Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Generation of Pericytic-Vascular Progenitors from Tankyrase/PARP-Inhibitor-Regulated Naïve (TIRN) Human Pluripotent Stem Cells

doi: 10.1007/978-1-0716-1908-7_10

Figure Lengend Snippet: Generation of embryonic pericytic-endothelial VP from primed hPSC. (a) Primed H9 hESC were differentiated to vascular mesoderm. CD31+ CD146+ CXCR4+ VP were further FACS-purified (red gate), and culture-expanded in EGM-2 medium into populations that contained distinct (b) endothelial CD144+ CD105+ CD34+ (purple-gated) and pericytic CD140b+ CD34− CD105lo (green-gated) populations. Expanded VP were assayed for endothelial function with (c) Ac-Dil-LDL uptake assays, and (d) In vivo Matrigel plug chimeric vascular network formation in NOD/SCID mice. Sections were stained with anti-human CD31 antibodies (brown). (e) Primed cord blood-hiPSC-derived CD31+ CD146+ VP formed assembled vascular tubes in collagen gel. (f) These tubes were imaged with transmission electron microscopy, which revealed cooperating endothelial-pericytic-like cells with bifurcations. L: lumen, n: nuclei

Article Snippet: We use Cy3-conjugated anti-HNA antibody from Millipore (MAB1281C3), rabbit anti-mouse Collagen IV from Millipore (AB756P), rat anti-mouse CD31 from Biosciences (550274), mouse anti-human CD34 from BD Biosciences (clone My10, #347660), mouse anti-human CD31 from Dako (M0823), rabbit anti-mouse collagen type IV from Millipore (AB756P; 1:100), and rat anti-mouse CD31 from BD Biosciences. (550274; 1:50).

Techniques: Purification, In Vivo, Staining, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Generation of Pericytic-Vascular Progenitors from Tankyrase/PARP-Inhibitor-Regulated Naïve (TIRN) Human Pluripotent Stem Cells

doi: 10.1007/978-1-0716-1908-7_10

Figure Lengend Snippet: (a) Waddington landscape model for TIRN reversion (red) of primed hiPSC (blue), adapted from [2]. Epigenetic obstacles to VP differentiation may be overcome with molecular reversion to a TIRN epiblast-like state (red) with a developmentally more primitive epigenetic configuration. (b) Schema of paradigm for generating cGMP-grade, HLA-defined TIRN-hiPSC (from reprogrammed CD34+ hematopoietic progenitors) with improved multi-lineage vascular, neural, and retinal pigmented epithelial (RPE) differentiation for comprehensive regeneration of ischemic neural retina

Article Snippet: We use Cy3-conjugated anti-HNA antibody from Millipore (MAB1281C3), rabbit anti-mouse Collagen IV from Millipore (AB756P), rat anti-mouse CD31 from Biosciences (550274), mouse anti-human CD34 from BD Biosciences (clone My10, #347660), mouse anti-human CD31 from Dako (M0823), rabbit anti-mouse collagen type IV from Millipore (AB756P; 1:100), and rat anti-mouse CD31 from BD Biosciences. (550274; 1:50).

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Generation of Pericytic-Vascular Progenitors from Tankyrase/PARP-Inhibitor-Regulated Naïve (TIRN) Human Pluripotent Stem Cells

doi: 10.1007/978-1-0716-1908-7_10

Figure Lengend Snippet: Method of human CD31+ engraftment quantitation in the vasculature of the neural layers (ILM, GCL, IPL, INL, OPL, ONL) of ischemia-injured mouse retinae. Shown is a representative example of ROI defining the neural retinal layers evaluated for human chimeric vascularization with murine blood vessels. A method of regional separation of neural retinal layers was employed for quantification of human CD31+ (hCD31) or alternatively CD34+ cells via automated counts (Analyze particles function) using the Fiji distribution of ImageJ software

Article Snippet: We use Cy3-conjugated anti-HNA antibody from Millipore (MAB1281C3), rabbit anti-mouse Collagen IV from Millipore (AB756P), rat anti-mouse CD31 from Biosciences (550274), mouse anti-human CD34 from BD Biosciences (clone My10, #347660), mouse anti-human CD31 from Dako (M0823), rabbit anti-mouse collagen type IV from Millipore (AB756P; 1:100), and rat anti-mouse CD31 from BD Biosciences. (550274; 1:50).

Techniques: Quantitation Assay, Software

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Generation of Pericytic-Vascular Progenitors from Tankyrase/PARP-Inhibitor-Regulated Naïve (TIRN) Human Pluripotent Stem Cells

doi: 10.1007/978-1-0716-1908-7_10

Figure Lengend Snippet: Generation of embryonic pericytic-endothelial VP from primed hPSC. (a) Primed H9 hESC were differentiated to vascular mesoderm. CD31+ CD146+ CXCR4+ VP were further FACS-purified (red gate), and culture-expanded in EGM-2 medium into populations that contained distinct (b) endothelial CD144+ CD105+ CD34+ (purple-gated) and pericytic CD140b+ CD34− CD105lo (green-gated) populations. Expanded VP were assayed for endothelial function with (c) Ac-Dil-LDL uptake assays, and (d) In vivo Matrigel plug chimeric vascular network formation in NOD/SCID mice. Sections were stained with anti-human CD31 antibodies (brown). (e) Primed cord blood-hiPSC-derived CD31+ CD146+ VP formed assembled vascular tubes in collagen gel. (f) These tubes were imaged with transmission electron microscopy, which revealed cooperating endothelial-pericytic-like cells with bifurcations. L: lumen, n: nuclei

Article Snippet: We use Cy3-conjugated anti-HNA antibody from Millipore (MAB1281C3), rabbit anti-mouse Collagen IV from Millipore (AB756P), rat anti-mouse CD31 from Biosciences (550274), mouse anti-human CD34 from BD Biosciences (clone My10, #347660), mouse anti-human CD31 from Dako (M0823), rabbit anti-mouse collagen type IV from Millipore (AB756P; 1:100), and rat anti-mouse CD31 from BD Biosciences. (550274; 1:50).

