Human induced pluripotent stem cells (hiPSCs) provide a powerful platform for disease modeling and have unlocked new possibilities for understanding the mechanisms governing human biology, ...physiology, and genetics. However, hiPSC-derivatives have traditionally been utilized in two-dimensional monocultures, in contrast to the multi-systemic interactions that influence cells in the body. We will discuss recent advances in generating more complex hiPSC-based systems using three-dimensional organoids, tissue-engineering, microfluidic organ-chips, and humanized animal systems. While hiPSC differentiation still requires optimization, these next-generation multi-lineage technologies can augment the biomedical researcher’s toolkit and enable more realistic models of human tissue function.
Sharma et al. review recent advances in multi-cell-lineage, hiPSC-derived platforms for disease modeling and drug screening. These complex model systems, which include organoids, assembloids, organ-chips, and tissue-engineered constructs, are more realistic representations of human tissues containing multiple cell types that interact during development, aging, and disease.
Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that ...CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (RhoS334) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of RhoS334, which prevented retinal degeneration and improved visual function.
The blood-brain barrier (BBB) tightly regulates the entry of solutes from blood into the brain and is disrupted in several neurological diseases. Using Organ-Chip technology, we created an entirely ...human BBB-Chip with induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (iBMECs), astrocytes, and neurons. The iBMECs formed a tight monolayer that expressed markers specific to brain vasculature. The BBB-Chip exhibited physiologically relevant transendothelial electrical resistance and accurately predicted blood-to-brain permeability of pharmacologics. Upon perfusing the vascular lumen with whole blood, the microengineered capillary wall protected neural cells from plasma-induced toxicity. Patient-derived iPSCs from individuals with neurological diseases predicted disease-specific lack of transporters and disruption of barrier integrity. By combining Organ-Chip technology and human iPSC-derived tissue, we have created a neurovascular unit that recapitulates complex BBB functions, provides a platform for modeling inheritable neurological disorders, and advances drug screening, as well as personalized medicine.
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•iPSC and microfluidic technologies were combined to generate a human BBB-Chip•Flow-induced shear and co-cultures enhance barrier performance•The BBB-Chip exhibits physiologically relevant TEER and can predict CNS penetrance•Personalized BBB-Chips can detect interindividual variability in BBB performance
The blood-brain barrier (BBB) is a multicellular neurovascular unit that tightly regulates brain homeostasis and is disturbed in several neurological diseases. Combining patient-specific stem cells and microfluidic technologies, Vatine et al. have generated a personalized human BBB-Chip, which recapitulates the human BBB and can predict variability between individuals.
The blood-brain barrier (BBB) is a critical component of the central nervous system that protects neurons and other cells of the brain parenchyma from potentially harmful substances found in ...peripheral circulation. Gaining a thorough understanding of the development and function of the human BBB has been hindered by a lack of relevant models given significant species differences and limited access to in vivo tissue. However, advances in induced pluripotent stem cell (iPSC) and organ-chip technologies now allow us to improve our knowledge of the human BBB in both health and disease. This review focuses on the recent progress in modeling the BBB in vitro using human iPSCs.
ABSTRACT
Active galactic nucleus (AGN)-driven outflows are now routinely used in models of galaxy evolution as a feedback mechanism; however, many of their properties remain highly uncertain. Perhaps ...the greatest source of uncertainty is the electron density of the outflowing gas, which directly affects derived kinetic powers and mass outflow rates. Here we present spatially resolved, wide spectral-coverage Xshooter observations of the nearby active galaxy IC 5063 (z = 0.001131), which shows clear signatures of outflows being driven by shocks induced by a radio jet interacting with the ISM. For the first time, we use the higher-critical-density transauroral (TR) S ii and O ii lines to derive electron densities in spatially resolved observations of an active galaxy, and present evidence that the lines are emitted in the same spatial regions as other key diagnostic lines. In addition, we find that the post-shock gas is denser than the pre-shock gas, possibly due to shock compression effects. We derive kinetic powers for the warm ionized outflow phase and find them to be below those required by galaxy evolution models; however, other studies of different gas phases in IC 5063 allow us to place our results in a wider context in which the cooler gas phases constitute most of the outflowing mass. We investigate the dominant ionization and excitation mechanisms and find that the warm ionized outflow phase is dominated by AGN-photoionization, while the warm molecular phase has composite AGN-shock excitation. Overall, our results highlight the importance of robust outflow diagnostics and reinforce the utility of the TR lines for future studies of outflows in active galaxies.
The blood–brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line ...the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human‐induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC‐derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor‐like EZ‐spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC‐derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co‐cultured with human iPSC‐derived BMECs, the iPSC‐derived neurons and astrocytes significantly elevated trans‐endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p‐glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans.
Read the Editorial Highlight for this article on page 843.
