RESUMO
Mouse embryonic fibroblast (MEF) cells are commonly used as feeder cells to maintain the pluripotent state of stem cells. MEFs produce growth factors and provide adhesion molecules and extracellular matrix (ECM) compounds for cellular binding. In the present study, we compared the expression levels of Fgf2, Bmp4, ActivinA, Lif and Tgfb1 genes at the mRNA level and the level of Fgf2 protein secretion and Lif cytokine secretion at passages one, three and five of MEFs isolated from 13.5-day-old and 15.5-day-old embryos of NMRI and C57BL/6 mice using real-time PCR and enzyme-linked immunosorbent assay. We observed differences in the expression levels of the studied genes and secretion of the two growth factors in the three passages of MEFs isolated from 13.5-day-old and 15.5-day-old embryos, respectively. These differences were also observed between the NMRI and C57BL/6 strains. The results of this study suggested that researchers should use mice embryos that have different genetic backgrounds and ages, in addition to different MEF passages, when producing MEFs based on the application and type of their study.
Assuntos
Fator 2 de Crescimento de Fibroblastos , Fibroblastos , Animais , Diferenciação Celular , Células Cultivadas , Células Alimentadoras/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Patrimônio Genético , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Engineered epithelial cell sheets for clinical replacement of non-functional upper aerodigestive tract mucosa are regulated as medicinal products and should be manufactured to the standards of good manufacturing practice (GMP). The current gold standard for growth of epithelial cells for research utilises growth arrested murine 3T3 J2 feeder layers, which are not available for use as a GMP compliant raw material. Using porcine mucosal tissue, we demonstrate a new method for obtaining and growing non-keratinised squamous epithelial cells and fibroblast cells from a single biopsy, replacing the 3T3 J2 with a growth arrested primary fibroblast feeder layer and using pooled Human Platelet lysate (HPL) as the media serum supplement to replace foetal bovine serum (FBS). The initial isolation of the cells was semi-automated using an Octodissociator and the resultant cell suspension cryopreservation for future use. When compared to the gold standard of 3T3 J2 and FBS containing medium there was no reduction in growth, viability, stem cell population or ability to differentiate to mature epithelial cells. Furthermore, this method was replicated with Human buccal tissue, providing cells of sufficient quality and number to create a tissue engineered sheet.
Assuntos
Células Epiteliais/citologia , Fibroblastos/citologia , Mucosa Bucal/citologia , Engenharia Tecidual/métodos , Células 3T3 , Animais , Automação Laboratorial/instrumentação , Automação Laboratorial/métodos , Células Cultivadas , Criopreservação/métodos , Criopreservação/normas , Meios de Cultura/química , Células Epiteliais/metabolismo , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Fibroblastos/metabolismo , Humanos , Camundongos , Guias de Prática Clínica como Assunto , Engenharia Tecidual/normasRESUMO
Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS). The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.
Assuntos
Estenose da Valva Aórtica/patologia , Valva Aórtica/citologia , Valva Aórtica/patologia , Buffy Coat/efeitos da radiação , Calcinose/patologia , Separação Celular/métodos , Células Alimentadoras/citologia , Citometria de Fluxo/métodos , Linfócitos T/citologia , Valva Aórtica/metabolismo , Células Cultivadas , Células Alimentadoras/metabolismo , Humanos , Linfócitos T/metabolismoRESUMO
Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.
Assuntos
Antineoplásicos/metabolismo , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Células Matadoras Naturais/citologia , Ligante 4-1BB/metabolismo , Antígenos CD34/metabolismo , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Alimentadoras/metabolismo , Células HEK293 , Mobilização de Células-Tronco Hematopoéticas , Humanos , Interleucinas/metabolismo , Células Matadoras Naturais/metabolismo , Fenótipo , Doadores de TecidosRESUMO
Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. In this study, we investigated the impact of replacing irradiated 3T3 (i3T3) murine fibroblast feeder cells by irradiated human corneal fibroblasts (iHFL) on the expression of Sp1 and NFI and evaluated their contribution to the proliferative properties of human corneal epithelial cells (hCECs) in both monolayer cultures and human tissue engineered corneas (hTECs). hCECs co-cultured with iHFL could be maintained for up to two more passages than when they were grown with i3T3. Western Blot and electrophoretic mobility shift assays (EMSAs) revealed no significant difference in the feeder-layer dependent increase in Sp1 at both the protein and DNA binding level, respectively, between HCECs grown with either i3T3 or iHFL. On the other hand, a significant increase in the expression and DNA binding of NFI was observed at each subsequent passage when hCECs were co-cultured along with i3T3. These changes were found to result from an increased expression of the NFIA and NFIB isoforms in hCECs grown with i3T3. Exposure of hCECs to cycloheximide revealed an increased stability of NFIB that likely resulted from post-translational glycosylation of this protein when these cells were co-cultured with i3T3. In addition, iHFL were as efficient as i3T3 at preserving corneal, slow-cycling, epithelial stem cells in the basal epithelium of the reconstructed hTECs. Furthermore, we observed an increased expression of genes whose encoded products promote hCECs differentiation along several passages in hCECs co-cultured with either type of feeder layer. Therefore, the iHFL feeder layer appears to be the most effective at maintaining the proliferative properties of hCECs in culture most likely by preserving high levels of Sp1 and low levels of NFIB, which is known for its gene repressor and cell differentiation properties.
