A differentiating neural stem cell-derived astrocytic population mitigates the inflammatory effects of TNF-α and IL-6 in an iPSC-based blood-brain barrier model

doi:10.1016/j.nbd.2018.07.030

Neurobiology of Disease

Volume 119, November 2018, Pages 113-120

https://doi.org/10.1016/j.nbd.2018.07.030 Get rights and content

Highlights

•
iPSC-brain endothelial cells and NSC-astrocytes form an in vitro BBB model.
•
TNF-α and IL-6 cause BBB dysfunction via an increase in transcellular permeability.
•
With NSC-derived astrocytes, more proinflammatory cytokines are secreted.
•
Despite cytokines, NSC-astrocytes help barrier breakdown caused by TNF-α and IL-6.
•
This stem-cell derived BBB model can be used to understand disease progression.

Abstract

Inflammation can be a risk factor for neurodegenerative diseases such as Alzheimer's disease (AD) and may also contribute to the progression of AD. Here, we sought to understand how inflammation affects the properties of the brain microvascular endothelial cells (BMECs) that compose the blood-brain barrier (BBB), which is impaired in AD. A fully human in vitro BBB model with brain microvascular endothelial cells derived from induced pluripotent stem cells and differentiating neural stem cell (NSC)-derived astrocytic cells was used to investigate the effects of neuroinflammation on barrier function. The cytokines TNF-α and IL-6 directly cause BBB dysfunction measured by a decrease in transendothelial electrical resistance, an increase in sodium fluorescein permeability, and a decrease in cell polarity, providing a link between neuroinflammation and specific aspects of BBB breakdown. An NSC-derived astrocytic cell population was added to the model and secreted cytokines and chemokines were quantified in monoculture and coculture both in the presence and absence of TNF-α and IL-6. Increased concentrations of pro-inflammatory cytokines known to be secreted by astrocytes or endothelial cells such as MCP-1, IL-8, IP-10, MIP-1β, IL-1 β, MIG, and RANTES peaked in inflammatory conditions when NSC-astrocytic cells were present. Despite the presence of several pro-inflammatory cytokines, the NSC-derived astrocytic cells mitigated the effects of inflammation measured by a restoration of transendothelial electrical resistance and IgG permeability. These results also suggest a breakdown in transcellular transport that precedes any increase in paracellular permeability in neuroinflammation. This model has the potential to resolve questions about neurodegenerative disease progression and delivery of therapeutics to the brain.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disease that is the most common cause of dementia. AD pathology is characterized by extracellular amyloid-β (Aβ) plaques and neurofibrillary tangles in neurons which together lead to neuronal death and cognitive loss. Once believed to be a secondary response in AD, there is increasing evidence that inflammation also contributes to AD progression (reviewed by Heppner et al., 2015). Both systemic inflammation (e.g. from chronic disease) and central nervous system inflammation (e.g. after traumatic brain injury) can be risk factors for AD (Holmes et al., 2009; Kyrkanides et al., 2011; Mayeux et al., 1993).

Tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 are two of the most commonly studied cytokines with respect to neuroinflammation in AD. A meta-analysis by Brosseron et al. (2014) revealed a correlation between increased TNF-α and IL-6 in blood or cerebrospinal fluid (CSF) in patients with severe AD compared to patients with mild cognitive impairment (MCI) or less severe AD (although several studies reviewed by Brosseron et al. revealed no correlation). Additionally, brain microvessels from patients with AD have been shown to secrete increased levels of inflammatory molecules, including IL-6 and TNF-α, compared with age matched healthy individuals (Grammas and Ovase, 2001).

In addition to neuroinflammation, vascular pathology may also contribute to AD. Human and animal model studies suggest that dysfunction of the blood-brain barrier (BBB) plays a critical role in the progression of AD and may precede the onset of neurodegeneration and cognitive decline (Bell and Zlokovic, 2009). The blood-brain barrier comprises the brain microvascular endothelial cells (BMECs) that line cerebral capillaries and these BMECs restrict and control the movement of molecules between the blood and the brain. Together with other cells of the neurovascular unit (NVU) such as astrocytes and pericytes, BMECs tightly regulate the neuronal microenvironment for proper function (Abbott et al., 2006).

