Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Mar 5;12(3):451-460.
doi: 10.1016/j.stemcr.2019.01.005. Epub 2019 Feb 7.

Induction of Mesoderm and Neural Crest-Derived Pericytes From Human Pluripotent Stem Cells to Study Blood-Brain Barrier Interactions

Affiliations
Free PMC article

Induction of Mesoderm and Neural Crest-Derived Pericytes From Human Pluripotent Stem Cells to Study Blood-Brain Barrier Interactions

Tannaz Faal et al. Stem Cell Reports. .
Free PMC article

Abstract

In the CNS, perivascular cells ("pericytes") associate with endothelial cells to mediate the formation of tight junctions essential to the function of the blood-brain barrier (BBB). The BBB protects the CNS by regulating the flow of nutrients and toxins into and out of the brain. BBB dysfunction has been implicated in the progression of Alzheimer's disease (AD), but the role of pericytes in BBB dysfunction in AD is not well understood. In the developing embryo, CNS pericytes originate from two sources: mesoderm and neural crest. In this study, we report two protocols using mesoderm or neural crest intermediates, to generate brain-specific pericyte-like cells from induced pluripotent stem cell (iPSC) lines created from healthy and AD patients. iPSC-derived pericytes display stable expression of pericyte surface markers and brain-specific genes and are functionally capable of increasing vascular tube formation and endothelial barrier properties.

Keywords: Alzheimer's disease; blood-brain barrier; endothelial cells; human pluripotent stem cells; mesoderm; neural crest; pericytes.

