“Necessity Is the Mother of Invention” or Inexpensive, Reliable, and Reproducible Protocol for Generating Organoids

Eremeev, Artem V.; Volovikov, Egor; Shuvalova, L.D.; Davidenko, A. V.; Khomyakova, E. A.; Bogomiakova, Margarita E.; Lebedeva, Olga; Zubkova, Olga A.; Lagarkova, Maria A.

doi:10.1134/s0006297919030143

Cited by 7 publications

(4 citation statements)

References 37 publications

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“…The next day, 3D spheroids were harvested into F1 medium containing 3/1 DMEM/F12 (Gibco), 5% FBS (Gibco), 0,1 μg/ml hydrocortisone (Gedeon Richter Ltd), 10 ng/ml EGF (Miltenyi Biotec) and 5 μg/ml insulin (Sigma). Further cultivation was carried out in special homemade mini-bioreactors [ 46 ] on an orbital shaker (70 rpm). From day 4, 2 ng/ml BMP-4 (R&D Systems) were added to the F1 medium.…”

Section: Methodsmentioning

confidence: 99%

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

et al. 2023

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Background Dozens of transplants generated from pluripotent stem cells are currently in clinical trials. The creation of patient-specific iPSCs makes personalized therapy possible due to their main advantage of immunotolerance. However, some reports have claimed recently that aberrant gene expression followed by proteome alterations and neoantigen formation can result in iPSCs recognition by autologous T-cells. Meanwhile, the possibility of NK-cell activation has not been previously considered. This study focused on the comparison of autologous and allogeneic immune response to iPSC-derived cells and isogeneic parental somatic cells used for reprogramming. Methods We established an isogeneic cell model consisting of parental dermal fibroblasts, fibroblast-like iPSC-derivatives (iPS-fibro) and iPS-fibro lacking beta-2-microglobulin (B2M). Using the cells obtained from two patients, we analyzed the activation of autologous and allogeneic T-lymphocytes and NK-cells co-cultured with target cells. Results Here we report that cells differentiated from iPSCs can be recognized by NK-cells rather than by autologous T-cells. We observed that iPS-fibro elicited a high level of NK-cell degranulation and cytotoxicity, while isogeneic parental skin fibroblasts used to obtain iPSCs barely triggered an NK-cell response. iPSC-derivatives with B2M knockout did not cause an additional increase in NK-cell activation, although they were devoid of HLA-I, the major inhibitory molecules for NK-cells. Transcriptome analysis revealed a significant imbalance of ligands for activating and inhibitory NK-cell receptors in iPS-fibro. Compared to parental fibroblasts, iPSC-derivatives had a reduced expression of HLA-I simultaneously with an increased gene expression of major activating ligands, such as MICA, NECTIN2, and PVR. The lack of inhibitory signals might be due to insufficient maturity of cells differentiated from iPSCs. In addition, we showed that pretreatment of iPS-fibro with proinflammatory cytokine IFNγ restored the ligand imbalance, thereby reducing the degranulation and cytotoxicity of NK-cells. Conclusion In summary, we showed that iPSC-derived cells can be sensitive to the cytotoxic potential of autologous NK-cells regardless of HLA-I status. Thus, the balance of ligands for NK-cell receptors should be considered prior to iPSC-based cell therapies. Trial registration Not applicable.

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Section: Methodsmentioning

confidence: 99%

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

et al. 2023

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“…This variability limits organoid use in screening new therapeutic drugs or in preclinical studies, requiring high standardization. Several research groups published protocols for creating large quantities of standardized organoids for use in drug screening and electrophysiological studies [ 49 , 88 , 89 ].…”

Section: The Specificities Of Organoids Creation and Cultivationmentioning

confidence: 99%

Cerebral Organoids—Challenges to Establish a Brain Prototype

Eremeev

Lebedeva

Bogomiakova

et al. 2021

Cells

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The new cellular models based on neural cells differentiated from induced pluripotent stem cells have greatly enhanced our understanding of human nervous system development. Highly efficient protocols for the differentiation of iPSCs into different types of neural cells have allowed the creation of 2D models of many neurodegenerative diseases and nervous system development. However, the 2D culture of neurons is an imperfect model of the 3D brain tissue architecture represented by many functionally active cell types. The development of protocols for the differentiation of iPSCs into 3D cerebral organoids made it possible to establish a cellular model closest to native human brain tissue. Cerebral organoids are equally suitable for modeling various CNS pathologies, testing pharmacologically active substances, and utilization in regenerative medicine. Meanwhile, this technology is still at the initial stage of development.

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“…Such variability precludes atlas creation for cerebral organoids (Zaslavsky et al, 2014). In order to reproduce the brain cyto-architectural development with a higher reproducibility, some studies investigated the addition of specific factor to the media (WNT, SSH, FGF) (Krefft et al, 2018;Cederquist et al, 2019;Kim et al, 2019;Sivitilli et al, 2020), whereas others used bioreactors (Qian et al, 2016;Eremeev et al, 2019;Velasco et al, 2019) or changed the type of culture (Berger et al, 2018;Nickels et al, 2020).…”

Section: Variability In 3d Brain Culturesmentioning

confidence: 99%

Recent Trends and Perspectives in Cerebral Organoids Imaging and Analysis

Martin

Chane

Clouchoux³

et al. 2021

Front. Neurosci.

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Purpose: Since their first generation in 2013, the use of cerebral organoids has spread exponentially. Today, the amount of generated data is becoming challenging to analyze manually. This review aims to overview the current image acquisition methods and to subsequently identify the needs in image analysis tools for cerebral organoids.Methods: To address this question, we went through all recent articles published on the subject and annotated the protocols, acquisition methods, and algorithms used.Results: Over the investigated period of time, confocal microscopy and bright-field microscopy were the most used acquisition techniques. Cell counting, the most common task, is performed in 20% of the articles and area; around 12% of articles calculate morphological parameters. Image analysis on cerebral organoids is performed in majority using ImageJ software (around 52%) and Matlab language (4%). Treatments remain mostly semi-automatic. We highlight the limitations encountered in image analysis in the cerebral organoid field and suggest possible solutions and implementations to develop.Conclusions: In addition to providing an overview of cerebral organoids cultures and imaging, this work highlights the need to improve the existing image analysis methods for such images and the need for specific analysis tools. These solutions could specifically help to monitor the growth of future standardized cerebral organoids.

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“Necessity Is the Mother of Invention” or Inexpensive, Reliable, and Reproducible Protocol for Generating Organoids

Cited by 7 publications

References 37 publications

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

Cerebral Organoids—Challenges to Establish a Brain Prototype

Recent Trends and Perspectives in Cerebral Organoids Imaging and Analysis

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