High levels of endothelial ICAM-1 prohibit natalizumab mediated abrogation of CD4+ T cell arrest on the inflamed BBB under flow in vitro.

INTRODUCTION: The humanized anti-α4 integrin blocking antibody natalizumab (NTZ) is an effective treatment for relapsing-remitting multiple sclerosis (RRMS) that is associated with the risk of progressive multifocal leukoencephalopathy (PML). While extended interval dosing (EID) of NTZ reduces the risk for PML, the minimal dose of NTZ required to maintain its therapeutic efficacy remains unknown. OBJECTIVE: Here we aimed to identify the minimal NTZ concentration required to inhibit the arrest of human effector/memory CD4+ T cell subsets or of PBMCs to the blood-brain barrier (BBB) under physiological flow in vitro. RESULTS: Making use of three different human in vitro BBB models and in vitro live-cell imaging we observed that NTZ mediated inhibition of α4-integrins failed to abrogate T cell arrest to the inflamed BBB under physiological flow. Complete inhibition of shear resistant T cell arrest required additional inhibition of ß2-integrins, which correlated with a strong upregulation of endothelial intercellular adhesion molecule (ICAM)-1 on the respective BBB models investigated. Indeed, NTZ mediated inhibition of shear resistant T cell arrest to combinations of immobilized recombinant vascular cell adhesion molecule (VCAM)-1 and ICAM-1 was abrogated in the presence of tenfold higher molar concentrations of ICAM-1 over VCAM-1. Also, monovalent NTZ was less potent than bivalent NTZ in inhibiting T cell arrest to VCAM-1 under physiological flow. In accordance with our previous observations ICAM-1 but not VCAM-1 mediated T cell crawling against the direction of flow. CONCLUSION: Taken together, our in vitro observations show that high levels of endothelial ICAM-1 abrogate NTZ mediated inhibition of T cell interaction with the BBB. EID of NTZ in MS patients may thus require consideration of the inflammatory status of the BBB as high levels of ICAM-1 may provide an alternative molecular cue allowing for pathogenic T cell entry into the CNS in the presence of NTZ.

Intrinsic blood-brain barrier dysfunction contributes to multiple sclerosis pathogenesis.

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the CNS are early hallmarks of multiple sclerosis (MS). The mechanisms leading to BBB dysfunction are incompletely understood and generally thought to be a consequence of neuroinflammation. Here, we have challenged this view and asked if intrinsic alterations in the BBB of MS patients contribute to MS pathogenesis. To this end, we made use of human induced pluripotent stem cells derived from healthy controls and MS patients and differentiated them into brain microvascular endothelial cell (BMEC)-like cells as in vitro model of the BBB. MS-derived BMEC-like cells showed impaired junctional integrity, barrier properties and efflux pump activity when compared to healthy controls. Also, MS-derived BMEC-like cells displayed an inflammatory phenotype with increased adhesion molecule expression and immune cell interactions. Activation of Wnt/ß-catenin signalling in MS-derived endothelial progenitor cells enhanced barrier characteristics and reduced the inflammatory phenotype. Our study provides evidence for an intrinsic impairment of BBB function in MS patients that can be modelled in vitro. Human iPSC-derived BMEC-like cells are thus suitable to explore the molecular underpinnings of BBB dysfunction in MS and will assist in the identification of potential novel therapeutic targets for BBB stabilization.

Brain Barriers and Multiple Sclerosis: Novel Treatment Approaches from a Brain Barriers Perspective.

Multiple sclerosis (MS) is considered a prototypic organ specific autoimmune disease targeting the central nervous system (CNS). Blood-brain barrier (BBB) breakdown and enhanced immune cell infiltration into the CNS parenchyma are early hallmarks of CNS lesion formation. Therapeutic targeting of immune cell trafficking across the BBB has proven a successful therapy for the treatment of MS, but comes with side effects and is no longer effective once patients have entered the progressive phase of the disease. Beyond the endothelial BBB, epithelial and glial brain barriers establish compartments in the CNS that differ in their accessibility to the immune system. There is increasing evidence that brain barrier abnormalities persist during the progressive stages of MS. Here, we summarize the role of endothelial, epithelial, and glial brain barriers in maintaining CNS immune privilege and our current knowledge on how impairment of these barriers contributes to MS pathogenesis. We discuss how therapeutic stabilization of brain barriers integrity may improve the safety of current therapeutic regimes for treating MS. This may also allow for the development of entirely novel therapeutic approaches aiming to restore brain barriers integrity and thus CNS homeostasis, which may be specifically beneficial for the treatment of progressive MS.

