Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function and shuttling of drugs and antibodies

The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cell-derived human brain microvascular endothelium interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB

In vitro models of the blood brain-barrier using iPSC-derived cells

The blood-brain barrier (BBB) constitutes the interface between the blood and the brain tissue. Its primary function is to maintain the tightly controlled microenvironment of the brain. Models of the BBB are useful for studying the development and maintenance of the BBB as well as diseases affecting it. Furthermore, BBB models are important tools in drug development and support the evaluation of the brain-penetrating properties of novel drug molecules. Currently used in vitro models of the BBB include immortalized brain endothelial cell lines and primary brain endothelial cells of human and animal origin. Unfortunately, these cell lines and primary cells have failed to recreate physiologically relevant control of transport in vitro. Human-induced pluripotent stem cell (iPSC)-derived brain endothelial cells have proven a promising alternative source of brain endothelial-like cells that replicate tight cell layers with low para-cellular permeability. Given the possibility to generate large amounts of iPSC-derived brain endothelial cells they are a feasible alternative when modelling the BBB in vitro. This thesis aimed to develop iPSC-derived models of the BBB that display a barrier like phenotype and characterize these models in terms of specific properties. The BBB model development was based on investigations into mechanisms important for barrier formation in iPSC-derived endothelial cells and development of high-quality supporting cells. The possibilities to use the model in drug discovery, and in determination of brain penetrating capacity of drug substances were specifically considered. These studies have increased knowledge of molecular mechanisms behind the restricted permeability across iPSC-derived endothelial cells and identified transcriptional changes that occur in iPSC-derived endothelial cells upon coculture with relevant cell types of the neurovascular unit. Furthermore, high quality iPSC-derived astrocytic cells were developed, and the biological relevance and model diversity between astrocytic models were evaluated. Both astrocytes and brain endothelial cells have been adapted to xeno-free culture conditions and used in the BBB models, demonstrating a xeno-free BBB model. Finally, a more biologically relevant microphysiological dynamic BBB model was generated. This model demonstrated improved permeability modelling and compatibility with high-throughput substance permeability screening. Taken together these results show that iPSC-derived BBB models are useful for studying BBB-specific properties in vitro and that both marker expression and functional evaluation of iPSC-derived cells are important in assessing cell identity and cell quality. In addition, these results show that iPSC derived BBB models are feasible for high-throughput permeability studies

Water birth

Bakgrund: Forskningsläget när det gäller vattenförlossning visar att det inte finns ökade risker för kvinnan och barnet att föda i vatten i jämförelse med konventionell förlossning när det föreligger en lågriskförlossning. Resultatet tolkas dock olika internationellt beroende på studiernas evidensvärde vilket innebär att vattenförlossning kan tillåtas eller ej. I Sverige saknas idag nationella riktlinjer och Socialstyrelsen har tidigare avrått från denna förlossningsmetod. Syfte: Syftet med studien var att beskriva det maternella och neonatala utfallet vid vattenförlossning vid ett universitetssjukhus i Sverige. Metod: Retrospektiv journalgranskning inom ramen för ett kvalitetsprojekt genomfördes med en kvantitativ ansats. Journaler diagnossatta med vattenförlossning under år 2017 – 2018 inkluderades i studien. Resultatet presenteras med deskriptiv statistik. Resultat: Totalt granskades 64 journaler. En förstföderska drabbades av en grad 3-bristning och de kvinnor som blödde över en liter fram till två timmar efter förlossningen uppgick till 4,9 %. Det utfördes en manuell placentalösning. I de fall där upplevelsen fanns beskriven i journalen hade en klar majoritet en bra förlossningsupplevelse. Det förekom ingen postpartuminfektion, episiotomi eller navelsträngsruptur. Ett barn hade Apgar Score <7 vid fem minuters ålder. Ett barn vårdades på neonatalvårdsavdelning. Önskemål om att få föda i vatten fanns dokumenterat i 15 journaler. Riskbedömning var endast journalfört i 28,2 % av journalerna. Slutsats: Även om denna studie är i form av ett kvalitetsprojekt har en jämförelse gjorts med tidigare forskning som visar att resultatet inte skiljer sig från de senaste årens forskning inom området som menar att det inte föreligger några ökade risker med vattenförlossning för lågriskpatienter. Dock bör fler studier utföras i en svensk kontext samt ett rutindokument med kriterier och handläggning tas fram som en trygghet både för personalen och de födande kvinnorna.Background: The current collective research about water birth shows no increased risk for the mother and the child as compared to low risk conventional birth. Internationally, the evidence value of the existing research can be interpreted differently, which can lead to differences in guidelines regarding water birth. There are no national guidelines for water birth in Sweden yet and The National Board of Health and Welfare in Sweden has previously discouraged from this birthing method. Aim: The aim of this study was to describe maternal and neonatal outcome following water birth at a Swedish university hospital. Method: This quality control project used a quantitative retrospective study design collecting data through medical record review. Journals containing the diagnose code water birth during 2017 – 2018 were included. The result is presented with descriptive statistics. Result: A total of 64 journals were reviewed. One primipara was diagnosed with a third-degree perineal tear and 4,9% of the women had a blood loss >1000 ml two hours postpartum. One manual placenta removal was conducted. In those journals where the birth experience was described, the majority of the women were satisfied. There were no findings of postpartum infection, episiotomy or umbilical cord avulsion. One newborn had an Apgar Score <7 in five minutes. One newborn was admitted to a neonatal intensive care unit. In 15 journals a note was found that they wished to give birth in water. Documented detection of risk level was only found in 28,2% of the journals. Conclusions: Although this study is a quality control project, comparisons have been made with previous research, showing that the result shows no increased risk for low risk patients birthing in water. However, further studies needs to be conducted in the Swedish setting and local guidelines for practice, including criteria, needs to be developed as an assurance for the care givers and the expecting mothers. Keywords: Water birth, maternal outcome, neonatal outcome, informed choice