Considerable progress has been made towards modeling the NVU in vitro, particularly from human stem cell sources, which mitigate availability and variability issues inherent to primary cell sources. BMECs derived from human induced pluripotent stem cells (iPSCs) exhibit an in vivo-like barrier phenotype, characterized by the presence of BMEC-specific proteins, functional and polarized molecular transport via proteins such as P-glycoprotein, high transendothelial electrical resistance (TEER), and low permeability to most molecules (Lippmann et al., 2014, Lippmann et al., 2012). The addition of other cell types of the NVU, such as astrocytes and pericytes (Lippmann et al., 2014), iPSC-derived astrocytes and neurons (Canfield et al., 2017), differentiating neural progenitor cells (Lim et al., 2007; Lippmann et al., 2011; Weidenfeller et al., 2007) and neural stem cells (NSCs) (Appelt-Menzel et al., 2017) improve the barrier phenotype of BMECs grown in vitro. Such in vitro BBB models can be used with different levels of complexity to elucidate contributions of different cell types and to further understand how different cell types can mitigate or exacerbate neurodegenerative disease.

This work aims to investigate the effects of neuroinflammation and crosstalk between cells of the NVU towards understanding the contributions of these two factors on BBB function in neurodegenerative diseases such as AD. Through the use of an in vitro model system with all cell components entirely derived from human stem cells, we first investigated the effects of inflammation via TNF-α and IL-6 on the barrier properties of iPSC-derived BMECs (Lippmann et al., 2014, Lippmann et al., 2012; Stebbins et al., 2016). Human NSC-derived astrocytic cells (Kleiderman et al., 2016) were added to the in vitro model and molecular crosstalk between NSC-astrocytic cells and BMECs in inflammation via secretion of cytokines and chemokines was measured. Finally, we investigated the ability of NSC-derived astrocytic cells to mitigate the effects of inflammation on the barrier function of the BBB.

Section snippets

NSC culture & differentiation

iPSC-derived BC1 HIP™ Neural Stem Cells (MTI-GlobalStem) were maintained on 6-well plates (Corning, Corning, NY) coated with a 1:200 solution of Corning2Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific) in Dulbecco Modified Eagle's Medium:Nutrient Mixture F-12 (DMEM/F12) with HEPES (Thermo Fisher Scientific) and incubated at room temperature for 1 h prior to use. NSCs were maintained in NSC Maintenance Medium (NSCMM) consisting of NeuralX NSC Medium

TNF-α and IL-6 treatment impairs barrier integrity

To investigate the effects of inflammation directly on the endothelial cells of the BBB, iPSC-derived BMECs were incubated with IL-6, TNF-α or both cytokines for 24 h. TEER, a measure of barrier integrity, was unchanged after the addition of IL-6 alone but was reduced by 13% and 16% after the addition of TNF-α and both cytokines respectively (Fig. 1A; unpaired t-test; p = .033 and p = .007). Permeability to sodium fluorescein, a measure of paracellular permeability, was 2- to 2.5-fold higher

Discussion

In vitro BBB models facilitate the study of transport phenomena at the cellular level and allow for different levels of complexity through the incorporation of different cell types. To investigate the effects of astrocytes on BMECs during inflammation, we used a fully human BBB model with cells derived entirely from stem cell sources. iPSCs were differentiated into BMECs while in parallel NSCs were differentiated into an astrocytic population and these were combined in a non-contact coculture.

Conclusions

These results demonstrate that the cytokines TNF-α and IL-6 act directly on the BMECs of the BBB and a transcellular breakdown occurs before paracellular permeability is impaired. This effect is mitigated by the presence of an NSC-derived astrocytic population, even though there is a significant increase in several pro-inflammatory cytokines known to impair BBB function. This model mimics cellular responses to inflammation at the BBB and can provide a way to study the contributions of

Conflict of interest

The authors declare no competing financial interests.

Acknowledgements

This work was funded in part by the National Science Foundation (Award Number 1144726). We wish to thank John Ruano-Salguero for his assistance with neural stem cell culture and differentiation.