Advancing human induced pluripotent stem cell-derived blood-brain barrier models for studying immune cell interactions.

Human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier (BBB) models established to date lack expression of key adhesion molecules involved in immune cell migration across the BBB in vivo. Here, we introduce the extended endothelial cell culture method (EECM), which differentiates hiPSC-derived endothelial progenitor cells to brain microvascular endothelial cell (BMEC)-like cells with good barrier properties and mature tight junctions. Importantly, EECM-BMEC-like cells exhibited constitutive cell surface expression of ICAM-1, ICAM-2, and E-selectin. Pro-inflammatory cytokine stimulation increased the cell surface expression of ICAM-1 and induced cell surface expression of P-selectin and VCAM-1. Co-culture of EECM-BMEC-like cells with hiPSC-derived smooth muscle-like cells or their conditioned medium further increased the induction of VCAM-1. Functional expression of endothelial ICAM-1 and VCAM-1 was confirmed by T-cell interaction with EECM-BMEC-like cells. Taken together, we introduce the first hiPSC-derived BBB model that displays an adhesion molecule phenotype that is suitable for the study of immune cell interactions.

GRP78 antibodies damage the blood-brain barrier and relate to cerebellar degeneration in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the neuromuscular junction caused by autoantibodies binding to P/Q-type voltage-gated calcium channels. Breakdown of the blood-brain barrier and diffusion of cerebellar granule/Purkinje cell-reactive autoantibodies into the CNS are critical for the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome. We recently found evidence that glucose-regulated protein 78 (GRP78) autoantibodies in the plasma of patients with neuromyelitis optica promote the CNS access of AQP4 autoantibodies. In the present study, we investigated whether the GRP78 autoantibodies in PCD-LEMS IgG boost the brain uptake of cerebellar cell-reactive antibodies across the blood-brain barrier and facilitate cerebellar dysfunction. We first evaluated the effects of purified IgG from PCD-LEMS or PCD patients on the blood-brain barrier function in human brain microvascular endothelial cells using a high content imaging system with nuclear factor κB p65 and intracellular adhesion molecule 1 (ICAM1) immunostaining. Next, we identified GRP78 autoantibodies causing blood-brain barrier permeability in PCD-LEMS IgG by co-immunoprecipitation and the living cell-based antibody binding assays. Exposure of brain microvascular endothelial cells to IgG from PCD-LEMS patients induced nuclear factor κB p65 nuclear translocation, ICAM1 upregulation, reduced claudin-5 expression, increased permeability and increased autocrine IL-1ß and IL-8 secretion; the IgG from patients with Lambert-Eaton myasthenic syndrome did not have these effects. We detected GRP78 autoantibodies in the IgG of LEMS-PCD (83.3%, n = 18), but observed fewer in patients with LEMS (6.6%, n = 15) and none were observed in the control subjects (n = 8). The depletion of GRP78 autoantibodies reduced the biological effect of LEMS-PCD IgG on brain microvascular endothelial cells. These findings suggest that GRP78 autoantibodies play a role beyond neuromyelitis optica and that they have direct implications in the phenotypic differences between PCD-LEMS and LEMS.

Increased IP-10 production by blood-nerve barrier in multifocal acquired demyelinating sensory and motor neuropathy and multifocal motor neuropathy.

OBJECTIVE: Dysfunction of the blood-nerve barrier (BNB) plays important roles in chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN). The aim of the present study was to identify the candidate cytokines/chemokines that cause the breakdown of the BNB using sera from patients with CIDP and MMN. METHODS: We determined the levels of 27 cytokines and chemokines in human peripheral nerve microvascular endothelial cells (PnMECs) after exposure to sera obtained from patients with CIDP variants (typical CIDP and multifocal acquired demyelinating sensory and motor neuropathy [MADSAM]), MMN and amyotrophic lateral sclerosis (ALS), and healthy controls (HC), using a multiplexed fluorescent bead-based immunoassay system. RESULTS: The induced protein (IP)10 level in the cells in both the MADSAM and MMN groups was markedly increased in comparison with the typical CIDP, ALS and HC groups. The other cytokines, including granulocyte colony-stimulating factor,vascular endothelial growth factor (VEGF) and interleukin-7, were also significantly upregulated in the MADSAM group. The increase of IP-10 produced by PnMECs was correlated with the presence of conduction block in both the MADSAM and MMN groups. CONCLUSION: The autocrine secretion of IP-10 induced by patient sera in PnMECs was markedly upregulated in both the MADSAM and MMN groups. The overproduction of IP-10 by PnMECs leads to the focal breakdown of the BNB and may help to mediate the transfer of pathogenic T cells across the BNB, thereby resulting in the appearance of conduction block in electrophysiological studies of patients with MADSAM and MMN.