Assuntos
Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Células Alimentadoras/metabolismo , Fibroblastos/metabolismo , Células-Tronco/metabolismo , Engenharia Tecidual , Células 3T3 , Animais , Diferenciação Celular , Proliferação de Células , Técnicas de Cocultura , Células Epiteliais/citologia , Epitélio Corneano/citologia , Células Alimentadoras/citologia , Fibroblastos/citologia , Humanos , Camundongos , Células-Tronco/citologiaRESUMO
The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell's fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.
Assuntos
Técnicas de Cultura de Células/métodos , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Queratinócitos/fisiologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Derme/citologia , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Fibroblastos/citologia , Humanos , Queratinócitos/transplante , Transplante de Pele/métodosRESUMO
NK cell adoptive therapy is a promising cancer therapeutic approach, but there are significant challenges that limiting its feasibility and clinical efficacy. One difficulty is the paucity of clinical grade manufacturing platforms to support the large scale expansion of highly active NK cells. We created an NK cell feeder cell line termed 'NKF' through overexpressing membrane bound IL-21 that is capable of inducing robust and sustained proliferation (>10,000-fold expansion at 5 weeks) of highly cytotoxic NK cells. The expanded NK cells exhibit increased cytotoxic function against a panel of blood cancer and solid tumor cells as compared to IL-2-activated non-expanded NK cells. The NKF-expanded NK cells also demonstrate efficacy in mouse models of human sarcoma and T cell leukemia. Mechanistic studies revealed that membrane-bound IL-21 leads to an activation of a STAT3/c-Myc pathway and increased NK cell metabolism with a shift towards aerobic glycolysis. The NKF feeder cell line is a promising new platform that enables the large scale proliferation of highly active NK cells in support of large scale third party NK cell clinical studies that have been recently intiatied. These results also provide mechanistic insights into how membrane-bound IL-21 regulates NK cell expansion.
Assuntos
Células Alimentadoras/metabolismo , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Cultura Primária de Células/métodos , Animais , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Proliferação de Células , Técnicas de Cocultura , Voluntários Saudáveis , Humanos , Interleucinas/imunologia , Interleucinas/metabolismo , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/transplante , Camundongos , Neoplasias/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aim: To examine whether AKT-modified stromal cells expand human CD34+ hematopoietic stem cells (HSCs). Methods: Coculture, in vitro functional assays, immuno-fluorescence microscopy, flow cytometry. Results: M2-10B4 stromal cells (M2) modified with AKT1 (M2-AKT) expanded primitive CD34+38- HSCs, but affected their functionality. A chimeric feeder layer comprising naive human bone marrow-derived mesenchymal stromal cells and M2-AKT not only overcame the negative effects of M2-AKT, but, unexpectedly, also gave a synergistic effect on the growth and functionality of the HSCs. Conditioned medium of bone marrow stromal cells worked as effectively, but cell-cell contact between HSCs and M2-AKT cells was necessary for the synergistic effect of M2-AKT and bone marrow-derived mesenchymal stromal cells or their CM. Conclusion: Chimeric feeders expand HSCs.
Assuntos
Proliferação de Células , Células Alimentadoras/metabolismo , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Mesenquimais/metabolismo , Proteínas Proto-Oncogênicas c-akt , Animais , Técnicas de Cocultura , Células Alimentadoras/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/genética , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Human umbilical cord blood (HUCB) is a suitable source of hematopoietic stem cells (HSCs) for therapeutic transplantation. Different approaches have been used to expand the number of HSCs to increase the rate of HSC transplantation success in patients, such as using different cocktails of cytokines, feeder cell layers, and biocompatible scaffolds. microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. They play crucial roles in hematopoiesis including stem cell proliferation, differentiation, stemness, and self-renewal properties. Here, we studied the UCB-derived CD34+ cell expansion and the miRNA signatures of CD34+ cells on two- and three-dimensional (2D and 3D) culture conditions. We successfully expanded the UCB-derived CD34+ cells in both liquid culture (2D) and on aminated polyethersulfone nanofiber scaffolds (3D). Next, we identified the miRNA signature of CD34+ cells and their target genes. We found 58 dysregulated miRNAs in 3D culture condition and 34 dysregulated miRNAs in 2D culture condition when compared to the freshly isolated CD34+ cells. Various types of target genes were also predicted in both conditions using two online databases.
