In vitro culture models of the blood‐brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to test
Brain pericytes regulate diverse aspects of neurovascular development and function, including blood‐brain barrier (BBB) induction and maintenance. Primary brain pericytes have been widely employed in coculture‐based in vitro models of the BBB, and a method to generate brain pericytes from human pluripotent stem cells (hPSCs) could provide a renewable, genetically tractable source of cells for BBB modeling and studying pericyte roles in development and disease. Here, we describe a protocol to differentiate hPSCs to NG2+PDGFRβ+αSMAlowbrain pericyte‐like cells in 22‐25 days through a p75‐NGFR+HNK‐1+neural crest intermediate, which mimics the developmental origin of forebrain pericytes. The resulting brain pericyte‐like cells have molecular and functional attributes of brain pericytes. We also provide protocols for maintenance, cryopreservation, and recovery of the neural crest intermediate, and for molecular and functional characterization of the resulting cells. © 2021 Wiley Periodicals LLC.
This article was corrected on 18 July 2022. See the end of the full text for details.
- NSF-PAR ID:
- 10238647
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 1
- Issue:
- 1
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract Mycobacterium tuberculosis (Mtb) dissemination across brain endothelial cells. One‐third of the global population is infected with Mtb, and in 1%‐2% of cases bacteria invade the CNS through a largely unknown process. The “Trojan horse” theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB‐granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination. © 2020 Wiley Periodicals LLC.Basic Protocol 1 : Isolation of primary mouse brain astrocytes and endothelial cellsBasic Protocol 2 : Isolation of primary mouse bone marrow–derived dendritic cellsSupport Protocol 1 : Validation of dendritic cell purity by flow cytometryBasic Protocol 3 : Isolation of primary mouse peripheral blood mononuclear cellsSupport Protocol 2 : Isolation of primary mouse spleen cellsSupport Protocol 3 : Purification and validation of CD4+ T cells from PBMCs and spleen cellsBasic Protocol 4 : Isolation of liver granuloma supernatant and determination of organ loadSupport Protocol 4 : In vivo and in vitro infection with mycobacteriaBasic Protocol 5 : Assembly of the BBB co‐culture modelBasic Protocol 6 : Assembly of the combined in vitro granuloma and BBB model -
more » « less
Abstract The brain vasculature maintains brain homeostasis by tightly regulating ionic, molecular, and cellular transport between the blood and the brain parenchyma. These blood–brain barrier (BBB) properties are impediments to brain drug delivery, and brain vascular dysfunction accompanies many neurological disorders. The molecular constituents of brain microvascular endothelial cells (BMECs) and pericytes, which share a basement membrane and comprise the microvessel structure, remain incompletely characterized, particularly in humans. To improve the molecular database of these cell types, we performed RNA sequencing on brain microvessel preparations isolated from snap-frozen human and mouse tissues by laser capture microdissection (LCM). The resulting transcriptome datasets from LCM microvessels were enriched in known brain endothelial and pericyte markers, and global comparison identified previously unknown microvessel-enriched genes. We used these datasets to identify mouse-human species differences in microvessel-associated gene expression that may have relevance to BBB regulation and drug delivery. Further, by comparison of human LCM microvessel data with existing human BMEC transcriptomic datasets, we identified novel putative markers of human brain pericytes. Together, these data improve the molecular definition of BMECs and brain pericytes, and are a resource for rational development of new brain-penetrant therapeutics and for advancing understanding of brain vascular function and dysfunction.
-
more » « less
Abstract The chytrid fungus
Batrachochytrium dendrobatidis (Bd ) is a causative agent of chytridiomycosis, a skin disease associated with amphibian population declines around the world. Despite the major impactBd is having on global ecosystems, much ofBd ’s basic biology remains unstudied. In addition to revealing mechanisms driving the spread of chytridiomycosis, studyingBd can shed light on the evolution of key fungal traits because chytrid fungi, includingBd , diverged before the radiation of the Dikaryotic fungi (multicellular fungi and yeast). StudyingBd in the laboratory is, therefore, of growing interest to a wide range of scientists, ranging from herpetologists and disease ecologists to molecular, cell, and evolutionary biologists. This protocol describes how to maintain developmentally synchronized liquid cultures ofBd for use in the laboratory, how to growBd on solid media, as well as cryopreservation and revival of frozen stocks. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1 : Reviving cryopreservedBd culturesBasic Protocol 2 : Establishing synchronized liquid cultures ofBd Basic Protocol 3 : Regular maintenance of synchronousBd in liquid cultureAlternate Protocol 1 : Regular maintenance of asynchronousBd in liquid cultureBasic Protocol 4 : Regular maintenance of synchronousBd on solid mediumAlternate Protocol 2 : Starting a culture on solid medium from a liquid cultureBasic Protocol 5 : Cryopreservation ofBd -
The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases such as PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRβ (sPDGFRβ) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRβ variants, and specifically during tissue homeostasis. Here, we found sPDGFRβ protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRβ isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRβ by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRβ transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRβ protein was detected throughout the brain parenchyma in distinct regions, such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRβ variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRβ variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRβ likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRβ in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion—critical processes underlying neuronal health and function, and in turn, memory and cognition.more » « less
-
more » « less
Abstract Nervous system development has been intensely studied in insects (especially
Drosophila melanogaster ), providing detailed insights into the genetic regulatory network governing the formation and maintenance of the neural stem cells (neuroblasts) and the differentiation of their progeny. Despite notable advances over the last two decades, neurogenesis in other arthropod groups remains by comparison less well understood, hampering finer resolution of evolutionary cell type transformations and changes in the genetic regulatory network in some branches of the arthropod tree of life. Although the neurogenic cellular machinery in malacostracan crustaceans is well described morphologically, its genetic molecular characterization is pending. To address this, we established an in situ hybridization protocol for the crayfishProcambarus virginalis and studied embryonic expression patterns of a suite of key genes, encompassing threeSoxB group transcription factors, twoachaete –scute homologs, aSnail family member, the differentiation determinantsProspero andBrain tumor , and the neuron markerElav . We document cell type expression patterns with notable similarities to insects and branchiopod crustaceans, lending further support to the homology of hexapod–crustacean neuroblasts and their cell lineages. Remarkably, in the crayfish head region, cell emigration from the neuroectoderm coupled with gene expression data points to a neuroblast‐independent initial phase of brain neurogenesis. Further,SoxB group expression patterns suggest an involvement ofDichaete in segmentation, in concordance with insects. Our target gene set is a promising starting point for further embryonic studies, as well as for the molecular genetic characterization of subregions and cell types in the neurogenic systems in the adult crayfish brain.
