RESUMO
Human extended pluripotent stem cells (EPSCs), with bidirectional chimeric ability to contribute to both embryonic and extraembryonic lineages, can be obtained and maintained by converting conventional pluripotent stem cells using chemicals. However, the transition system is based on inactivated mouse fibroblasts, and the underlying mechanism is not clear. Here we report a Matrigel-based feeder-free method to convert human embryonic stem cells and induced pluripotent stem cells into EPSCs and demonstrate the extended pluripotency in terms of molecular features, chimeric ability, and transcriptome. We further identify chemicals targeting glycolysis and histone methyltransferase to facilitate the conversion to and maintenance of feeder-free EPSCs. Altogether, our data not only establish a feeder-free system to generate human EPSCs, which should facilitate the mechanistic studies of extended pluripotency and further applications, but also provide additional insights into the transitions among different pluripotent states.
Assuntos
Células Alimentadoras/citologia , Células-Tronco Pluripotentes/citologia , Linhagem Celular , Quimera/fisiologia , Células Alimentadoras/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Indóis/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Piridonas/farmacologiaRESUMO
The present study aimed to investigate the ability of a novel form of the anti-angiogenic molecular antibody drug to induce Myeloma cell death after cultivation upon endothelial feeder cells. Bevacizumab-loaded chitosan (BCS) nanoparticles (NPs) were prepared by the ionic gelation method. Human U266 cell line and human umbilical vein endothelial cells were co-cultured for 72 h and treated with BCS nanoparticles (10µM) to study their impact on inhibition of cell growth and induction of apoptosis. Death assessments, P53 pro-apoptotic marker expression and the VEGF level were investigated by flow-cytometric analyses of the Annexin V, immunocytochemistry and ELISA, respectively. The endothelial monolayer co-culture showed protection of myeloma cells from apoptosis when exposed to NPs or without any treatment. In present of bevacizumab, the VEGF factor was effectively suppressed, and the p53 expression was significantly increased in bevacizumab-treated myeloma cells co-cultured with HUVECs, compared to other groups. BCS was capable of disturbing the effective interconnections of myeloma cells and HUVECs as supportive cells. Disruptions of tumor cells' connections with their microenvironment and blocking their possible supportive pathways might be a potential strategy to eradicate tumor cells and finally cure such types of cancer.
Assuntos
Apoptose , Bevacizumab/farmacologia , Células Alimentadoras/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Mieloma Múltiplo/patologia , Nanopartículas/administração & dosagem , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Proliferação de Células , Técnicas de Cocultura , Humanos , Mieloma Múltiplo/tratamento farmacológico , Nanopartículas/química , Neovascularização Patológica/patologiaRESUMO
The development of porcine expanded potential stem cells (pEPSCs) provides an invaluable tool for investigation of porcine stem cell pluripotency and opens a venue for research in biotechnology, agriculture, and regenerative medicine. Since the derivation of pEPSC from porcine pre-implantation embryos has been demanding in resource supply and technical challenges, it is more feasible and convenient for most laboratories to derive this new type of porcine stem cells by reprogramming somatic cells. In this chapter, we describe the detailed procedures for reprogramming porcine fetal fibroblast cells to EPSCiPSC with the eight reprogramming factors cloned on the piggyBac vectors followed by a selection for pluripotent cells independent of transgene expression using the EPSC media. This technique allows the generation of pEPSCs for stem cell research, genome editing, biotechnology, and agriculture.
