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

Abstract

Summary The studies 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 the 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.

Endothelial lipid phosphate phosphatase-3 deficiency that disrupts the endothelial barrier function is a modifier of cardiovascular development

“…Next, we isolated ECs from E12.5 WT- and MT -embryos as described in the methods; the procedure was as reported in previous studies. 30 – 32 Endothelial identity was confirmed using flow cytometry analysis with control IgG, anti-CD31, and anti-Flk1 antibodies ( Figure 5 E ). Next, using these isolated ECs, we performed a qRT-PCR assay to identify the target genes involved in Lpp3-regulated angiogenesis.…”

“…ECs were isolated from E13.5-day-old embryos using anti-Flk1 and anti-CD31 + beads (please see methods) and as previously described. 30 – 32 . ( E ) Purity of isolated mouse ECs: representative FACS profiles of mouse ECs with control IgG, anti-CD31, and anti-Flk1 antibodies.…”

“…Fluorescence-activated cell sorting (FACS) and isolation of ECs from mouse embryonic stem cells and induced pluripotent stem cells have been previously described by us. 30 – 32 Briefly, for isolation of mouse ECs, we used anti-mouse-Flk1 (CD309) and anti-mouse-CD31 microbeads (Miltenyi Biotec GmbH, San Diego, CA, USA). Mouse embryos at developmental stage at E11.5 were collected, washed three times with cold 1× PBS, pH 7.4; heart, lungs, brains, and livers were enzymatically dissociated in DMEM containing collagenase/dispase/0.1% trypsin/1.0 mM EDTA, for 30 min at 37°C in a CO 2 incubator.…”

“…These cells were immediately used for flow cytometry, biochemical experiments, and gene expression analyses as previously described. 30 – 32 …”

“… 33 , 34 Embryos harvested by caesarean section from E11.5, E12.5, and E13.5 were fixed in freshly prepared 2% p -formaldehyde in PBS for 4 h at 4°C. 32 , 33 Whole mount immunohistochemistry protocol has been described by Courtney Griffin lab. 35 Fixed embryos were treated with PBS containing 0.15% H 2 O 2 , 0.1% NaN 3 for 1 h at 4°C, thereafter incubated in blocking solution (PBS-MBT, 2% carnation dry milk, 0.2% BSA, 0.6% goat serum, and 0.1% Triton X-100 in PBS, pH 7.4) at 4°C for 1 h. Next, the samples were incubated in biotinylated rat anti-mouse CD31 antibody (1:50 in PBS-MBT) for 48 h, followed by wash in PBS-MT (PBS, pH 7.4 containing 2% carnation dry milk and 0.1% Triton X-100) for five times, 1 h each at room temperature.…”