Three-dimensional induced pluripotent stem-cell models of human brain angiogenesis

Abstract Background: During brain development, chemical cues released by developing neurons, cellular signaling with pericytes, and mechanical cues within the brain extracellular matrix (ECM) promotes angiogenesis occurs of brain microvascular endothelial cells (BMECs). During brain disease, angiogenesis can also occur due to pathological chemical, cellular, and mechanical signaling. Existing in vitro and in vivo models of brain angiogenesis have key limitations. Methods: Here, we develop a high-throughput in vitro BBB bead assay of brain angiogenesis utilizing 150 μm diameter beads coated with induced pluripotent stem-cell (iPSC)-derived human BMECs (dhBMECs). After embedding the beads within a 3D matrix, we introduce various chemical cues and extracellular matrix components to explore their effects on angiogenic behavior. Based on the results from the bead assay, we generate a multi-scale model of the human cerebrovasculature within perfusable three-dimensional tissue-engineered blood-brain barrier (BBB) microvessels. Results: A sprouting phenotype is optimized in confluent monolayers of dhBMECs using chemical treatment with vascular endothelial growth factor (VEGF) and wnt ligands, and the inclusion of pro-angiogenic ECM components. As a proof-of-principle that the bead angiogenesis assay can be applied to study pathological angiogenesis, we show that oxidative stress can exert concentration-dependent effects on angiogenesis. Finally, we demonstrate the formation of a hierarchical microvascular model of the human blood-brain barrier displaying key structural hallmarks. Conclusions: We develop two in vitro models of brain angiogenesis: the BBB bead assay and the tissue-engineered BBB microvessel model. These platforms provide a tool kit for studies of physiological and pathological brain angiogenesis, with key advantages over existing two-dimensional models.

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Three-dimensional induced pluripotent stem-cell models of human brain angiogenesis

10.21203/rs.2.23105/v1 ◽

2020 ◽

Author(s):

Raleigh M. Linville ◽

Diego Arevalo ◽

Joanna C. Maressa ◽

Nan Zhao ◽

Peter Searson

Keyword(s):

Blood Brain Barrier ◽

Chemical Cues ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Brain Barrier ◽

Blood Brain ◽

Induced Pluripotent ◽

The Brain ◽

Brain Angiogenesis

Abstract Background: During brain development, chemical cues released by developing neurons, cellular signaling with pericytes, and mechanical cues within the brain extracellular matrix (ECM) promote angiogenesis of brain microvascular endothelial cells (BMECs). Angiogenesis is also associated with diseases of the brain due to pathological chemical, cellular, and mechanical signaling. Existing in vitro and in vivo models of brain angiogenesis have key limitations. Methods: Here, we develop a high-throughput in vitro blood-brain barrier (BBB) bead assay of brain angiogenesis utilizing 150 μm diameter beads coated with induced pluripotent stem-cell (iPSC)-derived human BMECs (dhBMECs). After embedding the beads within a 3D matrix, we introduce various chemical cues and extracellular matrix components to explore their effects on angiogenic behavior. Based on the results from the bead assay, we generate a multi-scale model of the human cerebrovasculature within perfusable three-dimensional tissue-engineered blood-brain barrier microvessels.Results: A sprouting phenotype is optimized in confluent monolayers of dhBMECs using chemical treatment with vascular endothelial growth factor (VEGF) and wnt ligands, and the inclusion of pro-angiogenic ECM components. As a proof-of-principle that the bead angiogenesis assay can be applied to study pathological angiogenesis, we show that oxidative stress can exert concentration-dependent effects on angiogenesis. Finally, we demonstrate the formation of a hierarchical microvascular model of the human blood-brain barrier displaying key structural hallmarks. Conclusions: We develop two in vitro models of brain angiogenesis: the BBB bead assay and the tissue-engineered BBB microvessel model. These platforms provide a tool kit for studies of physiological and pathological brain angiogenesis, with key advantages over existing two-dimensional models.

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Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02089-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Gabriel Peinkofer ◽

Martina Maass ◽

Kurt Pfannkuche ◽

Agapios Sachinidis ◽

Stephan Baldus ◽

...

Keyword(s):

Extracellular Matrix ◽

Stem Cell ◽

Cell Adhesion ◽

Developmental Stage ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Induced Pluripotent

Abstract Background Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. Methods To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6–7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. Results The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. Conclusion The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

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Human induced pluripotent stem cell based in vitro models of the blood-brain barrier: the future standard?

Neural Regeneration Research ◽

10.4103/1673-5374.217326 ◽

2017 ◽

Vol 12 (10) ◽

pp. 1607 ◽

Cited By ~ 4

Author(s):

Winfried Neuhaus

Keyword(s):

Stem Cell ◽

Blood Brain Barrier ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

In Vitro Models ◽

Brain Barrier ◽

Blood Brain ◽

The Future ◽

Induced Pluripotent

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Three-dimensional induced pluripotent stem-cell models of human brain angiogenesis

Microvascular Research ◽

10.1016/j.mvr.2020.104042 ◽

2020 ◽

Vol 132 ◽

pp. 104042 ◽

Cited By ~ 1

Author(s):

Raleigh M. Linville ◽

Diego Arevalo ◽

Joanna C. Maressa ◽

Nan Zhao ◽

Peter C. Searson

Keyword(s):

Stem Cell ◽

Human Brain ◽

Pluripotent Stem Cell ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Cell Models ◽

Induced Pluripotent ◽

Stem Cell Models ◽

Brain Angiogenesis

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Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype

PLoS ONE ◽

10.1371/journal.pone.0245571 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0245571

Author(s):

Junya Yokoyama ◽

Shigeru Miyagawa ◽

Takami Akagi ◽

Mitsuru Akashi ◽

Yoshiki Sawa

Keyword(s):

