaRESUMO
INTRODUCTION: Ex vivo expansion of umbilical cord blood (UCB) is attempted to increase cell numbers to overcome the limitation of cell dose. Presently, suspension cultures or feeder mediated co-cultures are performed for expansion of hematopoietic stem cells (HSCs). Mesenchymal stem cells (MSCs) have proved to be efficient feeders for the maintenance of HSCs. Here, we have established MSCs-HSCs co-culture system with MSCs isolated from less invasive and ethically acceptable sources like umbilical cord tissue (C-MSCs) and placenta (P-MSCs). MSCs derived from these tissues are often compared with bone marrow derived MSCs (BM-MSCs) which are considered as a gold standard. However, so far none of the studies have directly compared C-MSCs with P-MSCs as feeders for ex vivo expansion of HSCs. Thus, we for the first time performed a systematic comparison of hematopoietic supportive capability of C and P-MSCs using paired samples. METHODS: UCB-derived CD34(+) cells were isolated and co-cultured on irradiated C and P-MSCs for 10 days. C-MSCs and P-MSCs were isolated from the same donor. The cultures comprised of serum-free medium supplemented with 25 ng/ml each of SCF, TPO, Flt-3 L and IL-6. After 10 days cells were collected and analyzed for phenotype and functionality. RESULTS: C-MSCs and P-MSCs were found to be morphologically and phenotypically similar but exhibited differential ability to support ex vivo hematopoiesis. Cells expanded on P-MSCs showed higher percentage of primitive cells (CD34(+)CD38(-)), CFU (Colony forming unit) content and LTC-IC (Long term culture initiating cells) ability. CD34(+) cells expanded on P-MSCs also exhibited better in vitro adhesion to fibronectin and migration towards SDF-1α and enhanced NOD/SCID repopulation ability, as compared to those grown on C-MSCs. P-MSCs were found to be closer to BM-MSCs in their ability to expand HSCs. P-MSCs supported expansion of functionally superior HSCs by virtue of reduction in apoptosis of primitive HSCs, higher Wnt and Notch activity, HGF secretion and cell-cell contact. On the other hand, C-MSCs facilitated expansion of progenitors (CD34(+)CD38(+)) and differentiated (CD34(-)CD38(+)) cells by secretion of IL1-α, ß, MCP-2, 3 and MIP-3α. CONCLUSIONS: P-MSCs were found to be better feeders for ex vivo maintenance of primitive HSCs with higher engraftment potential than the cells expanded with C-MSCs as feeders.
Assuntos
Proliferação de Células , Células-Tronco Mesenquimais/fisiologia , Animais , Antígenos CD34/metabolismo , Comunicação Celular , Diferenciação Celular , Técnicas de Cocultura , Células Alimentadoras/fisiologia , Feminino , Sangue Fetal/citologia , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Humanos , Transplante de Células-Tronco Mesenquimais , Camundongos Endogâmicos NOD , Camundongos SCID , Placenta/citologia , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Lesões Experimentais por Radiação/terapia , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
We demonstrated the possibility of enrichment of umbilical cord blood mononuclear fraction with early non-differentiated precursors under conditions of co-culturing with mesenchymal stromal cells from the human adipose tissue. It was established that umbilical cord blood mononuclear cells adhered to mesenchymal stromal cell feeder and then proliferate and differentiate into hemopoietic cells. In comparison with the initial umbilical cord blood mononuclear fraction, the cell population obtained after 7-day expansion contained 2-fold more CFU and 33.4 ± 9.5 and 24.2 ± 11.2% CD34(+) and CD133(+) cells, respectively, which corresponds to enrichment of precursor cell population by 148 ± 60. The proposed scheme of expansion of hemopoietic cells from umbilical cord blood is economically expedient and can widely used in biology and medicine.
Assuntos
Sangue Fetal/citologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Mesenquimais/fisiologia , Tecido Adiposo/citologia , Proliferação de Células , Separação Celular , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Células Alimentadoras/fisiologia , Feminino , HumanosRESUMO
Cell fate commitment during development is achieved through the expression of lineage-specific transcription factors. Recent studies have suggested that the expression of combinations of these lineage-specific transcription factors can convert adult somatic cells from one type to another. Here we report that the combination of p63, a master regulator of epidermal development and differentiation, and KLF4, a regulator of epidermal differentiation, is sufficient to convert dermal fibroblasts to a keratinocyte phenotype. Induced keratinocytes (KCs) expressed KC-specific proteins and had a transcriptome similar to KCs. Reprogramming to a KC phenotype was rapid and efficient with a vast majority of cells morphologically resembling and expressing KC-specific genes within a week of p63 and KLF4 transduction. Furthermore, p63 and KLF4 are capable of inducing a KC phenotype even in a cancerous cell line, highlighting their importance for epidermal specification. The robustness of the conversion process also allows the use of this as a model system to study the mechanisms of reprogramming.
