Fluorescence-conjugated monoclonal antibodies used for FACS analysis

<p>Fluorescence-conjugated monoclonal antibodies used for FACS analysis</p

Primers for reverse transcription-polymerase chain reaction

1<p>Ref. 21.</p>2<p>We used a single pair of PCR primers that cover the sequence specific to SM2, because these two isoforms are produced from a single gene by alternative splicing.</p>3<p>Ref. 22.</p>4<p>Ref. 23.</p

Characterization of transplanted human VPC-derived vascular cells.

<p>a) Flow cytometric analysis of cell surface markers on expanded human VPC-derived VEGF-R2⁺VE-cadherin⁺ cells ( = EC). b) Immunofluorescence image of CD31 (green) and αSMA (red) with nuclear staining (blue) in expanded EC. Scale bar: 100 µm. c) Immunostaining of mural cell markers (brown) with hematoxyline counter-staining of expanded VPC-derived VEGF-R2⁺VE⁻cadherin- cells ( = MC). Scale bar: 100 µm. d, e) RT-PCR analysis of mural cell (d) and skeletal/cardiac specific (e) markers in human VPC-derived vascular cells.</p

Smooth muscle specific antibodies used for analysis

<p>Smooth muscle specific antibodies used for analysis</p

Augmented vascular regeneration by intra-arterial transplantation of human VPC-derived vascular cells in a murine hindlimb ischemia model.

<p>a) Serial LDPI analysis in hindlimb ischemia mice. At day 14, the blood flow of ischemic limbs in all cell transplanted groups increased significantly compared to the control group (white arrowhead). After 42 days, significant blood flow recovery was observed in the uEPC and human VPC-derived EC and/or MC-transplanted groups (red arrowhead), but not in pEPC. b) Quantitative analysis of hindlimb blood flow by calculating the ischemic/normal limb perfusion ratios after the induction of hindlimb ischemia. *<i>P</i><0.05 vs. control, †<i>P</i><0.05 vs. pEPC, ††<i>P</i><0.05 vs. uEPC, ‡<i>P</i><0.05 vs. MC, §<i>P</i><0.05 vs. EC.</p

Characterization of peripheral blood and umbilical cord-derived EPC (pEPC and uEPC, respectively) by flow cytometer.

<p>a) Representative forward and side scatter profile of cultured pEPC. b-d) Flow cytometric analysis of ulex-lectin binding/acLDL uptake (b, c) and various cell surface markers (d) in pEPC. e) Flow cytometric analysis of cell surface markers in uEPC.</p

Incorporated human VPC-derived vascular cells at the sites of vascular regeneration.

<p>a) Transplanted CM-DiI (red) labeled pEPC or VPC-derived vascular cells in ischemic hindlimbs at day 7 were detected by the fluorescence stereomicroscope. Scale bar: 500 µm. b, c) Immunostaining of frozen sections harvested from ischemic limb tissues at day 14. Fluorescence staining of GSL I-isolectin B4 (green) and human CD31 (blue) with nuclear staining (red) in human VPC-derived EC+MC (b), pEPC, and uEPC (c) transplanted mice. Scale bar: 20 µm. d) Immunostaining of αSMA (green)/human SM1 (blue) with nuclear staining (red) in human VPC-derived EC+MC-transplanted mice at day 14. Scale bar: 20 µm.</p

Possible differentiation pathway of vascular cells from human ES cells via VPC.

<p>Possible differentiation pathway of vascular cells from human ES cells via VPC.</p

Immunohistochemical analysis of human VPC-derived vascular cells-transplanted murine hindlimb tissues.

<p>a) Representative fluorescent photographs of ischemic hindlimb stained for human (red) and mouse (green) CD31 at day 42. Overlapped-stained capillaries are shown in arrowhead. Scale bar: 100 µm. b) Quantitative analysis of the endothelial cell marker positive capillary density in ischemic hindlimbs at day 42. c) Representative αSMA immunostaining (brown) of ischemic hindlimbs at day 42. Scale bar. 100 µm. d) Quantitative analysis of αSMA positive capillary density in ischemic hindlimbs at day 42. e) Quantitative analysis of αSMA positive arterioles (black arrowhead) at day 42. *<i>P</i><0.05 vs. control, †<i>P</i><0.05 vs. pEPC, ††<i>P</i><0.05 vs. uEPC, ‡<i>P</i><0.05 vs. MC, §<i>P</i><0.05 vs. EC.</p