Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells

Abstract

The study of stem cell behavior and differentiation in a developmental context is complex, time-consuming, and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation of embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture.

Keywords: Angiogenesis; CD31; Endothelial cells; Flk1; Klf4; Nanog; Oct4; Sox2; VE-cadherin; iPS cells.

Figure 1. Induction of Flk1⁺VE-cadherin⁺ vascular EC progenies from iPS and ES cells

Timeline of emergence of Flk1+VE-cadherin+ vascular ECs. (A). 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. (Reprinted from Kohler EE et al., PloS One 2013 Dec 30;8(12):e85549).

Figure 2. FACS analysis of emerging vascular EC progenies from mES and iPS cells

Adherent cells (2×10⁵) 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. (Reprinted from Kohler EE et al., PloS One 2013 Dec 30;8(12):e85549).