RESUMO
Hematopoietic development is spatiotemporally tightly regulated by defined cell-intrinsic and extrinsic modifiers. The role of cytokines has been intensively studied in adult hematopoiesis; however, their role in embryonic hematopoietic specification remains largely unexplored. Here, we used induced pluripotent stem cell (iPSC) technology and established a 3-dimensional, organoid-like differentiation system (hemanoid) maintaining the structural cellular integrity to evaluate the effect of cytokines on embryonic hematopoietic development. We show, that defined stages of early human hematopoietic development were recapitulated within the generated hemanoids. We identified KDR+/CD34high/CD144+/CD43-/CD45- hemato-endothelial progenitor cells (HEPs) forming organized, vasculature-like structures and giving rise to CD34low/CD144-/CD43+/CD45+ hematopoietic progenitor cells. We demonstrate that the endothelial to hematopoietic transition of HEPs is dependent on the presence of interleukin 3 (IL-3). Inhibition of IL-3 signalling blocked hematopoietic differentiation and arrested the cells in the HEP stage. Thus, our data suggest an important role for IL-3 in early human hematopoiesis by supporting the endothelial to hematopoietic transition of hemato-endothelial progenitor cells and highlight the potential of a hemanoid-based model to study human hematopoietic development.
Assuntos
Células-Tronco Pluripotentes Induzidas , Interleucina-3 , Células-Tronco Pluripotentes , Adulto , Diferenciação Celular , Hematopoese , HumanosRESUMO
Human pluripotent stem cells (hPSCs), both embryonic (hESCs) and induced (hiPSCs), can be differentiated into derivatives of the three germ layers and are promising tools in regenerative medicine. Cardiovascular diseases are the top-ranking cause of premature death worldwide, and cell replacement therapies based on in vitro differentiated cardiomyocytes might provide a promising perspective to cure patients in the future. The molecular processes during hPSC cardiomyogenesis are far from being fully understood, and we thus have focused here on characterizing the proteome along hESC in vitro differentiation into cardiomyocytes (CMs). Stable isotope labeling of amino acids in cell culture was applied to quantitatively assess the proteome throughout defined stages of hESC cardiomyogenesis. Genetically enriched, >90% pure CM populations were used for shotgun proteomics, leading to the identification and quantitative determination of several thousand proteins. Pathway analysis revealed alterations in energy metabolism during cardiomyogenesis. Enzymes of glycolysis were identified as up-regulated upon differentiation, whereas enzymes involved in oxidative phosphorylation were down-regulated in aggregates on day 20 of differentiation (<10% CMs) and reconstituted on day 35 in >90% pure CMs. A structural protein that attracted our attention was the PDZ and LIM domain containing protein 5 (PDLIM5), which was strongly up-regulated during cardiomyogenesis and for which we detected novel stage-specific isoforms. Notably, expression of the 53 kDa isoforms b and g (corresponding to transcript variants 2 and 7) of PDLIM5 occurred simultaneously to the onset of expression of the early cardiac transcription factor NKX2.5, known to play a key role in cardiac development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Enzimas/metabolismo , Regulação da Expressão Gênica , Proteínas com Domínio LIM/metabolismo , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Proteômica/métodos , Proteínas Adaptadoras de Transdução de Sinal/química , Diferenciação Celular , Células Cultivadas , Metabolismo Energético , Glicólise , Proteína Homeobox Nkx-2.5/análise , Humanos , Marcação por Isótopo , Proteínas com Domínio LIM/química , Redes e Vias Metabólicas , Fosforilação Oxidativa , Isoformas de Proteínas/genética , Medicina RegenerativaRESUMO
Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular ß-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive ß-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-ß-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and ß-MyHC-protein isoforms. Yet, 35 and 75 days of cultivation on laminin-coated glass switched 66 and 87 % of all cardiomyocytes to exclusively express ß-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only ß-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of ß-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure ß-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.
Assuntos
Técnicas de Cultura de Células/métodos , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/biossíntese , Miosinas Ventriculares/biossíntese , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/citologia , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.
