3D culture of sorted Flk-1⁺ cells <i>in vitro</i>.

<p>(A) Representative images of tube formation assay <i>in vitro</i> (upper). Sorted Flk-1⁺ cells derived from young and old iPS cells were cultured alone for 24 hours on Matrigel. Quantitative analysis of network projections formed on Matrigel for each experimental group (lower) (n = 3 in each group). (B) Representative images of HUVEC co-cultured with Flk-1⁺ cells (upper). Sorted Flk-1⁺ cells derived from young and old iPS cells were co-cultured with HUVEC for 24 hours on Matrigel. Flk-1⁺ cells derived from young and old iPS cells (white arrow head) were confirmed. The bar indicates 200 µm. Quantitative analysis of the number of Flk-1⁺ cells derived from young and old iPS cells into HUVEC on Matrigel (lower) (n = 3 in each group).</p

Senescence assay <i>in vitro</i>.

<p>(A) Undifferentiated and differentiated iPS cells were stained with a senescence detection kit to detect senescence associated-β-galactosidase (SA-β-Gal) around the nuclear area. (B) Quantitative analysis of the number of SA-β-Gal positive cells in undifferentiated and differentiated iPS cells. Expression of (C) SIRT and senescence associated genes such as (D) ARF and (E) p21 in Flk-1⁺ cells from young and old murine iPS cells determined by real-time PCR. SIRT, ARF and p21 mRNA levels were expressed relative to GAPDH mRNA levels (n = 3 in each group).</p

Effects of cell transplantation on blood flow recovery in the ischemic hindlimb.

<p>(A) Representative LDBF images. A low perfusion signal (dark blue) was observed in the ischemic left hindlimb of control mice (PBS), whereas high perfusion signals (white to red) were detected in the ischemic left hindlimb of mice transplanted with Flk-1⁺ cells derived from young and old mice (2×10⁵ cells) on postoperative days 3, 7 and 14. (B) Quantitative analysis of the ischemic to non-ischemic limb LDBF ratio on pre- (Day-1) and postoperative days 0, 3, 7 and 14 (Control: n = 8, Young: n = 4, Old: n = 4). *p<0.05 for mice injected with Flk1⁺ cells (2×10⁵) vs. control mice. (C) Capillary density analysis. Capillary density was determined at day 21 after surgery. Collected ischemic hindlimb muscle was stained with VE-cadherin. Capillary density was calculated as below. The number of VE-cadherin positive cells per field was divided by the number of muscle fibers per field (n = 5 in each group). (D) VEGF, HGF and IGF synthesis in ischemic tissue determined by real-time PCR at day 7 after surgery following transplantation of Flk-1⁺ cells or PBS. VEGF, HGF or IGF mRNA levels were expressed relative to GAPDH mRNA levels (n = 5 in each group). N.S. = no significant difference between groups.</p

Differentiation into mature vascular cells <i>in vitro</i>.

<p>Sorted Flk-1⁺ cells derived from young and old iPS cells successfully differentiated into (A) mature endothelial cells (VE-cadherin positive) and (B) smooth muscle cells (α-SMA positive) 5 to 7 days after re-culture <i>in vitro</i>. Total nuclei were identified by DAPI counterstaining (blue). (C) Representative images of FACS analysis in differentiated cells (upper). FACS analysis was performed 5 to 7 days after re-plating of sorted Flk-1⁺ cells derived from young and old iPS cells on type IV collagen-coated dishes. Quantitative analysis of α-SMA, VE-cadherin and Ki-67 positive cells in differentiated cells (n = 5 in each group) (lower).</p

Tracking Flk-1⁺ cells during the chronic phase <i>in vivo</i>.

<p>(A) PKH26 labeled Flk-1⁺ cells from young iPS cells (red) and EGFP labeled Flk-1⁺ cells from old iPS cells (green) in ischemic muscle on postoperative day 21. Double fluorescence staining of VE-cadherin and labeled Flk-1⁺ cells in ischemic muscle. Co-localization is indicated by yellow in the merged images (magnification, ×200; bar indicates 200 µm). Total nuclei was identified by DAPI counterstaining (blue). (B) Quantitative analysis of the number of implanted Flk-1⁺ cells from young and old murine iPS cells in the chronic phase (n = 4 in each group).</p