Flk1+ and VE-Cadherin+ Endothelial Cells Derived from iPSCs Recapitulates Vascular Development during Differentiation and Display Similar Angiogenic Potential as ESC-Derived Cells

<div><p>Rationale</p><p>Induced pluripotent stem (iPS) cells have emerged as a source of potentially unlimited supply of autologous endothelial cells (ECs) for vascularization. However, the regenerative function of these cells relative to adult ECs and ECs derived from embryonic stem (ES) cells is unknown. The objective was to define the differentiation characteristics and vascularization potential of Fetal liver kinase (Flk)1⁺ and Vascular Endothelial (VE)-cadherin⁺ ECs derived identically from mouse (m)ES and miPS cells. </p> <p>Methods and Results</p><p>Naive mES and miPS cells cultured in type IV collagen (IV Col) in defined media for 5 days induced the formation of adherent cell populations, which demonstrated similar expression of Flk1 and VE-cadherin and the emergence of EC progenies. FACS purification resulted in 100% Flk1⁺ VE-cadherin⁺ cells from both mES and miPS cells. Emergence of Flk1⁺VE-cadherin⁺ cells entailed expression of the vascular developmental transcription factor <i>Er71</i>, which bound identically to <i>Flk1, VE-cadherin</i>, and <i>CD31</i> promoters in both populations. Immunostaining with anti-VE-cadherin and anti-CD31 antibodies and microscopy demonstrated the endothelial nature of these cells. Each cell population (unlike mature ECs) organized into well-developed vascular structures <i>in</i><i>vitro</i> and incorporated into CD31⁺ neovessels in matrigel plugs implanted in nude mice <i>in</i><i>vivo</i>.</p> <p>Conclusion</p><p>Thus, iPS cell-derived Flk1⁺VE-cadherin⁺ cells expressing the Er71 are as angiogenic as mES cell-derived cells and incorporate into CD31⁺ neovessels. Their vessel forming capacity highlights the potential of autologous iPS cells-derived EC progeny for therapeutic angiogenesis.</p> </div

miPS derived ECs in the Matrigel plug are not mobilized to subsequent areas of injury.

<p>Mice were subjected to hindlimb ischemia 7 days after injection of Matrigel containing either no cells, mouse ECs or iPS cell-derived ECs. Cells were transduced with mCherry lentivirus particles before mixing with Matrigel. Ischemic tibialis anterior (TA) muscles were harvested 14 days after the surgery (21 days after Matrigel implantation). Cross sections of frozen tissues were observed under laser confocal microscopy. Images acquired with appropriate filter set for mCherry detection show fluorescent signals (20X objective) (A). Quantification demonstrated no appreciable difference in the numbers of fluorescent cells between the groups, suggesting that the obtained signals were likely due to autofluorescence and not due to a mobilization of the Matrigel-engrafted iPS-ECs (B). Fluorescence spectrum images were acquired by excitation with 405 and 562 nm and detection wave length with 470 nm and from 577 to 684 by 9-10 nm steps. The spectral analysis confirmed that the fluorescent signals from the hindlimbs of mice containing Matrigel plugs with mCherry labeled cells and those containing no cells were overlapping. Therefore, the fluorescent signals seen in (A) were indicative of autofluorescence of endogenous cells and did not demonstrate mobilization of iPS-ECs into the ischemic TA muscle (C). Experiments were repeated 3 times.</p

Er71 binds to <i>Flk1-, VE-cadherin-</i>, and CD31-promoters in mES and iPS cells.