Assuntos
Fígado , Organoides , Humanos , Animais , Camundongos , Engenharia Tecidual , Hepatócitos , Células CultivadasRESUMO
Macrophages derived from human induced pluripotent stem cells (iPSCs) have the potential to enable the development of cell-based therapies for numerous disease conditions. We here provide a detailed protocol for the mass production of iPSC-derived macrophages (iPSC-Mac) in scalable suspension culture on an orbital shaker or in stirred-tank bioreactors (STBRs). This strategy is straightforward, robust and characterized by the differentiation of primed iPSC aggregates into 'myeloid-cell-forming-complex' intermediates by means of a minimal cytokine cocktail. In contrast to the 'batch-like differentiation approaches' established for other iPSC-derived lineages, myeloid-cell-forming-complex-intermediates are stably maintained in suspension culture and continuously generate functional and highly pure iPSC-Mac. Employing a culture volume of 120 ml in the STBR platform, ~1-4 × 107 iPSC-Mac can be harvested at weekly intervals for several months. The STBR technology allows for real-time monitoring of crucial process parameters such as biomass, pH, dissolved oxygen, and nutrition levels; the system also promotes systematic process development, optimization and linear upscaling. The process duration, from the expansion of iPSC until the first iPSC-Mac harvest, is 28 d. Successful application of the protocol requires expertise in pluripotent stem cell culture, differentiation and analytical methods, such as flow cytometry. Fundamental know-how in biotechnology is also advantageous to run the process in the STBR platform. The continuous, scalable production of well-defined iPSC-Mac populations is highly relevant to various fields, ranging from developmental biology, immunology and cell therapies to industrial applications for drug safety and discovery.
Assuntos
Células-Tronco Pluripotentes InduzidasRESUMO
Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study, we focus on the properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing (scRNAseq) of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17high PTENlow dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2-/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNAseq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrate that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model.
Assuntos
Fibrose Pulmonar Idiopática , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibrose , Humanos , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Camundongos , FenótipoRESUMO
Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.
Assuntos
Coração/embriologia , Intestinos/embriologia , Organoides/embriologia , Padronização Corporal , Desenvolvimento Embrionário , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Fator 4 Nuclear de Hepatócito/genética , Proteína Homeobox Nkx-2.5/genética , Humanos , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXF/genética , Análise de Sequência de RNARESUMO
The ideal cell culture model should mimic the cell physiology and the mechanical and the chemical cues that are present in specific tissues and organs, within a convenient high-throughput format. A possible key feature for such models is to recapture the cell polarity, the interactions between cells, and the interactions between the cells and the elastic extracellular matrix (ECM) by orienting the cells in a three-dimensional (3D) matrix. A common method to create 3D cell environments is to let the cells aggregate into spheroids with a diameter of around 200 µm. A major challenge for 3D cell cultures is to perform quick and easy imaging of the dense cell population, especially noninvasively. This protocol explains how to take advantage of the number of cells growing out from cell spheroids over time as a readout of the effect of a drug. The assay is compatible with standard imaging techniques and can be performed noninvasively using light microscopy or as a complement to other fluorescent imaging assays.
Assuntos
Técnicas de Cultura de Células/métodos , Miócitos Cardíacos/citologia , Esferoides Celulares/efeitos dos fármacos , Amiodarona/farmacologia , Aspirina/farmacologia , Bioensaio , Doxorrubicina/farmacologia , Matriz Extracelular , Ensaios de Triagem em Larga Escala , Humanos , Esferoides Celulares/citologiaRESUMO
Loss-of-function mutations of the SCN5A gene encoding for the sodium channel α-subunit NaV1.5 result in the autosomal dominant hereditary disease Brugada Syndrome (BrS) with a high risk of sudden cardiac death in the adult. We here engineered human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRISPR/Cas9 introduced BrS-mutation p.A735V-NaV1.5 (g.2204C > T in exon 14 of SCN5A) as a novel model independent of patient´s genetic background. Recent studies raised concern regarding the use of hiPSC-CMs for studying adult-onset hereditary diseases due to cells' immature phenotype. To tackle this concern, long-term cultivation of hiPSC-CMs on a stiff matrix (27-42 days) was applied to promote maturation. Patch clamp recordings of A735V mutated hiPSC-CMs revealed a substantially reduced upstroke velocity and sodium current density, a prominent rightward shift of the steady state activation curve and decelerated recovery from inactivation as compared to isogenic hiPSC-CMs controls. These observations were substantiated by a comparative study on mutant A735V-NaV1.5 channels heterologously expressed in HEK293T cells. In contrast to mutated hiPSC-CMs, a leftward shift of sodium channel inactivation was not observed in HEK293T, emphasizing the importance of investigating mechanisms of BrS in independent systems. Overall, our approach supports hiPSC-CMs' relevance for investigating channelopathies in a dish.
