Effects of injection of GM-IMs to clodronate-liposome treated mice.

<div><p>(A) Schematic outline of clodronate-liposome and GM-IMs treatment. Two-month old female C57BL/6 mice were intravenously injected with clodronate liposomes. After two days, 5 x 10⁶ GM-IMs were injected intravenously.</p> <p>(B) Spleens were taken after 14 days or two months of clodronate treatment. (C) The spleen weight per body weight of mice for day 15 to one month after clodronate-treatment. Data shown are the mean ratios ± SE of seven mice.</p> <p>(D) Frozen sections of spleens two months after clodronate treatment were stained with H and E. Scale Bars: 300 µm.</p> <p>(E) Frozen sections of spleen two months after injection of clodronate were assessed for F4/80 expression by DAB staining (upper). Colors indicate the strength of DAB staining: orange is highest, yellow is mid-level and blue is lowest (lower). Total DAB optical densities are the mean rations ±SE of three independent experiments.</p> <p>(F) The expression of F4/80 in the spleen on days seven or 14 after injection of clodronate. F4/80 was detected by flow cytometry.</p> <p>(G) GM-IMs from EGFP-C57BL/6 mice were injected intravenously after two days of clodronate injection. GFP-positive tissue cells were analyzed by flow cytometry.</p> <p>(H) Expression of <i>Csf1</i> and <i>Csf2</i> in the spleen. Spleens were taken from C57BL/6 mice after seven days of clodronate injection or clodronate and GM-IMs injection. The expression of <i>Csf1</i> and <i>Csf2</i> was analyzed by real-time RT-PCR. The data were normalized to <i>Gapdh</i>.</p> <p>(I) Expression of <i>Csf1</i> and <i>Csf2</i> in stimulated GM-IMs and PMφ. GM-IMs were stimulated with 20 ng/mL IFNγ or 20 ng/mL IL-4 and 20 ng/mL IL-13 for 24 hr. The expression was analyzed as in (H). Data shown are the mean ratios ± SE of three independent experiments (H, I). P value: *<0.05, **<0.01, ***<0.001.</p></div

Continuous proliferation of GM-IMs was dependent upon GM-CSF.

<div><p>GM-IMs were plated in ten cm non-coated plastic dishes. They were cultured in RPMI-1640 supplemented with 10% FBS with or without 10% GM-CSF-CM.</p> <p>(A) GM-IMs (3x10⁵) were cultured in ten cm non-coated dishes and after six days of culture, the cell number was counted by trypan blue dye exclusion. Data shown are the mean ratios ± SE of four independent experiments. P value: **<0.001.</p> <p>(B) On days one and three of culture, cells were stained with BrdU-FITC and 7-AAD and analyzed by flow cytometry. Data are representative of three independent experiments.</p> <p>(C) Comparison of the average telomere length ratios (ATLR) between BM cells cultured in 10% GM-CSF-CM for the indicated times during which cells became GM-IMs and indicated cells or murine tails. Data shown are the mean ratios ± SE of three independent experiments. P value: *<0.01, **<0.001 in comparison to that of tail from 8 weeks old murine.</p></div

Establishment of GM-IMs from murine BM.

<div><p>(A) BM cells were taken from two-month old C57BL/6 or EGFP mice. Suspensions of BM cells (3x10⁶ cells) were plated in ten cm diameter non-coated plastic culture dishes. They were cultured in RPMI-1640 supplemented with 10% GM-CSF-CM and 10% FBS. Lower panel shows phase contrast photograph of BM cultured with 10% GM-CSF-CM for six days or six months. Scale bars: 50 µm.</p> <p>(B) Growth curve of GM-IMs. BM cells cultured with 10% GM-CSF-CM for the indicated times, during which cells became GM-IMs. Data shown are the mean ratios ± SE of four independent experiments.</p> <p>(C) SA-β-gal staining of BM cells cultured with 10% GM-CSF-CM for the indicated times, during which cells became GM-IMs. Scale bars: 100 µm.</p> <p>(D) Expression of cellular senescent marker proteins. Cell lysates were Western blotted for p16. Β-actin was used as a control.</p> <p>(E) May-Grunwald-Giemsa staining of GM-IMs. Five months cultured GM-IMs was compared with days zero and six of BM culture in 10% GM-CSF-CM, BMMφ and peritoneal macrophages (PMφ). Scale bars: 50 µm.</p> <p>Data are representative of three independent experiments with similar results (A, C-E).</p></div

Functional analysis of GM-IMs.

<div><p>(A) Bacterial killing assay. <i>S. aureus</i> (left) or <i>E. coli</i> (right) were serially diluted in 96-well plates. They were mixed with 5 x 10⁴ PMφ or GM-IMs and cultured overnight in 100 µL RPMI-1640 and 10% FCS and 3% GM-CSF-CM. One µL of mixed culture at appropriate dilution (which showed the clear differences in the number of bacteria) was plated on an agar plate. After 24 hr, the number of colony forming units (CFU) was counted. Data shown are the mean ratios ± SE of three independent experiments. P value: * <0.01, **<0.001.</p> <p>(B) May-Grunwald-Giemsa stain of GM-IMs cultured with <i>E. coli</i> for 24 hr. Left shows GM-IMs without bacteria, and right shows GM-IMs with bacteria. Scale bars: 100 µm. Data are representative of three independent experiments.</p></div

Gene expression profiles of GM-IMs compared to BMDCs.

<div><p>Comparison of gene expression levels between ten months cultured GM-IMs and BMDCs cultured for six days, both in 10% GM-CSF-CM. They were analyzed with a GeneChip mouse genome 430 2.0 microarray.</p> <p>(A) Relative chemokine expression of GM-IMs compared to BMDCs.</p> <p>(B-D) Relative chemokine receptor (B), cytokine (C) and cytokine receptor (D) expression, as in (A).</p></div

Potentials of GM-IMs to differentiate to M1 or M2 macrophages.

<div><p>GM-IMs or PMφ were stimulated with 20 ng/mL IFNγ, 20 ng/mL IL-4 and 20 ng/mL IL-13 or ong µg/mL LPS for 24 hr.</p> <p>(A) Phase contrast photographs of GM-IMs after stimulation. Scale Bar: 100 µm.</p> <p>(B) After stimulation by IFNγ for 24hr, expression of M1 marker genes was analyzed by RT-PCR.</p> <p>(C) After stimulation by LPS, RNA was extracted and expression of M1 marker genes was analyzed by RT-PCR.</p> <p>(D) After stimulation by IL-4 and IL-13, expression of M2 marker genes was analyzed by RT-PCR.</p> <p>(E) GM-IMs or BMMφ (1 x 10⁶) were cultured in six-well plastic plates with RPMI-1640 containing 10% FBS and 3% GM-CSF-CM. They were stimulated with one µg/mL LPS for indicated times. Supernatants were taken and cytokine expression of IL-6, IL-1β or TNFα was analyzed by ELISA. Data shown are the mean ratios ± SE of three separate experiments.</p> <p>(F) The dorsal skin of two-month old female C57BL/6 mice (n=4) was punctured through two layers of skin with a sterile disposable three mm biopsy punch (Kai Industries). GM-IMs (3 x 10⁴) were directly injected into each wound on the right side. PBS was dropped on the other side as a control. The changes in the percentages of wound areas at each time point were compared to the initial wound area. Data shown are the mean ratios ± SE of three independent experiments. P value: * < 0.05, **<0.01, ***<0.001, GM-IMs injected area versus PBS injected area.</p> <p>Data are representative of three independent experiments with similar results (A-D).</p></div

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

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

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