Techniques: Purification, In Vivo, Staining, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Generation of Pericytic-Vascular Progenitors from Tankyrase/PARP-Inhibitor-Regulated Naïve (TIRN) Human Pluripotent Stem Cells

doi: 10.1007/978-1-0716-1908-7_10

Figure Lengend Snippet: Stepwise transition of isogenic primed hPSC to Tankyrase/PARP inhibitor-regulated naïve (TIRN)-hPSC culture conditions, and subsequent vascular directed differentiation protocol to N-VP. (a) Schematic of stepwise protocol for LIF-3i TIRN reversion of primed hPSC. (b) Schematic of APEL vascular progenitor (VP) differentiation system. AA: Activin A; B: BMP4; CHIR: CHIR99021; V: VEGF. Differentiated CD31-expressing N-VP are enriched with magnetic-activated cell sorting (MACS)

Article Snippet: We use Cy3-conjugated anti-HNA antibody from Millipore (MAB1281C3), rabbit anti-mouse Collagen IV from Millipore (AB756P), rat anti-mouse CD31 from Biosciences (550274), mouse anti-human CD34 from BD Biosciences (clone My10, #347660), mouse anti-human CD31 from Dako (M0823), rabbit anti-mouse collagen type IV from Millipore (AB756P; 1:100), and rat anti-mouse CD31 from BD Biosciences. (550274; 1:50).

Techniques: Expressing, FACS

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Generation of Pericytic-Vascular Progenitors from Tankyrase/PARP-Inhibitor-Regulated Naïve (TIRN) Human Pluripotent Stem Cells

doi: 10.1007/978-1-0716-1908-7_10

Figure Lengend Snippet: In vitro differentiation of N-VP from TIRN-hPSC and in vivo engraftment into a humanized murine model of ischemic retinopathy. (a) Representative images of primed VP versus N-VP from a fibroblast-hiPSC after 1 passage (1 week) in EGM-2 medium post-CD31+ MACS purification, demonstrating higher human CD31 (hCD31) and Ki-67 proliferation antigen expression. (b) Humanized murine model of ischemic retinal disease for quantifying N-VP engraftment (schematic adapted from [2]). Left panel, I/R location at anterior chamber and site of human cell injections into the vitreous body. Right panel, timeline of in vivo engraftment analysis. Immunodeficient NOG mice with retinal ischemia-reperfusion (I/R) injury can be leveraged for testing human N-VP therapies by injection directly into the vitreous body. Intraocular pressure (IOP) is elevated to 120 mm Hg for 90 min in the anterior chamber, followed by vascular reperfusion, which results in retinal vasculature loss, and apoptotic death of retinal neurons ~7 days following I/R injury. Although this model does not exactly simulate the sequence of events of diabetic retinopathy, it produces the same pathology seen in all retinopathies: acellular capillaries and ischemic neuronal degeneration. (c) Representative stitched image of a whole-mount retina demonstrating survival of HNA+ N-VP cells in the superficial vascular layers of the I/R damaged retina at 7 days following intra-vitreal injection

Article Snippet: We use Cy3-conjugated anti-HNA antibody from Millipore (MAB1281C3), rabbit anti-mouse Collagen IV from Millipore (AB756P), rat anti-mouse CD31 from Biosciences (550274), mouse anti-human CD34 from BD Biosciences (clone My10, #347660), mouse anti-human CD31 from Dako (M0823), rabbit anti-mouse collagen type IV from Millipore (AB756P; 1:100), and rat anti-mouse CD31 from BD Biosciences. (550274; 1:50).

Techniques: In Vitro, In Vivo, Purification, Expressing, Injection, Sequencing

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Generation of Pericytic-Vascular Progenitors from Tankyrase/PARP-Inhibitor-Regulated Naïve (TIRN) Human Pluripotent Stem Cells

doi: 10.1007/978-1-0716-1908-7_10

Figure Lengend Snippet: Method of human CD31+ engraftment quantitation in the vasculature of the neural layers (ILM, GCL, IPL, INL, OPL, ONL) of ischemia-injured mouse retinae. Shown is a representative example of ROI defining the neural retinal layers evaluated for human chimeric vascularization with murine blood vessels. A method of regional separation of neural retinal layers was employed for quantification of human CD31+ (hCD31) or alternatively CD34+ cells via automated counts (Analyze particles function) using the Fiji distribution of ImageJ software

Article Snippet: We use Cy3-conjugated anti-HNA antibody from Millipore (MAB1281C3), rabbit anti-mouse Collagen IV from Millipore (AB756P), rat anti-mouse CD31 from Biosciences (550274), mouse anti-human CD34 from BD Biosciences (clone My10, #347660), mouse anti-human CD31 from Dako (M0823), rabbit anti-mouse collagen type IV from Millipore (AB756P; 1:100), and rat anti-mouse CD31 from BD Biosciences. (550274; 1:50).

Techniques: Quantitation Assay, Software