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18 pages, 4364 KiB

Open AccessArticle

Reactive Oxygen Species Mediate Transcriptional Responses to Dopamine and Cocaine in Human Cerebral Organoids

by Thomas T. Rudibaugh, Samantha R. Stuppy and Albert J. Keung

Int. J. Mol. Sci. 2023, 24(22), 16474; https://doi.org/10.3390/ijms242216474 - 18 Nov 2023

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Dopamine signaling in the adult ventral forebrain regulates behavior, stress response, and memory formation and in neurodevelopment regulates neural differentiation and cell migration. Excessive dopamine levels, including those due to cocaine use in utero and in adults, could lead to long-term adverse consequences. [...] Read more.

Dopamine signaling in the adult ventral forebrain regulates behavior, stress response, and memory formation and in neurodevelopment regulates neural differentiation and cell migration. Excessive dopamine levels, including those due to cocaine use in utero and in adults, could lead to long-term adverse consequences. The mechanisms underlying both homeostatic and pathological changes remain unclear, in part due to the diverse cellular responses elicited by dopamine and the reliance on animal models that exhibit species-specific differences in dopamine signaling. In this study, we use the human-derived ventral forebrain organoid model of Xiang–Tanaka and characterize their response to cocaine or dopamine. We explore dosing regimens of dopamine or cocaine to simulate acute or chronic exposure. We then use calcium imaging, cAMP imaging, and bulk RNA-sequencing to measure responses to cocaine or dopamine exposure. We observe an upregulation of inflammatory pathways in addition to indicators of oxidative stress following exposure. Using inhibitors of reactive oxygen species (ROS), we then show ROS to be necessary for multiple transcriptional responses of cocaine exposure. These results highlight novel response pathways and validate the potential of cerebral organoids as in vitro human models for studying complex biological processes in the brain. Full article

(This article belongs to the Special Issue Diversity of Induced Pluripotent Stem Cells)

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19 pages, 9553 KiB

Open AccessArticle

Derivation of Arbas Cashmere Goat Induced Pluripotent Stem Cells in LCDM with Trophectoderm Lineage Differentiation and Interspecies Chimeric Abilities

by Fang Liu, Jing Wang, Yongli Yue, Chen Li, Xuemin Zhang, Jinzhu Xiang, Hanning Wang and Xueling Li

Int. J. Mol. Sci. 2023, 24(19), 14728; https://doi.org/10.3390/ijms241914728 - 29 Sep 2023

Cited by 2 | Viewed by 892

Abstract

The Arbas cashmere goat is a unique biological resource that plays a vital role in livestock husbandry in China. LCDM is a medium with special small molecules (consisting of human LIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride) for generation pluripotent stem cells (PSCs) [...] Read more.

The Arbas cashmere goat is a unique biological resource that plays a vital role in livestock husbandry in China. LCDM is a medium with special small molecules (consisting of human LIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride) for generation pluripotent stem cells (PSCs) with bidirectional developmental potential in mice, humans, pigs, and bovines. However, there is no report on whether LCDM can support for generation of PSCs with the same ability in Arbas cashmere goats. In this study, we applied LCDM to generate goat induced PSCs (giPSCs) from goat fetal fibroblasts (GFFs) by reprogramming. The derived giPSCs exhibited stem cell morphology, expressing pluripotent markers, and could differentiate into three germ layers. Moreover, the giPSCs differentiated into the trophectoderm lineage by spontaneous and directed differentiation in vitro. The giPSCs contributed to embryonic and extraembryonic tissue in preimplantation blastocysts and postimplantation chimeric embryos. RNA-sequencing analysis showed that the giPSCs were very close to goat embryos at the blastocyst stage and giPSCs have similar properties to typical extended PSCs (EPSCs). The establishment of giPSCs with LCDM provides a new way to generate PSCs from domestic animals and lays the foundation for basic and applied research in biology and agriculture. Full article

(This article belongs to the Special Issue Diversity of Induced Pluripotent Stem Cells)