Assuntos
Síndrome de Brugada/genética , Células-Tronco Pluripotentes Induzidas/citologia , Mutação , Miócitos Cardíacos/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adulto , Síndrome de Brugada/patologia , Sistemas CRISPR-Cas , Células HEK293 , Humanos , Técnicas de Patch-ClampRESUMO
Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding â¼1.3 × 108 CM in 150 mL and â¼4.0 × 108 CMs in 350-500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Reatores Biológicos , Antígenos CD13/genética , Antígenos CD13/metabolismo , Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Humanos , Mesoderma/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismoRESUMO
The envisioned routine application of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) for therapies and industry-compliant screening approaches will require efficient and highly reproducible processes for the mass production of well-characterized CM batches.On their way toward beating CMs, hPSCs initially undergo an epithelial-to-mesenchymal transition into a primitive-streak (PS)-like population that later gives rise to all endodermal and mesodermal lineages, including cardiovascular progenies (CVPs). CVPs are multipotent and possess the capability to give rise to all major cell types of the heart, including CMs, endothelial cells, cardiac fibroblasts, and smooth muscle cells. This article provides an historical overview and describes the stepwise development of protocols that typically result in the appearance of beating CMs within 7-12 days of hPSC differentiation.We describe the development of directed and closely controlled cardiomyogenic differentiation, which now enables the induction of >90% CM purity without further lineage enrichment. Although secreted lineage specifiers (revealed from developmental biology) were initially used, we outline the advantages of chemical pathway modulators, as defined by more recent screening approaches. Subsequently, we discuss the use of defined culture media for upscaling the production of hPSC-CMs in controlled bioreactors and how this, in principle, unlimited source of human CMs can be used to progress heart regeneration and stimulate the drug discovery pipeline. Graphical Abstract.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Miócitos Cardíacos/metabolismo , Animais , Coração/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Regeneração/efeitos dos fármacosRESUMO
Three-dimensional (3D) models with cells arranged in clusters or spheroids have emerged as valuable tools to improve physiological relevance in drug screening. One of the challenges with cells cultured in 3D, especially for high-throughput applications, is to quickly and non-invasively assess the cellular state in vitro. In this article, we show that the number of cells growing out from human induced pluripotent stem cell (hiPSC)-derived cardiac spheroids can be quantified to serve as an indicator of a drug’s effect on spheroids captured in a microfluidic device. Combining this spheroid-on-a-chip with confocal high content imaging reveals easily accessible, quantitative outgrowth data. We found that effects on outgrowing cell numbers correlate to the concentrations of relevant pharmacological compounds and could thus serve as a practical readout to monitor drug effects. Here, we demonstrate the potential of this semi-high-throughput “cardiac cell outgrowth assay” with six compounds at three concentrations applied to spheroids for 48 h. The image-based readout complements end-point assays or may be used as a non-invasive assay for quality control during long-term culture.
RESUMO
With their capability to self-renew and differentiate into derivatives of all three germ layers, human pluripotent stem cells (hPSCs) offer a unique model to study aspects of human development in vitro. Directed differentiation towards mesendodermal lineages is a complex process, involving transition through a primitive streak (PS)-like stage. We have recently shown PS-like patterning from hPSCs into definitive endoderm, cardiac as well as presomitic mesoderm by only modulating the bulk cell density and the concentration of the GSK3 inhibitor CHIR99021, a potent activator of the WNT pathway. The patterning process is modulated by a complex paracrine network, whose identity and mechanistic consequences are poorly understood. To study the underlying dynamics, we here applied mathematical modeling based on ordinary differential equations. We compared time-course data of early hPSC differentiation to increasingly complex model structures with incremental numbers of paracrine factors. Model simulations suggest at least three paracrine factors being required to recapitulate the experimentally observed differentiation kinetics. Feedback mechanisms from both undifferentiated and differentiated cells turned out to be crucial. Evidence from double knock-down experiments and secreted protein enrichment allowed us to hypothesize on the identity of two of the three predicted factors. From a practical perspective, the mathematical model predicts optimal settings for directing lineage-specific differentiation. This opens new avenues for rational stem cell bioprocessing in more advanced culture systems, e.g. in perfusion-fed bioreactors enabling cell therapies.
Assuntos
Diferenciação Celular/fisiologia , Modelos Teóricos , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Piridinas/farmacologia , Pirimidinas/farmacologiaRESUMO
Biologically active human bone morphogenetic protein-4 (hBMP-4) was successfully produced in a prokaryotic host. For this aim, hBMP-4 cDNA was cloned in Escherichia coli (E. coli) and the protein was produced in a non-active aggregated form. After washing and solubilization, in vitro refolding of the rhBMP-4 monomer was performed using rapid dilution. In this study, different refolding conditions were tested for the dimerization of rhBMP-4 by one-factor-at-a-time variation. The dimerization process was found to be sensitive to pH, protein concentration and the presence of aggregation suppressors. In contrast, redox conditions and ionic strength did not impact refolding as expected. The dimer was separated from the remaining monomer, aggregates and host cell contaminants in a single step using cation-exchange membrane chromatography. The rhBMP-4 dimer produced in E. coli was biologically active as demonstrated by its capability to induce trophoblast differentiation and primitive streak induction of human pluripotent stem cells (hPSCs).
