Abstract
Meninges, or the membranous coverings of the brain and spinal cord, play host to dozens of morbid pathologies. In this study we provide a method to isolate the leptomeningeal cell layer, identify leptomeninges in histologic slides, and maintain leptomeningeal fibroblasts in in vitro culture. Using an array of transcriptomic, histological, and cytometric analyses, we identified ICAM1 and SLC38A2 as two novel markers of leptomeningeal cells in vivo and in vitro. Our results confirm the fibroblastoid nature of leptomeningeal cells and their ability to form a sheet-like layer that covers the brain and spine parenchyma. These findings will enable researchers in central nervous system barriers to describe leptomeningeal cell functions in health and disease.
Graphical Abstract
1. Introduction
The meninges are complex connective tissue structures that surround the brain and spinal cord. Meninges are anatomically divided into the pachymeninges, represented by dura mater and leptomeninges, consisting of the arachnoid mater and pia mater. Embryologically, meningeal layers are derived from mesenchymal tissue that surrounds the neural tube during development. The dura mater is a well-innervated, highly vascularized, tough, collagenous membrane that also contains lymphatics 1–5. Lying just below the subdural space, the arachnoid mater, forms a multilayer membrane that covers the cerebrospinal fluid-filled subarachnoid space, creating a cellular barrier through its tight junctions 6–10. Proximal to the brain parenchyma, the pia mater, a single cell-layer membrane, tightly covers the brain and spinal cord and extends into sulci and fissures 11–14.
The leptomeninges host a wide variety of physiologic and pathologic processes, including meningitis, immunoinflammatory disorders, leptomeningeal metastasis, and others 15. However, our current understanding of the pathophysiology of these processes is largely limited to events occurring in the cerebrospinal fluid. The role of leptomeninges in these pathologies remains largely under-explored. Many studies highlight the importance of the leptomeninges during development 16,17, but it is unclear how these cells might directly interact with the parenchyma. Interestingly, pathology affecting the parenchyma, being infectious or non-infectious, is almost always preceded by meningeal inflammation 18–22. A recent study identifies numerous novel markers for pial, arachnoid and dural cells in embryonic meningeal tissue 23. However, the durability of these markers in adult mice or humans remains unknown. Interest in the leptomeninges has been on the rise due to its implication in metastases from several tumors, inflicting dismal prognoses 24,25. In order to study this essential component of this system, examination of normal leptomeningeal cells is crucial. Described here is a detailed method for isolation and primary culture of leptomeningeal cells from adult mice.
2. Results
2.1. Characterization and in vitro culture of leptomeningeal fibroblasts
The meningeal layers isolated from vertebrates contain a variety of cell types including immune, endothelial and other cells. The dominant cell type comprising these layers are collagen-producing fibroblasts, visualized in red, using picrosirius red staining or in blue, using Masson’s trichome staining in human leptomeninges and dura and mouse cranial and spinal meninges (Figure 1A–D). However, the distinct and brain-specific markers of adult meningeal fibroblasts that would allow for identification of these discrete populations are not yet known.
Figure 1. Characterization and isolation of leptomeningeal fibroblasts.
A-D Representative images of human (A-B) and mouse meninges (C-D) stained with picrosirius red and Masson’s trichome, visualizing collagen fibers. Human scale bar = 100 μm; mouse scale bar = 50 μm.
E Schematic of incision lines across lambdoid, sagittal and coronal sutures of a mouse skull, preceding leptomeninges isolation.
F Microdissection and collection of mouse leptomeninges from skull. Scale bar = 5 mm. See also Movie 1.
G Schematic of processing of microdissected tissue into single-cell suspension and in vitro culture.
H Representative images of murine leptomeningeal and dural fibroblasts in in vitro culture, stained for nuclei (DAPI) and filamentous actin (Phalloidin). Scale bar = 50 μm.
I Representative dot plot showing the relative compositon of microdissected and dissociated murine leptomeninges and bar plot showing quantification of flow cytometric analysis of dissociated leptomeninges (n = 3 biological replicates, each containing tissue from 5 mice).
J Schematic showing the identification of six putative leptomeningeal markers using bulk transcriptomics (see also Table S1). Abbreviations: Ar – arachnoid, Bo – bone, BM – bone marrow, D- dura, DBC – dural border cells, mD – meningeal dura, L – leptomeninges, P – pia, Par – parenchyma, poD – periosteal dura, Sk – skull, Sp – spine, V – vessel.