References (38)

A. Appelt-Menzel et al.
Establishment of a human blood-brain barrier co-culture model mimicking the neurovascular unit using induced pluri- and multipotent stem cells
Stem Cell Rep.
(2017)
P. Grammas et al.
Inflammatory factors are elevated in brain microvessels in Alzheimer's disease
Neurobiol. Aging
(2001)
P. Grammas et al.
Cerebrovascular transforming growth factor-β contributes to inflammation in the Alzheimer's disease brain
Am. J. Pathol.
(2002)
K.S. Lee et al.
Bioplex analysis of plasma cytokines in Alzheimer's disease and mild cognitive impairment
Immunol. Lett.
(2008)
J.C. Lim et al.
Neural precursor cell influences on blood-brain barrier characteristics in rat brain endothelial cells
Brain Res.
(2007)
R.M. Ransohoff et al.
Neuroinflammation: ways in which the immune system affects the brain
Neurotherapeutics
(2015)
M.J. Stebbins et al.
Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells
Methods
(2016)
M.Q. Xia et al.
Expression of the chemokine receptor CXCR3 on neurons and the elevated expression of its ligand IP-10 in reactive astrocytes: in vitro ERK1/2 activation and role in Alzheimer's disease
J. Neuroimmunol.
(2000)
N.J. Abbott et al.
Astrocyte-endothelial interactions at the blood-brain barrier
Nat. Rev. Neurosci.
(2006)
R.D. Bell et al.
Neurovascular mechanisms and blood-brain barrier disorder in Alzheimer's disease
Acta Neuropathol.
(2009)

F. Brosseron et al.

Body fluid cytokine levels in mild cognitive impairment and Alzheimer's disease: a comparative overview

Mol. Neurobiol.

(2014)

B. Broux et al.

Glial regulation of the blood-brain barrier in health and disease

Semin. Immunopathol.

(2015)

A.M. Butt et al.

Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study

J. Physiol.

(1990)

S.G. Canfield et al.

An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells

J. Neurochem.

(2017)

M. De Bock et al.

Into rather unexplored terrain—transcellular transport across the blood-brain barrier

Glia

(2016)

R. Deane et al.

IgG-assisted age-dependent clearance of Alzheimer's amyloid beta peptide by the blood-brain barrier neonatal Fc receptor 1

J. Neurosci.

(2005)

M.A. Deli et al.

Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology

Cell. Mol. Neurobiol.

(2005)

A. Garg et al.

Investigation of the influence of FcRn on the distribution of IgG to the brain

AAPS J.

(2009)

F.L. Heppner et al.

Immune attack: the role of inflammation in Alzheimer disease

Nat. Rev. Neurosci.

(2015)

Cited by (44)