Enhanced xeno-free differentiation of hiPSC-derived astroglia applied in a blood-brain barrier model

Background Human induced pluripotent stem cells (hiPSC) hold great promise for use in cell therapy applications and for improved in vitro models of human disease. So far, most hiPSC differentiation protocols to astroglia use undefined, animal-containing culture matrices. Laminins, which play an essential role in the regulation of cell behavior, offer a source of defined, animal-free culture matrix. Methods In order to understand how laminins affect astroglia differentiation, recombinant human laminin-521 (LN521), was compared to a murine Engelbreth-Holm-Swarm sarcoma derived laminin (L2020). Astroglia expression of protein and mRNA together with glutamate uptake and protein secretion function, were evaluated. Finally, these astroglia were evaluated in a coculture model of the blood-brain barrier (BBB). Results Astroglia of good quality were generated from hiPSC on both LN521 and L2020. However, astroglia differentiated on human LN521 showed higher expression of several astroglia specific mRNAs and proteins such as GFAP, S100B, Angiopoietin-1, and EAAT1, compared to astroglia differentiated on murine L2020. In addition, glutamate uptake and ability to induce expression of junction proteins in endothelial cells were affected by the culture matrix for differentiation. Conclusion Our results suggest that astroglia differentiated on LN521 display an improved phenotype and are suitable for coculture in a hiPSC-derived BBB model. This provides a starting point for a more defined and robust derivation of astroglia for use in BBB coculture models

Truncation of the TAR DNA-binding protein 43 is not a prerequisite for cytoplasmic relocalization, and is suppressed by caspase inhibition and by introduction of the A90V sequence variant

The RNA-binding and -processing protein TAR DNA-binding protein 43 (TDP-43) is heavily linked to the underlying causes and pathology of neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In these diseases, TDP-43 is mislocalized, hyperphosphorylated, ubiquitinated, aggregated and cleaved. The importance of TDP-43 cleavage in the disease pathogenesis is still poorly understood. Here we detail the use of D-sorbitol as an exogenous stressor that causes TDP-43 cleavage in HeLa cells, resulting in a 35 kDa truncated product that accumulates in the cytoplasm within one hour of treatment. We confirm that the formation of this 35 kDa cleavage product is mediated by the activation of caspases. Inhibition of caspases blocks the cleavage of TDP-43, but does not prevent the accumulation of full-length protein in the cytoplasm. Using D-sorbitol as a stressor and caspase activator, we also demonstrate that the A90V variant of TDP-43, which lies adjacent to the caspase cleavage site within the nuclear localization sequence of TDP-43, confers partial resistance against caspase-mediated generation of the 35 kDa cleavage product.CC BY 4.0</p