We optimized a microdissection and dissociation technique that allows for isolation of mouse leptomeninges that includes pial and arachnoid fibroblasts (Figure 1E–G and Movie 1). In in vitro culture, these cells show fibroblastoid morphology with flat oval nuclei and spindle shaped cell bodies, reminiscent of dural fibroblasts (Figure 1H, dura was isolated using microdissection techniques described elsewhere 26 and dissociated to single-cell suspension similarly to leptomeninges). To assess the feasibility of maintaining leptomeningeal cells in culture, we assessed the viability of leptomeningeal cells with in vitro passaging and repetitive freeze-thaw cycles. Given the primary nature of these cells, viability of leptomeningeal cells decreased by about 5% with every passage reaching 42% viability after 10 passages. Viability further decreased with increasing numbers of freeze-thaw cycles leptomeningeal cells were subjected to (Figure S1A–B). These leptomeningeal cells also display increased cellular senescence with in vitro passaging shown, by accumulation of Cdkn2a expression with increased passage numbers (Figure S1C).
2.2. Identification of cell surface markers of meningeal fibroblasts
To identify a set of putative markers of adult meningeal cells, double-negative (DN; CD45/CD31−) cells from isolated mouse leptomeninges were sorted and subjected to bulk RNA-sequencing (Figure 1I–J). Filtering for relative abundance and cell membrane localization led to identification of six candidate genes (Figure 1J and Table S1). The relative abundance of these genes and localization of their gene products were screened with quantitative PCR, flow cytometry and immunohistochemistry techniques, complemented with a comprehensive database search (Panther, UniProt). Using this strategy, we selected ICAM1 and SLC38A2 for further validation in in vitro fibroblast culture and human and mouse meninges.
ICAM1 and SLC38A2 were robustly expressed by both cranial (meningeal and dural) and extra-cranial fibroblasts (pulmonary and mammary) in human and mouse in vitro cultures (Figure 2). Lineage-negative mouse leptomeningeal cells sorted for Icam1 show homogenous fibroblastoid morphology upon attachment, unlike the unsorted, heterogenous leptomeningeal pool (Figure S1D). Both leptomeningeal and dural fibroblasts retain their characteristic marker gene expression in in vitro conditions (Figure S2). Staining of formalin-fixed paraffin-embedded tissue sections of human meninges and mouse skulls confirmed relatively increased expression of ICAM1 and SLC38A2 in leptomeningeal fibroblasts (pial and arachnoid) in comparison to fibroblasts in dural layers (Figure 3). Importantly, the expression of ICAM1 and SLC38A2 remained detectable and relatively stable across sexes and ages in human meninges (Figure 4).
Figure 2. Expression of ICAM1 and SLC38A2 in in vitro cultured fibroblasts.
A Representative confocal images of cultured human fibroblasts stained for ICAM1, SLC38A2, filamentous actin and nuclei. Scale bar = 100 μm.
B Quantification of mean pixel intensity of ICAM1 and SLC38A2 in human fibroblasts cultured in vitro. n = 6, three ROIs per two biological replicates were quantified.
C Representative fluorescent microscopy images of cultured murine fibroblasts stained for ICAM1, SLC38A2, filamentous actin and nuclei. Scale bar = 100 μm.
D Quantification of mean pixel intensity of ICAM1 and SLC38A2 in human fibroblasts cultured in vitro. n = 9, three ROIs per three biological replicates were quantified.
Figure 3. Expression of ICAM1 and SLC38A2 in human and mouse meninges.
A-B Representative images of human leptomeninges (A) and dura (B) stained for ICAM1 and SLC38A2. Scale bar = 100 μm.
C-D Representative images of mouse cranial (C) and basilar meninges (D) stained for Icam1 and Slc38a2. Scale bar = 50 μm.
Abbreviations: Ar – arachnoid, BM – bone marrow, D- dura,, P – pia, V – vessel.
Figure 4. Expression of ICAM1 and SLC38A2 in human leptomeninges across sexes and ages.
Staining for ICAM1 and SLC38A2 in leptomeninges four human donors showing strong and stable expression of these two proteins across sexes and ages. Scale bar = 50 μm.
Abbreviations: Ar – arachnoid, P – pia, V – vessel.
Using an alternative orthogonal approach to confirm these findings, we took advantage of the Allen Brain Map single-cell transcriptomic atlas. We identified five distinct transcriptomic states in a small cluster of mouse vascular-leptomeningeal cells (VLMC), fraction of which is reminiscent of leptomeningeal fibroblasts. Taking into account the mRNA drop-out during single-cell RNA-seq, both Icam1 and Slc38a2 mRNA were detected in all of these subsets (Figure 5A). The presence of Icam1 and Slc38a2 mRNA in the leptomeningeal tissues was further verified in publicly available RNA in-situ hybridization (ISH) data (Figure 5B). To clarify the function of these VLMC, we assessed the cellular functions overrepresented within the highly variable genes in both mouse and human VLMC single-cell transcriptomes (Figure 5C and Tables S2–3). In both organisms, VLMC are involved in a wide spectrum of physiological functions, including the transport of small molecules, synthesis of extracellular matrix components, and matrix-mediated interactions. The cell surface markers ICAM1 and SLC38A2, identified in this manuscript as fibroblast markers (Figure 6), may in future help to further explore the heterogeneity and biological function of leptomeningeal fibroblasts in adult tissues.
