Human iPSCs and their uses in developmental toxicology

Amyotrophic lateral sclerosis (ALS) is a progressive disease leading to the degeneration of motor neurons (MNs). Neuroinflammation is involved in the pathogenesis of ALS; however, interactions of specific immune cell types and MNs are not well studied. We recently found a shift toward T helper (Th)1/Th17 cell-mediated, pro-inflammatory immune responses in the peripheral immune system of ALS patients, which positively correlated with disease severity and progression. Whether Th17 cells or their central mediator, Interleukin-17 (IL-17), directly affects human motor neuron survival is currently unknown. Here, we evaluated the contribution of Th17 cells and IL-17 on MN degeneration using the co-culture of iPSC-derived MNs of fused in sarcoma (FUS)-ALS patients and isogenic controls with Th17 lymphocytes derived from ALS patients, healthy controls, and multiple sclerosis (MS) patients (positive control). Only Th17 cells from MS patients induced severe MN degeneration in FUS-ALS as well as in wildtype MNs. Their main effector, IL-17A, yielded in a dose-dependent decline of the viability and neurite length of MNs. Surprisingly, IL-17F did not influence MNs. Importantly, neutralizing IL-17A and anti-IL-17 receptor A treatment reverted all effects of IL-17A. Our results offer compelling evidence that Th17 cells and IL-17A do directly contribute to MN degeneration.

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Generation of functional conjunctival epithelium, including goblet cells, from human iPSCs

Cell Reports ◽

10.1016/j.celrep.2021.108715 ◽

2021 ◽

Vol 34 (5) ◽

pp. 108715

Author(s):

Kimihito Nomi ◽

Ryuhei Hayashi ◽

Yuki Ishikawa ◽

Yuki Kobayashi ◽

Tomohiko Katayama ◽

...

Keyword(s):

Goblet Cells ◽

Conjunctival Epithelium ◽

Human Ipscs

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An Efficient Method for the Differentiation of Human iPSC-Derived Endoderm toward Enterocytes and Hepatocytes

Cells ◽

10.3390/cells10040812 ◽

2021 ◽

Vol 10 (4) ◽

pp. 812

Author(s):

Shimeng Qiu ◽

Yaling Li ◽

Yuki Imakura ◽

Shinji Mima ◽

Tadahiro Hashita ◽

...

Keyword(s):

Small Intestine ◽

Activin A ◽

Double Positive ◽

Differentiation Method ◽

Drug Metabolizing Enzyme ◽

Human Ipscs ◽

Novel Method ◽

Induced Pluripotent ◽

Metabolizing Enzyme ◽

Human Ipsc

The endoderm, differentiated from human induced pluripotent stem cells (iPSCs), can differentiate into the small intestine and liver, which are vital for drug absorption and metabolism. The development of human iPSC-derived enterocytes (HiEnts) and hepatocytes (HiHeps) has been reported. However, pharmacokinetic function-deficiency of these cells remains to be elucidated. Here, we aimed to develop an efficient differentiation method to induce endoderm formation from human iPSCs. Cells treated with activin A for 168 h expressed higher levels of endodermal genes than those treated for 72 h. Using activin A (days 0–7), CHIR99021 and PI−103 (days 0–2), and FGF2 (days 3–7), the hiPSC-derived endoderm (HiEnd) showed 97.97% CD−117 and CD−184 double-positive cells. Moreover, HiEnts derived from the human iPSC line Windy had similar or higher expression of small intestine-specific genes than adult human small intestine. Activities of the drug transporter P-glycoprotein and drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5 were confirmed. Additionally, Windy-derived HiHeps expressed higher levels of hepatocyte- and pharmacokinetics-related genes and proteins and showed higher CYP3A4/5 activity than those derived through the conventional differentiation method. Thus, using this novel method, the differentiated HiEnts and HiHeps with pharmacokinetic functions could be used for drug development.

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The CAD risk locus 9p21 increases the risk of vascular calcification in an iPSC-derived VSMC model

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02229-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Anja Trillhaase ◽

Beatrice Schmidt ◽

Marlon Märtens ◽

Undine Haferkamp ◽

Jeanette Erdmann ◽

...