Assuntos
Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição/metabolismo , Animais , Criopreservação/métodos , Células Alimentadoras/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mitomicina/farmacologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Suínos , Fatores de Transcrição/genética , TransfecçãoRESUMO
BACKGROUND AIMS: Natural killer (NK) cells are promising cells for immunotherapy of cancer, and there are ongoing efforts to improve their ex vivo expansion to clinically relevant numbers. This study focused on the development of a C1-, C2-, Bw4 killer cell immunoglobulin-like receptor (KIR) ligand and NKG2A ligand-containing feeder cell line for autonomous expansion of functional NK cells. METHODS: PC3PSCA-derived feeder cells expressing IL-2, 4-1BBL and membrane-bound IL-15-mutDAP12 (mIL-15d) fusion protein in combinations or alone were generated and used for expansion. Expanded NK cells were analyzed with respect to subpopulations, expression of NK cell receptors and immune checkpoint molecules as well as their cytotoxicity against K562 cells, cetuximab-marked tumor cells and autologous B cells. RESULTS: Only combinatorial expression of IL-2 plus 4-1BBL or IL-2, 4-1BBL plus mIL-15d in feeder cells efficiently expanded NK cells and supported selective outgrowth of NK cells from peripheral blood mononuclear cell samples. Best expansion of NK cells was achieved using PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells. Such expanded NK cells exhibited upregulation of natural cytotoxicity receptors, DNAM-1 and NKG2C and induced expression of high affinity IL-2 receptor, which were paralleled by attenuated KIR and increased expression of NKG2A and ILT2. In addition, elevated TIM-3 levels were noted and PD-1 and T cell immunoreceptor with Ig and ITIM domain (TIGIT) levels remained low. Expanded NK cells were highly cytolytic when encountering K562 cells and cetuximab-marked target cells but remained unresponsive to autologous B cells and target cells with protective levels of human leukocyte antigen. CONCLUSIONS: Collectively, the results demonstrate the feasibility of PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells for robust expansion of NK cells, which remain tolerant to self and could be used in the future for adoptive cell therapy of cancer.
Assuntos
Autoantígenos/imunologia , Células Alimentadoras/citologia , Tolerância Imunológica , Células Matadoras Naturais/citologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos CD/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cetuximab/farmacologia , Células Alimentadoras/efeitos dos fármacos , Células HEK293 , Humanos , Tolerância Imunológica/efeitos dos fármacos , Interleucina-2/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , LigantesRESUMO
BACKGROUND: Optimal expansion of therapeutic natural killer (NK) cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-cell expansion, but few SFM systems have been developed for NK cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum-containing medium for their ability to support NK cell expansion and function. METHODS: Human peripheral blood NK cells were expanded in selected media by recursive weekly stimulation with K562-based feeder cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts. RESULTS: TexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production. DISCUSSION: The AIM V + ICSR SFM system supports excellent ex vivo expansion of clinical-grade NK cells with the phenotype and function needed for adoptive immunotherapy.
Assuntos
Meios de Cultura Livres de Soro/farmacologia , Células Alimentadoras/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos T/metabolismo , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Meios de Cultura Livres de Soro/química , Citotoxicidade Imunológica , Proteína Ligante Fas/metabolismo , Humanos , Células K562 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Adoptive cell therapy using ex vivo expanded lymphocytes has shown remarkable efficacy in tumor immunotherapy recently. Among various transfused immune cells, T lymphocytes are the most widely used since they are critical mediators of the immune system and have the capacity to kill tumor cells. However, there are drawbacks in the expanded T cells for transfusion including limited cytotoxicity, limited proliferation and lack of specificity. To improve the quality of these ex vivo expanded T cells, we have designed a new method to expand a group of T cells which are named bispecific antibodies activated T cells. It is the first time that such cells are induced by introducing the bispecific antibody drug (blinatumomab) and feeder cells (normal B cells and irradiated B cell originated lymphoma cells) to the traditional T cells culture system. Culture of freshly isolated human peripheral blood mononuclear cells in this newly designed cell culture system enabled these expanded T cells that (a) displayed a robust proliferation ability; (b) showed fully activated phenotype and enhanced cytokines production; (c) had a low proportion of CD4+CD25+ T regulatory cells and (d) exhibited strengthened cytotoxicity at relatively low effector: target ratios. This work further confirmed the feasibility of rapid induction and expansion of large amounts of human T cells in vitro by using bispecific antibodies and feeder cells. This strategy could also be used for other immune cells rapid expansion and help to improve the quality of these expanded immune cells for adoptive transfusion.