Figure 5: Expression of Icam1 and Slc38a2 in vascular and leptomeningeal cells (VLMC).
A tSNE plot showing the expression of Icam1 and Slc38a2 at single-cell resolution in the cluster of mouse VLMC in Allen Brain Map.
B RNA in situ hybridization of Icam1 and Slc38a2 in adult mouse brain section. Scale bar = 1.7 mm. Arrows in inserts point to the pia mater and arachnoid layer.
C Functional annotation of highly variable genes in mouse and human VLMC, plots show top ten cellular processes ranked based on P value (see also Tables S2, 3; FDR < 0.05).
Figure 6. Expression of ICAM1, SLC38A2 and other meningeal markers across the meninges.
Scheme illustrating relative expression of known meningeal markers across the cranial meninges.
3. Discussion
3.1. Applications of the protocol
Despite their critical role in physiology and various pathologies, our understanding of the structure and function of adult leptomeninges remains incomplete. The presented detailed protocol enables investigators to reliably establish a primary leptomeningeal culture with fibroblast-like characteristics and that retains two putative adult leptomeningeal markers in vitro: ICAM1 and SLC38A2. The isolated primary leptomeningeal cells enable the in vitro modelling of conditions that affect leptomeninges, such as infections, autoimmune conditions or cancer. In order to assess the mechanistic and biochemical features of this niche, the primary leptomeningeal cells can be isolated, cultured and maintained in vitro for a limited number of passages, preserving the characteristic markers described in this manuscript. These markers can be used to purify or identify leptomeningeal cells for additional downstream applications. Importantly, these markers have a functional role as well, allowing for the interaction of meningeal cells with other cells within their environment.
3.2. Comparison with other methods
Other protocols exist for the bulk isolation of the meninges from mouse or rat brains 3,26,27. However, these protocols fail to distinguish between the different meningeal layers and are usually used to study bulk meningeal immunity or vasculature in response to parenchymal or meningeal diseases 28. Moreover, the pachymeninges host a distinct range of pathologies apart from the leptomeninges 29. Isolation of a pure leptomeningeal population enables dedicated study of this unique anatomic compartment. Using our protocol, researchers can reliably extract and study leptomeningeal fibroblasts with the help of the identified markers.
3.3. Applications of the protocol
We provide a step-by-step protocol allowing the investigators to specifically dissect mouse leptomeninges that can be further used for a wide variety of downstream applications, including primary cell culture. The major obstacle in this procedure is the identification and gross dissection of leptomeninges. The method in this manuscript provides guidance to the researcher without prior knowledge of mouse meningeal anatomy to reliably isolate this delicate tissue. Freshly isolated leptomeningeal tissue can be digested into single-cell suspension and seeded into cell culture that can be maintained for a limited number of passages, thereby providing opportunities for increased mechanistic and biochemical understanding of this tissue using tissue culture-based methodologies. Alternatively, single-cell suspensions can be analyzed with flow cytometry or sort-purified. Such sorted leptomeningeal cells from normal mice and mice with meningeal pathologies can be assessed with qPCR or RNA-sequencing to uncover pathology-induced aberrances in signaling pathways or genes-of-interest. Freshly isolated leptomeninges can be, after brief fixation, stain for antigens-of-interest in whole-mount preparations.
Using strategy described in this protocol, we identified and validated two putative leptomeningeal markers, ICAM1 and SLC8A2. These markers can be used separately or in tandem to identify the leptomeningeal layer in ex vivo mouse brains or human autopsy specimens.
3.4. Limitations
One of the limitations of this method is the requirement of deep anatomical understanding of murine meninges that is critical for precise leptomeninges identification and extraction. This will ensure maximal efficiency of leptomeningeal cell recovery without cross-contamination with cells derived from pachymeninges or brain tissue. To avoid cross-contamination with surrounding tissue, the experimenter will need to carefully peel the leptomeninges from the brain parenchyma while laterally retracting the skull. To ensure sufficient viability of leptomeningeal cells, dissection must occur within few minutes after euthanasia and tissues must be placed into ice-cold saline. In summary, this technique requires balance between speed and precision to achieve a successful leptomeningeal fibroblast isolation.
4. Materials and Methods
Mice.