Assuntos
Diferenciação Celular/fisiologia , Fibroblastos/citologia , Prepúcio do Pênis/citologia , Queratinócitos/citologia , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Membrana/genética , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Células Alimentadoras/fisiologia , Fibroblastos/fisiologia , Expressão Gênica/fisiologia , Células HCT116 , Humanos , Recém-Nascido , Queratinócitos/fisiologia , Fator 4 Semelhante a Kruppel , Masculino , Fenótipo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Cultura Primária de Células , Proteínas Repressoras/genética , Fatores de Transcrição/genéticaRESUMO
Human induced pluripotent stem cells (hiPSCs) need to be generated and expanded under clinically applicable culture conditions before they can be used for clinical application. In this study, we demonstrate that inactivated human mesenchymal stem cells (hMSCs) from different donors can be used as feeder cells to support the establishment and maintenance of hiPSCs. The hiPSCs we generated and expanded on hMSCs exhibited the typical morphology of human embryonic stem cells (hESCs), expressed undifferentiated pluripotent cell markers and genes, differentiated into all three germ layers via embryoid body and teratoma formation, and retained a normal chromosomal karyotype after 14 passages. However, we found that the rate of hiPSCs generation on hMSCs was 7.26%±2.09% compared with that on mouse embryonic fibroblasts (MEFs), and the calculated expansion efficiency of hiPSCs on hMSCs was lower than that on MEFs. hMSCs from various donors and different passages did not influence the results. These findings suggest that hMSCs can be used as feeder cells to derive and maintain hiPSCs, and thus provide another clinically feasible method for generating and expanding hiPSCs. However, the cytokines and adhesion molecules in this system should be identified to develop a preferable clinical culture condition for hiPSCs.
Assuntos
Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Proliferação de Células , Células Alimentadoras/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Adulto , Animais , Células da Medula Óssea/fisiologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Células-Tronco Embrionárias/citologia , Células Alimentadoras/fisiologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Cariótipo , Masculino , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Modelos AnimaisRESUMO
PURPOSE: Mouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application. METHODS: A novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2-3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells. RESULTS: ESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation. CONCLUSIONS: Human feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.
Assuntos
Comunicação Celular/fisiologia , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Endométrio/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Técnicas de Cocultura/métodos , Embrião de Mamíferos , Endométrio/fisiologia , Células Alimentadoras/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The use of unrestricted somatic stem cells (USSCs) holds great promise for future clinical applications. Conventionally, mouse embryonic fibroblasts (MEFs) or other animal-based feeder layers are used to support embryonic stem cell (ESC) growth; the use of such feeder cells increases the risk of retroviral and other pathogenic infection in clinical trials. Implementation of a human-based feeder layer, such as hUSSCs that are isolated from human sources, lowers such risks. Isolated cord blood USSCs derived from various donors were used as a novel, supportive feeder layer for growth of C4mES cells (Royan C4 ESCs). Complete cellular characterization using immunocytochemical and flow cytometric methods were performed on murine ESCs (mESCs) and hUSSCs. mESCs cultured on hUSSCs showed similar cellular morphology and presented the same cell markers of undifferentiated mESC as would have been observed in mESCs grown on MEFs. Our data revealed these cells had negative expression of Stat3, Sox2, and Fgf4 genes while showing positive expression for Pou5f1, Nanog, Rex1, Brachyury, Lif, Lifr, Tert, B2m, and Bmp4 genes. Moreover, mESCs cultured on hUSSCs exhibited proven differentiation potential to germ cell layers showing normal karyotype. The major advantage of hUSSCs is their ability to be continuously cultured for at least 50 passages. We have also found that hUSSCs have the potential to provide ESC support from the early moments of isolation. Further study of hUSSC as a novel human feeder layer may lead to their incorporation into clinical methods, making them a vital part of the application of human ESCs in clinical cell therapy.