Keyword(s):

Coronary Artery ◽

Vascular Calcification ◽

Alizarin Red ◽

Vitro Assay ◽

Human Ipscs ◽

Increased Risk ◽

Risk Locus ◽

9P21 Locus ◽

Cad Risk

Abstract Background Coronary artery disease (CAD) is the leading cause of death worldwide. Chromosome locus 9p21 was the first to be associated with increased risk of CAD and coronary artery calcification (CAC). Vascular calcification increases the risk for CAD. Vascular smooth muscle cells (VSMCs) are one of the major cell types involved in the development of vascular calcification. Methods So far, mainly animal models or primary SMCs have been used to model human vascular calcification. In this study, a human in vitro assay using iPSC-derived VSMCs was developed to examine vascular calcification. Human iPSCs were derived from a healthy non-risk (NR) and risk (R) donor carrying SNPs in the 9p21 locus. Additionally, 9p21 locus knockouts of each donor iPSC line (NR and R) were used. Following differentiation, the iPSC-derived VSMCs were characterized based on cell type, proliferation, and migration rate, along with calcium phosphate (CaP) deposits. CaP deposits were confirmed using Calcein and Alizarin Red S staining and then quantified. Results The data demonstrated significantly more proliferation, migration, and CaP deposition in VSMCs derived from the R and both KO iPSC lines than in those derived from the NR line. Molecular analyses confirmed upregulation of calcification markers. These results are consistent with recent data demonstrating increased calcification when the 9p21 murine ortholog is knocked-out. Conclusion Therefore, in conclusion, genetic variation or deletion of the CAD risk locus leads to an increased risk of vascular calcification. This in vitro human iPSC model of calcification could be used to develop new drug screening strategies to combat CAC.

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Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia

International Journal of Molecular Sciences ◽

10.3390/ijms22094334 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4334

Author(s):

Katrina Albert ◽

Jonna Niskanen ◽

Sara Kälvälä ◽

Šárka Lehtonen

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

Neurodegenerative Disease ◽

Induced Pluripotent Stem Cells ◽

Glial Cells ◽

Pluripotent Stem Cells ◽

Cell Types ◽

Human Ipscs ◽

Induced Pluripotent

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

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A non-invasive method to generate induced pluripotent stem cells from primate urine

Scientific Reports ◽

10.1038/s41598-021-82883-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Johanna Geuder ◽

Lucas E. Wange ◽

Aleksandar Janjic ◽

Jessica Radmer ◽

Philipp Janssen ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Sendai Virus ◽

Cell Types ◽

Urine Samples ◽

Comparative Approach ◽

Non Invasive ◽

Human Ipscs ◽

Induced Pluripotent

AbstractComparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that suspension- Sendai Virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to non-invasively generate iPSCs from primate urine. This will extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.

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Human iPSC-Based Modeling of Central Nerve System Disorders for Drug Discovery

International Journal of Molecular Sciences ◽

10.3390/ijms22031203 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1203

Author(s):

Lu Qian ◽

Julia TCW

Keyword(s):

Drug Discovery ◽

Disease Modeling ◽

Three Dimensional ◽

Structural Complexity ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Central Nerve System ◽

Human Ipscs ◽

Nerve System

A high-throughput drug screen identifies potentially promising therapeutics for clinical trials. However, limitations that persist in current disease modeling with limited physiological relevancy of human patients skew drug responses, hamper translation of clinical efficacy, and contribute to high clinical attritions. The emergence of induced pluripotent stem cell (iPSC) technology revolutionizes the paradigm of drug discovery. In particular, iPSC-based three-dimensional (3D) tissue engineering that appears as a promising vehicle of in vitro disease modeling provides more sophisticated tissue architectures and micro-environmental cues than a traditional two-dimensional (2D) culture. Here we discuss 3D based organoids/spheroids that construct the advanced modeling with evolved structural complexity, which propels drug discovery by exhibiting more human specific and diverse pathologies that are not perceived in 2D or animal models. We will then focus on various central nerve system (CNS) disease modeling using human iPSCs, leading to uncovering disease pathogenesis that guides the development of therapeutic strategies. Finally, we will address new opportunities of iPSC-assisted drug discovery with multi-disciplinary approaches from bioengineering to Omics technology. Despite technological challenges, iPSC-derived cytoarchitectures through interactions of diverse cell types mimic patients’ CNS and serve as a platform for therapeutic development and personalized precision medicine.

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Book Review: Developmental Toxicology: Mechanisms and Risk

Alternatives to Laboratory Animals ◽

10.1177/026119298801600118 ◽

1988 ◽

Vol 16 (1) ◽

pp. 96-97

Author(s):

Karen Atkinson

Keyword(s):

Developmental Toxicology

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Simplified 89Zr-Labeling Protocol of Oxine (8-Hydroxyquinoline) Enabling Prolonged Tracking of Liposome-Based Nanomedicines and Cells

Pharmaceutics ◽

10.3390/pharmaceutics13071097 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1097

Author(s):

Andras Polyak ◽

Jens P. Bankstahl ◽

Karen F. W. Besecke ◽

Constantin Hozsa ◽

Wiebke Triebert ◽

...

Keyword(s):

Reaction Times ◽

Extraction Methods ◽

Cell Labeling ◽

Size Exclusion ◽

Cell Therapies ◽

Biodistribution Studies ◽

Positron Emission ◽

Human Ipscs ◽

Radiochemical Yields

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.

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