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16 pages, 19398 KiB

Open AccessArticle

A Microfluidic Experimental Method for Studying Cell-to-Cell Exosome Delivery–Taking Stem Cell–Tumor Cell Interaction as a Case

by Xing Yue (Larry) Peng, Pengxiang Su, Yaxin Guo, Jing Zhang, Linghan Peng and Rongrong Zhang

Int. J. Mol. Sci. 2023, 24(17), 13419; https://doi.org/10.3390/ijms241713419 - 30 Aug 2023

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Abstract

Cell-to-cell communication must occur through molecular transport in the intercellular fluid space. Nanoparticles, such as exosomes, diffuse or move more slowly in fluids than small molecules. To find a microfluidic technology for real-time exosome experiments on intercellular communication between living cells, we use [...] Read more.

Cell-to-cell communication must occur through molecular transport in the intercellular fluid space. Nanoparticles, such as exosomes, diffuse or move more slowly in fluids than small molecules. To find a microfluidic technology for real-time exosome experiments on intercellular communication between living cells, we use the microfluidic culture dish’s quaternary ultra-slow microcirculation flow field to accumulate nanoparticles in a specific area. Taking stem cell–tumor cell interaction as an example, the ultra-slow microcirculatory flow field controls stem cell exosomes to interfere with tumor cells remotely. Under static coculture conditions (without microfluidics), the tumor cells near stem cells (<200 µm) show quick breaking through from its Matrigel drop to meet stem cells, but this ‘breaking through’ quickly disappears with increasing distance. In programmed ultra-slow microcirculation, stem cells induce tumor cells 5000 μm far at the site of exosome deposition (according to nanoparticle simulations). After 14 days of programmed coculture, the glomeration and migration of tumor cells were observed in the exosome deposition area. This example shows that the ultra-slow microcirculation of the microfluidic culture dish has good prospects in quantitative experiments to study exosome communication between living cells and drug development of cancer metastasis. Full article

(This article belongs to the Special Issue Diversity of Induced Pluripotent Stem Cells)

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17 pages, 3296 KiB

Open AccessArticle

Modulation of Notch Signaling at Early Stages of Differentiation of Human Induced Pluripotent Stem Cells to Dopaminergic Neurons

by Nataliia V. Katolikova, Aleksandr A. Khudiakov, Daria D. Shafranskaya, Andrey D. Prjibelski, Alexey E. Masharskiy, Mikael S. Mor, Alexey S. Golovkin, Anastasia K. Zaytseva, Irina E. Neganova, Evgeniya V. Efimova, Raul R. Gainetdinov and Anna B. Malashicheva

Int. J. Mol. Sci. 2023, 24(2), 1429; https://doi.org/10.3390/ijms24021429 - 11 Jan 2023

Cited by 2 | Viewed by 1964

Abstract

Elaboration of protocols for differentiation of human pluripotent stem cells to dopamine neurons is an important issue for development of cell replacement therapy for Parkinson’s disease. A number of protocols have been already developed; however, their efficiency and specificity still can be improved. [...] Read more.

Elaboration of protocols for differentiation of human pluripotent stem cells to dopamine neurons is an important issue for development of cell replacement therapy for Parkinson’s disease. A number of protocols have been already developed; however, their efficiency and specificity still can be improved. Investigating the role of signaling cascades, important for neurogenesis, can help to solve this problem and to provide a deeper understanding of their role in neuronal development. Notch signaling plays an essential role in development and maintenance of the central nervous system after birth. In our study, we analyzed the effect of Notch activation and inhibition at the early stages of differentiation of human induced pluripotent stem cells to dopaminergic neurons. We found that, during the first seven days of differentiation, the cells were not sensitive to the Notch inhibition. On the contrary, activation of Notch signaling during the same time period led to significant changes and was associated with an increase in expression of genes, specific for caudal parts of the brain, a decrease of expression of genes, specific for forebrain, as well as a decrease of expression of genes, important for the formation of axons and dendrites and microtubule stabilizing proteins. Full article