All animal studies were approved by the MSKCC IACUC, protocol #18–01-002. Female and male C57BL/6J and female Balb/C mice were acquired from Jackson Laboratory and housed in the MSKCC vivarium. Mice used in this study were at 8–16 weeks of age.
Human tissues.
Human FFPE autopsy tissue was collected under MSKCC IRB #18–065 and #18–292 from patients that provided written informed consent and obtained from MSKCC Last Wish Program. Tissues used in this study was derived from donors that deceased due to extracranial complications of cancer.
Preparation of single-cell suspensions from mouse leptomeninges, dura, lungs and mammary fat pads.
Leptomeningeal layer was isolated from mouse skulls as described in detailed below. Briefly, the transparent tissue adherent to the cerebral cortex was extracted using Dumont #7 forceps, starting from the superior sagittal sinus and moving caudally in a medial to lateral fashion. Cerebral meninges were removed piecewise.
Pieces of dissected leptomeninges were placed immediately in the dissociation solution containing collagenase (200 u/mL; Worthington, Cat. LS005273), dispase (StemCell Technologies, Cat. 07913) and DNase I (50 U/mL; Worthington, Cat. LS006333) in serum-free high-glucose DMEM (MSKCC Media Core, Cat. HG DME) or serum-free MenCM (Sciencell, Cat. 1400) and kept on ice. Tissue in microtubes was placed on rotator for 2 hours at 37°C until completely dissociated. Suspension was filtered through the 35 μm cell strainer cap into a new 5 mL polystyrene tube (Falcon, Cat. 352235). To prevent cell loss, tube can be coated with 1% BSA. If necessary, red blood cell can be lysed with 1x ACK buffer (Thermo Fisher, Cat. A1049201). Mouse dura was peeled off from below the cranial bone, as described in 3 and processed to single-cell suspensions as described above. Mouse lungs and mammary fat pads were dissected, minced to 1 mm pieces, and digested and processed to single-cell suspensions as described above. Single-cell suspensions were used for flow cytometry, flow sorting, and in vitro culture.
In vitro culture of cranial and extracranial fibroblasts.
For mouse fibroblasts, single-cell suspensions from various body sites were prepared as described above. To enrich for fibroblasts in vitro, we took advantage of relatively fast attachment of these cells to the cell culture plastic 30. Single cell suspensions were seeded into 6-well plates containing high-glucose DMEM, supplemented with 15% FBS and 1% penicillin-streptomycin. Two hours after seeding, the non-attached content of the well was carefully aspirated, surface of the well was washed once, and the well was again filled with complete cell culture medium. Medium was replenished every other day. Murine fibroblasts were cultured for 10–14 days and passaged once before using them in the experiments. Human fibroblasts were obtained from commercial sources (all from Stemcell; meningeal Cat. 1400, dural Cat. 1420, pulmonary Cat. 3300, mammary Cat. 7630) and maintained in meningeal or fibroblast medium (Stemcell, Cat. 1401 and 2301) as recommended.
Flow cytometry and sorting.
Cells were stained using primary fluorochrome-conjugated antibodies at 4°C for 20 min. Following antibodies were used in this study: PE/Dazzle 594 anti-mouse CD31 antibody (Biolegend, Cat. 102429), BUV395 Rat anti-mouse CD45 antibody (BD Biosciences, Cat. 564279), FITC anti-mouse CD45 antibody (Biolegend, Cat. 103108), FITC anti-mouse CD31 antibody (Biolegend, Cat. 102406), FITC anti-mouse Ter119 antibody (Biolegend, Cat. 116206), PE/Dazzle 594 anti-mouse CD54/Icam1 antibody (Biolegend, Cat.116129). Dead cells were excluded using Live/Dead fixable dead cell stain kits, used as recommended (Invitrogen, Green - Cat. L34969 and Far Red - Cat. L34973). For sorting, viable single cells were sorted using Aria SORP (BD) through 150 μm nozzle directly into RNA lysis buffer (Qiagen, RLT) or complete cell culture medium. Cytometric data were processed as described previously 31.
Bulk transcriptomics and single-cell transcriptomics data analysis.