Assuntos
Anticorpos Biespecíficos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Imunoterapia Adotiva , Ativação Linfocitária , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Antígenos CD19/análise , Antígenos CD19/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Antígenos CD4/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Alimentadoras/efeitos dos fármacos , Células Alimentadoras/imunologia , Humanos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Single-cell cloning during hybridoma production ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can be stably maintained. Cloning of hybridoma cells in semisolid medium is one of the most commonly used methods for producing single-cell clones. The technique is easy, but, because it is performed in two stages, it does take longer than other methods. Not all cells will grow in soft agar, and there may be a bias on the type of colony that appears. However, most of the commonly used myeloma fusion partners have relatively good cloning efficiencies in soft agar, and, consequently, so do most hybridomas. Even though every attempt is made to ensure that the cells are in a single-cell suspension before plating, there is no way to guarantee that the colonies do not arise from two cells that were stuck together. Therefore, single-cell cloning in soft agar should be repeated at least twice before the cells are considered clonal.
Assuntos
Ágar/farmacologia , Clonagem Molecular/métodos , Hibridomas/citologia , Análise de Célula Única , Animais , Proliferação de Células/efeitos dos fármacos , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , HumanosRESUMO
Hair follicles (HFs) are a wellcharacterized niche for adult stem cells (SCs), and include epithelial and melanocytic SCs. HF cells are an accessible source of multipotent adult SCs for the generation of the interfollicular epidermis, HF structures and sebaceous glands in addition to the reconstitution of novel HFs in vivo. In the present study, it was demonstrated that HF cells are able to be induced to differentiate into cardiomyocytelike cells in vitro under specific conditions. It was determined that HF cells cultured on OP9 feeder cells in KnockOutDulbecco's modified Eagle's medium/B27 in the presence of vascular endothelial growth factors differentiated into cardiomyocytelike cells that express markers specific to cardiac lineage, but do not express noncardiac lineage markers including neural stem/progenitor cell, HF bulge cells or undifferentiated spermatogonia markers. These cardiomyocytelike cells exhibited a spindle and filamentshaped morphology similar to that presented by cardiac muscles and exhibited spontaneous beating that persisted for over 3 months. These results demonstrate that SC reprogramming and differentiation may be induced without resulting in any genetic modification, which is important for the clinical applications of SCs including tissue and organ regeneration.
Assuntos
Folículo Piloso/citologia , Miócitos Cardíacos/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/farmacologia , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Fenótipo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
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
Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells' phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes' proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer.
Assuntos
Toxina da Cólera/farmacologia , Células Alimentadoras/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Isoproterenol/farmacologia , Queratinócitos/efeitos dos fármacos , Células 3T3 , Adulto , Idoso , Animais , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultura/farmacologia , AMP Cíclico/farmacologia , Feminino , Fibroblastos/citologia , Humanos , Queratinócitos/citologia , Camundongos , Pessoa de Meia-Idade , Modelos EstatísticosRESUMO
Purpose: Daratumumab and its use in combination with other agents is becoming a new standard of care for the treatment of multiple myeloma. We mechanistically studied how daratumumab acts on natural killer (NK) cells.Experimental Design: Quantities of NK cells in peripheral blood and/or bone marrow of patients with multiple myeloma or healthy donors were examined by flow cytometry. NK-cell apoptosis and the associated mechanism were assessed by flow cytometry and immunoblotting. Patients' NK cells were expanded in vitro using feeder cells. Combination treatment of daratumumab and expanded NK cells was performed using an MM.1S xenograft animal model.Results: CD38-/low NK cells survived, whereas CD38+ NK cells were almost completely eliminated, in peripheral blood and bone marrow of daratumumab-treated multiple myeloma patients. NK-cell depletion occurred due to daratumumab-induced NK-cell fratricide via antibody-dependent cellular cytotoxicity. Consequently, CD38-/low NK cells were more effective for eradicating multiple myeloma cells than were CD38+ NK cells in the presence of daratumumab. Blockade of CD38 with the F(ab)2 fragments of daratumumab inhibited the antibody-mediated NK-cell