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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Comparative Angiogenic Activities of Induced Pluripotent Stem Cells Derived from Young and Old Mice

Advanced age is associated with decreased stem cell activity. However, the effect of aging on the differentiation capacity of induced pluripotent stem (iPS) cells into cardiovascular cells has not been fully clarified. We investigated whether iPS cells derived from young and old mice are equally capable of differentiating into vascular progenitor cells, and whether these cells regulate vascular responses in vivo. iPS cells from mouse embryonic fibroblasts (young) or 21 month-old mouse bone marrow (old) were used. Fetal liver kinase-1 positive (Flk-1+) cells, as a vascular progenitor marker, were induced after 3 to 4 days of culture from iPS cells derived from young and old mice. These Flk-1+ cells were sorted and shown to differentiate into VE-cadherin+ endothelial cells and α-SMA+ smooth muscle cells. Tube-like formation was also successfully induced in both young and old murine Flk-1+ cells. Next, hindlimb ischemia was surgically induced, and purified Flk-1+ cells were directly injected into ischemic hindlimbs of nude mice. Revascularization of the ischemic hindlimb was significantly accelerated in mice transplanted with Flk-1+ cells derived from iPS cells from either young or old mice, as compared to control mice as evaluated by laser Doppler blood flowmetry. The degree of revascularization was similar in the two groups of ischemic mice injected with iPS cell-derived Flk-1+ cells from young or old mice. Transplantation of Flk-1+ cells from both young and old murine iPS cells also increased the expression of VEGF, HGF and IGF mRNA in ischemic tissue as compared to controls. iPS cell-derived Flk-1+ cells differentiated into vascular progenitor cells, and regulated angiogenic vascular responses both in vitro and in vivo. These properties of iPS cells derived from old mice are essentially the same as those of iPS cells from young mice, suggesting the functionality of generated iPS cells themselves to be unaffected by aging

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

Mesenchymal Stem Cell-Like Cells Derived from Mouse Induced Pluripotent Stem Cells Ameliorate Diabetic Polyneuropathy in Mice

Background. Although pathological involvements of diabetic polyneuropathy (DPN) have been reported, no dependable treatment of DPN has been achieved. Recent studies have shown that mesenchymal stem cells (MSCs) ameliorate DPN. Here we demonstrate a differentiation of induced pluripotent stem cells (iPSCs) into MSC-like cells and investigate the therapeutic potential of the MSC-like cell transplantation on DPN. Research Design and Methods. For induction into MSC-like cells, GFP-expressing iPSCs were cultured with retinoic acid, followed by adherent culture for 4 months. The MSC-like cells, characterized with flow cytometry and RT-PCR analyses, were transplanted into muscles of streptozotocin-diabetic mice. Three weeks after the transplantation, neurophysiological functions were evaluated. Results. The MSC-like cells expressed MSC markers and angiogenic/neurotrophic factors. The transplanted cells resided in hindlimb muscles and peripheral nerves, and some transplanted cells expressed S100β in the nerves. Impairments of current perception thresholds, nerve conduction velocities, and plantar skin blood flow in the diabetic mice were ameliorated in limbs with the transplanted cells. The capillary number-to-muscle fiber ratios were increased in transplanted hindlimbs of diabetic mice. Conclusions. These results suggest that MSC-like cell transplantation might have therapeutic effects on DPN through secreting angiogenic/neurotrophic factors and differentiation to Schwann cell-like cells

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

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