(This article belongs to the Special Issue Diversity of Induced Pluripotent Stem Cells)

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12 pages, 1205 KiB

Open AccessArticle

Quality Control of Human Pluripotent Stem Cell Colonies by Computational Image Analysis Using Convolutional Neural Networks

by Anastasiya Mamaeva, Olga Krasnova, Irina Khvorova, Konstantin Kozlov, Vitaly Gursky, Maria Samsonova, Olga Tikhonova and Irina Neganova

Int. J. Mol. Sci. 2023, 24(1), 140; https://doi.org/10.3390/ijms24010140 - 21 Dec 2022

Cited by 2 | Viewed by 2245

Abstract

Human pluripotent stem cells are promising for a wide range of research and therapeutic purposes. Their maintenance in culture requires the deep control of their pluripotent and clonal status. A non-invasive method for such control involves day-to-day observation of the morphological changes, along [...] Read more.

Human pluripotent stem cells are promising for a wide range of research and therapeutic purposes. Their maintenance in culture requires the deep control of their pluripotent and clonal status. A non-invasive method for such control involves day-to-day observation of the morphological changes, along with imaging colonies, with the subsequent automatic assessment of colony phenotype using image analysis by machine learning methods. We developed a classifier using a convolutional neural network and applied it to discriminate between images of human embryonic stem cell (hESC) colonies with “good” and “bad” morphological phenotypes associated with a high and low potential for pluripotency and clonality maintenance, respectively. The training dataset included the phase-contrast images of hESC line H9, in which the morphological phenotype of each colony was assessed through visual analysis. The classifier showed a high level of accuracy (89%) in phenotype prediction. By training the classifier on cropped images of various sizes, we showed that the spatial scale of ~144 μm was the most informative in terms of classification quality, which was an intermediate size between the characteristic diameters of a single cell (~15 μm) and the entire colony (~540 μm). We additionally performed a proteomic analysis of several H9 cell samples used in the computational analysis and showed that cells of different phenotypes differentiated at the molecular level. Our results indicated that the proposed approach could be used as an effective method of non-invasive automated analysis to identify undesirable developmental anomalies during the propagation of pluripotent stem cells. Full article

(This article belongs to the Special Issue Diversity of Induced Pluripotent Stem Cells)

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Review

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12 pages, 1096 KiB

Open AccessReview

Overcoming the Variability of iPSCs in the Manufacturing of Cell-Based Therapies

by Suman C. Nath, Laura Menendez and Inbar Friedrich Ben-Nun

Int. J. Mol. Sci. 2023, 24(23), 16929; https://doi.org/10.3390/ijms242316929 - 29 Nov 2023

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Abstract

Various factors are known to contribute to the diversity of human induced pluripotent stem cells (hiPSCs). Among these are the donor’s genetic background and family history, the somatic cell source, the iPSC reprogramming method, and the culture system of choice. Moreover, variability is [...] Read more.

Various factors are known to contribute to the diversity of human induced pluripotent stem cells (hiPSCs). Among these are the donor’s genetic background and family history, the somatic cell source, the iPSC reprogramming method, and the culture system of choice. Moreover, variability is seen even in iPSC clones, generated in a single reprogramming event, where the donor, somatic cell type, and reprogramming platform are the same. The diversity seen in iPSC lines often translates to epigenetic differences, as well as to differences in the expansion rate, iPSC line culture robustness, and their ability to differentiate into specific cell types. As such, the diversity of iPSCs presents a hurdle to standardizing iPSC-based cell therapy manufacturing. In this review, we will expand on the various factors that impact iPSC diversity and the strategies and tools that could be taken by the industry to overcome the differences amongst various iPSC lines, therefore enabling robust and reproducible iPSC-based cell therapy manufacturing processes. Full article

(This article belongs to the Special Issue Diversity of Induced Pluripotent Stem Cells)