Myocardial Infarction ◽

Extracellular Matrix ◽

Pluripotent Stem Cell ◽

Heart Function ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Left Ventricular ◽

Induced Pluripotent

The extracellular matrix (ECM) plays a key role in the viability and survival of implanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We hypothesized that coating of three-dimensional (3D) cardiac tissue-derived hiPSC-CMs with the ECM protein fibronectin (FN) would improve the survival of transplanted cells in the heart and improve heart function in a rat model of ischemic heart failure. To test this hypothesis, we first explored the tolerance of FN-coated hiPSC-CMs to hypoxia in an in vitro study. For in vivo assessments, we constructed 3D-hiPSC cardiac tissues (3D-hiPSC-CTs) using a layer-by-layer technique, and then the cells were implanted in the hearts of a myocardial infarction rat model (3D-hiPSC-CTs, n = 10; sham surgery control group (without implant), n = 10). Heart function and histology were analyzed 4 weeks after transplantation. In the in vitro assessment, cell viability and lactate dehydrogenase assays showed that FN-coated hiPSC-CMs had improved tolerance to hypoxia compared with the control cells. In vivo, the left ventricular ejection fraction of hearts implanted with 3D-hiPSC-CT was significantly better than that of the sham control hearts. Histological analysis showed clear expression of collagen type IV and plasma membrane markers such as desmin and dystrophin in vivo after implantation of 3D-hiPSC-CT, which were not detected in 3D-hiPSC-CMs in vitro. Overall, these results indicated that FN-coated 3D-hiPSC-CT could improve distressed heart function in a rat myocardial infarction model with a well-expressed cytoskeletal or basement membrane matrix. Therefore, FN-coated 3D-hiPSC-CT may serve as a promising replacement for heart transplantation and left ventricular assist devices and has the potential to improve survivability and therapeutic efficacy in cases of ischemic heart disease.

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Prediction of Drug Permeability Using In Vitro Blood–Brain Barrier Models with Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells

BioResearch Open Access ◽

10.1089/biores.2019.0026 ◽

2019 ◽

Vol 8 (1) ◽

pp. 200-209 ◽

Cited By ~ 9

Author(s):

Makiko Ohshima ◽

Shota Kamei ◽

Hideo Fushimi ◽

Shinji Mima ◽

Tadanori Yamada ◽

...

Keyword(s):

Endothelial Cells ◽

Stem Cell ◽

Blood Brain Barrier ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Drug Permeability ◽

Induced Pluripotent

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Honing the Double-Edged Sword: Improving Human iPSC-Microglia Models

Frontiers in Immunology ◽

10.3389/fimmu.2020.614972 ◽

2020 ◽

Vol 11 ◽

Author(s):

Anne Hedegaard ◽

Szymon Stodolak ◽

William S. James ◽

Sally A. Cowley

Keyword(s):

Extracellular Matrix ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Media Composition ◽

Physiological Relevance ◽

Key Features ◽

Induced Pluripotent ◽

Human Ipsc

Human induced Pluripotent Stem Cell (hiPSC) models are a valuable new tool for research into neurodegenerative diseases. Neuroinflammation is now recognized as a key process in neurodegenerative disease and aging, and microglia are central players in this. A plethora of hiPSC-derived microglial models have been published recently to explore neuroinflammation, ranging from monoculture through to xenotransplantation. However, combining physiological relevance, reproducibility, and scalability into one model is still a challenge. We examine key features of the in vitro microglial environment, especially media composition, extracellular matrix, and co-culture, to identify areas for improvement in current hiPSC-microglia models.

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Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype

10.1101/2021.01.05.425387 ◽

2021 ◽

Author(s):

Junya Yokoyama ◽

Shigeru Miyagawa ◽

Takami Akagi ◽

Mitsuru Akashi ◽

Yoshiki Sawa

Keyword(s):

Myocardial Infarction ◽

Extracellular Matrix ◽

Pluripotent Stem Cell ◽

Heart Function ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Left Ventricular ◽

Induced Pluripotent

AbstractThe extracellular matrix (ECM) plays a key role in the viability and survival of implanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We hypothesized that coating of three-dimensional (3D) cardiac tissue-derived hiPSC-CMs with the ECM protein fibronectin (FN) would improve the survival of transplanted cells in the heart and improve heart function in a rat model of ischemic heart failure. To test this hypothesis, we first explored the tolerance of FN-coated hiPSC-CMs to hypoxia in an in vitro study. For in vivo assessments, we constructed 3D-hiPSC cardiac tissues (3D-hiPSC-CTs) using a layer-by-layer technique, and then the cells were implanted in the hearts of a myocardial infarction rat model (3D-hiPSC-CTs, n = 10; sham surgery control group (without implant), n = 10). Heart function and histology were analyzed 4 weeks after transplantation. In the in vitro assessment, cell viability and lactate dehydrogenase assays showed that FN-coated hiPSC-CMs had improved tolerance to hypoxia compared with the control cells. In vivo, the left ventricular ejection fraction of hearts implanted with 3D-hiPSC-CT was significantly better than that of the sham control hearts. Histological analysis showed clear expression of collagen type IV and plasma membrane markers such as desmin and dystrophin in vivo after implantation of 3D-hiPSC-CT, which were not detected in 3D-hiPSC-CMs in vitro. Overall, these results indicated that FN-coated 3D-hiPSC-CT could improve distressed heart function in a rat myocardial infarction model with a well-expressed cytoskeletal or basement membrane matrix. Therefore, FN-coated 3D-hiPSC-CT may serve as a promising replacement for heart transplantation and left ventricular assist devices and has the potential to improve survivability and therapeutic efficacy in cases of ischemic heart disease.

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