Assuntos
Antígenos CD34/metabolismo , Sangue Fetal/metabolismo , MicroRNAs/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Células Alimentadoras/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Nanofibras/químicaRESUMO
Adipose stromal/progenitor cells (ASCs) can differentiate into adipocytes in the course of adipogenesis. This process is governed by systemic factors and signals of the adipose stem cell niche. ASCs isolated from fat tissues and amplified in vitro provide an essential and reliable model system to study adipogenesis. However, current cell culture models routinely grow ASCs on plastic surfaces largely missing niche parameters. In the present communication, we employed human foreskin fibroblasts (HFFs) monolayers as feeder cells for ASCs, which were isolated from human subcutaneous white adipose tissue and amplified in vitro. We found that PPARγ2 and several adipocyte markers were significantly higher expressed in differentiated ASCs growing on feeder layers relative to plastic dishes. Moreover, a significant higher number of adipocytes was generated from ASCs cultured on feeder layer and these adipocytes contained larger fat droplets. Insulin strongly stimulated glucose uptake into adipocytes produced on feeder layer suggesting that these cells show characteristic metabolic features of fat cells. Finally, we show that the HFF feeder layer allows adipogenic differentiation of low-density-seeded ASCs. In conclusion, we demonstrate that the HFF feeder layer increases adipocyte differentiation of ASCs and allows differentiation of low density seeded progenitor cells into functional adipocytes.
Assuntos
Adipogenia , Tecido Adiposo/citologia , Células Alimentadoras/metabolismo , Fibroblastos/metabolismo , Prepúcio do Pênis/citologia , Células-Tronco Mesenquimais/citologia , Adulto , Técnicas de Cocultura/métodos , Meios de Cultivo Condicionados/farmacologia , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-IdadeRESUMO
Ex vivo expansion of hematopoietic progenitors is considered as an attractive tool to increase the number of stem and progenitor cells (HSPCs) for cell therapy. The efficacy of ex vivo expansion is strongly depends on the feeder cell activity to mimic hematopoietic microenvironment. Here we demonstrated, that combination of mitomycin C-induced growth arrest and tissue-related O2 (physiological hypoxia) modulated stromal capacity of adipose tissue derived stromal cells (ASCs). Growth arrest did not affect viability, stromal phenotype and multilineage potential of ASCs permanently expanded at tissue-related O2. Meanwhile, the PCR analysis revealed an up-regulation of genes, encoded molecules of cell-cell (ICAM1, HCAM/CD44) and cell-matrix adhesion (ITGs), extracellular matrix production (COLs) and remodeling (MMPs, HAS1) in growth-arrested ASCs at physiological hypoxia in comparison with ambient O2 (20%). The number of ICAM-1 positive ASCs was increased under low O2 as well. These alterations contributed into the ex vivo expansion of cord blood HSPCs providing the preferential production of primitive HSPCs. The number of cobblestone area forming cell (CAFC) colonies was 1.5-fold higher at physiological hypoxia (p < 0.05). CAFCs considered as long-term culture-initiating cells (LTC-IC) known to support long-term hematopoiesis restoration in vivo. The presented data may be applicable in the development of upscale protocols of HSPC expansion.
Assuntos
Tecido Adiposo/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Oxigênio/metabolismo , Células Estromais/citologia , Tecido Adiposo/metabolismo , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Células Cultivadas , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Oxigênio/análise , Células Estromais/metabolismoRESUMO
Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-ß signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.