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20 pages, 4806 KiB

Open AccessReview

Corneal Endothelial-like Cells Derived from Induced Pluripotent Stem Cells for Cell Therapy

by Xiao Yu Ng, Gary S. L. Peh, Gary Hin-Fai Yam, Hwee Goon Tay and Jodhbir S. Mehta

Int. J. Mol. Sci. 2023, 24(15), 12433; https://doi.org/10.3390/ijms241512433 - 04 Aug 2023

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Abstract

Corneal endothelial dysfunction is one of the leading causes of corneal blindness, and the current conventional treatment option is corneal transplantation using a cadaveric donor cornea. However, there is a global shortage of suitable donor graft material, necessitating the exploration of novel therapeutic [...] Read more.

Corneal endothelial dysfunction is one of the leading causes of corneal blindness, and the current conventional treatment option is corneal transplantation using a cadaveric donor cornea. However, there is a global shortage of suitable donor graft material, necessitating the exploration of novel therapeutic approaches. A stem cell-based regenerative medicine approach using induced pluripotent stem cells (iPSCs) offers a promising solution, as they possess self-renewal capabilities, can be derived from adult somatic cells, and can be differentiated into all cell types including corneal endothelial cells (CECs). This review discusses the progress and challenges in developing protocols to induce iPSCs into CECs, focusing on the different media formulations used to differentiate iPSCs to neural crest cells (NCCs) and subsequently to CECs, as well as the characterization methods and markers that define iPSC-derived CECs. The hurdles and solutions for the clinical application of iPSC-derived cell therapy are also addressed, including the establishment of protocols that adhere to good manufacturing practice (GMP) guidelines. The potential risks of genetic mutations in iPSC-derived CECs associated with long-term in vitro culture and the danger of potential tumorigenicity following transplantation are evaluated. In all, this review provides insights into the advancement and obstacles of using iPSC in the treatment of corneal endothelial dysfunction. Full article

(This article belongs to the Special Issue Diversity of Induced Pluripotent Stem Cells)

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20 pages, 5222 KiB

Open AccessReview

Induced Pluripotent Stem Cells for Tissue-Engineered Skeletal Muscles

by Shudong Zhao, Jishizhan Chen, Lei Wu, Xin Tao, Naheem Yaqub and Jinke Chang

Int. J. Mol. Sci. 2023, 24(14), 11520; https://doi.org/10.3390/ijms241411520 - 15 Jul 2023

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Abstract

Skeletal muscle, which comprises a significant portion of the body, is responsible for vital functions such as movement, metabolism, and overall health. However, severe injuries often result in volumetric muscle loss (VML) and compromise the regenerative capacity of the muscle. Tissue-engineered muscles offer [...] Read more.

Skeletal muscle, which comprises a significant portion of the body, is responsible for vital functions such as movement, metabolism, and overall health. However, severe injuries often result in volumetric muscle loss (VML) and compromise the regenerative capacity of the muscle. Tissue-engineered muscles offer a potential solution to address lost or damaged muscle tissue, thereby restoring muscle function and improving patients’ quality of life. Induced pluripotent stem cells (iPSCs) have emerged as a valuable cell source for muscle tissue engineering due to their pluripotency and self-renewal capacity, enabling the construction of tissue-engineered artificial skeletal muscles with applications in transplantation, disease modelling, and bio-hybrid robots. Next-generation iPSC-based models have the potential to revolutionize drug discovery by offering personalized muscle cells for testing, reducing reliance on animal models. This review provides a comprehensive overview of iPSCs in tissue-engineered artificial skeletal muscles, highlighting the advancements, applications, advantages, and challenges for clinical translation. We also discussed overcoming limitations and considerations in differentiation protocols, characterization methods, large-scale production, and translational regulations. By tackling these challenges, iPSCs can unlock transformative advancements in muscle tissue engineering and therapeutic interventions for the future. Full article

(This article belongs to the Special Issue Diversity of Induced Pluripotent Stem Cells)

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