<p>Schematics of promoter/enhancer regions of <i>Flk1</i>- (-1.4 kb), VE-cadherin- (-1.0 kb), and CD31- (-1.0 kb) harboring GGAA sites (triangles represent GGAA sites on the forward strand and asterisks represent these in the reverse strand), and the number within parentheses indicates the number of putative Er71 binding sites; TSS, transcription start site (A). Total lysates were subjected to Western blot analysis with indicated antibodies (B). The primers used for the amplification of the induced promoter/enhancer in the mouse genome (C). Flk1⁺VE-cadherin⁺ cells derived from mES- and miPS-cells were cultured overnight. ChIP was performed with antibodies (Abs) specific for Glut-1 (control), Sp1, and Er71. Representative images of PCR products from the mouse Flk1, VE-cadherin, and CD31 promoter/enhancer with the use of input chromatin prepared from indicated ECs. PCR products are as shown (D). The experiments were carried out 3 times.</p

Mouse iPS-derived Flk1⁺VE-cadherin⁺ cells incorporate into CD31-positive vessels.

<p>Time line of the Matrigel experiment (A). Representative images of H&E stained Matrigel plugs obtained from nude mice receiving control mouse ECs or mES- or iPS-derived Flk1⁺VE-cadherin⁺ ECs (2x10⁵) (see Methods for details) (B,C,D). Immunohistochemistry of indicating that Matrigel sections were stained with anti-CD31 (green) and mCherry (red, arrowheads) (E-M). Autofluorescent leukocytes indicated perfused vessels (L). Quantification of number of neovessels in Matrigel sections incorporating the Flk1⁺VE-cadherin⁺ cells expressing mCherry (N). Scale bar 100 µm. Experiments were repeated at least 3 times. </p

Induction of Flk1⁺VE-cadherin⁺ vascular EC progenies from iPS and ES cells.

<p>Timeline of emergence of Flk1⁺VE-cadherin⁺ vascular ECs (<b>A</b>). Undifferentiated mES (J1 line) or miPS (iMZ-21) cells were cultured for 5 d in IV Col-coated dishes in media containing BMP4, bFGF, and VEGF¹⁶⁵ to induce generation of vascular EC progenies. (B&C) Representative phase contrast microscopy of mES cell-derived adherent vascular progenies after d 5 in culture at indicated magnifications (B&C). Representative phase contrast microscopy of miPS cell-derived vascular progenies after day 5 in culture at the indicated magnifications (E&F). FACS profile of the emergence of Flk1⁺VE-cadherin⁺ vascular progenies (D&G). All experiments were repeated >5 times. Data indicate the mean±S.E.M. n=5.</p

FACS analysis of emerging vascular EC progenies from mES and iPS cells.

<p>Adherent cells (2x10⁵) were detached and subjected to two-step FACS-aided purification. Control FACS profile on day 5 of cells derived from mES (J1) cells (A). Representative FACS profiles of day 5, with vascular progenies assessed using anti-Flk1 and anti-VE-cadherin antibodies obtained from mES (J1) cells (B) and derived from miPS (iMZ-21) cells (C); Control FACS profile on day 5 of cells derived from miPS (iMZ-21) cells (D). Representative FACS after the second step of purification derived from mES (E) and iPS cells (F). The yield of Flk1⁺VE-cadherin⁺ after the second round of FACS was 100% for both mES and miPS-derived vascular progenies. Morphology of mES- and miPS-derived vascular ECs (G-J). Flk1⁺VE-cadherin⁺ vascular progenies derived from mES and miPS cells were cultured overnight in IV Col-coated dishes, immunostained with anti-VE-cadherin (green) and anti-CD31 (red) of cells derived from mES cells (G&H) and miPS cells (I&J). DAPI, nucleus (blue). Magnifications are as indicated; the scale bar is 200 µm. Experiments were repeated 3 times.</p

Angiogenic potential of Flk1⁺VE-cadherin⁺ ECs derived from mES and iPS cells.

<p>Quantification of branching point structures; control mouse ECs and mES- or iPS-derived Flk1⁺VE-cadherin⁺ ECs (2x10⁵) were plated onto Matrigel in the presence of VEGF¹⁶⁵ (50 ng/ml). After 18 hr, the numbers of branching points were counted. Data are expressed as percentage of branching points (n=3, *P <0.05 vs. control or as indicated) (A); Representative phase contrast images of branching point structures. The experiments were repeated 3 times with the use of triplicate wells. Scale bar, 200 µm. The arrows indicate branching points (B-G). </p