The blood-brain barrier, a key bridge to treat neurodegenerative diseases
2023, Ageing Research Reviews
As a highly selective and semi-permeable barrier that separates the circulating blood from the brain and central nervous system (CNS), the blood-brain barrier (BBB) plays a critical role in the onset and treatment of neurodegenerative diseases (NDs). To delay or reverse the NDs progression, the dysfunction of BBB should be improved to protect the brain from harmful substances. Simultaneously, a highly efficient drug delivery across the BBB is indispensable. Here, we summarized several methods to improve BBB dysfunction in NDs, including knocking out risk geneAPOE4, regulating circadian rhythms, restoring the gut microenvironment, and activating the Wnt/β-catenin signaling pathway. Then we discussed the advances in BBB penetration techniques, such as transient BBB opening, carrier-mediated drug delivery, and nasal administration, which facilitates drug delivery across the BBB. Furthermore, various in vivo and in vitro BBB models and research methods related to NDs are reviewed. Based on the current research progress, the treatment of NDs in the long term should prioritize the integrity of the BBB. However, a treatment approach that combines precise control of transient BBB permeability and non-invasive targeted BBB drug delivery holds profound significance in improving treatment effectiveness, safety, and clinical feasibility during drug therapy. This review involves the cross application of biology, materials science, imaging, engineering and other disciplines in the field of BBB, aiming to provide multi-dimensional research directions and clinical ideas for the treating NDs.
Granulin loss of function in human mature brain organoids implicates astrocytes in TDP-43 pathology
2023, Stem Cell Reports
Loss of function (LoF) of TAR-DNA binding protein 43 (TDP-43) and mis-localization, together with TDP-43-positive and hyperphosphorylated inclusions, are found in post-mortem tissue of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those carrying LoF variants in the progranulin gene (GRN). Modeling TDP-43 pathology has been challenging in vivo and in vitro. We present a three-dimensional induced pluripotent stem cell (iPSC)-derived paradigm—mature brain organoids (mbOrg)—composed of cortical-like-astrocytes (iA) and neurons. When devoid of GRN, mbOrgs spontaneously recapitulate TDP-43 mis-localization, hyperphosphorylation, and LoF phenotypes. Mixing and matching genotypes in mbOrgs showed that GRN^−/− iA are drivers for TDP-43 pathology. Finally, we rescued TDP-43 LoF by adding exogenous progranulin, demonstrating a link between TDP-43 LoF and progranulin expression. In conclusion, we present an iPSC-derived platform that shows striking features of human TDP-43 proteinopathy and provides a tool for the mechanistic modeling of TDP-43 pathology and patient-tailored therapeutic screening for FTD and ALS.
Recent advances in drug delivery and targeting to the brain
2022, Journal of Controlled Release
Our body keeps separating the toxic chemicals in the blood from the brain. A significant number of drugs do not enter the central nervous system (CNS) due to the blood-brain barrier (BBB). Certain diseases, such as tumor growth and stroke, are known to increase the permeability of the BBB. However, the heterogeneity of this permeation makes it difficult and unpredictable to transport drugs to the brain. In recent years, research has been directed toward increasing drug penetration inside the brain, and nanomedicine has emerged as a promising approach. Active targeting requires one or more specific ligands on the surface of nanoparticles (NPs), which brain endothelial cells (ECs) recognize, allowing controlled drug delivery compared to conventional targeting strategies. This review highlights the mechanistic insights about different cell types contributing to the development and maintenance of the BBB and summarizes the recent advancement in brain-specific NPs for different pathological conditions. Furthermore, fundamental properties of brain-targeted NPs will be discussed, and the standard lesion features classified by neurological pathology are summarized.
Organ-specific endothelial cell heterogenicity and its impact on regenerative medicine and biomedical engineering applications
2022, Advanced Drug Delivery Reviews
Citation Excerpt :
Further refinement of this protocol over the years led to the development of human pluripotent stem cell-derived BECs and iBECs with a TEER >5000 Ω/cm2, which is higher than the TEER seen in primary human BECs or any human cell lines [109,120,121]. Based on the Lippman protocol, Mantle et al. developed a fully human in vitro BBB model using hiPSC-derived neural astrocytic cells to study the effect of neuroinflammation on barrier properties [122]. The barrier properties were significantly enhanced using hypoxic conditions during the cell differentiation.
Endothelial cells (ECs) are a key cellular component of the vascular system as they form the inner lining of the blood vessels. Recent findings highlight that ECs express extensive phenotypic heterogenicity when following the vascular tree from the major vasculature down to the organ capillaries. However, in vitro models, used for drug development and testing, or to study the role of ECs in health and disease, rarely acknowledge this EC heterogenicity. In this review, we highlight the main differences between different EC types, briefly summarize their different characteristics and focus on the use of ECs in in vitro models. We introduce different approaches on how ECs can be utilized in co-culture test systems in the field of brain, pancreas, and liver research to study the role of the endothelium in health and disease. Finally, we discuss potential improvements to current state-of-the-art in vitro models and future directions.
Upscaling biological complexity to boost neuronal and oligodendroglia maturation and improve in vitro developmental neurotoxicity (DNT) evaluation
2022, Reproductive Toxicology
Citation Excerpt :
Furthermore, Pamies and colleagues have shown that human iPSC-derived brain microphysiological 3D systems can successfully be cultured yielding a large proportion of both neurons and glial cells (astrocytes and oligodendrocytes), characterized by the generation of neuronal spontaneous electrical activity and axon myelination [13–16]. The availability of heterogeneous in vitro test systems able to recapitulate neuronal-glial cell interaction, myelin formation and neuronal network functionality is particularly important, especially when studying chemical-induced neuroinflammation or neuroinflammatory disorders [17], neurodegeneration [18,19], or stroke [20]. In the present study, we compared the differentiation potential of a previously described human iPSC-derived neuronal and glial cell model [6] cultured as 3D neurospheres maintained in suspension for up to 6 weeks vs 2D monolayers differentiated for the same period of time.
Human induced pluripotent stem cell (iPSC)-derived neuronal and glial cell models are suitable to assess the effects of environmental chemicals on the developing brain. Such test systems can recapitulate several key neurodevelopmental features, such as neural stem cell formation and differentiation towards different neuronal subtypes and astrocytes, neurite outgrowth, synapse formation and neuronal network formation and function, which are crucial for brain development. While monolayer, two-dimensional (2D) cultures of human iPSC-neuronal or glial derivatives are generally suited for high-throughput testing, they also show some limitations. In particular, differentiation towards myelinating oligodendrocytes can only be achieved after extended periods in differentiation. In recent years, the implementation of three-dimensional (3D) neuronal and glial models obtained from human iPSCs has been shown to compensate for such limitations, enabling robust differentiation towards both neuronal and glial cell populations, myelination and formation of more mature neuronal network activity. Here we compared the differentiation capacity of human iPSC-derived neural stem cells cultured either as 2D monolayer or as 3D neurospheres, and assessed chlorpyrifos (CPF) effects. Data indicate that 3D neurospheres differentiate towards neurons and oligodendroglia more rapidly than 2D cultures; however, the 2D model is more suitable to assess neuronal functionality by analysis of spontaneous electrical activity using multielectrode array. Moreover, 2D and 3D test systems are diversely susceptible to CPF treatment. In conclusion, the selection of the most suitable in vitro test system (either 2D or 3D) should take into account the context of use and intended research goals (‘fit for purpose’ principle).
Drug Discovery in Induced Pluripotent Stem Cell Models
2022, Comprehensive Pharmacology
Historically, cellular context was never an issue in drug development as the focus of such efforts was on animal models and tissue explants in relevant models of disease. The switch to heterologous systems, while simplifying drug discovery and increasing throughput, made many assumptions about its translatability to more relevant contexts such as primary cells or in vivo models of disease. To achieve personalized and precision medicine in a real sense, drug discovery and disease modeling will ultimately need to be done on a patient-specific basis. Here, we focus of induced pluripotent stem cells (iPSCs) as a means to develop 2D and 3D models to understand cardiovascular and neuronal disorders and to use these models for purposes of drug discovery. The combination of modern screening technologies for drug discovery combined with disease modeling in iPSCs forms the basis of a nascent approach to patient-based drug discovery.