RNA from flow-sorted CD31/CD45− cells from freshly isolated, 5 male C57Bl/6 mice-derived leptomeningeal tissue was extracted using RNeasy Micro Kit (Qiagen, #74034). After RiboGreen quantification and quality control by Agilent BioAnalyzer, 2 ng total RNA with RNA integrity numbers ranging from 9.7 to 10 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit (Clonetech catalog # 63488), with 12 cycles of amplification. Subsequently, 10ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) using 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4000 in a 50bp/50bp paired end run, using the HiSeq 3000/4000 SBS Kit (Illumina). An average of 36 million paired reads were generated per sample. Reads from generated FASTQ files were quality checked and mapped to the mouse reference genome (mm10) using STAR2.5.0.a. The expression count matrix of uniquely mapped reads was computed with HTseq v0.5.3. Raw counts were normalized by library size using DESeq2 pipeline in R v3.6.0 running in RStudio v1.0.143 (see Table S1). The single-cell data from the Allen Brain Map were analyzed via UCSC Cell Browser (mouse cortex, v2019/2020, 75k cells, accessed in May 2020; see Tables S2–3) 32,33. Transcriptomic signatures of VLMC clusters were determined with Reactome 34.
Quantitative PCR.
qPCR analysis of genes-of-interest was performed as described previously 25. Briefly, total RNA was isolated from sorted samples with the RNeasy Plus Micro kit and from in vitro cultured cells with RNeasy Plus Micro kit (Qiagen, Cat. 74034 and 74136) as recommended. cDNA was synthesized with High Capacity Reverse transcription kit (Thermo Fisher Scientific #4368814). PCR was performed with TaqMan probes (Thermo Scientific) and Applied Biosystems TaqMan Advanced Master Mix (Applied Biosystems #4444963) on the ViiA 7 system (Applied Biosystems). The experiment was run using three biological replicates and data were processed using the 2−ΔΔCq method. TaqMan probes used in this study include: Cdkn2a (Mm00494449_m1), Icam1 (Mm00516023_m1), Slc38a2 (Mm00628416_m1), Actb (Mm00437762_m1), Ngfr (Mm00446296_m1), Mgp (Mm00485009_m1), Fxyd5 (Mm00435435_m1), Crabp2 (Mm00801691_m1), Aldh1a2 (Mm00501306_m1), Pltp (Mm00448202_m1), Ogn (Mm01349370_m1), Polr2a (Mm00839502_m1).
Histology and immunofluorescence.
Mice were euthanized and mouse skulls were dissected, placed to 10% neutral formalin (Sigma, Cat. HT4501128) for three days, decalcified with 0.5M EDTA (MSKCC Media Core) for one week and then fixed for one more day in 10% formalin before embedding into paraffin. Sections of human meninges and mouse skulls were stained with picrosirius red and Masson’s trichome using routine histology techniques 35. For immunofluorescence staining, slides were de-paraffinized and rehydrated through xylene and series of ethanols. Antigens were retrieved in steamer using 1x Dako Target retrieval solution (Agilent, S1699). Cultures of in vitro fibroblasts were seeded into glass 8-well microscopy chambers (EMD Millipore or Nunc Lab-Tek) coated with 0.1% collagen (MSKCC Media Core) or laminin in case of sorted cells (Corning, Cat. CB-40232) and fixed after 24 hours with 4% formaldehyde (Electron Microscopy, Cat. 15710). Stainings were performed as described previously 24. Antibodies and probes used in this study include: anti-mouse and anti-human ICAM1 (R&D, Cat. AF796 and BBA17), anti-mouse/human SLC38A2 (Bioss, Cat. bs-12125R), DAPI (Thermo Fisher, D1306), Phalloidin AF647 (Thermo Fisher, A22287). Slides were mounted in Prolong Diamond (Thermo Fisher, P36961) and scanned with Mirax slide scanner (Zeiss) or SP5 confocal microscope (Leica). The mean pixel intensity was analyzed with Fiji/ImageJ. Mouse ISH data were retrieved from Allen Brain Atlas 36.
Software and data analysis.
Histological images were accessed through CaseViewer v2.4, 3Dhistech. Plots were generated in Prism v9, GraphPad. Figures were assembled in InDesign 2021, Adobe.
5. Step-by-step protocol of mouse leptomeninges microdissection
-
Prepare the tissue dissociation solution: 200 U/mL Collagenase and 1.5 U/mL Dispase in 1mL total of serum-free MenCM media. Keep on ice until leptomeningeal tissue extraction.
CRITICAL: Leptomeningeal tissue should always be kept on ice to maximize live cell yield. The tissue dissociation solution is optimized for fibroblastoid cell dissociation.
Euthanize mice according to the approved animal protocol.
Sterilize the mouse head with 70% ethanol. Using large scissors, cut through the skin from the back of the head towards the eyes. Detach as much skin and muscles as possible off the skull and down the cervical spine approximately to the C7 level.
Sever mouse head just above the shoulders (C7).
Cut caudally to the coronal suture to remove the nasal bone while preserving underlying olfactory lobes.
-
Using a posterior approach, slide scissors through the oral cavity and sever muscles connecting the lower jaw. Discard the lower jaw with attaching muscles.