Assuntos
Técnicas de Cocultura/métodos , Células-Tronco Embrionárias/fisiologia , Células Alimentadoras/fisiologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Corpos Embrioides/citologia , Células-Tronco Embrionárias/citologia , Células Alimentadoras/citologia , Sangue Fetal/citologia , Citometria de Fluxo , Marcadores Genéticos/genética , Humanos , Imunofenotipagem , Cariótipo , Camundongos , Camundongos SCID , Teratoma/químicaRESUMO
Human embryonic stem cells maintained on human amniotic epithelial cells (hESCs(hAEC)) are better preserved in an undifferentiated state and express pluripotency genes Oct4, Nanog, and Sox2 at higher levels compared with growth on mitotically inactivated mouse embryonic fibroblasts (hESCs(MEF)). Here we report that this correlates with the absence of the tumor suppressor and metabolic balancer gene, LKB1 expression in hESCs(hAEC). RNA interference knockdown of LKB1 in hESCs(MEF) resulted in upregulation of pluripotency marker genes of Oct4 and Nanog, while downregulation of differentiation markers (Runx1, AFP, GATA, Brachyury, Sox17 and Nestin). As in somatic cells, LKB1 controls p21/WAF1 expression by promoter binding in hESCs(MEF). Our results suggested that the absence of LKB1-mediated signaling is an important determinant of feeder cell-mediated support of hESC renewal.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Quinases Proteína-Quinases Ativadas por AMP , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células Alimentadoras/efeitos dos fármacos , Células Alimentadoras/metabolismo , Células Alimentadoras/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/farmacologiaRESUMO
We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as α-fetoprotein, albumin, α1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: indocyanine green dye uptake (≈ 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin E1 attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.
Assuntos
Ductos Biliares/fisiologia , Técnicas de Cultura de Células/métodos , Proliferação de Células , Meios de Cultura Livres de Soro/farmacologia , Hepatócitos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Células-Tronco/fisiologia , Ductos Biliares/citologia , Ductos Biliares/efeitos dos fármacos , Técnicas de Cultura de Células/estatística & dados numéricos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citotoxinas/farmacologia , Células Alimentadoras/citologia , Células Alimentadoras/fisiologia , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/citologia , Fígado/fisiologia , Testes de Função Hepática/métodos , Especificidade de Órgãos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.
Assuntos
Técnicas de Cultura de Células , Células Alimentadoras/fisiologia , Engenharia Genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Embrionárias/metabolismo , Células Alimentadoras/metabolismo , Feminino , Loci Gênicos , Recombinação Homóloga , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Cariótipo , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Neoplasias Experimentais/patologia , Telomerase/genética , Teratoma/patologia , Transdução Genética , Proteína Wnt3A/biossíntese , Proteína Wnt3A/genéticaRESUMO
Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and Iß1 and p63 (putative stem cell markers) in all culture conditions. The level of Iß1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.
Assuntos
Epitélio Corneano/fisiologia , Células Alimentadoras/fisiologia , Fibroblastos/fisiologia , Limbo da Córnea/citologia , Mucosa Bucal/citologia , Células 3T3 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Âmnio/citologia , Animais , Proliferação de Células , Forma Celular , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Derme/citologia , Epitélio Corneano/metabolismo , Oftalmopatias/terapia , Humanos , Queratina-3/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Proteínas de Neoplasias/metabolismo , Técnicas de Cultura de TecidosRESUMO
We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.
Assuntos
Amidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Alimentadoras/fisiologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Mama/citologia , Técnicas de Cultura de Células , Reprogramação Celular/efeitos dos fármacos , Colágeno , Combinação de Medicamentos , Células Epiteliais/citologia , Células Alimentadoras/citologia , Feminino , Humanos , Laminina , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Próstata/citologia , Neoplasias da Próstata/patologia , Proteoglicanas , Transplante HeterólogoRESUMO
Synergistic effects of mesenchymal stem cells (MSCs) isolated from bone marrow (BM), umbilical cord blood (UCB) and periosteum, and fibroblasts as mixed feeder cells (MFCs) on the expansion of hematopoietic progenitor cells (HPCs) were investigated in serum- and exogenous cytokine-free conditions. Enriched CD34(+) cells were cultured for 2weeks over the cell lines alone, individually, or selected combinations of them. When the cells were cultured over MFCs, the maximum increase in expansion of total nucleated cells and CD34(+)/CD38(-) cells was 157.3- and 128.6-fold, respectively. Furthermore, hematopoietic cytokine such as IL-6 and chemokines (e.g., IL-8, growth related oncogene (GRO), GRO-alpha, matrix metalloproteinase (MMP)-1, and MMP-3) were significantly increased in mixed feeder cells. Based on these results, MFCs can be more efficient for the ex vivo expansion of HPCs. These results strongly suggest that MFCs are more suitable for HPCs mass production.