Assuntos
Células Epiteliais/citologia , Miosina Tipo II/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Células-Tronco/citologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , Células Alimentadoras/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
There is considerable interest in the ex vivo propagation of primary human basal epithelial stem/progenitor cells with a view to their use in drug development, toxicity testing and regenerative medicine. These cells can be expanded in co-culture with mitotically inactivated 3T3-J2 murine embryonic feeder cells but, similar to other epithelial cell culture systems employing 3T3-J2 cells, the aspects of cross-talk between 3T3-J2 cells and human airway basal cells that are critical for their expansion remain largely unknown. In this study, we investigated secreted growth factors that are produced by 3T3-J2 cells and act upon primary human airway basal cells. We found robust production of hepatocyte growth factor (HGF) from fibroblast feeder cells following mitotic inactivation. Consistent with the limited cross-species reactivity of murine HGF on the human HGF receptor (MET; HGFR), MET inhibition did not affect proliferative responses in human airway basal cells and HGF could not replace feeder cells in this culture system. However, we found that murine HGF is not completely inactive on human airway epithelial cells or cancer cell lines but stimulates the phosphorylation of GRB2-associated-binding protein 2 (GAB2) and signal transducer and activator of transcription 6 (STAT6). Although HGF induces phosphorylation of STAT6 tyrosine 641 (Y641), there is no subsequent STAT6 nuclear translocation or STAT6-driven transcriptional response. Overall, these findings highlight the relevance of cross-species protein interactions between murine feeder cells and human epithelial cells in 3T3-J2 co-culture and demonstrate that STAT6 phosphorylation occurs in response to MET activation in epithelial cells. However, STAT6 nuclear translocation does not occur in response to HGF, precluding the transcriptional activity of STAT6.
Assuntos
Comunicação Celular , Células Epiteliais/metabolismo , Células Alimentadoras/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Mucosa Respiratória/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura , Ativação Enzimática , Células Epiteliais/citologia , Células Alimentadoras/citologia , Humanos , Camundongos , Mucosa Respiratória/citologia , Fator de Transcrição STAT6/metabolismoRESUMO
Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αß T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)-engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting.
Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/citologia , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/metabolismo , Complexo CD3/imunologia , Proliferação de Células/efeitos dos fármacos , Células Alimentadoras/metabolismo , Humanos , Imunofenotipagem , Interleucina-2/farmacologia , Células K562 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Coculture of hematopoietic stem cells (HSC) with primary stromal cells from HSC niches supports the maintenance and expansion of HSC and progenitors ex vivo. However, a major drawback is the availability of primary human samples for research and clinical applications. We investigated the use of in vitro derived osteoblasts as a new source of feeder cells and characterized the molecular pathways that mediate their growth-promoting activities. First, we compared the growth and differentiation modulating activities of mesenchymal stromal cells (MSC)-derived osteoblasts (M-OST) with those of their undifferentiated precursor on umbilical cord blood (UCB) progenitors. Feeder-free cultures were also included as baseline control. Cell growth and expansion of hematopoietic progenitors were significantly enhanced by both feeder cell types. However, progenitor cell growth was considerably greater with M-OST. Coculture also promoted the maintenance of immature CD34+ progenitor subsets and modulated in a positive fashion the expression of several homing-related cell surface receptors, in a feeder-specific fashion. Serial transplantation experiments revealed that M-OST coculture supported the maintenance of long-term lympho-myeloid reconstituting HSC that provided engraftment levels that were generally superior to those from MSC cocultures. Mechanistically, we found that coculture with M-OST was associated with enhanced beta-catenin (ß-Cat) activity in UCB cells and that abrogation of ß-Cat/T-cell factor activity blunted the growth-promoting activity of the M-OST coculture. Conversely, Notch inhibition reduced UCB cell expansion, but to a much lesser extent. In conclusion, this study demonstrates that M-OST are excellent feeder cells for HSC and progenitors, and it identifies key molecular pathways that are responsible for the growth-enhancing activities of osteoblasts on UCB progenitors.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Animais , Antígenos CD34/metabolismo , Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Técnicas de Cocultura/métodos , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Osteoblastos/metabolismoRESUMO
The developmental progression of conventional DC has been quite well defined, yet the developmental pathway of monocyte-derived, GM-CSF-driven DC is less well understood. We addressed this issue by establishing an isolation strategy that identifies five distinct GM-CSF derived cell types. Expression of Ly6C and CD115 (Csf-1R) was used to identify and isolate four populations. One of the populations could be further separated based on CD11c expression, distinguishing five populations. We further defined these cells based on expression of transcription factors and markers of early and later stages of myeloid development. These discreet developmental stages corresponded well with previously defined populations: Common Myeloid Progenitors (CMP), Granulocyte/Macrophage Progenitors (GMP), Monocytes, as well as Monocyte-derived macrophages (moMac) and Monocyte-derived DC (moDC). Finally, within the moMac population we also identified moDC precursor activity (moDP) that could be distinguished from moMac and moDC based on their level of MHC class II expression and developmental plasticity.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células Mieloides/citologia , Células Mieloides/metabolismo , Animais , Antígenos Ly/metabolismo , Biomarcadores/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Separação Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , Células Alimentadoras/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Inflamação/genética , Inflamação/patologia , Cinética , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Células Mieloides/efeitos dos fármacos , Fenótipo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismoRESUMO
Gene transfer into normal quiescent human B cells is a challenging procedure. The present study aimed to investigate whether it is possible to increase the levels of transgene expression by using various types of promoters to drive the expression of selected genesofinterest. To produce lentiviral particles, the present study used the 2nd generation psPAX2 packaging vector and the vesicular stomatitis virus expressing envelope vector pMD2.G. Subsequently, lentiviral vectors were generated containing various promoters, including cytomegalovirus (CMV), elongation factor1 alpha (EF1α) and spleen focusforming virus (SFFV). The present study was unable to induce satisfactory transduction efficiency in quiescent normal B cells; however, infection of normal B cells with EpsteinBarr virus resulted in increased susceptibility to lentiviral transduction. In addition, the SFFV promoter resulted in a higher level of transgene expression compared with CMV or EF1α promoters. As a proofof concept that this approach allows for stable gene expression in normal B cells, the present study used bicistronic lentiviral vectors with genes encoding fluorescent reporter proteins, as well as Xbox binding protein1 and binding immunoglobulin protein.
