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2.
J Exp Med ; 219(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34779829

RESUMO

Helminth parasites are adept manipulators of the immune system, using multiple strategies to evade the host type 2 response. In the intestinal niche, the epithelium is crucial for initiating type 2 immunity via tuft cells, which together with goblet cells expand dramatically in response to the type 2 cytokines IL-4 and IL-13. However, it is not known whether helminths modulate these epithelial cell populations. In vitro, using small intestinal organoids, we found that excretory/secretory products (HpES) from Heligmosomoides polygyrus blocked the effects of IL-4/13, inhibiting tuft and goblet cell gene expression and expansion, and inducing spheroid growth characteristic of fetal epithelium and homeostatic repair. Similar outcomes were seen in organoids exposed to parasite larvae. In vivo, H. polygyrus infection inhibited tuft cell responses to heterologous Nippostrongylus brasiliensis infection or succinate, and HpES also reduced succinate-stimulated tuft cell expansion. Our results demonstrate that helminth parasites reshape their intestinal environment in a novel strategy for undermining the host protective response.


Assuntos
Células Epiteliais/metabolismo , Células Caliciformes/metabolismo , Intestino Delgado/citologia , Organoides/metabolismo , Infecções por Strongylida/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Epiteliais/parasitologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células Caliciformes/parasitologia , Proteínas de Helminto/metabolismo , Proteínas de Helminto/farmacologia , Interações Hospedeiro-Parasita , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Intestino Delgado/parasitologia , Camundongos Endogâmicos C57BL , Nematospiroides dubius/metabolismo , Nematospiroides dubius/fisiologia , Nippostrongylus/metabolismo , Nippostrongylus/fisiologia , Organoides/citologia , Organoides/parasitologia , Infecções por Strongylida/parasitologia , Ácido Succínico/farmacologia , Transcriptoma/efeitos dos fármacos
3.
Nat Commun ; 12(1): 7302, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34911939

RESUMO

Three-dimensional brain organoids have emerged as a valuable model system for studies of human brain development and pathology. Here we establish a midbrain organoid culture system to study the developmental trajectory from pluripotent stem cells to mature dopamine neurons. Using single cell RNA sequencing, we identify the presence of three molecularly distinct subtypes of human dopamine neurons with high similarity to those in developing and adult human midbrain. However, despite significant advancements in the field, the use of brain organoids can be limited by issues of reproducibility and incomplete maturation which was also observed in this study. We therefore designed bioengineered ventral midbrain organoids supported by recombinant spider-silk microfibers functionalized with full-length human laminin. We show that silk organoids reproduce key molecular aspects of dopamine neurogenesis and reduce inter-organoid variability in terms of cell type composition and dopamine neuron formation.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Dopamina/metabolismo , Neurônios/metabolismo , Organoides/crescimento & desenvolvimento , Encéfalo/citologia , Humanos , Neurogênese , Neurônios/citologia , Organoides/citologia , Organoides/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Transcriptoma
4.
Elife ; 102021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34698018

RESUMO

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments.


Assuntos
Axônios/ultraestrutura , Tomografia com Microscopia Eletrônica , Organoides/citologia , Encéfalo/citologia , Encéfalo/ultraestrutura , Microscopia Crioeletrônica , Células HeLa , Humanos , Substâncias Macromoleculares/metabolismo , Microscopia , Microscopia de Fluorescência , Organoides/ultraestrutura
5.
Nature ; 598(7879): 205-213, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616060

RESUMO

During mammalian development, differences in chromatin state coincide with cellular differentiation and reflect changes in the gene regulatory landscape1. In the developing brain, cell fate specification and topographic identity are important for defining cell identity2 and confer selective vulnerabilities to neurodevelopmental disorders3. Here, to identify cell-type-specific chromatin accessibility patterns in the developing human brain, we used a single-cell assay for transposase accessibility by sequencing (scATAC-seq) in primary tissue samples from the human forebrain. We applied unbiased analyses to identify genomic loci that undergo extensive cell-type- and brain-region-specific changes in accessibility during neurogenesis, and an integrative analysis to predict cell-type-specific candidate regulatory elements. We found that cerebral organoids recapitulate most putative cell-type-specific enhancer accessibility patterns but lack many cell-type-specific open chromatin regions that are found in vivo. Systematic comparison of chromatin accessibility across brain regions revealed unexpected diversity among neural progenitor cells in the cerebral cortex and implicated retinoic acid signalling in the specification of neuronal lineage identity in the prefrontal cortex. Together, our results reveal the important contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical development.


