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Background: Social anxiety disorder (SAD) and major depressive disorder (MDD) are both associated with diminished global positive affect. However, little is known about which specific positive emotions are affected, and which positive emotions differentiate MDD from SAD. Methods: Four groups of adults recruited from the community were examined (N = 272): control group (no psychiatric history; n = 76), SAD without MDD group (n = 76), MDD without SAD group (n = 46), and comorbid group (diagnoses of both SAD and MDD; n = 74). Discrete positive emotions were measured with the Modified Differential Emotions Scale, which asked about the frequency of 10 different positive emotions experienced during the past week. Results: The control group had higher scores on all positive emotions compared to all three clinical groups. The SAD group had higher scores on awe, inspiration, interest, and joy compared to the MDD group, and higher scores on those emotions, as well as amusement, hope, love, pride, and contentment, than the comorbid group. MDD and comorbid groups did not differ on any positive emotions. Gratitude did not differ significantly between clinical groups. Conclusion: Adopting a discrete positive emotion approach revealed shared and distinct features across SAD, MDD, and their comorbidity. We consider possible mechanisms underlying transdiagnostic vs. disorder-specific emotion deficits. Supplementary Information: The online version contains supplementary material available at 10.1007/s10608-023-10355-y.
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Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature.
Assuntos
Perfilação da Expressão Gênica/métodos , Células-Tronco Pluripotentes/metabolismo , Proteoma/análise , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Espectrometria de Massas , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/citologia , Proteoma/genética , Proteoma/metabolismoRESUMO
Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one antibody (mAb 84) is cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complement-independent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Células-Tronco Embrionárias/citologia , Sialoglicoproteínas/análise , Animais , Anticorpos , Anticorpos Monoclonais , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Células-Tronco Embrionárias/fisiologia , Citometria de Fluxo , Células HeLa , Humanos , Camundongos , Sialoglicoproteínas/imunologiaRESUMO
An automated vision system, TeratomEye, was developed for the identification of three representative tissue types: muscle, gut and neural epithelia which are commonly found in teratomas formed from human embryonic stem cells. Muscle tissue, a common structure was identified with an accuracy of 90.3% with high specificity and sensitivity greater than 90%. Gut epithelia were identified with an accuracy of 87.5% with specificity and sensitivity greater than 80%. Neural epithelia which were the most difficult structures to distinguish gave an accuracy of 47.6%. TeratomEye is therefore useful for the automated identification of differentiated tissues in teratoma sections.
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Diferenciação Celular , Células-Tronco Embrionárias/citologia , Mucosa Intestinal/citologia , Células Musculares/citologia , Células Neuroepiteliais/citologia , Técnicas Citológicas/métodos , HumanosRESUMO
Monoclonal antibodies (mAbs) are used as targeted therapies against cancers. These mAbs kill cancer cells via various mechanisms of actions. In this study, human embryonic stem cells (hESCs) was used as the immunogen to generate a panel of antibodies. From this panel of mAbs, A19 was found to bind both hESC and various cancer cell lines. The antigen target of A19 was identified as Erbb-2 and glycan analysis showed that A19 binds to a N-glycan epitope on the antigen. A19 was elucidated to internalize into cancer cells following binding to Erbb-2 and hence developed as an antibody-drug conjugate (ADC). Using ADC as the mechanism of action, A19 was able to kill cancer cells in vitro and delayed the onset of tumour formation in mice xenograft model. When compared to Herceptin, A19 binds to different isoforms of Erbb-2 and does not compete with Herceptin for the same epitope. Hence, A19 has the potential to be developed as an alternative targeted therapeutic agent for cancers expressing Erbb-2.
