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1.
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.

2.
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.

4.
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
5.
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.

6.
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
7.
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.

8.
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
9.
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
10.
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
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