fratricide. CD38-/low NK cells displayed a significantly better potential for expansion than CD38+ NK cells, and the expanded NK cells derived from the former population were more cytotoxic than those derived from the latter against multiple myeloma cells. Therefore, infusion of ex vivo-expanded autologous NK cells from daratumumab-treated patients may improve the antibody therapy.Conclusions: We unravel a fratricide mechanism for daratumumab-mediated NK-cell depletion and provide a potential therapeutic strategy to overcome this side effect in daratumumab-treated patients with multiple myeloma. Clin Cancer Res; 24(16); 4006-17. ©2018 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Mieloma Múltiplo/tratamento farmacológico , ADP-Ribosil Ciclase 1/imunologia , Animais , Anticorpos Monoclonais/efeitos adversos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Células Alimentadoras/efeitos dos fármacos , Citometria de Fluxo , Xenoenxertos , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/patologia , Glicoproteínas de Membrana/imunologia , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologiaRESUMO
Graphene oxide (GO), with good hydrophilicity and biocompatibility, is widely explored as a carrier for various factors in the field of stem cell differentiation. However, its function of sustaining the stemness of mouse embryonic stem cells (mESCs) and the underlying mechanisms of this process remains undiscovered. Herein, we explored the biofunction of GO on mESCs and revealed the involved signaling pathways and key gene. The alkaline phosphatase activity detection, pluripotency genes quantification and the teratomas formation in vivo confirmed that GO nanosheets could sustain the self-renewal ability of mESCs instead of influencing its pluripotency. The underlying signaling pathways were uncovered by RNA-seq that integrin signaling pathway was involved in the biofunction of GO on mESCs and Vinculin turned to be a key gene for the effect of GO. Further experiments confirmed that the downregulation of Vinculin influenced the fate of mESCs through decreasing the expression of MEK1. Altogether, the study demonstrated for the first time that GOs hold the potential in sustaining the self-renewal of mESCs and clarified the mechanism of this function, which make it play a new role in stem cell research and regenerative medicine.
Assuntos
Autorrenovação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Grafite/farmacologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Nanoestruturas/química , Vinculina/genética , Fosfatase Alcalina/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos/citologia , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Integrinas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Nanoestruturas/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Vinculina/metabolismoRESUMO
In this chapter, we describe a protocol to induce at a high rate the differentiation of brown/brown like adipocyte progenitors (BAPs) derived from human induced pluripotent stem cells (hiPSCs). We also describe culture conditions to maintain hiPSCs and to derive hiPSC-BAPs.This novel culture system provides an unlimited source of human brown adipocytes and a unique means for studying events regulating the generation and recruitment of human BAPs.
Assuntos
Adipócitos Marrons/citologia , Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Amidas/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Alimentadoras/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Camundongos , Mitomicina/farmacologia , Piridinas/farmacologiaRESUMO
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
Cultured epidermal autografts have been used worldwide since 1981 for patients with extensive third-degree burn wounds and limited skin donor sites. Despite significant progress in techniques toward improving clinical outcome of skin grafts, the long in vitro preparation time of cultured autografts has remained a major factor limiting its widespread use. Here, we show that pharmacological inhibition of TGF-ß signaling promotes the expansion of human epidermal keratinocytes (HEKs) with high proliferative potential in co-cultures with both murine 3T3-J2 cells and human feeder cells, including dermal fibroblasts and preadipocytes. In contrast, TGF-ß signaling inhibition does not enhance the growth of HEKs in a serum- and feeder-free condition, an alternative approach to propagate HEKs for subsequent autograft production. Our results have important implications for the use of TGF-ß signaling inhibition as a viable therapeutic strategy for improving Green's methodology and for more efficient production of customized skin autografts with human feeder cells.
Assuntos
Técnicas de Cocultura , Células Epidérmicas , Células Alimentadoras/citologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Células 3T3 , Animais , Modelos Animais de Doenças , Epiderme/efeitos dos fármacos , Células Alimentadoras/efeitos dos fármacos , Humanos , Camundongos , Transplante de Pele/métodosRESUMO
Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.