Assuntos
Linfócitos B/metabolismo , Técnicas de Transferência de Genes , Regiões Promotoras Genéticas , Adulto , Linfócitos B/virologia , Biomarcadores/metabolismo , Ligante de CD40/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Células Alimentadoras/metabolismo , Feminino , Fluorescência , Expressão Gênica , Células HEK293 , Herpesvirus Humano 4/fisiologia , Humanos , Sítios Internos de Entrada Ribossomal/genética , Lentivirus/genética , Masculino , Pessoa de Meia-Idade , Vírus Formadores de Foco no Baço/fisiologia , Transdução Genética , TransgenesRESUMO
BACKGROUND: Mechanical stimuli play important roles in the proliferation and differentiation of adult stem cells. However, few studies on their effects on induced pluripotent stem cells (iPSCs) have been published. METHODS: Human dermal fibroblasts were seeded onto flexible membrane-bottom plates, and infected with retrovirus expressing the four reprogramming factors OCT4, SOX2, KLF, and c-MYC (OSKM). The cells were subjected to equiaxial stretching (3% or 8% for 2, 4, or 7 days) and seeded on feeder cells (STO). The reprogramming into iPSCs was evaluated by the expression of pluripotent markers, in vitro differentiation into three germ layers, and teratoma formation. RESULTS: Equiaxial stretching enhanced reprogramming efficiency without affecting the viral transduction rate. iPSCs induced by transduction of four reprogramming factors and application of equiaxial stretching had characteristics typical of iPSCs in terms of pluripotency and differentiation potentials. CONCLUSIONS: This is the first study to show that mechanical stimuli can increase reprogramming efficiency. However, it did not enhance the infection rate, indicating that mechanical stimuli, defined as stretching in this study, have positive effects on reprogramming rather than on infection. Additional studies should evaluate the mechanism underlying the modulation of reprogramming of somatic cells into iPSCs.
Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Estresse Mecânico , Biomarcadores/metabolismo , Proliferação de Células/genética , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
Pluripotent stem cells have been proposed as an unlimited source of pancreatic ß cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional ß cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ ß-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes.
Assuntos
Autorrenovação Celular/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pâncreas/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/metabolismo , Transativadores/metabolismo , Transplante HeterólogoRESUMO
Ex vivo expansion of hematopoietic stem cells (HSCs) with most current methods can hardly satisfy clinical application requirement. While in vivo, HSCs efficiently self-renew in niche where they interact with 3D extracellular matrix and stromal cells. Therefore, co-cultures of CD34+ cells and mesenchyme stem cells derived from human amniotic membrane (hAMSCs) on the basis of biomimetic macroporous three-dimensional (3D) poly(ε-caprolactone) (PCL) scaffolds are developed, where scaffolds and hAMSCs are applied to mimic structural and cellular microenvironment of HSCs. The influence of scaffolds, feeder cells, and contact manners on expansion and stemness maintenance of CD34+ cells is investigated in this protocol. Biomimetic scaffolds-dependent co-cultures of CD34+ cells and hAMSCs can effectively promote the expansion of CD34+ cells; meanwhile, indirect contact is superior to direct contact. The combination of biomimetic scaffolds and hAMSCs represents a new strategy for achieving clinical-scale ex vivo expansion of CD34+ cells.