Establishment of a new conditionally immortalized human skeletal muscle microvascular endothelial cell line.

In skeletal muscle, the capillaries have tight junctions (TJs) that are structurally similar to those in the blood-brain barrier (BBB) and blood-nerve barrier (BNB). Although many findings have been clarified in the territory of BBB and BNB, few have so far examined the TJs of capillaries in the skeletal muscle. In addition, no in vitro human skeletal muscle microvasculature models have been reported thus far. We newly established a new human skeletal muscle microvascular endothelial cell (HSMMEC) line. HSMMECs were isolated from human skeletal muscle and were infected with retroviruses harboring temperature-sensitive SV40 T antigen and telomerase genes. This cell line, termed TSM15, showed a spindle fiber-shaped morphology, an immunoreactivity to anti-factor VIII and anti-VE-cadherin antibodies, and a temperature-sensitive growth. TSM15 cells grew stably for more than 40 passages when they were cultured at 33°C, thereby retaining their spindle fiber-shaped morphology and contact inhibition at confluence. The cells expressed tight junctional molecules such as claudin-5, occludin, and zonula occludens-1, as well as transporters such as a glucose transporter 1. The transendothelial electrical resistance of TSM15 was as high as those of the human brain microvascular endothelial cell line. This novel cell line might facilitate the analyses of the pathophysiology of inflammatory myopathy, such as dermatomyositis, and can improve our understanding of the physiological and biochemical properties of the microvasculature in human skeletal muscle.

Identification of galectin-3 as a possible antibody target for secondary progressive multiple sclerosis.

BACKGROUND: Recent studies have revealed that the disruption of the blood-brain barrier (BBB) might contribute to the induction of neurodegeneration in the progressive stage of multiple sclerosis (MS). OBJECTIVE: We investigated a potential target for the serum auto-antibodies responsible for the BBB impairment in patients with secondary progressive MS (SPMS). METHODS: We identified undetermined target antigens in human brain microvascular endothelial cells (BMECs) that reacted with auto-antibodies in sera from SPMS patients using a proteomic approach. In addition, we examined how the identified auto-antibodies compromise the BBB integrity. RESULTS: We found that 10 of 11 SPMS sera had auto-antibodies against galectin-3, although the patients with other neurological diseases did not have these antibodies. Downregulation of galectin-3 led to elevated intercellular adhesion molecule-1 (ICAM-1) and phospho-nuclear factor-kappa (NFκ) B p65 expression in the BMECs. Exposure to SPMS patients' sera also increased the protein levels of ICAM-1 and phospho-NFκB p65 in BMECs, but these effects induced by anti-galectin-3 immunoreactivity were canceled by the downregulation of galectin-3. CONCLUSION: Galectin-3 is a possible immunological target molecule of the pathogenic auto-antibodies and contributes to the persistent BBB breakdown in patients with SPMS. These antibodies may also serve as a novel biomarker for SPMS.

[Therapeutic Strategies for Inflammatory Neuropathy].

Most cases of inflammatory neuropathy can be treated. It is important to treat patients before axonal degeneration can cause irreversible damage. Conventional treatments include corticosteroids, intravenous immunoglobulin (IVIg), and plasma exchange. Recently, the efficacy of various immunosuppressive and biological agents has increased. Drug efficacy varies depending on the disease and the underlying pathomechanisms. In addition, patients often respond differently to each treatment; therefore, selecting the most appropriate treatment for each patient by assessing the severity of the disease and the efficacy of drugs at appropriate time points is necessary.

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype.

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/ß-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Modeling Brain Vasculature Immune Interactions In Vitro.

The endothelial blood-brain barrier (BBB) protects central nervous system (CNS) neurons from the changeable milieu of the bloodstream by strictly controlling the movement of molecules and immune cells between the blood and the CNS. Immune cell migration across the vascular wall is a multistep process regulated by the sequential interaction of different signaling and adhesion molecules on the endothelium and the immune cells. Accounting for its unique barrier properties and trafficking molecule expression profile, particular adaptions in immune cell migration across the BBB have been observed. Thus, in vitro models of the BBB are desirable to explore the precise cellular and molecular mechanisms involved in immune cell trafficking across the BBB. The challenge to overcome is that barrier properties of brain microvascular endothelial cells are not intrinsic and readily lost in culture. With a focus on human in vitro BBB models, we here discuss the suitability of available in vitro models for the BBB for exploring the specific mechanisms involved in immune cell trafficking across the BBB.

