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1.
Nat Protoc ; 16(12): 5652-5672, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34759383

RESUMO

Heart-forming organoids (HFOs) derived from human pluripotent stem cells (hPSCs) are a complex, highly structured in vitro model of early heart, foregut and vasculature development. The model represents a potent tool for various applications, including teratogenicity studies, gene function analysis and drug discovery. Here, we provide a detailed protocol describing how to form HFOs within 14 d. In an initial 4 d preculture period, hPSC aggregates are individually formed in a 96-well format and then Matrigel-embedded. Subsequently, the chemical WNT pathway modulators CHIR99021 and IWP2 are applied, inducing directed differentiation. This highly robust protocol can be used on many different hPSC lines and be combined with manipulation technologies such as gene targeting and drug testing. HFO formation can be assessed by numerous complementary methods, ranging from various imaging approaches to gene expression studies. Here, we highlight the flow cytometry-based analysis of individual HFOs, enabling the quantitative monitoring of lineage formation.

2.
Blood Adv ; 2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34649271

RESUMO

Primary or secondary immunodeficiencies are characterized by disruption of the cellular and humoral immunity. Respiratory infections are a major cause of morbidity and mortality among immunodeficient or immunocompromised patients with Staphylococcus aureus being a common offending organism. We here propose an adoptive macrophage transfer approach aiming to enhance impaired pulmonary immunity against S. aureus. Our studies, using human induced pluripotent stem cells (iPSC)-derived macrophages (iMφ) demonstrate efficient antimicrobial potential against Methicillin-sensitive and Methicillin-resistant clinical isolates of S. aureus. Using an S. aureus airway infection model in immunodeficient mice, we demonstrate that the adoptive transfer of iMφ is able to reduce the bacterial load more than 10-fold within 20 hours. This effect was associated with reduced granulocyte infiltration and less damage in lung tissue of transplanted animals. Whole transcriptome analysis of iMφ compared to monocyte-derived macrophages indicates a more profound upregulation of inflammatory genes early after infection and faster normalization 24 hours post-infection. Our data demonstrate high therapeutic efficacy of iMφ-based immunotherapy against S. aureus infections and offers an alternative treatment stratgey for immunodeficient or immunocompromised patients.

3.
Pharmaceutics ; 13(7)2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34371788

RESUMO

In this work, a method for the preparation of the highly lipophilic labeling synthon [89Zr]Zr(oxinate)4 was optimized for the radiolabeling of liposomes and human induced pluripotent stem cells (hiPSCs). The aim was to establish a robust and reliable labeling protocol for enabling up to one week positron emission tomography (PET) tracing of lipid-based nanomedicines and transplanted or injected cells, respectively. [89Zr]Zr(oxinate)4 was prepared from oxine (8-hydroxyquinoline) and [89Zr]Zr(OH)2(C2O4). Earlier introduced liquid-liquid extraction methods were simplified by the optimization of buffering, pH, temperature and reaction times. For quality control, thin-layer chromatography (TLC), size-exclusion chromatography (SEC) and centrifugation were employed. Subsequently, the 89Zr-complex was incorporated into liposome formulations. PET/CT imaging of 89Zr-labeled liposomes was performed in healthy mice. Cell labeling was accomplished in PBS using suspensions of 3 × 106 hiPSCs, each. [89Zr]Zr(oxinate)4 was synthesized in very high radiochemical yields of 98.7% (96.8% ± 2.8%). Similarly, high internalization rates (≥90%) of [89Zr]Zr(oxinate)4 into liposomes were obtained over an 18 h incubation period. MicroPET and biodistribution studies confirmed the labeled nanocarriers' in vivo stability. Human iPSCs incorporated the labeling agent within 30 min with ~50% efficiency. Prolonged PET imaging is an ideal tool in the development of lipid-based nanocarriers for drug delivery and cell therapies. To this end, a reliable and reproducible 89Zr radiolabeling method was developed and tested successfully in a model liposome system and in hiPSCs alike.

5.
Int J Mol Sci ; 22(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918735

RESUMO

Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.