Assuntos
Técnicas de Cultura de Células/métodos , Células Alimentadoras/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/fisiologia , Antígenos CD34/metabolismo , Células da Medula Óssea/fisiologia , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , HumanosRESUMO
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes that produce mAbs often reactive with microbial or autoantigens. Long-term culture of B-CLL clones would permit the collection and characterization of B-CLL mAbs to study antigen specificity and of B-CLL DNA to investigate molecular mechanisms promoting the disease. However, the derivation of long-term cell lines (eg, by EBV), has not been efficient. We have improved the efficiency of EBV B-CLL transformation of CpG oligonucleotide-stimulated cells by incubating patient peripheral blood mononuclear cells in the presence of an irradiated mouse macrophage cell line, J774A.1. Using this approach, peripheral blood mononuclear cells isolated from 13 of 21 B-CLL patients were transformed as documented by IGHV-D-J sequencing. Four clones grew and retained CD5 expression in culture for 2 to 4 months. However, despite documentation of EBV infection by expression of EBNA2 and LMP1, B-CLL cells died after removal of macrophage feeder cells. Nevertheless, using electrofusion technology, we generated 6 stable hetero-hybridoma cell lines from EBV-transformed B-CLL cells, and these hetero-hybridomas produced immunoglobulin. Thus, we have established enhanced methods of B-CLL culture that will enable broader interrogation of B-CLL cells at the genetic and protein levels.
Assuntos
Processos de Crescimento Celular/fisiologia , Transformação Celular Viral , Células Alimentadoras/citologia , Herpesvirus Humano 4/fisiologia , Leucemia Linfocítica Crônica de Células B/patologia , Macrófagos/citologia , Animais , Linhagem Celular Transformada , Transformação Celular Viral/genética , Transformação Celular Viral/fisiologia , Células Cultivadas , Técnicas de Cocultura , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células Alimentadoras/fisiologia , Herpesvirus Humano 4/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Macrófagos/fisiologia , Camundongos , Regulação para Cima , Proteínas Virais/genéticaRESUMO
Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal.
Assuntos
Âmnio/citologia , Células Epiteliais/fisiologia , Células Alimentadoras/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , MicroRNAs/genética , Fatores de Transcrição SOXB1/genética , Animais , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura/métodos , Regulação para Baixo/genética , Embrião de Mamíferos , Células Epiteliais/citologia , Células Alimentadoras/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , MicroRNAs/metabolismo , Gravidez , Fatores de Transcrição SOXB1/metabolismo , Fatores de TempoRESUMO
Kidney development is regulated by a coordinated reciprocal induction of metanephric mesenchymal (MM) and ureteric bud (UB) cells. Here, established MM and UB progenitor cell lines were recombined in three-dimensional Matrigel implants in SCID mice. Differentiation potential was examined for changes in phenotype, organization, and the presence of specialized proteins using immunofluorescence and bright-field and electron microscopy. Both cell types, when grown alone, did not develop into specialized structures. When combined, the cells organized into simple organoid structures of polarized epithelia with lumens surrounded by capillary-like structures. Tracker experiments indicated the UB cells formed the tubuloid structures, and the MM cells were the source of the capillary-like cells. The epithelial cells stained positive for pancytokeratin, the junctional complex protein ZO-1, collagen type IV, as well as UB and collecting duct markers, rearranged during transfection (RET), Dolichos biflorus lectin, EndoA cytokeratin, and aquaporin 2. The surrounding cells expressed α-smooth muscle actin, vimentin, platelet endothelial cell adhesion molecule 1 (PECAM), and aquaporin 1, a marker of vasculogenesis. The epithelium exhibited apical vacuoles, microvilli, junctional complexes, and linear basement membranes. Capillary-like structures showed endothelial features with occasional pericytes. UB cell epithelialization was augmented in the presence of MM cell-derived conditioned medium, glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), or fibronectin. MM cells grown in the presence of UB-derived conditioned medium failed to undergo differentiation. However, UB cell-derived conditioned medium induced MM cell migration. These studies indicate that tubulogenesis and vasculogenesis can be partially recapitulated by recombining individual MM and UB cell lineages, providing a new model system to study organogenesis ex vivo.