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Anticorpos Monoclonais Murinos , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/farmacologia , Células-Tronco Embrionárias Humanas/imunologia , Neoplasias Experimentais , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Antineoplásicos Imunológicos/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Monoclonal antibodies (mAbs) play an increasingly important role in cancer therapy. To address the wide heterogeneity of the disease, the identification of novel antigen targets and the development of mAbs against them are needed. Our lab previously generated a panel of mAbs against human embryonic stem cells (hESC) using a whole cell immunization approach in mice. These mAbs can potentially target oncofetal antigens and be repurposed for antibody or antibody drug conjugate (ADC) therapy. From this panel, the novel IgG1 2448 was found to bind surface antigens on hESC and multiple cancer cell lines. Here, we show 2448 targets a unique glycan epitope on annexin A2 (ANXA2) and can potentially monitor the Epithelial-Mesenchymal Transition (EMT) in ovarian and breast cancer. To evaluate 2448 as a potential drug, 2448 was engineered and expressed as a chimeric IgG1. Chimeric 2448 (ch2448) demonstrated efficient and specific killing when conjugated to cytotoxic payloads as an ADC. In addition, ch2448 elicited potent antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro and in vivo. Further engineering of ch2448 to remove fucose in the Fc domain enhanced ADCC. Overall, these findings indicate that embryonic ANXA2 is an attractive target and suggest that ch2448 is a promising candidate for further therapeutic development.
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The maintenance of undifferentiated human embryonic stem cells (hESC) requires feeder cells, either in co-culture or feeder-free with conditioned medium (CM) from the feeders. In this study, we compared the CM of a supporting primary mouse embryonic feeder (MEF) and an isogenic but non-supporting MEF line (DeltaE-MEF) in order to gain an insight to the differential expression profile of secreted factors. Using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight (MALDI) tandem mass spectrometry, 13 protein identities were found to be downregulated in DeltaE-MEF compared to MEF, of which 4 were found to be soluble factors and 3 proteins were membrane-associated or related to the extracellular matrix. In addition, four other proteins were identified to be differentially expressed in MEF-CM using high pressure liquid chromatography (HPLC) and cytokine arrays. In functional experiments where CM was replaced with six of the factors identified, hESC were able to proliferate for five continuous passages whilst maintaining 68-82% and 74-98% expression of pluripotent markers, Oct-4 and Tra-1-60, respectively. Using proteomic tools, important proteins from CM that supports hESC culture have been identified, which when replaced with recombinant proteins, continue to support undifferentiated hESC growth in a feeder-free culture platform.
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Células-Tronco Embrionárias/metabolismo , Proteínas/análise , Animais , Células Cultivadas , Meios de Cultivo Condicionados , Meios de Cultura Livres de Soro , Citocinas/metabolismo , Eletroforese em Gel Bidimensional , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Camundongos , Fatores de Transcrição de Octâmero/metabolismo , Proteínas/genética , Proteínas Recombinantes/metabolismoRESUMO
In this study we report observations that mouse embryonic fibroblasts (MEF) capable of supporting expansion of pluripotent, human embryonic stem cells (hESC) fail to support after immortalization using E6/E7 oncogenes in serum conditions; however this can be reversed following addition of exogenous TGF-beta2. Microarray analysis of immortalized and non-immortalized MEF revealed differential gene expression of several TGF-beta related genes. By supplementing TGF-beta2 into E6/E7 immortalized MEF cultures, this enabled proliferation of undifferentiated, pluripotent hESC as demonstrated by marker expression (Oct-4, SSEA-4, alkaline phosphatase) and teratoma formation representing three germ layers following hESC injection into immuno-deficient mice. Subsequent investigation using quantitative real-time PCR highlighted differential gene expression of several extracellular matrix related transcripts in primary and immortal (+/-TGF-beta2) feeder cells including the induction of osteopontin following addition of TGF-beta2. Our results demonstrate that TGF-beta2 and its related genes in MEF play a role in the support of pluripotent hESC expansion.
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Células-Tronco Pluripotentes/citologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Humanos , Camundongos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Osteopontina , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Sialoglicoproteínas/biossíntese , Fator de Crescimento Transformador beta2RESUMO
Human embryonic stem cells (hESC) are pluripotent cells that proliferate indefinitely in culture, whilst retaining their capacity for differentiation into different cell types. However, hESC cultures require culture in direct contact with feeder cells or conditioned medium (CM) from feeder cells. The most common source of feeders has been primary mouse embryonic fibroblast (MEF). In this study, we immortalized a primary MEF line with the E6 and E7 genes from HPV16. The immortal line, DeltaE-MEF, was able to proliferate beyond 7-9 passages and has an extended lifespan beyond 70 passages. When tested for its ability to support hESC growth, it was found that hESC continue to maintain the undifferentiated morphology for >40 passages both in co-culture with DeltaE-MEF and in feeder-free cultures supplemented with CM from DeltaE-MEF. The cultures also continue to express the pluripotent markers, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, alkaline phosphatase and maintain a normal karyotype. In addition, these hESC formed teratomas when injected into SCID mice. Lastly, we demonstrated the feasibility of scaling-up significant quantities of undifferentiated hESC (>10(8) cells) using DeltaE-MEF in cell factories. The results from this study suggest that immortalized feeders can provide a consistent and reproducible source of feeders for hESC expansion and research.