Assuntos
Vesículas Extracelulares/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neovascularização Fisiológica , Transporte Biológico , Biomarcadores , Hipóxia Celular , Autorrenovação Celular , Células Cultivadas , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Medicina Regenerativa/métodos
6.
Stem Cells Transl Med ; 10(7): 1063-1080, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33660952

RESUMO

To harness the full potential of human pluripotent stem cells (hPSCs) we combined instrumented stirred tank bioreactor (STBR) technology with the power of in silico process modeling to overcome substantial, hPSC-specific hurdles toward their mass production. Perfused suspension culture (3D) of matrix-free hPSC aggregates in STBRs was applied to identify and control process-limiting parameters including pH, dissolved oxygen, glucose and lactate levels, and the obviation of osmolality peaks provoked by high density culture. Media supplements promoted single cell-based process inoculation and hydrodynamic aggregate size control. Wet lab-derived process characteristics enabled predictive in silico modeling as a new rational for hPSC cultivation. Consequently, hPSC line-independent maintenance of exponential cell proliferation was achieved. The strategy yielded 70-fold cell expansion in 7 days achieving an unmatched density of 35 × 106 cells/mL equivalent to 5.25 billion hPSC in 150 mL scale while pluripotency, differentiation potential, and karyotype stability was maintained. In parallel, media requirements were reduced by 75% demonstrating the outstanding increase in efficiency. Minimal input to our in silico model accurately predicts all main process parameters; combined with calculation-controlled hPSC aggregation kinetics, linear process upscaling is also enabled and demonstrated for up to 500 mL scale in an independent bioreactor system. Thus, by merging applied stem cell research with recent knowhow from industrial cell fermentation, a new level of hPSC bioprocessing is revealed fueling their automated production for industrial and therapeutic applications.

7.
Nat Biotechnol ; 39(6): 737-746, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33558697

RESUMO

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 RNA
8.
Mol Ther ; 29(4): 1395-1410, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33388418

RESUMO

Doxorubicin is one of the most potent chemotherapeutic agents. However, its clinical use is restricted due to the severe risk of cardiotoxicity, partially attributed to elevated production of reactive oxygen species (ROS). Telomerase canonically maintains telomeres during cell division but is silenced in adult hearts. In non-dividing cells such as cardiomyocytes, telomerase confers pro-survival traits, likely owing to the detoxification of ROS. Therefore, we hypothesized that pharmacological overexpression of telomerase may be used as a therapeutic strategy for the prevention of doxorubicin-induced cardiotoxicity. We used adeno-associated virus (AAV)-mediated gene therapy for long-term expression of telomerase in in vitro and in vivo models of doxorubicin-induced cardiotoxicity. Overexpression of telomerase protected the heart from doxorubicin-mediated apoptosis and rescued cardiac function, which was accompanied by preserved cardiomyocyte size. At the mechanistic level, we observed altered mitochondrial morphology and dynamics in response to telomerase expression. Complementary in vitro experiments confirmed the anti-apoptotic effects of telomerase overexpression in human induced pluripotent stem cell-derived cardiomyocytes after doxorubicin treatment. Strikingly, elevated levels of telomerase translocated to the mitochondria upon doxorubicin treatment, which helped to maintain mitochondrial function. Thus, telomerase gene therapy could be a novel preventive strategy for cardiotoxicity by chemotherapy agents such as the anthracyclines.


Assuntos
Cardiotoxicidade/genética , Doxorrubicina/efeitos adversos , Neoplasias/tratamento farmacológico , Telomerase/genética , Animais , Apoptose/efeitos dos fármacos , Cardiotoxicidade/prevenção & controle , Cardiotoxicidade/terapia , Dependovirus/genética , Doxorrubicina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Miócitos Cardíacos/efeitos dos fármacos , Neoplasias/complicações , Neoplasias/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Telomerase/farmacologia
9.
Cell Rep ; 32(9): 108090, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32877672

RESUMO

MYO18B loss-of-function mutations and depletion significantly compromise the structural integrity of striated muscle sarcomeres. The molecular function of the encoded protein, myosin-18B (M18B), within the developing muscle is unknown. Here, we demonstrate that recombinant M18B lacks motor ATPase activity and harbors previously uncharacterized N-terminal actin-binding domains, properties that make M18B an efficient actin cross-linker and molecular brake capable of regulating muscle myosin-2 contractile forces. Spatiotemporal analysis of M18B throughout cardiomyogenesis and myofibrillogenesis reveals that this structural myosin undergoes nuclear-cytoplasmic redistribution during myogenic differentiation, where its incorporation within muscle stress fibers coincides with actin striation onset. Furthermore, this analysis shows that M18B is directly integrated within the muscle myosin thick filament during myofibril maturation. Altogether, our data suggest that M18B has evolved specific biochemical properties that allow it to define and maintain sarcomeric organization from within the thick filament via its dual actin cross-linking and motor modulating capabilities.