RESUMO
Endothelial cells (ECs) are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor) cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs) represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Endoteliais da Veia Umbilical Humana/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Biomarcadores/metabolismo , Reatores Biológicos , Proliferação de Células , Células Cultivadas , Xenoenxertos , Humanos , Cariótipo , Modelos Animais , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Suspensões , Peixe-ZebraRESUMO
Induced pluripotent stem cell (iPSC)-derived hematopoietic cells represent a highly attractive source for cell and gene therapy. Given the longevity, plasticity, and self-renewal potential of distinct macrophage subpopulations, iPSC-derived macrophages (iPSC-Mφ) appear of particular interest in this context. We here evaluated the airway residence, plasticity, and therapeutic efficacy of iPSC-Mφ in a murine model of hereditary pulmonary alveolar proteinosis (herPAP). We demonstrate that single pulmonary macrophage transplantation (PMT) of 2.5-4 × 106 iPSC-Mφ yields efficient airway residence with conversion of iPSC-Mφ to an alveolar macrophage (AMφ) phenotype characterized by a distinct surface marker and gene expression profile within 2 months. Moreover, PMT significantly improves alveolar protein deposition and other critical herPAP disease parameters. Thus, our data indicate iPSC-Mφ as a source of functional macrophages displaying substantial plasticity and therapeutic potential that upon pulmonary transplantation will integrate into the lung microenvironment, adopt an AMφ phenotype and gene expression pattern, and profoundly ameliorate pulmonary disease phenotypes.
Assuntos
Subunidade beta Comum dos Receptores de Citocinas/genética , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/transplante , Proteinose Alveolar Pulmonar/terapia , Animais , Células Cultivadas , Deleção de Genes , Hematopoese , Camundongos , Camundongos Knockout , Proteinose Alveolar Pulmonar/genética , Proteinose Alveolar Pulmonar/patologiaRESUMO
The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45+CD11b+CD14+CD163+ cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies.
Assuntos
Infecções Bacterianas/prevenção & controle , Reatores Biológicos , Imunoterapia/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos/citologia , Infecções Respiratórias/prevenção & controle , Animais , Infecções Bacterianas/imunologia , Técnicas de Cultura de Células , Humanos , Macrófagos/fisiologia , Camundongos , Microscopia Eletrônica de Varredura , Pseudomonas aeruginosa/patogenicidade , Infecções Respiratórias/imunologiaRESUMO
Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.
Assuntos
Transgenes/genética , Animais , Linhagem Celular , Células Cultivadas , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução Genética , Transgenes/fisiologiaRESUMO
Controlled large-scale production of human pluripotent stem cells (hPSCs) is indispensable for their envisioned clinical translation. Aiming at advanced process development in suspension culture, the sensitivity of hPSC media to continuous peristaltic pump-based circulation, a well-established technology extensively used in hydraulically-driven bioreactors, was investigated. Unexpectedly, conditioning of low protein media (i.e. E8 and TeSR-E8) in a peristaltic pump circuit induced severe viability loss of hPSCs cultured as aggregates in suspension. Optical, biochemical, and cytological analyses of the media revealed that the applied circulation mode resulted in the reduction of the growth hormone insulin by precipitation of micro-sized particles. Notably, in contrast to insulin depletion, individual withdrawal of other medium protein components (i.e. bFGF, TGFß1 or transferrin) provoked minor reduction of hPSC viability, if any. Supplementation of the surfactant glycerol or the use of the insulin analogue Aspart did not overcome the issue of insulin precipitation. In contrast, the presence of bovine or human serum albumin (BSA or HSA, respectively) stabilized insulin rescuing its content, possibly by acting as molecular chaperone-like protein, ultimately supporting hPSC maintenance. This study highlights the potential and the requirement of media optimization for automated hPSC processing and has broad implications on media development and bioreactor-based technologies.
Assuntos
Técnicas de Cultura de Células/métodos , Insulina/análise , Células-Tronco Pluripotentes/fisiologia , Reatores Biológicos , Agregação Celular , Sobrevivência Celular , Meios de Cultivo Condicionados , HumanosRESUMO
Subtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO), a chemical modulator of small-/intermediate-conductance Ca2+-activated potassium channels (SKs 1-4). Investigating EBIO in human pluripotent stem cells (PSCs), we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs, timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs, including an increase in nodal- and atrial-like phenotypes. However, our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and, subsequently, CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO, presumably via an SK-independent mechanism. Together, EBIO did not promote cardiogenic differentiation of PSCs, opposing previous findings, but triggered lineage-selective survival at a cardiac progenitor stage, which we propose as a pharmacological strategy to modulate CM subtype composition.