Journal: bioRxiv

Article Title: A newly-recognized population of residual neural crest cells in the adult leptomeninges is re-activated for vascular repair

doi: 10.1101/2022.12.30.522316

Figure Lengend Snippet: A. In normal adult male C57BL/6J mice, CD271+ cells (Subset 5) were present in the meninges, cranial sutures, and skull bone marrow, but not in the cerebral cortex. B-C. CD271 cells isolated from leptomeninges-enriched tissue highly expressed Tfap2, Foxd3, Sox10 compared to cells from the cortex. D, E. Not just the mouse meninges but also human leptomeninges contain CD271+ NSCSs. From primary human. D. A suspension of dissociated primary human leptomeninges were passed through magnetic beads conjugated with a CD271 antibody. The CD271+ cells were cultured for 4 days in DMEM/F12 plus 1% N2, 2% B27, FGF2 (100 ng/ml), and IGF1 (100 ng/ml). E. Immunocytochemistry confirmed that the purified human leptomeningeal CD271+ cells expressed the neural crest markers HNK-1, Sox10, and CXCR4. F. Immunostaining of the infarcted mouse brain showed that CD271+ cells were increased in the ipsilateral cortex at 3 days after transient (60 min) focal MCAO (tMCAO) or permanent distal MCAO (dMCAO). G. At day 3, after tMCAO, genes consistent with a neural crest identity and perivascular function, such as Ngfr, Tfap2, Foxd3, Prrx2 , and Twist1 , were upregulated in CD271+ cells while Sox10 (a gene involved in neural crest’s melanocyte-yielding role) was decreased (Uninjured control mice, n=3; tMCAO mice, n=4). H. AAV-CAG-tdTomato (5×10 9 viral genomes) was applied onto the ipsilateral leptomeninges via a cranial window in adult male C57BL/6J mice and two-photon imaging was conducted before and 3 days after tMCAO. i. Flow cytometry analysis confirmed that CD271+ cells (the label most specific for NC) were labeled with tdTomato. I-K. Some tdTomato+ cells migrated down to the ipsilateral cortex juxtaposed with brain penetrating vessels 3 days post-tMCAO. L. Immunostaining confirmed accumulation of CD271+ cells co-expressing tdTomato. M. CD271+ cells were isolated by MACS from the injured cortex after tMCAO. N. Leptomeninges-enriched fractions were extracted 3 days post-stroke. From this fraction, CD271+ cells were isolated by MACS. These CD271+ cells were capable of forming floating spheres that highly expressed vimentin and Foxd3. O. The multipotency of the CD271+ cells was confirmed by their ability to yield entirely different neural crest-derived lineages under different culture conditions. The addition of nerve growth factor (NGF; 50 ng/mL) and brain-derived neurotrophic factor (BDNF; 50 ng/mL) on 7 consecutive days (in the absence of serum) induced the emergence of peripheral or autonomic neurons (as detected by NeuroTrace-fluorescent dye obtained by Thermo Fisher Scientific) whereas TGF-β (5 ng/mL) and FBS (5%) in DMEM/F12 media for 7 days produced smooth muscle (as identified by the expression of smooth muscle actin [SMA]). P. Exogenous CD271+ cells were labeled ex vivo with the red fluorescent tracker PKH26 and transplanted onto the cortical surface at day 1 post-stroke. Q. At 3 days post-stroke, PKH26-labeled cells were in close proximity to blood vessels (recognized by their CD31 immunopositivity) in cortical layers II/III and IV. All data are shown as mean ± SD.

Article Snippet: Relative levels of genes were determined by amplifying FOXD3 (Applied Biosystems, Mm02384867_s1), TFAP2 (Applied Biosystems, Mm00493468_m1), SOX10 (Applied Biosystems, Mm00569909_m1), NGFR (Applied Biosystems, Mm00446296_m1), PRRX2 (Applied Biosystems, Mm00436428_m1), TWIST1 (Applied Biosystems, Mm00442036_m1) and normalized by housekeeping gene HPRT (Applied Biosystems, Mm01545399_m1).

Techniques: Isolation, Suspension, Magnetic Beads, Cell Culture, Immunocytochemistry, Purification, Immunostaining, Control, Imaging, Flow Cytometry, Labeling, Expressing, Derivative Assay, Produced, Ex Vivo

Journal: Stem Cells and Development

Article Title: Subfractionation of Differentiating Human Embryonic Stem Cell Populations Allows the Isolation of a Mesodermal Population Enriched for Intermediate Mesoderm and Putative Renal Progenitors

doi: 10.1089/scd.2010.0017

Figure Lengend Snippet: List of Predesigned Taqman ® Probes for QPCR (Applied Biosystems)

Article Snippet: For validation of microarrays, gene expression levels normalized to the housekeeping gene 18S rRNA were calibrated against a large stock of cDNA of an in-house undifferentiated human ES cell line [ 39 ]. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Catalog Number Gene Symbol Description Hs00742896_s1 POU5F1 Homo sapiens POU domain, class 5, transcription factor 1 transcript variant 1, mRNA Hs00220998_m1 GDF3 Homo sapiens growth differentiation factor 3, mRNA Hs00232144_m1 LHX1 Homo sapiens LIM homeobox 1, mRNA Hs00240858_m1 PAX2 Homo sapiens paired box 2, mRNA Hs00240913_m1 WT1 Homo sapiens Wilms' tumor 1, mRNA Hs00377071_m1 OSR1 Homo sapiens odd skipped related 1 ( Drosophila ), mRNA Hs00244943_m1 MEOX1 Homo sapiens mesenchyme homeobox 1, mRNA Hs00361186_m1 TWIST1 Homo sapiens twist homolog 1, mRNA Hs00156373_m1 CD34 Homo sapiens CD34 molecule, mRNA Hs00171403_m1 GATA4 Homo sapiens GATA-binding protein 4, mRNA Hs00172991_m1 IKZF1 Homo sapiens IKAROS family zinc finger 1 (Ikaros), mRNA Hs00230962_m1 FOXF1 Homo sapiens forkhead box F1, mRNA Hs00174344_m1 CDH5 Homo sapiens cadherin 5, type 2, (vascular endothelium), mRNA Hs00246256_m1 FST Homo sapiens follistatin, mRNA Hs00365539_m1 TBX6 Homo sapiens T-box 6, mRNA Hs00231821_m1 TCF15 Homo sapiens transcription factor 15 (basic helix-loop-helix), mRNA Open in a separate window List of Predesigned Taqman ® Probes for QPCR (Applied Biosystems)

Techniques: Variant Assay, Wilms Tumor Assay