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Sistema Livre de Células/metabolismo , Técnicas de Cocultura/métodos , Fibroblastos/citologia , Fibroblastos/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Comunicação Celular/fisiologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Tamanho Celular , Sobrevivência Celular , Humanos , Camundongos , Camundongos SCID , Projetos PilotoRESUMO
Cell surface antigens are important targets for monoclonal antibodies, but they are often difficult to work with due to their association with the cell membrane. Phage display is a versatile technique that can be applied to generate binders against difficult targets. Here we used antibody phage display to isolate a binder for a rare and specialized cell, the human corneal endothelial cell. The human corneal endothelium is a medically important cell layer; defects in this layer account for about half of all corneal transplants. Despite its importance, no specific antigens have been found to mark this cell type. By panning a phage library directly on human corneal endothelial cells, we isolated an antibody that bound to these cells and not the other types of corneal cells. Subsequently, we identified the antibody's putative target to be CD166 by immunoprecipitation and mass spectrometry. This approach can be used to isolate antibodies against other poorly-characterized cell types, such as stem cells or cancer cells, without any prior knowledge of their discriminating markers.
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Anticorpos Monoclonais/isolamento & purificação , Antígenos CD/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Células Endoteliais/metabolismo , Endotélio Corneano/metabolismo , Proteínas Fetais/metabolismo , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Antígenos CD/genética , Biomarcadores/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Células Endoteliais/citologia , Endotélio Corneano/citologia , Proteínas Fetais/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Imunoprecipitação , Especificidade de Órgãos , Cultura Primária de Células , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismoRESUMO
Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good 'cobblestone-like' morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC.
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Anticorpos Monoclonais/imunologia , Cegueira/imunologia , Células Endoteliais/imunologia , Endotélio Corneano/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Cegueira/metabolismo , Cegueira/terapia , Cadáver , Moléculas de Adesão Celular Neuronais/imunologia , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio Corneano/citologia , Endotélio Corneano/metabolismo , Proteínas Fetais/imunologia , Proteínas Fetais/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos Endogâmicos BALB C , Peroxirredoxina VI/imunologia , Peroxirredoxina VI/metabolismo , Ligação Proteica/imunologiaRESUMO
Four commercially available serum-free and defined culture media tested on 2 human embryonic stem cell (hESC) lines were all found to support undifferentiated growth for >10 continuous passages. For hESC cultured with defined StemPro and mTeSR1 media, the cells were maintained feeder-free on culture dishes coated with extracellular matrices (ECMs) with no requirement of feeder-conditioned media (CM). For xeno-free serum replacer (XSR), HEScGRO, and KnockOut media, mitotically inactivated human foreskin feeders (hFFs) were required for hESC growth. Under the different media conditions, cells continued to exhibit alkaline phosphatase activity and expressed undifferentiated hESC markers Oct-4, stage-specific embryonic antigens 4 (SSEA-4), and Tra-1-60. In addition, hESC maintained the expression of podocalyxin-like protein-1 (PODXL), an antigen recently reported in another study to be present in undifferentiated hESC. The cytotoxic antibody mAb 84 binds via PODXL expressed on hESC surface and kills >90% of hESC within 45 min of incubation. When these cells were spontaneously differentiated to form embryoid bodies, derivatives representing the 3 germ layers were obtained. Injection of hESC into animal models resulted in teratomas and the formation of tissue types indicative of ectodermal, endodermal, and mesodermal lineages were observed. Our data also suggested that StemPro and mTeSR1 media were more optimal for hESC proliferation compared to cells grown on CM because the growth rate of hESC increased by 30%-40%, higher split ratio was thus required for weekly passaging. This is advantageous for the large-scale cultivation of hESC required in clinical applications.
