In vitro techniques for assessing differentiation and functionality of human pluripotent stem cell ‐derived RPE cells

Abstract Human pluripotent stem cell (hPSC)-derived organoids and differentiated cells have similar characteristics, such as cell types, structure, and functions, to human organs and tissues. Thus, in vitro human organoids and tissue-specific cells serve as a superior alternative to conventional cell lines and animal models in drug development and regenerative medicine. However, since hPSC-derived organoids and differentiated cells show fetal-like features, further differentiation and maturation methods have been developed for the generation of high-quality in vitro models of the corresponding human organs and tissues. Therefore, for a simple and reproducible analysis of the quality of organoids and cells to compensate for the shortcomings of existing experimental validation studies, a quantitative evaluation method should be developed. In this study, using the GTEx database (a total of 8,555 samples in 53 tissues), we constructed a quantitative calculation system (organ-specific panels and calculation algorithm) to assess the similarity to the human lung, stomach, and heart and confirmed the algorithm using in-house RNA-seq data (total RNA from 20 tissues). To evaluate our system, we generated hPSC-derived lung organoids, gastric organoids, and cardiomyocytes and detected 33.4%, 51.7%, and 83.4% similarity, respectively, to the corresponding human target organs. To facilitate access and use of our system for researchers, we developed the web-based user interface (Web-based Similarity Analysis System, W-SAS; for liver, lung, stomach, and heart) presenting similarity to the appropriate organs as percentages and specific gene expression patterns. Thus, the W-SAS system could provide valuable information for the generation of high-quality and readily available organoids/cells differentiated from hPSCs and a strategy to guide proper lineage-oriented differentiation.

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Functional characterization of human pluripotent stem cell-derived cortical networks differentiated on laminin-521 substrate: comparison to rat cortical cultures

Scientific Reports ◽

10.1038/s41598-019-53647-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Tanja Hyvärinen ◽

Anu Hyysalo ◽

Fikret Emre Kapucu ◽

Laura Aarnos ◽

Andrey Vinogradov ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Microelectrode Array ◽

Functional Characterization ◽

Principal Component ◽

Network Activity ◽

Human Pluripotent Stem Cell ◽

Cortical Networks ◽

Cortical Cultures

AbstractHuman pluripotent stem cell (hPSC)-derived neurons provide exciting opportunities for in vitro modeling of neurological diseases and for advancing drug development and neurotoxicological studies. However, generating electrophysiologically mature neuronal networks from hPSCs has been challenging. Here, we report the differentiation of functionally active hPSC-derived cortical networks on defined laminin-521 substrate. We apply microelectrode array (MEA) measurements to assess network events and compare the activity development of hPSC-derived networks to that of widely used rat embryonic cortical cultures. In both of these networks, activity developed through a similar sequence of stages and time frames; however, the hPSC-derived networks showed unique patterns of bursting activity. The hPSC-derived networks developed synchronous activity, which involved glutamatergic and GABAergic inputs, recapitulating the classical cortical activity also observed in rodent counterparts. Principal component analysis (PCA) based on spike rates, network synchronization and burst features revealed the segregation of hPSC-derived and rat network recordings into different clusters, reflecting the species-specific and maturation state differences between the two networks. Overall, hPSC-derived neural cultures produced with a defined protocol generate cortical type network activity, which validates their applicability as a human-specific model for pharmacological studies and modeling network dysfunctions.

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Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro – Correlation between contraction force and electrophysiology

Biomaterials ◽

10.1016/j.biomaterials.2015.01.067 ◽

2015 ◽

Vol 51 ◽

pp. 138-150 ◽

Cited By ~ 119

Author(s):

Marcelo C. Ribeiro ◽

Leon G. Tertoolen ◽

Juan A. Guadix ◽

Milena Bellin ◽

Georgios Kosmidis ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Human Pluripotent Stem Cell ◽

Contraction Force ◽

Functional Maturation

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Human Pluripotent Stem Cell-Derived Neurons Are Functionally Mature In Vitro and Integrate into the Mouse Striatum Following Transplantation

Molecular Neurobiology ◽

10.1007/s12035-020-01907-4 ◽

2020 ◽

Vol 57 (6) ◽

pp. 2766-2798 ◽

Cited By ~ 2

Author(s):

Andrea Comella-Bolla ◽

Javier G. Orlandi ◽

Andrés Miguez ◽

Marco Straccia ◽

María García-Bravo ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Human Pluripotent Stem Cell ◽

Mouse Striatum

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Regulation of JAM2 Expression in the Lungs of Streptozotocin-Induced Diabetic Mice and Human Pluripotent Stem Cell-Derived Alveolar Organoids

Biomedicines ◽

10.3390/biomedicines8090346 ◽

2020 ◽

Vol 8 (9) ◽

pp. 346 ◽

Cited By ~ 2

Author(s):

Roya Rasaei ◽

Eunbi Kim ◽

Ji-Young Kim ◽

Sunghun Na ◽

Jung-Hyun Kim ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Causative Factor ◽