We developed an isogenic human blood–brain barrier (BBB) model comprising induced pluripotent stem cell (iPSC) derived brain endothelial cells (BMECs), astrocytes, and neurons. We have demonstrated that iPSC‐derived neurons and astrocytes can improve the tight junctions and induce barrier tightening in iPSC‐derived BMECs. An isogenic BBB model where each cell type is derived from the same patient iPSC source would enable new approaches in brain disease modeling and drug development.
Read the Editorial Highlight for this article on page 843.
Angiotensin II is an emerging therapy for septic acute kidney injury, but it is unknown if its vasoconstrictor action induces renal hypoxia. We therefore examined the effects of angiotensin II on ...intrarenal PO2 in ovine sepsis. We also assessed the validity of urinary PO2 as a surrogate measure of medullary PO2.
Interventional study.
Research Institute.
Sixteen adult Merino ewes (n = 8/group).
Sheep were instrumented with fiber-optic probes in the renal cortex, medulla, and within a bladder catheter to measure PO2. Conscious sheep were infused with Escherichia coli for 32 hours. At 24-30 hours, angiotensin II (0.5-33.0 ng/kg/min) or saline vehicle was infused.
Septic acute kidney injury was characterized by hypotension and a 60% ± 6% decrease in creatinine clearance. During sepsis, medullary PO2 decreased from 36 ± 1 to 30 ± 3 mm Hg after 1 hour and to 20 ± 2 mm Hg after 24 hours; at these times, urinary PO2 was 42 ± 2, 34 ± 2, and 23 ± 2 mm Hg. Increases in urinary neutrophil gelatinase-associated lipocalin (12% ± 3%) and serum creatinine (60% ± 23%) were only detected at 8 and 24 hours, respectively. IV infusion of angiotensin II, at 24 hours of sepsis, restored arterial pressure and improved creatinine clearance, while not exacerbating medullary or urinary hypoxia.
In septic acute kidney injury, renal medullary and urinary hypoxia developed several hours before increases in currently used biomarkers. Angiotensin II transiently improved renal function without worsening medullary hypoxia. In septic acute kidney injury, angiotensin II appears to be a safe, effective therapy, and urinary PO2 may be used to detect medullary hypoxia.
Corneal wound healing is a complex process that occurs in response to various injuries and commonly used refractive surgery. It is a significant clinical problem, which may lead to serious ...complications due to either incomplete (epithelial) or excessive (stromal) healing. Epithelial stem cells clearly play a role in this process, whereas the contribution of stromal and endothelial progenitors is less well studied. The available evidence on stem cell participation in corneal wound healing is reviewed, together with the data on the use of corneal and non‐corneal stem cells to facilitate this process in diseased or postsurgical conditions. Important aspects of corneal stem cell generation from alternative cell sources, including pluripotent stem cells, for possible transplantation upon corneal injuries or in disease conditions are also presented. Stem Cells 2017;35:2105–2114
Schematic of corneal wounds and current stem cell treatments. Epithelial wounds (left): limbal biopsies or limbal epithelial stem cell (LESC)‐enriched cultures are used clinically. As an alternative source, iPSC‐derived LESC are examined in animals. Stromal wounds (right): mesenchymal stem cell injections are tested in animals to reduce haze and scarring. Endothelial wounds (bottom): progenitor cell injections are tested preclinically.
Through mechanisms that remain poorly defined, defects in nucleocytoplasmic transport and accumulations of specific nuclear-pore-complex-associated proteins have been reported in multiple ...neurodegenerative diseases, including C9orf72 Amyotrophic Lateral Sclerosis and Frontotemporal Dementia (ALS/FTD). Using super-resolution structured illumination microscopy, we have explored the mechanism by which nucleoporins are altered in nuclei isolated from C9orf72 induced pluripotent stem-cell-derived neurons (iPSNs). Of the 23 nucleoporins evaluated, we observed a reduction in a subset of 8, including key components of the nuclear pore complex scaffold and the transmembrane nucleoporin POM121. Reduction in POM121 appears to initiate a decrease in the expression of seven additional nucleoporins, ultimately affecting the localization of Ran GTPase and subsequent cellular toxicity in C9orf72 iPSNs. Collectively, our data suggest that the expression of expanded C9orf72 ALS/FTD repeat RNA alone affects nuclear POM121 expression in the initiation of a pathological cascade affecting nucleoporin levels within neuronal nuclei and ultimately downstream neuronal survival.
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•Expression of eight nucleoporins is reduced in human C9orf72 neuronal nuclei•Reduction in POM121 affects nuclear pore complex composition•Nucleoporin alterations diminish nucleocytoplasmic transport and neuronal survival•G4C2 repeat RNA initiates the pathogenic cascades, leading to decreased nuclear POM121
Coyne et al. demonstrate that G4C2 repeat RNA initiates a reduction of POM121 expression within C9orf72 neuronal nuclear pore complexes. Decreased nuclear POM121 affects the expression of seven additional nucleoporins, resulting in altered nuclear pore composition. This combined nucleoporin reduction affects the localization of nucleocytoplasmic transport proteins and neuronal survival.
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