[Clinical features of 8 patients with multifocal motor neuropathy in the long-term follow-up].

OBJECTIVE: To clarify the clinical and long-term characteristics of multifocal motor neuropathy (MMN). METHODS: We retrospectively evaluated data from 8 consecutive MMN patients in Yamaguchi University Hospital from 2005 to 2020. Clinical information including dominant hand, occupations, hobbies, nerve conduction data, protein level in cerebrospinal fluid (CSF), responsiveness to intravenous immunoglobulin (IVIg) therapy as initial therapy as well as maintenance therapy were collected. RESULTS: Unilateral upper limb was initially affected in all patients and a dominant upper extremity was affected in six of them. Seven patients had occupations or hobbies which were associated with overuse of their dominant upper extremity. CSF protein level was normal or slightly elevated. Nerve conduction studies showed conduction blocks in 4 cases. Effectiveness of IVIg treatment as initial therapy was observed in all patients. Maintenance therapy was not needed in 2 patients because of mild symptoms with stable clinical course. Long-term maintenance therapy with immunoglobulin was effective in 5 patients during the follow-up period. CONCLUSION: Dominant upper extremity was frequently affected and most patients had job or habit associated with its overuse, suggesting that physical overload induces inflammation or demyelination in MMN. IVIg was commonly effective as both introduction and long-term maintenance therapies. Complete remission was achieved after several IVIg treatments in some patients.

Antineutrophil cytoplasmic antibody-associated vasculitis with predominant truncal muscle weakness: a retrospective case series.

Introduction: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) frequently leads to mononeuritis multiplex, which are characterized by distal weakness associated with sensory disturbances. Although AAV has also been reported to be associated with myopathy, the pathogenesis and characteristics remain unclear. We aimed to show the clinical and laboratory findings in AAV-associated myopathy. Methods: This retrospective single-center study included patients with the diagnosis of AAV who had been admitted to the neurology department and had biopsy specimens of muscle and/or nerve tissue. Results: We identified four patients with a distinct clinical presentation of muscle weakness in the trunk and proximal limbs. The weakness resembled that of inflammatory muscle disease. These patients denied symptoms associated with neuropathy, and had normal serum creatine kinase (CK) levels. Needle electromyography (needle EMG) showed spontaneous electrical activity at rest, and results of magnetic resonance imaging (MRI) suggested inflammatory myopathy. Muscle biopsy specimens from all four patients revealed vasculitis and inflammation in proximity to the affected vessels, without any discernible characteristics of other myopathies. The patients also complained of symptoms affecting other organs, such as the ears and kidneys, which is typical of AAV cases. Remission induction therapy, such as cyclophosphamide pulse therapy in addition to oral prednisolone, were effective for all four patients. However, relapses occurred in two patients during maintenance therapy and two patients died of aspiration pneumonia. Discussion: The clinical course of our patients might represent a subtype of AAV that is characterized by muscle weakness of the trunk and proximal extremities and arises from vasculitis within the muscles. The clinical manifestations of our patients were similar to those of patients with inflammatory myopathy with regard to the distribution of muscle weakness, MRI and needle EMG findings. However, there are notable differences between AAV associated myopathy vs. inflammatory myositis like dermatomyositis; (1) the absence of elevated CK levels, (2) the presence of complications in other organs, (3) distinct pathological findings, and (4) severe outcomes. Awareness that AAV patients with muscle involvement could have a subtype of AAV that seriously affects various organs is critical for an accurate diagnosis and effective therapeutic management.

The choroid plexus acts as an immune cell reservoir and brain entry site in experimental autoimmune encephalomyelitis.