Assuntos
Reprogramação Celular , Técnicas de Cocultura/métodos , Neoplasias/patologia , Amidas/farmacologia , Animais , Transformação Celular Neoplásica , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Ratos , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
A series of experiments on co-culturing of Mel IL melanoma cells and mesenhymal stem cells showed that these cells do not influence proliferation of each other, but we observed weaker adhesion of stromal stem cells to plastic in cocultures where with melanoma cells were grown on mesenhymal stem cells feeder. Cell proliferation was also considerably influenced by experimental conditions, which should be taken into account for correct interpretation of obtained results. The principles of experiments on co-culturing of cancer and stromal cells are formulated that take into account the most important factors influencing cell behavior and minimize the probability of artifact results. It was concluded that co-culturing conditions cells significantly affect the experimental results and can be the source of conflicting conclusions on mutual influence of stromal and cancer cells in vitro.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Meios de Cultura/farmacologia , Células Alimentadoras/efeitos dos fármacos , Melanoma/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Tecido Adiposo/citologia , Animais , Comunicação Celular/efeitos dos fármacos , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Interpretação Estatística de Dados , Células Alimentadoras/citologia , Humanos , Melanoma/patologia , Células-Tronco Mesenquimais/citologia , Cultura Primária de Células , Ratos , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
Hepatocyte-like cells (HLCs) generated in vitro from human pluripotent stem cells (hPSCs) provide an invaluable resource for basic research, regenerative medicine, drug screening, toxicology, and modeling of liver disease and development. This unit describes a small-molecule-driven protocol for in vitro differentiation of hPSCs into HLCs without the use of growth factors. hPSCs are coaxed through a developmentally relevant route via the primitive streak to definitive endoderm (DE) using the small molecule CHIR99021 (a Wnt agonist), replacing the conventional growth factors Wnt3A and activin A. The small-molecule-derived DE is then differentiated to hepatoblast-like cells in the presence of dimethyl sulfoxide. The resulting hepatoblasts are then differentiated to HLCs with N-hexanoic-Tyr, Ile-6 aminohexanoic amide (Dihexa, a hepatocyte growth factor agonist) and dexamethasone. The protocol provides an efficient and reproducible procedure for differentiation of hPSCs into HLCs utilizing small molecules. © 2016 by John Wiley & Sons, Inc.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Hepatócitos/citologia , Células-Tronco Pluripotentes/citologia , Bibliotecas de Moléculas Pequenas/farmacologia , Técnicas de Cultura de Tecidos/métodos , Endoderma/citologia , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismoRESUMO
INTRODUCTION: Growth-arrested feeder cells following Mitomycin C treatment are instrumental in stem cell culture allowing development of regenerative strategies and alternatives to animal testing in drug discovery. The concentration of Mitomycin C and feeder cell type was described to affect feeder performance but the criticality of feeder cell exposure density was not addressed. We hypothesize that the exposure cell density influences the effectiveness of Mitomycin C in an arithmetic manner. METHODS: Three different exposure cell densities of Swiss 3T3 fibroblasts were treated with a range of Mitomycin C concentrations for 2h. The cells were replaced and the viable cells counted on 3, 6, 9, 12 and 20days. The cell extinctions were compared with doses per cell which were derived by dividing the product of concentration and volume of Mitomycin C solution with exposure cell number. RESULTS: The periodic post-treatment feeder cell extinctions were not just dependent on Mitomycin C concentration but also on dose per cell. Analysis of linearity between viable cell number and Mitomycin C dose per cell derived from the concentrations of 3 to 10µg/ml revealed four distinct categories of growth-arrest. Confluent cultures exposed to low concentration showed growth-arrest failure. DISCUSSION: The in vitro cell density titration can facilitate prediction of a compound's operational in vivo dosing. For containing the growth arrest failure, an arithmetic volume derivation strategy is proposed by fixing the exposure density to a safe limit. The feeder extinction characteristics are critical for streamlining the stem cell based pharmacological and toxicological assays.