Assuntos
Citoesqueleto de Actina/metabolismo , Miócitos Cardíacos/metabolismo , Miosinas/metabolismo , Sarcômeros/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Humanos , Domínios Proteicos , Proteínas Recombinantes/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-32793579

RESUMO

Human cardiomyocytes (CMs) have potential for use in therapeutic cell therapy and high-throughput drug screening. Because of the inability to expand adult CMs, their large-scale production from human pluripotent stem cells (hPSC) has been suggested. Significant improvements have been made in understanding directed differentiation processes of CMs from hPSCs and their suspension culture-based production at chemically defined conditions. However, optimization experiments are costly, time-consuming, and highly variable, leading to challenges in developing reliable and consistent protocols for the generation of large CM numbers at high purity. This study examined the ability of data-driven modeling with machine learning for identifying key experimental conditions and predicting final CM content using data collected during hPSC-cardiac differentiation in advanced stirred tank bioreactors (STBRs). Through feature selection, we identified process conditions, features, and patterns that are the most influential on and predictive of the CM content at the process endpoint, on differentiation day 10 (dd10). Process-related features were extracted from experimental data collected from 58 differentiation experiments by feature engineering. These features included data continuously collected online by the bioreactor system, such as dissolved oxygen concentration and pH patterns, as well as offline determined data, including the cell density, cell aggregate size, and nutrient concentrations. The selected features were used as inputs to construct models to classify the resulting CM content as being "sufficient" or "insufficient" regarding pre-defined thresholds. The models built using random forests and Gaussian process modeling predicted insufficient CM content for a differentiation process with 90% accuracy and precision on dd7 of the protocol and with 85% accuracy and 82% precision at a substantially earlier stage: dd5. These models provide insight into potential key factors affecting hPSC cardiac differentiation to aid in selecting future experimental conditions and can predict the final CM content at earlier process timepoints, providing cost and time savings. This study suggests that data-driven models and machine learning techniques can be employed using existing data for understanding and improving production of a specific cell type, which is potentially applicable to other lineages and critical for realization of their therapeutic applications.

11.
Stem Cell Reports ; 15(1): 13-21, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32531193

RESUMO

Pluripotency is tightly regulated and is crucial for stem cells and their implementation for regenerative medicine. Non-coding RNAs, especially long non-coding RNAs (lncRNAs) emerged as orchestrators of versatile (patho)-physiological processes on the transcriptional and post-transcriptional level. Cyrano, a well-conserved lncRNA, is highly expressed in stem cells suggesting an important role in pluripotency, which we aimed to investigate in loss-off-function (LOF) experiments. Cyrano was described previously to be essential for the maintenance of mouse embryonic stem cell (ESC) pluripotency. In contrast, using different genetic models, we here found Cyrano to be dispensable in murine and human iPSCs and in human ESCs. RNA sequencing revealed only a moderate influence of Cyrano on the global transcriptome. In line, Cyrano-depleted iPSCs retained the potential to differentiate into the three germ layers. In conclusion, different methods were applied for LOF studies to rule out potential off-target effects. These approaches revealed that Cyrano does not impact pluripotency.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Autorrenovação Celular/genética , Inativação Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos Knockout , RNA Longo não Codificante/genética , Transcriptoma/genética
12.
Stem Cell Reports ; 14(5): 788-802, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32302556

RESUMO

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent an attractive model to investigate CM function and disease mechanisms. One characteristic marker of ventricular specificity of human CMs is expression of the ventricular, slow ß-myosin heavy chain (MyHC), as opposed to the atrial, fast α-MyHC. The main aim of this study was to investigate at the single-cell level whether contraction kinetics and electrical activity of hESC-CMs are influenced by the relative expression of α-MyHC versus ß-MyHC. For effective assignment of functional parameters to the expression of both MyHC isoforms at protein and mRNA levels in the very same hESC-CMs, we developed a single-cell mapping technique. Surprisingly, α- versus ß-MyHC was not related to specific contractile or electrophysiological properties of the same cells. The multiparametric cell-by-cell analysis suggests that in hESC-CMs the expression of genes associated with electrical activity, contraction, calcium handling, and MyHCs is independently regulated.