The choroid plexus (ChP) has been suggested as an alternative central nervous system (CNS) entry site for CCR6+ Th17 cells during the initiation of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). To advance our understanding of the importance of the ChP in orchestrating CNS immune cell entry during neuroinflammation, we here directly compared the accumulation of CD45+ immune cell subsets in the ChP, the brain and spinal cord at different stages of EAE by flow cytometry. We found that the ChP harbors high numbers of CD45int resident innate but also of CD45hi adaptive immune cell subsets including CCR6+ Th17 cells. With the exception to tissue-resident myeloid cells and B cells, numbers of CD45+ immune cells and specifically of CD4+ T cells increased in the ChP prior to EAE onset and remained elevated while declining in brain and spinal cord during chronic disease. Increased numbers of ChP immune cells preceded their increase in the cerebrospinal fluid (CSF). Th17 but also other CD4+ effector T-cell subsets could migrate from the basolateral to the apical side of the blood-cerebrospinal fluid barrier (BCSFB) in vitro, however, diapedesis of effector Th cells including that of Th17 cells did not require interaction of CCR6 with BCSFB derived CCL20. Our data underscore the important role of the ChP as CNS immune cell entry site in the context of autoimmune neuroinflammation.

SARS-CoV-2 infects epithelial cells of the blood-cerebrospinal fluid barrier rather than endothelial cells or pericytes of the blood-brain barrier.

BACKGROUND: As a consequence of SARS-CoV-2 infection various neurocognitive and neuropsychiatric symptoms can appear, which may persist for several months post infection. However, cell type-specific routes of brain infection and underlying mechanisms resulting in neuroglial dysfunction are not well understood. METHODS: Here, we investigated the susceptibility of cells constituting the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP) to SARS-CoV-2 infection using human induced pluripotent stem cell (hiPSC)-derived cellular models and a ChP papilloma-derived epithelial cell line as well as ChP tissue from COVID-19 patients, respectively. RESULTS: We noted a differential infectibility of hiPSC-derived brain microvascular endothelial cells (BMECs) depending on the differentiation method. Extended endothelial culture method (EECM)-BMECs characterized by a complete set of endothelial markers, good barrier properties and a mature immune phenotype were refractory to SARS-CoV-2 infection and did not exhibit an activated phenotype after prolonged SARS-CoV-2 inoculation. In contrast, defined medium method (DMM)-BMECs, characterized by a mixed endothelial and epithelial phenotype and excellent barrier properties were productively infected by SARS-CoV-2 in an ACE2-dependent manner. hiPSC-derived brain pericyte-like cells (BPLCs) lacking ACE2 expression were not susceptible to SARS-CoV-2 infection. Furthermore, the human choroid plexus papilloma-derived epithelial cell line HIBCPP, modeling the BCSFB was productively infected by SARS-CoV-2 preferentially from the basolateral side, facing the blood compartment. Assessment of ChP tissue from COVID-19 patients by RNA in situ hybridization revealed SARS-CoV-2 transcripts in ChP epithelial and ChP stromal cells. CONCLUSIONS: Our study shows that the BCSFB of the ChP rather than the BBB is susceptible to direct SARS-CoV-2 infection. Thus, neuropsychiatric symptoms because of COVID-19 may rather be associated with dysfunction of the BCSFB than the BBB. Future studies should consider a role of the ChP in underlying neuropsychiatric symptoms following SARS-CoV-2 infection.

The Modular µSiM: A Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro.

Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support "on-chip" clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, an extension of the µSiM (microdevice featuring a silicon-nitride membrane) platform is introduced. The modular µSiM (m-µSiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. The utility of the m-µSiM is demonstrated by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and nonengineering, brain barriers focused laboratories. In situ and sampling-based assays of small molecule diffusion are developed and validated as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-µSiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. The ability to quickly reconfigure the m-µSiM for coculture and immune cell transmigration studies through addition of accessories and/or quick exchange of components is then demonstrated. Because the development of modified components and accessories is easily achieved, custom designs of the m-µSiM shall be accessible to any laboratory desiring a barrier-style tissue chip platform.

[A case of POEMS syndrome with enlarged pancreas due to IgG4-related autoimmune pancreatitis].

A 57-year-old man developed bilateral hands and feet numbness, followed by weakness with the legs and skin pigmentation. These symptoms became gradually worsened, and we made a diagnosis of POEMS syndrome because of progressive polyneuropathy, skin changes, IgG lambda type monoclonal proteinemia, and elevated level of serum vascular endothelial growth factor (VEGF). Diffusely enlarged pancreas was noticed in computed tomography. Serological, radiological, and histological findings revealed enlarged pancreas was due to IgG4-related autoimmune pathogenesis. After high dose chemotherapy with autologous peripheral stem cell transplantation, his clinical manifestations, IgG lambda type monoclonal proteinemia, and elevated level of serum VEGF were improved, whereas diffuse enlargement of the pancreas did not change. This is the first case report of POEMS syndrome accompanied with IgG4-related autoimmune pancreatitis. Co-existence of monoclonal and polyclonal plasma proliferative changes in the present patient may provide keys to clarify common mechanisms shared by these two rare disorders, POEMS syndrome and IgG4-related autoimmune disease.

