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
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)
- 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) - et al.
Inflammatory factors are elevated in brain microvessels in Alzheimer's disease
Neurobiol. Aging
(2001) - et al.
Cerebrovascular transforming growth factor-β contributes to inflammation in the Alzheimer's disease brain
Am. J. Pathol.
(2002) - et al.
Bioplex analysis of plasma cytokines in Alzheimer's disease and mild cognitive impairment
Immunol. Lett.
(2008) - et al.
Neural precursor cell influences on blood-brain barrier characteristics in rat brain endothelial cells
Brain Res.
(2007) - et al.
Neuroinflammation: ways in which the immune system affects the brain
Neurotherapeutics
(2015) - et al.
Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells
Methods
(2016) - 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) - et al.
Astrocyte-endothelial interactions at the blood-brain barrier
Nat. Rev. Neurosci.
(2006) - et al.
Neurovascular mechanisms and blood-brain barrier disorder in Alzheimer's disease
Acta Neuropathol.
(2009)
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.
Cited by (44)
The blood-brain barrier, a key bridge to treat neurodegenerative diseases
2023, Ageing Research ReviewsRecent advances in drug delivery and targeting to the brain
2022, Journal of Controlled ReleaseOrgan-specific endothelial cell heterogenicity and its impact on regenerative medicine and biomedical engineering applications
2022, Advanced Drug Delivery ReviewsCitation 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.
Upscaling biological complexity to boost neuronal and oligodendroglia maturation and improve in vitro developmental neurotoxicity (DNT) evaluation
2022, Reproductive ToxicologyCitation 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.
Drug Discovery in Induced Pluripotent Stem Cell Models
2022, Comprehensive Pharmacology