Figures

None
Figure 1
Figure 1
Differentiation and Characterization of hPSCs into Mesoderm and NC-Derived Pericytes (A) Schematic diagram of mesoderm (MIM) and NC differentiation protocols. Five days following MIM and NC induction, cells were passaged and maintained in pericyte medium (PM) to produce mesoderm-derived pericytes (mPC) and neural crest-derived pericytes (ncPC). (B and C) Representative flow cytometry analyses for surface expression of mesodermal marker KDR, and NC markers HNK-1 and CD271 in hPSCs after 5 days in MIM (B) or NC media (C) compared with fluorescence minus one (FMO) control stain. (D) qRT-PCR analysis of mesodermal genes TBXT and MIXL1 (left panel) and NC genes PAX3, PAX7, and TFAP2A expression (right panel) in hPSCs after 5 days in MIM (red) or NC media (blue). Gene expression was calculated relative to undifferentiated H9 hPSCs. Undifferentiated AD5 iPSCs showed similar expression as H9 hPSCs (data not shown). Mean ± SD was calculated from triplicate reactions of three to six biological replicates. Statistical significance in was determined using the Student's unpaired t test (∗∗p < 0.05, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001).
Figure 2
Figure 2
Gene Expression Analysis of Pericyte Genes in ncPCs and mPCs (A) Representative flow cytometry analysis of pericyte (PDGFRβ, NG2, CD13, and CD146) and hemato-endothelial (CD34) markers in human brain vascular pericytes (HBVPs) (green, top row), mPC (red, middle row), and ncPC (blue, bottom row). The percentage of differentiated cells positive for each marker is shown for the stained cell (colored histograms) compared with the FMO controls (gray histograms). mPCs and ncPCs shown were derived from AD5 iPSCs and are representative of all hPSC lines. (B) qRT-PCR of pericyte genes PDGFRB, ANGPT1, VTN, FOXC1, and FOXF2 in undifferentiated hPSCs (white), HBVPs (green), mPCs (red), and ncPCs (blue). Gene expression was normalized to RPLP0 and calculated relative to HBVPs. Mean ± SD was calculated from triplicate reactions of three to six biological replicates. (C) Western blot of FOXF2 (top row) and VTN (middle row) protein in undifferentiated iPSCs, HBVPs, mPCs, and ncPCs. GAPDH protein (bottom row) was used as a loading control. See also Figure S1.
Figure 3
Figure 3
iPSC-Derived Pericytes Promote EC Function (A) ECFCs (EC, red) and pericytes (PC, green) co-cultured in 3D tube formation assay. ECFCs were cultured alone or co-cultured with HBVPs, mPCs, and ncPCs and imaged after 1 day (top row) or 4 days (bottom row) in culture. Scale bars, 100 μm. (B) Percent (%) relative tube length of ECs cultured alone (gray) or with HBVPs (green), mPCs (red), or ncPCs (blue) was calculated relative to the tube length at day 1 of ECs cultured alone. Mean ± SD was calculated from three to five biological replicates. mPCs and ncPCs were derived from AD5 iPSCs and are representative of all hPSC-PCs. (C) Representative flow cytometry analysis of endothelial markers CD144 and GLUT1 in iPSC-derived brain microvessel endothelial cells (BMECs). (D) Schematic diagram of transwell system used to co-culture BMECs with pericytes to determine transendothelial electrical resistance (TEER). (E) Representative TEER values of BMECs that were cultured alone (gray) or with HBVPs (green), mPC (red, left graph), or ncPCs (blue, right graph). Mean ± SD was calculated from triplicate transwell reactions. mPCs and ncPCs were derived from AD5 iPSCs. (F) Quantification of peak TEER values at 48 h post co-culture of BMEC monocultures compared with co-culture with pericyte lines derived from multiple iPSC lines (H9, AD5, and AD22) and averaged across multiple experiments. Mean ± SD was calculated from three to six biological replicates in triplicate transwell reactions. Statistical significance in (B) and (F) was determined using the Student's unpaired t test (∗∗p < 0.05, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001). See also Figure S2.
Figure 4
Figure 4
Effect of WNT Inhibition on NC-Derived Pericyte Induction (A) Schematic diagram of NC differentiation protocol supplemented with the WNT activator CHIR99021 (top row) or with the WNT inhibitor DKK1 (bottom row) for 5 days, then passaged and maintained in pericyte medium (PM). (B) qRT-PCR analysis of NC gene PAX7 expression in undifferentiated hPSCs (gray) or hPSCs after 5 days in NC supplemented with CHIR99021 or DKK1. Mean ± SD was calculated from triplicate reactions of two biological replicates. (C) Representative flow cytometry analysis of pericyte marker expression in pericytes derived from NC supplemented with CHIR99021 (blue) or DKK1 (orange) after passage into PM for 3–7 days. The FMO control (gray histogram) was used to define the gate (dashed line) used to calculate the percentage of cells positive for each marker, which is shown. (D) qRT-PCR of pericyte genes VTN, FOXC1, and FOXF2 in cells derived from NC supplemented with CHIR99021 (blue) or DKK1 (orange) after passage into PM. Gene expression was normalized to RPLP0 and calculated relative to undifferentiated hPSCs (gray). Mean ± SD was calculated from triplicate reactions of two biological replicates. See also Figure S3.

Similar articles

See all similar articles

Cited by 7 articles

See all "Cited by" articles

References

    1. Appelt-Menzel A., Cubukova A., Günther K., Edenhofer F., Piontek J., Krause G., Stüber T., Walles H., Neuhaus W., Metzger M. Establishment of a human blood-brain barrier co-culture model mimicking the neurovascular unit using induced pluri- and multipotent stem cells. Stem Cell Reports. 2017;8:894–906. - PMC - PubMed
    1. Armulik A., Genové G., Mäe M., Nisancioglu M.H., Wallgard E., Niaudet C., He L., Norlin J., Lindblom P., Strittmatter K. Pericytes regulate the blood-brain barrier. Nature. 2010;468:557–561. - PubMed
    1. Armulik A., Genové G., Betsholtz C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell. 2011;21:193–215. - PubMed
    1. Cattoretti G., Schiró R., Orazi A., Soligo D., Colombo M.P. Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro. Blood. 1993;81:1726–1738. - PubMed
    1. Daneman R., Zhou L., Kebede A.A., Barres B.A. Pericytes are required for blood brain barrier integrity during embryogenesis. Nature. 2010;468:562–566. - PMC - PubMed

Publication types

MeSH terms

Feedback