Models of the blood-brain barrier using iPSC-derived cells

The blood-brain barrier (BBB) constitutes the interface between the blood and the brain tissue. Its primary function is to maintain the tightly controlled microenvironment of the brain. Models of the BBB are useful for studying the development and maintenance of the BBB as well as diseases affecting it. Furthermore, BBB models are important tools in drug development and support the evaluation of the brain-penetrating properties of novel drug molecules. Currently used in vitro models of the BBB include immortalized brain endothelial cell lines and primary brain endothelial cells of human and animal origin. Unfortunately, many cell lines and primary cells do not recreate physiological restriction of transport in vitro. Human-induced pluripotent stem cell (iPSC)-derived brain endothelial cells have proven a promising alternative source of brain endothelial-like cells that replicate tight cell layers with low paracellular permeability. Given the possibility to generate large amounts of human iPSC-derived brain endothelial cells they are a feasible alternative when modelling the BBB in vitro. iPSC-derived brain endothelial cells form tight cell layers in vitro and their barrier properties can be enhanced through coculture with other cell types of the BBB. Currently, many different models of the BBB using iPSC-derived cells are under evaluation to study BBB formation, maintenance, disruption, drug transport and diseases affecting the BBB. This review summarizes important functions of the BBB and current efforts to create iPSC-derived BBB models in both static and dynamic conditions. In addition, it highlights key model requirements and remaining challenges for human iPSC-derived BBB models in vitro.CC BY 4.0</p

D-sorbitol induces the formation of a 35 kDa cleavage product of TDP-43 that is localized in the cytosol.

<p>(A) Western blot of TDP-43 protein (C-terminal antibody) after treatment of HeLa cells with 0.4 M D-sorbitol or 1 mM hydrogen peroxide for one hour. (B), (C) Densitometry analysis of (B) full-length TDP-43, (C) 35 kDa fragment of TDP-43 (CTF35). Densitometry results represent mean signal ± SEM (N = 4). (D) Detection of CTF35 in cytoplasmic (Cyto) and nuclear (Nuc) fractions of HeLa cells after treatment with D-sorbitol or hydrogen peroxide. (E) Immunofluorescence detection of cytoplasmic TDP-43 after D-sorbitol treatment. Scale bar = 15 μm. (F) Quantification of cytoplasmic TDP-43 immunofluorescence signal. Results represent mean TDP-43 immunofluorescence signal in the cytoplasm as a percentage of total TDP-43 signal in the cell ± SEM (N = 4). ** p < 0.01, *** p < 0.001.</p

Inhibition of caspases prevents the formation of CTF35 of TDP-43, but does not prevent relocalization of the full-length protein to the cytosol.

<p>(A) TDP-43 Western blot of samples treated with or without D-sorbitol after pre-treatment with caspase inhibitor Z-VAD-FMK or DMSO control. α-tubulin was used as a loading control. FL = full-length TDP-43, CTF35 = 35 kDa C-terminal fragment of TDP-43. (B) Densitometry quantification of the 35 kDa fragment of TDP-43 after sorbitol stress in the absence or presence of caspase inhibitor (N = 5). (C) Immunofluorescence staining of nuclear and cytoplasmic TDP-43 using a C-terminal TDP-43 antibody. Cells were treated with or without D-sorbitol after pre-treatment with caspase inhibitor. Scale bar = 15 μm. (D) Quantification of nuclear and cytoplasmic TDP-43 fluorescence signals (N = 3). (E) Western blot of nuclear and cytoplasmic fractions of stressed HeLa cells. GAPDH was used as a control for the cytoplasmic fractions, histone H3 was chosen to confirm enrichment of the nuclear fraction. All results represent mean ± SEM; ** p < 0.01, *** p < 0.001.</p

The osmotic and oxidative stressor D-sorbitol triggers cleavage of endogenous TDP-43 in HeLa cells.

<p>Untransfected HeLa cells expressing endogenous TDP-43 were treated with 10 μM MG132 for 4 h, 1 mM hydrogen peroxide (H₂O₂) for 1 h, 0.5 mM sodium arsenite (SA) for 30 min, 1 μM thapsigargin (TH) for 1 h, 0.4 M D-sorbitol (Sorb) for 1 h or with respective H₂O and DMSO solvent controls. Western blot detection using a polyclonal TDP-43 antibody revealed a truncated product of TDP-43 with an approximate molecular weight of 35 kDa (CTF35, indicated by arrowhead) after treatment with D-sorbitol, but not with the other stressors.</p