Assuntos
Encéfalo/citologia , Epigenômica , Neurogênese , Análise de Célula Única , Atlas como Assunto , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Suscetibilidade a Doenças , Elementos Facilitadores Genéticos , Humanos , Neurônios/citologia , Neurônios/metabolismo , Organoides/citologia , Tretinoína/metabolismo
6.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638524

RESUMO

Human pluripotent stem cells (hPSCs) are grouped into two cell types; embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). hESCs have provided multiple powerful platforms to study human biology, including human development and diseases; however, there were difficulties in the establishment of hESCs from human embryo and concerns over its ethical issues. The discovery of hiPSCs has expanded to various applications in no time because hiPSCs had already overcome these problems. Many hPSC-based studies have been performed using two-dimensional monocellular culture methods at the cellular level. However, in many physiological and pathophysiological conditions, intra- and inter-organ interactions play an essential role, which has hampered the establishment of an appropriate study model. Therefore, the application of recently developed technologies, such as three-dimensional organoids, bioengineering, and organ-on-a-chip technology, has great potential for constructing multicellular tissues, generating the functional organs from hPSCs, and recapitulating complex tissue functions for better biological research and disease modeling. Moreover, emerging techniques, such as single-cell transcriptomics, spatial transcriptomics, and artificial intelligence (AI) allowed for a denser and more precise analysis of such heterogeneous and complex tissues. Here, we review the applications of hPSCs to construct complex organs and discuss further prospects of disease modeling and drug discovery based on these PSC-derived organs.


Assuntos
Sistema Nervoso Central/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Biologia Sintética/métodos , Engenharia Tecidual/métodos , Pesquisa Biomédica , Diferenciação Celular , Técnicas de Cocultura , Humanos
7.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638582

RESUMO

It is difficult to regenerate mammalian retinal cells once the adult retina is damaged, and current clinical approaches to retinal damages are very limited. The introduction of the retinal organoid technique empowers researchers to study the molecular mechanisms controlling retinal development, explore the pathogenesis of retinal diseases, develop novel treatment options, and pursue cell/tissue transplantation under a certain genetic background. Here, we revisit the historical background of retinal organoid technology, categorize current methods of organoid induction, and outline the obstacles and potential solutions to next-generation retinal organoids. Meanwhile, we recapitulate recent research progress in cell/tissue transplantation to treat retinal diseases, and discuss the pros and cons of transplanting single-cell suspension versus retinal organoid sheet for cell therapies.


Assuntos
Organoides/citologia , Retina/citologia , Transplante de Tecidos/métodos , Animais , Humanos , Doenças Retinianas/terapia
8.
Int J Mol Sci ; 22(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34639103

RESUMO

Various pathogens, such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, are threatening human health worldwide. The natural hosts of these pathogens are thought to be bats. The rousette bat, a megabat, is thought to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Additionally, the rousette bat showed a transient infection in the experimental inoculation of SARS-CoV-2. In the current study, we established and characterized intestinal organoids from Leschenault's rousette, Rousettus leschenaultii. The established organoids successfully recapitulated the characteristics of intestinal epithelial structure and morphology, and the appropriate supplements necessary for long-term stable culture were identified. The organoid showed susceptibility to Pteropine orthoreovirus (PRV) but not to SARS-CoV-2 in experimental inoculation. This is the first report of the establishment of an expandable organoid culture system of the rousette bat intestinal organoid and its sensitivity to bat-associated viruses, PRV and SARS-CoV-2. This organoid is a useful tool for the elucidation of tolerance mechanisms of the emerging rousette bat-associated viruses such as Ebola and Marburg virus.


Assuntos
COVID-19/virologia , Quirópteros/virologia , Organoides/virologia , Orthoreovirus/fisiologia , Infecções por Reoviridae/virologia , SARS-CoV-2/fisiologia , Animais , COVID-19/veterinária , Técnicas de Cultura de Células , Células Cultivadas , Quirópteros/fisiologia , Humanos , Intestinos/citologia , Intestinos/virologia , Organoides/citologia , Infecções por Reoviridae/veterinária
9.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638891

RESUMO

The biology of aging is focused on the identification of novel pathways that regulate the underlying processes of aging to develop interventions aimed at delaying the onset and progression of chronic diseases to extend lifespan. However, the research on the aging field has been conducted mainly in animal models, yeast, Caenorhabditis elegans, and cell cultures. Thus, it is unclear to what extent this knowledge is transferable to humans since they might not reflect the complexity of aging in people. An organoid culture is an in vitro 3D cell-culture technology that reproduces the physiological and cellular composition of the tissues and/or organs. This technology is being used in the cancer field to predict the response of a patient-derived tumor to a certain drug or treatment serving as patient stratification and drug-guidance approaches. Modeling aging with patient-derived organoids has a tremendous potential as a preclinical model tool to discover new biomarkers of aging, to predict adverse outcomes during aging, and to design personalized approaches for the prevention and treatment of aging-related diseases and geriatric syndromes. This could represent a novel approach to study chronological and/or biological aging, paving the way to personalized interventions targeting the biology of aging.


Assuntos
Envelhecimento/genética , Técnicas de Cultura de Células/métodos , Epigenômica/métodos , Instabilidade Genômica/genética , Genômica/métodos , Organoides/metabolismo , Envelhecimento/metabolismo , Animais , Humanos , Modelos Genéticos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Organoides/citologia
10.
Elife ; 102021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34528872

RESUMO

Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form three-dimensional organoids or 'alveolospheres' with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization and support the concept of thermodynamic entropy in aging biology.


Assuntos
Células Epiteliais Alveolares , Senescência Celular/fisiologia , Células-Tronco Mesenquimais , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/fisiologia , Animais , Células Cultivadas , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Organoides/citologia , Organoides/metabolismo , Estresse Oxidativo
11.
Nature ; 597(7875): 196-205, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497388

RESUMO

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.


Assuntos
Movimento Celular , Rastreamento de Células , Células/citologia , Biologia do Desenvolvimento/métodos , Embrião de Mamíferos/citologia , Feto/citologia , Disseminação de Informação , Organogênese , Adulto , Animais , Atlas como Assunto , Técnicas de Cultura de Células , Sobrevivência Celular , Visualização de Dados , Feminino , Humanos , Imageamento Tridimensional , Masculino , Modelos Animais , Organogênese/genética , Organoides/citologia , Células-Tronco/citologia
12.
Am J Physiol Cell Physiol ; 321(5): C798-C811, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34524930

RESUMO

Altered esophageal ion transport mechanisms play a key role in inflammatory and cancerous diseases of the esophagus, but epithelial ion processes have been less studied in the esophagus because of the lack of a suitable experimental model. In this study, we generated three-dimensional (3D) esophageal organoids (EOs) from two different mouse strains and characterized the ion transport processes of the EOs. EOs form a cell-filled structure with a diameter of 250-300 µm and were generated from epithelial stem cells as shown by FACS analysis. Using conventional PCR and immunostaining, the presence of Slc26a6 Cl-/HCO3- anion exchanger (AE), Na+/H+ exchanger (NHE), Na+/HCO3- cotransporter (NBC), cystic fibrosis transmembrane conductance regulator (CFTR), and anoctamin 1 Cl- channels was detected in EOs. Microfluorimetric techniques revealed high NHE, AE, and NBC activities, whereas that of CFTR was relatively low. In addition, inhibition of CFTR led to functional interactions between the major acid-base transporters and CFTR. We conclude that EOs provide a relevant and suitable model system for studying the ion transport mechanisms of esophageal epithelial cells, and they can be also used as preclinical tools to assess the effectiveness of novel therapeutic compounds in esophageal diseases associated with altered ion transport processes.


Assuntos
Células Epiteliais/metabolismo , Esôfago/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Organoides/metabolismo , Células-Tronco/metabolismo , Animais , Anoctamina-1/genética , Anoctamina-1/metabolismo , Antiporters/genética , Antiporters/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Esôfago/citologia , Feminino , Transporte de Íons , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos Endogâmicos C57BL , Organoides/citologia , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
13.
APMIS ; 129(11): 653-662, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34580927

RESUMO

Infective endocarditis (IE) is a heart valve infection with high mortality rates. IE results from epithelial lesions, inducing sterile healing vegetations consisting of platelets, leucocytes, and fibrin that are susceptible for colonization by temporary bacteremia. Clinical testing of new treatments for IE is difficult and fast models sparse. The present study aimed at establishing an in vitro vegetation simulation IE model for fast screening of novel treatment strategies. A healing promoting platelet and leucocyte-rich fibrin patch was used to establish an IE organoid-like model by colonization with IE-associated bacterial isolates Staphylococcus aureus, Streptococcus spp (S. mitis group), and Enterococcus faecalis. The patch was subsequently exposed to tobramycin, ciprofloxacin, or penicillin. Bacterial colonization was evaluated by microscopy and quantitative bacteriology. We achieved stable bacterial colonization on the patch, comparable to clinical IE vegetations. Microscopy revealed uneven, biofilm-like colonization of the patch. The surface-associated bacteria displayed increased tolerance to antibiotics compared to planktonic bacteria. The present study succeeded in establishing an IE simulation model with the relevant pathogens S. aureus, S. mitis group, and E. faecalis. The findings indicate that the IE model mirrors the natural IE process and has the potential for fast screening of treatment candidates.


Assuntos
Endocardite Bacteriana/microbiologia , Modelos Biológicos , Antibacterianos/farmacologia , Bacteriemia/microbiologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Tolerância a Medicamentos , Endocardite Bacteriana/tratamento farmacológico , Endocardite Bacteriana/patologia , Humanos , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/microbiologia
14.
Cells ; 10(9)2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34572137

RESUMO

Inherited retinal degenerations (IRD) affecting either photoreceptors or pigment epithelial cells cause progressive visual loss and severe disability, up to complete blindness. Retinal organoids (ROs) technologies opened up the development of human inducible pluripotent stem cells (hiPSC) for disease modeling and replacement therapies. However, hiPSC-derived ROs applications to IRD presently display limited maturation and functionality, with most photoreceptors lacking well-developed outer segments (OS) and light responsiveness comparable to their adult retinal counterparts. In this review, we address for the first time the microenvironment where OS mature, i.e., the subretinal space (SRS), and discuss SRS role in photoreceptors metabolic reprogramming required for OS generation. We also address bioengineering issues to improve culture systems proficiency to promote OS maturation in hiPSC-derived ROs. This issue is crucial, as satisfying the demanding metabolic needs of photoreceptors may unleash hiPSC-derived ROs full potential for disease modeling, drug development, and replacement therapies.


Assuntos
Bioengenharia/métodos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/citologia , Animais , Humanos , Degeneração Retiniana/patologia
15.
Am J Physiol Lung Cell Mol Physiol ; 321(4): L775-L786, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34378410

RESUMO

Air-liquid interface (ALI) cultures are frequently used in lung research but require substantial cell numbers that cannot readily be obtained from patients. We explored whether organoid expansion [three-dimensional (3D)] can be used to establish ALI cultures from clinical samples with low epithelial cell numbers. Airway epithelial cells were obtained from tracheal aspirates (TA) from preterm newborns and from bronchoalveolar lavage (BAL) or bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, cells were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to 1) cellular composition, 2) response to cigarette smoke exposure, and 3) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. We established well-differentiated ALI cultures from all samples. Cellular compositions (basal, ciliated, and goblet cells) were comparable. All 3D-expanded cultures showed a similar stress response following cigarette smoke exposure but differed from the 2D-expanded cultures. Higher peak levels of antioxidant genes HMOX1 and NQO1 and a more rapid return to baseline, and a lower unfolded protein response was observed after cigarette smoke exposure in 3D-derived cultures compared to 2D-derived cultures. In addition, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. Organoid-based expansion of clinical samples with low cell numbers, such as TA from preterm newborns is a valid method and tool to establish ALI cultures.


Assuntos
Brônquios/citologia , Células Epiteliais/citologia , Organoides/citologia , Mucosa Respiratória/citologia , Fumaça/efeitos adversos , Adulto , Líquido da Lavagem Broncoalveolar/citologia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células Cultivadas , Heme Oxigenase-1/metabolismo , Humanos , Recém-Nascido , Interleucina-13/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Receptores Notch/antagonistas & inibidores , Produtos do Tabaco/efeitos adversos , Resposta a Proteínas não Dobradas/efeitos dos fármacos
16.
Nat Commun ; 12(1): 4730, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354063

RESUMO

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Dispositivos Lab-On-A-Chip , Neurogênese/fisiologia , Organoides/crescimento & desenvolvimento , Organoides/fisiologia , Animais , Encéfalo/citologia , Meios de Cultura , Fenômenos Eletrofisiológicos , Matriz Extracelular/fisiologia , Estudos de Viabilidade , Perfilação da Expressão Gênica , Humanos , Hidrogéis , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Anatômicos , Modelos Neurológicos , Neurogênese/genética , Neuroglia/citologia , Neuroglia/fisiologia , Técnicas de Cultura de Órgãos/instrumentação , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Suínos
17.
Sci Rep ; 11(1): 15889, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354183

RESUMO

Enteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6-8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.