CRITICAL: Proceed with this step with caution to prevent puncturing the overlying palate and injuring the brain tissue.
Transfer the skull and attached vertebral column into ice-cold PBS for meningeal extraction.
Place skull so that ventral aspect of the jaw is facing down. Insert surgical scissors through spinal cord canal at C7, caudally, and cut vertebrae rostrally on dorsal surface adjacent to spinous processes to reach the base of the skull.
-
Cut the skull shallowly along the lambdoid, sagittal and coronal sutures with care taken not to puncture the underlying brain parenchyma.
CRITICAL: Consider using blunt tip scissors to prevent any damage to underlying meninges.
Place the skull and attached vertebra in a 100 mm dish with PBS to proceed with dissection under the stereomicroscope.
Place the skull on the side that will be dissected to allow visualization of structures beneath the skull when retracted.
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Using Cushing forceps, take hold of the skull from the sagittal suture and open laterally. Extraction of leptomeninges is accomplished while opening and carefully retracting the skull with slow motion.
CRITICAL: Handle the skull with care to prevent disruption of the underlying meninges. Retract the skull very slowly and carefully while extracting tissue from the cortical surface. Make sure not to puncture the brain parenchyma with the scissors to prevent the contamination of the tissue sample.
6. Troubleshooting
| Problem | Possible reason(s) | Possible solution(s) |
|---|---|---|
| Difficulty in holding and extracting leptomeningeal tissue | - Too much liquid in the dish - Forceps with teeth are being used |
- Drain as much liquid as possible without completely drying the brain - Use flat forceps to avid cutting the delicate leptomeningeal tissue |
| Low yield of cells after digestion | - Incomplete digestion of large tissue pieces - Elevated temperature during dissection - Tissue desiccation - Extensively prolonged tissue extraction - Cells lost during centrifugation or aspiration of supernatant |
- Add additional dissociation solution if large clumps are still seen after one hour of dissociation - Place tissue on ice for the whole duration of extraction - Time needed for the procedure will decrease with acquired expertise - Aspirate the supernatant from the side opposite to the cell pellet (if visible) |
| High percentage of dead cells Presence of debris |
- Wrong dissociation frequency, speed or time was used. Dissociation was too rigorous. - Extraction of myelinated tissue with the leptomeningeal sample |
- Use the settings provided in the protocol for optimal dissociation of the leptomeningeal tissue - Use forceps with narrow tips and extract leptomeningeal while retracting the skull |
Supplementary Material
Movie 1. Microdissection of mouse leptomeninges.
The skull is taken hold from the sagittal suture and open laterally. Extraction of leptomeninges (L) is accomplished while opening and carefully retracting the skull with slow motion. Note the collapsing dura (D) when the skull is widely open.
Supplementary Figure 1. Characterization of in vitro survival of mouse leptomeningeal fibroblasts.
A Plot showing the decrease of leptomeningeal fibroblast viability during long-term in vitro culture.
B Assessment of cell viability after freezing at passages 2 (F1), 3 (F2) and 4 (F3). Cell viability was assessed by trypan blue exclusion.
C Assessment of cellular senescence during in vitro culture of leptomeningeal fibroblasts. Cdkn2a (gene encoding p21) expression levels were determined with qPCR, n = 3.
D Fluorescent microscopy images of unsorted pool of digested leptomeninges of lineage-negative, Icam1+ sorted cells, adhered to a laminin-coated slide. Representative images are from two biological replicates. Scale bar = 20 μm.
Supplementary Figure 2. Marker gene expression and colocalization of Icam1 with known markers of pia and dura in mouse basilar meninges.
Gene expression of Icam1, Slc38a2 and meningeal markers in in vitro cultured mouse fibroblasts, as determined with qPCR. n = 3 for leptomeningeal, dural and mammary fibroblasts and n = 2 for pulmonary fibroblasts.
Highlights.
We provide a method for the reliable isolation and culture of mouse leptomeningeal fibroblasts.
We identify Icam1 and Slc38a2 as novel tissue identity markers in mouse and human leptomeninges.
Icam1 and Slc38a2 may be used to identify leptomeningeal cells in tandem with other known markers through an array of histological and transcriptomic techniques.
Acknowledgements
We are deeply grateful to the patients and families who donated clinical samples for this research to the MSKCC Last Wish Program. We acknowledge the use and help of the Flow Cytometry, Molecular Cytology and Integration Genomics Operation cores at MSKCC; we are in particular grateful to Eric Rosiek for help with image acquisition and analysis and to Ning Fan for help with classical histology staining. MSKCC is supported by the National Cancer Institute grant P30 CA008748. J.R. was supported by the American Brain Tumor Association Basic Research Fellowship, the Terri Brodeur Breast Cancer Foundation Fellowship, and the Druckenmiller Center for Lung Cancer Research. This work was further supported by the Pew Charitable Trusts grant GC241069, Damon Runyon Cancer Research Foundation grant GC240764, Pershing Square Sohn Cancer Research Alliance grant GC239280, the Baker Family Foundation (all to A.B.).