Differentiation of human pluripotent stem cells to brain microvascular endothelial cell-like cells suitable to study immune cell interactions.

We describe the extended endothelial cell culture method (EECM) for the differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cell (BMEC)-like cells. EECM-BMEC-like cells resemble primary human BMECs in morphology, molecular junctional architecture, and diffusion barrier characteristics. A mature immune phenotype with proper endothelial adhesion molecule expression makes this model distinct from any other hPSC-derived in vitro blood-brain barrier (BBB) model and suitable to study immune cell migration across the BBB in a disease relevant and personalized fashion. For complete details on the use and execution of this protocol, please refer to Lian et al. (2014), Nishihara et al. (2020a).

Wnt signaling mediates acquisition of blood-brain barrier properties in naïve endothelium derived from human pluripotent stem cells.

Endothelial cells (ECs) in the central nervous system (CNS) acquire their specialized blood-brain barrier (BBB) properties in response to extrinsic signals, with Wnt/ß-catenin signaling coordinating multiple aspects of this process. Our knowledge of CNS EC development has been advanced largely by animal models, and human pluripotent stem cells (hPSCs) offer the opportunity to examine BBB development in an in vitro human system. Here, we show that activation of Wnt signaling in hPSC-derived naïve endothelial progenitors, but not in matured ECs, leads to robust acquisition of canonical BBB phenotypes including expression of GLUT-1, increased claudin-5, decreased PLVAP, and decreased permeability. RNA-seq revealed a transcriptome profile resembling ECs with CNS-like characteristics, including Wnt-upregulated expression of LEF1, APCDD1, and ZIC3. Together, our work defines effects of Wnt activation in naïve ECs and establishes an improved hPSC-based model for interrogation of CNS barriergenesis.

The cells that line the inside of blood vessels are called endothelial cells. In the blood vessels of the brain, these cells form a structure called the 'blood-brain barrier', which allows nutrients to pass from the blood into the brain, while at the same time preventing harmful substances like toxins from crossing. Faults in the blood-brain barrier can contribute to neurological diseases, but the blood-brain barrier can also restrict drugs from accessing the brain, making it difficult to treat certain conditions. Understanding how the endothelial cells that form the blood-brain barrier develop may offer insight into new treatments for neurological diseases. During the development of the embryo, endothelial cells develop from stem cells. They can also be generated in the laboratory from human pluripotent stem cells or 'hPSCs', which are cells that can produce more cells like themselves, or differentiate into any cell type in the body. Scientists can treat hPSCs with specific molecules to make them differentiate into endothelial cells, or to modify their properties. This allows researchers to monitor how different types of endothelial cells form. Endothelial cells at the blood-brain barrier are one of these types. During their development, these cells gain distinct features, including the production of proteins called GLUT-1, claudin-5 and LSR. GLUT-1 transports glucose across endothelial cells' membranes, while claudin-5 and LSR tightly join adjacent cells together, preventing molecules from leaking into the brain through the space between cells. In mouse endothelial cells, a signaling protein called Wnt is responsible for turning on the genes that code for these proteins. But how does Wnt signaling impact human endothelial cells? Gastfriend et al. probed the effects of Wnt signaling on human endothelial cells grown in the lab as they differentiate from hPSCs. They found that human endothelial cells developed distinct blood-brain barrier features when Wnt signaling was activated, producing GLUT-1, claudin-5 and LSR. Gastfriend et al. also found that human endothelial cells were more responsive to Wnt signaling earlier in their development. Additionally, they identified the genes that became activated in human endothelial cells when Wnt signaling was triggered. These findings provide insight into the development and features of the endothelial cells that form the human blood-brain barrier. The results are a first step towards a better understanding of how this structure works in humans. This information may also allow researchers to develop new ways to deliver drugs into the brain.

Human CD4+ T cell subsets differ in their abilities to cross endothelial and epithelial brain barriers in vitro.

BACKGROUND: The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood-brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). OBJECTIVE: Analysis of the cellular and molecular mechanisms involved in the migration of different human CD4+ T-cell subsets across the BBB versus the BCSFB. METHODS: Human in vitro models of the BBB and BCSFB were employed to study the migration of circulating and CNS-entry experienced CD4+ T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. RESULTS: While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10- to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barrier-crossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. CONCLUSIONS: Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB.