Therapeutic angiogenesis by transplantation of induced pluripotent stem cell-derived Flk-1 positive cells

Abstract

Background: Induced pluripotent stem (iPS) cells are the novel stem cell population induced from somatic cells. It is anticipated that iPS will be used in the expanding field of regenerative medicine. Here, we investigated whether implantation of fetal liver kinase-1 positive (Flk-1+) cells derived from iPS cells could improve angiogenesis in a mouse hind limb model of ischemia.

Results: Flk-1+ cells were induced from iPS cells after four to five days of culture. Hind limb ischemia was surgically induced and sorted Flk-1+ cells were directly injected into ischemic hind limbs of athymic nude mice. Revascularization of the ischemic hind limb was accelerated in mice that were transplanted with Flk-1+ cells compared with control mice, which were transplanted with vehicle, as evaluated by laser Doppler blood flowmetry. Transplantation of Flk-1+ cells also increased expression of VEGF mRNA in ischemic tissue compared to controls.

Conclusions: Direct local implantation of iPS cell-derived Flk-1+ cells would salvage tissues from ischemia. These data indicate that iPS cells could be valuable in the therapeutic induction of angiogenesis.

Figure 1

Purification of Flk-1+ cells from iPS cells. A) Flk-1 expression profiles from 3.5 days to 7.5 days of cultivation as determined by flow cytometric analysis. (* p < 0.05 Day4.5 vs Day3.5, 5.5, 6.5 and 7.5). B) FACS analysis of pre and post MACS-sorted Flk-1⁺ cells at day 4.5. More than 99% of enriched cells were positive for Flk-1. Some of these purified Flk-1⁺ cells were positive for Nanog-GFP.

Figure 2

Characterization of Flk1+ cells. Differentiated iPS cells were analyzed by FACS. Differentiated iPS cells were stained with Flk1-APC and additional cell marker (c-kit, Sca-1, CD11b, CD31, VECAD, CD34, CD44, CD45, CD90, SSEA-1, CXCR4 and ECAD). Additional cell marker was analyzed after gating Flk1⁺ cells. Red line shows the result of the negative control and Blue line shows the result of the sample stained by each antibody.

Figure 3

Time course RT-CPR in vitro. The expression of Flk-1 was peaked at Day5 from differentiation. Undifferentiated iPS cells markers, Nanog and Oct3/4, were strongly expressed in early phase and started to gradually decrease with differentiation. We also observed the transient expression of c-myc at Day4 and Day5.

Figure 4

Effects of cell transplantation on blood flow recovery in the ischemic hind limb. A) Strategy to purify Flk-1⁺ cells and to transplant them into ischemic hind limb tissues. B) Representative LDBF images. A low perfusion signal (dark blue) was observed in the ischemic left hind limb of control mice, whereas high perfusion signals (white to red) were detected in Flk-1⁺ cell-transplanted animals (6 × 10⁴ cells) on postoperative days 3, 7 and 14. C) Quantitative analysis of the ischemic/nonischemic limb LDBF ratio on pre (Day-1) and post operative days 0, 3, 7 and 14 (n = 4). *p < 0.05 Flk1⁺ cells (6×10⁴) injected mice vs. control mice. D) Cell dose-dependent effect of transplantation seven days after surgery. Flk-1⁺ cells (2 × 10³, 2 × 10⁴ or 6 × 10⁴ cells) or PBS as a control were injected into the ischemic limb at postoperative day 1 (n = 4/each group, **p < 0.05 2×10⁴ or 6×10⁴ vs. control mice).

Figure 5

Flk-1+ cell implantation stimulated VEGF, basic FGF, HGF and IGF expression in ischemic tissue. VEGF, basic FGF, HGF and IGF synthesis in ischemic hind limb muscles was determined by real-time RT-PCR following transplantation of the Flk-1⁺ cells (2 × 10³, 2 × 10⁴ or 6 × 10⁴ cells) or PBS-injection as the control(CNT). Results are expressed as the level of VEGF, basic FGF, HGF and IGF mRNA to day three control. GAPDH mRNA levels were used as the internal control. *P < 0.05 6×10⁴ vs. CNT at each day (n = 4 in each groups). N.S. = not significant difference between groups at same day.

Figure 6

Tracking Flk-1⁺ cells in vitro and at chronic phase in vivo. A) Tube formation assay in vitro. PKH26 red-labeled Flk-1⁺ iPS cells were co-cultured with HUVECs for 24 hours on Matrigel. iPS cells (red) were confirmed to be incorporated into network structures. The bar indicates 200 μm. B) Double fluorescence staining of CD31 (green) and PKH26 (red) in ischemic adductor muscles on postoperative day 21. Co-localization is indicated by yellow in the merged images (magnification, ×400; bar indicates 50 μm). Flk-1⁺ cells (6 × 10⁴ cells) were stained with PKH26 red and then injected into ischemic adductor muscles. Double positive cells (▲) and single positive PKH26 cells(↑) are indicated.