Footnotes
Competing interests
A.B. is an inventor on U.S. provisional patent application 62/258,044, “Modulating Permeability of the Blood Cerebrospinal Fluid Barrier” filed by Memorial Sloan Kettering Cancer Center. A.B. is an unpaid member of the Scientific Advisory Board of EVREN Technologies.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Data availability statement
RNA-seq data were deposited to NCBI GEO under the accession number GSE150419. Source data are available from corresponding author upon reasonable request.
References
- 1.Protasoni M et al. The collagenic architecture of human dura mater. J Neurosurg 114, 1723–1730, doi: 10.3171/2010.12.JNS101732 (2011). [DOI] [PubMed] [Google Scholar]
- 2.Absinta M et al. Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. Elife 6, doi: 10.7554/eLife.29738 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Louveau A et al. Structural and functional features of central nervous system lymphatic vessels. Nature 523, 337–341, doi: 10.1038/nature14432 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Zwirner J, Scholze M, Waddell JN, Ondruschka B & Hammer N Mechanical Properties of Human Dura Mater in Tension - An Analysis at an Age Range of 2 to 94 Years. Sci Rep 9, 16655, doi: 10.1038/s41598-019-52836-9 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Kinaci A, Bergmann W, Bleys RL, van der Zwan A & van Doormaal TP Histologic Comparison of the Dura Mater among Species. Comp Med 70, 170–175, doi: 10.30802/AALAS-CM-19-000022 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Balin BJ, Broadwell RD, Salcman M & el-Kalliny M Avenues for entry of peripherally administered protein to the central nervous system in mouse, rat, and squirrel monkey. J Comp Neurol 251, 260–280, doi: 10.1002/cne.902510209 (1986). [DOI] [PubMed] [Google Scholar]
- 7.Yasuda K et al. Drug transporters on arachnoid barrier cells contribute to the blood-cerebrospinal fluid barrier. Drug Metab Dispos 41, 923–931, doi: 10.1124/dmd.112.050344 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Tohma Y, Yamashima T & Yamashita J Immunohistochemical localization of cell adhesion molecule epithelial cadherin in human arachnoid villi and meningiomas. Cancer Res 52, 1981–1987 (1992). [PubMed] [Google Scholar]
- 9.Zhang J, Smith D, Yamamoto M, Ma L & McCaffery P The meninges is a source of retinoic acid for the late-developing hindbrain. J Neurosci 23, 7610–7620 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Cha JH et al. Prompt meningeal reconstruction mediated by oxygen-sensitive AKAP12 scaffolding protein after central nervous system injury. Nat Commun 5, 4952, doi: 10.1038/ncomms5952 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Adeeb N et al. The pia mater: a comprehensive review of literature. Childs Nerv Syst 29, 1803–1810, doi: 10.1007/s00381-013-2044-5 (2013). [DOI] [PubMed] [Google Scholar]
- 12.Zhang ET, Inman CB & Weller RO Interrelationships of the pia mater and the perivascular (Virchow-Robin) spaces in the human cerebrum. J Anat 170, 111–123 (1990). [PMC free article] [PubMed] [Google Scholar]
- 13.Saboori P & Sadegh A Histology and Morphology of the Brain Subarachnoid Trabeculae. Anat Res Int 2015, 279814, doi: 10.1155/2015/279814 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.McLone DG & Bondareff W Developmental morphology of the subarachnoid space and contiguous structures in the mouse. Am J Anat 142, 273–293, doi: 10.1002/aja.1001420302 (1975). [DOI] [PubMed] [Google Scholar]
- 15.S H in Inflammatory Diseases of the Meninges (ed Rohde S) 169–183 (Springer, 2012). [Google Scholar]
- 16.Choe Y & Pleasure SJ Meningeal Bmps Regulate Cortical Layer Formation. Brain Plast 4, 169–183, doi: 10.3233/BPL-170048 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Zarbalis K et al. Cortical dysplasia and skull defects in mice with a Foxc1 allele reveal the role of meningeal differentiation in regulating cortical development. Proc Natl Acad Sci U S A 104, 14002–14007, doi: 10.1073/pnas.0702618104 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Schain AJ et al. Activation of pial and dural macrophages and dendritic cells by cortical spreading depression. Ann Neurol 83, 508–521, doi: 10.1002/ana.25169 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Russi AE & Brown MA The meninges: new therapeutic targets for multiple sclerosis. Transl Res 165, 255–269, doi: 10.1016/j.trsl.2014.08.005 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Benakis C et al. Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδ T cells. Nat Med 22, 516–523, doi: 10.1038/nm.4068 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Enzmann G et al. The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury. Acta Neuropathol 125, 395–412, doi: 10.1007/s00401-012-1076-3 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Remsik J et al. Inflammatory Leptomeningeal Cytokines Mediate COVID-19 Neurologic Symptoms in Cancer Patients. Cancer Cell 39, 276–283.e273, doi: 10.1016/j.ccell.2021.01.007 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.DeSisto J et al. Single-Cell Transcriptomic Analyses of the Developing Meninges Reveal Meningeal Fibroblast Diversity and Function. Dev Cell 54, 43–59.e44, doi: 10.1016/j.devcel.2020.06.009 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Chi Y et al. Cancer cells deploy lipocalin-2 to collect limiting iron in leptomeningeal metastasis. Science 369, 276–282, doi: 10.1126/science.aaz2193 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Remsik J et al. Leptomeningeal metastatic cells adopt two phenotypic states. Cancer Rep (Hoboken), e1236, doi: 10.1002/cnr2.1236 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Louveau A, Filiano AJ & Kipnis J Meningeal whole mount preparation and characterization of neural cells by flow cytometry. Curr Protoc Immunol 121, e50, doi: 10.1002/cpim.50 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Bowyer JF et al. A visual description of the dissection of the cerebral surface vasculature and associated meninges and the choroid plexus from rat brain. J Vis Exp, e4285, doi: 10.3791/4285 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Saunders NR, Habgood MD, Møllgård K & Dziegielewska KM The biological significance of brain barrier mechanisms: help or hindrance in drug delivery to the central nervous system? F1000Res 5, doi: 10.12688/f1000research.7378.1 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Coles JA, Stewart-Hutchinson PJ, Myburgh E & Brewer JM The mouse cortical meninges are the site of immune responses to many different pathogens, and are accessible to intravital imaging. Methods 127, 53–61, doi: 10.1016/j.ymeth.2017.03.020 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Melzer M, Beier D, Young PP & Saraswati S Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts. J Vis Exp, doi: 10.3791/60909 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Remsik J et al. Plasticity and intratumoural heterogeneity of cell surface antigen expression in breast cancer. Br J Cancer, doi: 10.1038/bjc.2017.497 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Speir ML et al. UCSC Cell Browser: Visualize Your Single-Cell Data. Bioinformatics, doi: 10.1093/bioinformatics/btab503 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Yao Z et al. A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation. Cell 184, 3222–3241.e3226, doi: 10.1016/j.cell.2021.04.021 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Jassal B et al. The reactome pathway knowledgebase. Nucleic Acids Res 48, D498–D503, doi: 10.1093/nar/gkz1031 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Remšík J et al. TGF-β regulates Sca-1 expression and plasticity of pre-neoplastic mammary epithelial stem cells. Sci Rep 10, 11396, doi: 10.1038/s41598-020-67827-4 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Lein ES et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176, doi: 10.1038/nature05453 (2007). [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Movie 1. Microdissection of mouse leptomeninges.
The skull is taken hold from the sagittal suture and open laterally. Extraction of leptomeninges (L) is accomplished while opening and carefully retracting the skull with slow motion. Note the collapsing dura (D) when the skull is widely open.
Supplementary Figure 1. Characterization of in vitro survival of mouse leptomeningeal fibroblasts.
A Plot showing the decrease of leptomeningeal fibroblast viability during long-term in vitro culture.
B Assessment of cell viability after freezing at passages 2 (F1), 3 (F2) and 4 (F3). Cell viability was assessed by trypan blue exclusion.
C Assessment of cellular senescence during in vitro culture of leptomeningeal fibroblasts. Cdkn2a (gene encoding p21) expression levels were determined with qPCR, n = 3.
D Fluorescent microscopy images of unsorted pool of digested leptomeninges of lineage-negative, Icam1+ sorted cells, adhered to a laminin-coated slide. Representative images are from two biological replicates. Scale bar = 20 μm.
Supplementary Figure 2. Marker gene expression and colocalization of Icam1 with known markers of pia and dura in mouse basilar meninges.
Gene expression of Icam1, Slc38a2 and meningeal markers in in vitro cultured mouse fibroblasts, as determined with qPCR. n = 3 for leptomeningeal, dural and mammary fibroblasts and n = 2 for pulmonary fibroblasts.
Data Availability Statement
RNA-seq data were deposited to NCBI GEO under the accession number GSE150419. Source data are available from corresponding author upon reasonable request.