Assuntos
Potenciais de Ação , Miosinas Cardíacas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosinas Cardíacas/genética , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análise de Célula Única
13.
Cells ; 8(12)2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31817235

RESUMO

For the production and bio-banking of differentiated derivatives from human pluripotent stem cells (hPSCs) in large quantities for drug screening and cellular therapies, well-defined and robust procedures for differentiation and cryopreservation are required. Definitive endoderm (DE) gives rise to respiratory and digestive epithelium, as well as thyroid, thymus, liver, and pancreas. Here, we present a scalable, universal process for the generation of DE from human-induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Optimal control during the differentiation process was attained in chemically-defined and xeno-free suspension culture, and high flexibility of the workflow was achieved by the introduction of an efficient cryopreservation step at the end of DE differentiation. DE aggregates were capable of differentiating into hepatic-like, pancreatic, intestinal, and lung progenitor cells. Scale-up of the differentiation process using stirred-tank bioreactors enabled production of large quantities of DE aggregates. This process provides a useful advance for versatile applications of DE lineages, in particular for cell therapies and drug screening.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Diferenciação Celular , Linhagem da Célula , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Técnicas de Cultura Celular por Lotes/instrumentação , Reatores Biológicos , Linhagem Celular , Criopreservação/métodos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo
15.
J Biomech ; 94: 99-106, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31376980

RESUMO

Three-dimensional dynamic suspension is becoming an effective cell culture method for a wide range of bioprocesses, with an increasing number of bioreactors proposed for this purpose. The complex hydrodynamics establishing within these devices affects bioprocess outcomes and efficiency, and usually expensive in vitro trial-and-error experiments are needed to properly set the working parameters. Here we propose a methodology to define a priori the hydrodynamic working parameters of a dynamic suspension bioreactor, selected as a test case because of the complex hydrodynamics characterizing its operating condition. A combination of computational and analytical approaches was applied to generate operational guideline graphs for defining a priori specific working parameters. In detail, 43 simulations were performed under pulsed flow regime to characterize advective transport within the device depending on different operative conditions, i.e., culture medium flow rate and its duty cycle, cultured particle diameter, and initial particle suspension volume. The operational guideline graphs were then used to set specific hydrodynamic working parameters for an in vitro proof-of-principle test, where human induced pluripotent stem cell (hiPSC) aggregates were cultured for 24 h within the bioreactor. The in vitro findings showed that, under the selected pulsed flow regime, sedimentation was avoided, hiPSC aggregate circularity and viability were preserved, and culture heterogeneity was reduced, thus confirming the appropriateness of the a priori method. This methodology has the potential to be adaptable to other dynamic suspension devices to support experimental studies by providing in silico-based a priori knowledge, useful to limit costs and to optimize culture bioprocesses.


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Teóricos , Simulação por Computador , Humanos , Hidrodinâmica
16.
Sci Rep ; 9(1): 11173, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371804

RESUMO

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-Clamp
17.
Stem Cell Reports ; 13(2): 366-379, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31353227

RESUMO

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 ∼1.3 × 108 CM in 150 mL and ∼4.0 × 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/metabolismo
18.
Methods Mol Biol ; 1994: 55-70, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31124104

RESUMO

Cardiomyocytes from human pluripotent stem cells (hPSCs) have the ability to advance specificity of in vitro assays for drug discovery and safety pharmacology. They may also provide a superior cell source for envisioned cell therapies to repair damaged hearts. All applications will require the production of cardiomyocytes (CMs) by robust upscalable bioprocesses via industry-compliant technologies. This paper describes a detailed procedure for producing hPSC-CMs in stirred tank bioreactors in 100 ml process scale. The strategy combines both hPSC expansion in suspension culture and, directly followed by, cardiogenic differentiation using small molecule-Wnt pathway modulators. We also provide a protocol describing how to plan and expand the pluripotent stem cells to enable parallel inoculation of 4× 150 ml parallel bioreactor differentiations, potentially producing more than 240 × 106 cardiomyocytes in 22 days.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Benzotiazóis/farmacologia , Reatores Biológicos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos
19.
Methods Mol Biol ; 1994: 79-91, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31124106

RESUMO

This chapter describes a detailed protocol on human pluripotent stem cells (hPSCs) cultivation as matrix-free cell-only aggregates in defined and xeno-free culture medium in stirred tank bioreactors (STBRs). Starting with a frozen stock pre-expanded on conventional culture dishes (2D), the ultimate process is performed in 150 mL culture scale in stirred tank bioreactors (3D) and is designed to produce up to 500 million pluripotent hPSC within 7 days. The culture strategy includes perfusion-based cell feeding facilitating process control, automation, and higher cell yields. Ultimately, this detailed protocol describes an important step for generating a defined starting cell population for directed lineage differentiation and subsequently fueling human cell-based assays and regenerative medicine approaches.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes/citologia , Automação/métodos , Reatores Biológicos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Meios de Cultura , Humanos
20.
Methods Mol Biol ; 1994: 185-193, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31124116

RESUMO

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/citologia
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