View all citing articles on Scopus

View full text

A differentiating neural stem cell-derived astrocytic population mitigates the inflammatory effects of TNF-α and IL-6 in an iPSC-based blood-brain barrier model

Highlights

Abstract

Introduction

Section snippets

NSC culture & differentiation

TNF-α and IL-6 treatment impairs barrier integrity

Discussion

Conclusions

Conflict of interest

Acknowledgements

Stem Cell Rep.

Neurobiol. Aging

Am. J. Pathol.

Immunol. Lett.

Brain Res.

Neurotherapeutics

Methods

J. Neuroimmunol.

Astrocyte-endothelial interactions at the blood-brain barrier

Nat. Rev. Neurosci.

Neurovascular mechanisms and blood-brain barrier disorder in Alzheimer's disease

Acta Neuropathol.

Body fluid cytokine levels in mild cognitive impairment and Alzheimer's disease: a comparative overview

Mol. Neurobiol.

Glial regulation of the blood-brain barrier in health and disease

Semin. Immunopathol.

Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study

J. Physiol.

An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells

J. Neurochem.

Into rather unexplored terrain—transcellular transport across the blood-brain barrier

Glia

IgG-assisted age-dependent clearance of Alzheimer's amyloid beta peptide by the blood-brain barrier neonatal Fc receptor 1

J. Neurosci.

Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology

Cell. Mol. Neurobiol.

Investigation of the influence of FcRn on the distribution of IgG to the brain

AAPS J.

Immune attack: the role of inflammation in Alzheimer disease

Nat. Rev. Neurosci.