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Diferenciação Celular , Meios de Cultura Livres de Soro/farmacologia , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Adaptação Fisiológica/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Instabilidade Cromossômica/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Embrionárias/enzimologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Cariotipagem , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Fatores de TempoRESUMO
The conventional method of culturing human embryonic stem cells (hESC) is on two-dimensional (2D) surfaces, which is not amenable for scale up to therapeutic quantities in bioreactors. We have developed a facile and robust method for maintaining undifferentiated hESC in three-dimensional (3D) suspension cultures on matrigel-coated microcarriers achieving 2- to 4-fold higher cell densities than those in 2D colony cultures. Stable, continuous propagation of two hESC lines on microcarriers has been demonstrated in conditioned media for 6 months. Microcarrier cultures (MC) were also demonstrated in two serum-free defined media (StemPro and mTeSR1). MC achieved even higher cell concentrations in suspension spinner flasks, thus opening the prospect of propagation in controlled bioreactors.
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Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Reatores Biológicos , Colágeno/química , Meios de Cultura Livres de Soro , Combinação de Medicamentos , Ectoderma/metabolismo , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Humanos , Cariotipagem , Laminina/química , Mesoderma/metabolismo , Proteoglicanas/químicaRESUMO
Embryonic stem cells (ESC) have the unique ability to differentiate into a variety of tissue types. However, the realization of regenerative medicine will require the production of large quantities of ESC which subsequently have to be differentiated into the final phenotype. Thus, we sought to develop a simple and scaleable bioprocess to increase densities of ESC to achieve this goal. Using mouse embryonic stem cells (mESC) as a model, by combining automated feeding and culture of mESC on petriperm dishes, cell densities were enhanced up to 6.4 x 10(6) cells/cm2 compared to conventional petri dish culture which only reached 0.2 to 1.4 x 10(6) cells/cm2. It was found that mESC from all experiments maintained excellent viability, pluripotency, and genetic stability after growing for 6 days in petriperm cultures with automated feeding. The expression of Oct-4 transcription factor was observed in all cultures, mESC formed embryoid bodies in differentiated cultures and teratomas in SCID mice, confirming their pluripotency, and karyotype of the cultures was normal. This culture method was stable for routine passaging and a second mESC cell line was shown to perform in a similar manner on petriperm with automated feeding. This work represents an important step towards achieving high density cultures of ESC.
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Técnicas de Cultura de Células/métodos , Embrião de Mamíferos/citologia , Células-Tronco/citologia , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Reatores Biológicos , Ciclo Celular , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Corantes Fluorescentes , Indóis , Cariotipagem , Antígenos CD15/metabolismo , Masculino , Camundongos , Camundongos SCID , Fator 3 de Transcrição de Octâmero , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Transplante de Células-Tronco , Células-Tronco/metabolismo , Teratoma/patologia , Fatores de Transcrição/metabolismoRESUMO
Human embryonic stem cells (HES) hold great potential for regenerative medicine because of their ability to differentiate to any cell type. However, a limitation is that HES cells require a feeder layer to stay undifferentiated. Routinely, mouse embryonic fibroblast is used. However, for therapeutic applications, contamination with mouse cells may be considered unacceptable. In this study, we evaluated three commercially available human foreskin feeder (HF) lines for their ability to support HES cell growth in media supplemented with serum or serum replacer. HES cells on HF in serum replacer-supplemented media were cultured for >30 passages. They remained undifferentiated, maintained a normal karyotype, and continued to be positive for the pluripotent markers Oct-4, SOX-2, SSEA-4, GCTM-2, Tra-1-60, Tra-1-81, and alkaline phosphatase. In vivo, HES cells formed teratomas in SCID mouse models that represent the three embryonic germ layers. In contrast, HES cells cultured on HF in serum-supplemented media differentiated after three passages. Morphologically, the cells became cystic with a loss of intracellular Oct-4. We have successfully adapted and cultured undifferentiated HES cells on three human feeder lines for >30 passages. No difficulties were observed with the exception of serum in the media. This study reveals a safe and accessible source for feeders for HES cell research and potential therapeutic applications.