Human Mesenchymal Stem Cell ◽

Therapeutic Effects ◽

Diabetic Mice ◽

Human Pluripotent Stem Cell ◽

Potential Indicator

Hyperglycemia is a causative factor in the pathogenesis of respiratory diseases, known to induce fibrosis and inflammation in the lung. However, little attention has been paid to genes related to hyperglycemic-induced lung alterations and stem cell applications for therapeutic use. In this study, our microarray data revealed significantly increased levels of junctional adhesion molecule 2 (JAM2) in the high glucose (HG)-induced transcriptional profile in human perivascular cells (hPVCs). The elevated level of JAM2 in HG-treated hPVCs was transcriptionally and epigenetically reversible when HG treatment was removed. We further investigated the expression of JAM2 using in vivo and in vitro hyperglycemic models. Our results showed significant upregulation of JAM2 in the lungs of streptozotocin (STZ)-induced diabetic mice, which was greatly suppressed by the administration of conditioned medium obtained from human mesenchymal stem cell cultures. Furthermore, JAM2 was found to be significantly upregulated in human pluripotent stem cell-derived multicellular alveolar organoids by exposure to HG. Our results suggest that JAM2 may play an important role in STZ-induced lung alterations and could be a potential indicator for predicting the therapeutic effects of stem cells and drugs in diabetic lung complications.

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Transcriptomic Profiling of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Over Time

10.1101/842328 ◽

2019 ◽

Author(s):

Grace E. Lidgerwood ◽

Anne Senabouth ◽

Casey J.A. Smith-Anttila ◽

Vikkitharan Gnanasambandapillai ◽

Dominik C. Kaczorowski ◽

...

Keyword(s):

Stem Cell ◽

Retinal Pigment Epithelium ◽

Pluripotent Stem Cell ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Embryonic Stem Cell Line ◽

Embryonic Stem ◽

Human Pluripotent Stem Cell ◽

Mesenchymal Transition ◽

Rpe Cells

AbstractHuman pluripotent stem cell (hPSC)-derived progenies are immature versions of cells, presenting a potential limitation to the accurate modelling of disease associated with maturity or age. Hence, it is important to characterise how closely cells used in culture resemble their native counterparts. In order to select appropriate points in time for RPE cultures to reflect native counterparts, we characterised the transcriptomic profiles of hPSC-derived retinal pigment epithelium (RPE) cells from 1- and 12-month cultures. We differentiated the human embryonic stem cell line H9 into RPE cells, performed single cell RNA-sequencing of a total of 16,576 cells, and analysed the resulting data to assess the molecular changes of RPE cells across these two culture time points. Our results indicate the stability of the RPE transcriptomic signature, with no evidence of an epithelial – mesenchymal transition, and with maturing populations of RPE observed with time in culture. Assessment of gene ontology pathways revealed that as cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions. This might reflect an increased ability to handle oxidative stress as cells mature. Comparison with native human RPE data confirmed a maturing transcriptional profile of RPE cells in culture. These results suggest that in vitro long-term culture of RPE cells allow the modelling of specific phenotypes observed in native mature tissue. Our work highlights the transcriptional landscape of hPSC-derived RPE as they age in culture, which provides a reference for native and patient-samples to be benchmarked against.

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Identification of Drugs Blocking SARS-CoV-2 Infection using Human Pluripotent Stem Cell-derived Colonic Organoids

10.1101/2020.05.02.073320 ◽

2020 ◽

Cited By ~ 4

Author(s):

Xiaohua Duan ◽

Yuling Han ◽

Liuliu Yang ◽

Benjamin E. Nilsson-Payant ◽

Pengfei Wang ◽

...

Keyword(s):

Stem Cell ◽

Viral Entry ◽

Pluripotent Stem Cell ◽

Cell Types ◽

Human Pluripotent Stem Cell ◽

Humanized Mouse Model ◽

Multiple Cell ◽

Approved Drugs

Summary ParagraphThe current COVID-19 pandemic is caused by SARS-coronavirus 2 (SARS-CoV-2). There are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of SARS-CoV-2 biology. As a result, there is an urgent need to create new disease models to study SARS-CoV-2 biology and to screen for therapeutics using human disease-relevant tissues. COVID-19 patients typically present with respiratory symptoms including cough, dyspnea, and respiratory distress, but nearly 25% of patients have gastrointestinal indications including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with worse COVID-19 outcomes1. Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of colonic cell types to SARS-CoV-2 infection. Single cell RNA-seq and immunostaining showed that the putative viral entry receptor ACE2 is expressed in multiple hESC-derived colonic cell types, but highly enriched in enterocytes. Multiple cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which was further validated in vivo using a humanized mouse model. We used hPSC-derived COs in a high throughput platform to screen 1280 FDA-approved drugs against viral infection. Mycophenolic acid and quinacrine dihydrochloride were found to block the infection of SARS-CoV-2 pseudo-entry virus in COs both in vitro and in vivo, and confirmed to block infection of SARS-CoV-2 virus. This study established both in vitro and in vivo organoid models to investigate infection of SARS-CoV-2 disease-relevant human colonic cell types and identified drugs that blocks SARS-CoV-2 infection, suitable for rapid clinical testing.

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