Assuntos
Colo/citologia , Pseudo-Obstrução Intestinal/terapia , Células-Tronco Neurais/citologia , Animais , Técnicas de Cultura de Células/métodos , Colo/fisiologia , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/fisiologia , Feminino , Pseudo-Obstrução Intestinal/fisiopatologia , Intestino Delgado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Crista Neural/citologia , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Organoides/citologia , Organoides/metabolismo , Transplante de Células-Tronco/métodos
18.
Cells ; 10(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34359871

RESUMO

The creation of a testis organoid (artificial testis tissue) with sufficient resemblance to the complex form and function of the innate testis remains challenging, especially using non-rodent donor cells. Here, we report the generation of an organoid culture system with striking biomimicry of the native immature testis tissue, including vasculature. Using piglet testis cells as starting material, we optimized conditions for the formation of cell spheroids, followed by long-term culture in an air-liquid interface system. Both fresh and frozen-thawed cells were fully capable of self-reassembly into stable testis organoids consisting of tubular and interstitial compartments, with all major cell types and structural details expected in normal testis tissue. Surprisingly, our organoids also developed vascular structures; a phenomenon that has not been reported in any other culture system. In addition, germ cells do not decline over time, and Leydig cells release testosterone, hence providing a robust, tunable system for diverse basic and applied applications.


Assuntos
Materiais Biomiméticos/farmacologia , Organoides/fisiologia , Testículo/irrigação sanguínea , Animais , Contagem de Células , Criopreservação , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Hormônio Luteinizante/metabolismo , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Especificidade de Órgãos , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Suínos , Testículo/citologia , Testículo/ultraestrutura , Testosterona/metabolismo
19.
Vet Res ; 52(1): 108, 2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34391473

RESUMO

Enteroid cultures are three-dimensional in vitro models that reflect the cellular composition and architecture of the small intestine. One limitation with the enteroid conformation is the enclosed lumen, making it difficult to expose the apical surface of the epithelium to experimental treatments. The present study was therefore conducted to generate cultures of equine enteroids and to develop methods for culture of enteroid-derived cells on a two-dimensional plane, enabling easy access to the apical surface of the epithelium. Equine enteroids were established from small intestinal crypts within 7-9 days of culture. Transcriptional analysis of cell type markers confirmed the presence of enterocytes, stem-, Paneth-, proliferative-, enteroendocrine-, goblet- and tuft cells. This cellular composition was maintained over multiple passages, showing that the enteroids can be kept for prolonged periods. The transfer from 3D enteroids to 2D monolayers slightly modified the relative expression levels of the cell type markers, indicating a decrease of goblet- and Paneth cells in the monolayers. Stimulation with the TLR2, 3 and 4 agonists Pam3CSK4, Poly I:C and LPS, respectively, induced the pro-inflammatory cytokines TNF-α and IL-8, while the TLR5 agonist FliC only induced TNF-α. In addition, an up-regulation of TGF-ß, IL-33 and IFN-ß was recorded after exposure to lipofected Poly I:C that also affected the monolayer integrity. Thus, the equine enteroid-derived 2D monolayers described in the present study show both genetic and functional similarities with the equine intestine making it an interesting in vitro model for studies demanding access to the apical surface, e.g. in studies of host-microbe interactions.


Assuntos
Técnicas de Cultura de Células/veterinária , Cavalos , Organoides/citologia , Animais , Técnicas de Cultura de Células/métodos , Feminino , Intestino Delgado/citologia
20.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445185

RESUMO

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for disease modeling and drug cardiotoxicity screening. To this end, we recently developed human cardiac organoids (hCOs) for modeling human myocardium. Here, we perform a transcriptomic analysis of various in vitro hiPSC-CM platforms (2D iPSC-CM, 3D iPSC-CM and hCOs) to deduce the strengths and limitations of these in vitro models. We further compared iPSC-CM models to human myocardium samples. Our data show that the 3D in vitro environment of 3D hiPSC-CMs and hCOs stimulates the expression of genes associated with tissue formation. The hCOs demonstrated diverse physiologically relevant cellular functions compared to the hiPSC-CM only models. Including other cardiac cell types within hCOs led to more transcriptomic similarities to adult myocardium. hCOs lack matured cardiomyocytes and immune cells, which limits a complete replication of human adult myocardium. In conclusion, 3D hCOs are transcriptomically similar to myocardium, and future developments of engineered 3D cardiac models would benefit from diversifying cell populations, especially immune cells.


Assuntos
Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Organoides/metabolismo , Transcriptoma , Adulto , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miocárdio/citologia , Miócitos Cardíacos/citologia , Organoides/citologia
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