![Figure 4. Shh and Dll-4 boost functional arterial hMAPC-EC differentiation in vivo. (A) Live in vivo imaging of a Matrigel plug containing VEGF165 and hMAPCs labeled with CFSE 10 days after subcutaneous implantation. Note the localized CFSE-labeled area (outlined by a dashed white line) located in the Matrigel in the vicinity of a large vascular tree (arrowheads) from the overlying host skin. (B-J) Histologic analysis on cross-sections through Matrigel plugs containing hMAPCs and VEFG165 (B-D) or hMAPCs and VEGF165 + Shh+ Dll-4 (“arterial cytokine mix” [E-J]). (B) Electron microscopy showing a capillary composed of a Resovist-labeled hMAPC-derived EC in Matrigel plugs. A semithin section (B, top panel), an ultrathin section (B, bottom panel), and a detail of iron particles (insets in lower panel of B) are shown. (C-F) Immunohistochemical staining of 3 μm paraffin cross-sections through Matrigel plugs for human-specific CD31 (C) and human-specific VE-cadherin (D) (both indicating their EC identity) and human-specific Hey-2 (E) and human-specific EphrinB1 (F) (both indicating their arterial EC identity). Arrows indicate red blood cells in vessel lumen. (G-H) Double confocal immunofluorescence staining of 40 μm cryopreserved cross-sections through Matrigel plugs with human endothelial-specific lectin UEA (green) and Hey-2 (red) (G) or UEA (green) and Ephrin B1 (red) (H). Topro (blue) was used for nuclear staining. (I) High-resolution live in vivo imaging of a Matrigel plug containing VEGF165 and hMAPCs labeled with CFSE 10 days after subcutaneous implantation and 30 minutes after intravenous injection of UEA lectin. Note colocalization (yellow; indicated by arrowheads) of CFSE-labeled cells (green) and UEA lectin (red) area, indicating that the vessels containing CFSE-labeled cells were connected to the host vascular system. (J) Double confocal immunofluorescence staining of 40 μm cryopreserved cross-sections through Matrigel plugs with human endothelial-specific lectin UEA (green) and α-actin (red), showing hMAPC-ECs (arrowheads) coated by α-actin–positive SMCs. Topro (blue) was used for nuclear staining. “L” in panels B and D-F indicates the vessel lumen. Magnifications ×63 (E-F), ×40 (C-D,J), and ×20 (G-I). Scale bars in panel B: 10 μm (semithin); 2.5 μm (ultrathin); 1 μm (upper inset); 0.5 μm (lower inset).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/109/6/10.1182_blood-2006-06-030411/4/zh80060709720004.jpeg?Expires=1723325213&Signature=BJ3uiUi1K0G0NvmvGpYyIpluIKXSFtC~40BK1tnWxkXUjCMrt82jA09oKg1ZuYiA14uA-6cGVnlHrmCafvVLKiHUmDE5QwquV1UTGk7CPIp6HFQR~VEH6hHLKmm6tu29IkDPYeRO2MoExYwFkw2HkWPxhKE1m~mB42vtCMm98FpnR1m0qoQr9vHdDfMwQHIdqkBCibZ~2Cp0ztHSgV78-fy73bv36xcYNZVBte89KMJ6M1-sNEuxgRqBZBVkf1JEYf9c4Y-lVpUFsyNcZoxrKjCWZYQKe-6XHflb0BG-KcLz62oJ5MElkjRHplwNbPfuIjEy8qCrnVzIQpgT-mgvJA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Shh and Dll-4 boost functional arterial hMAPC-EC differentiation in vivo. (A) Live in vivo imaging of a Matrigel plug containing VEGF165 and hMAPCs labeled with CFSE 10 days after subcutaneous implantation. Note the localized CFSE-labeled area (outlined by a dashed white line) located in the Matrigel in the vicinity of a large vascular tree (arrowheads) from the overlying host skin. (B-J) Histologic analysis on cross-sections through Matrigel plugs containing hMAPCs and VEFG165 (B-D) or hMAPCs and VEGF165 + Shh+ Dll-4 (“arterial cytokine mix” [E-J]). (B) Electron microscopy showing a capillary composed of a Resovist-labeled hMAPC-derived EC in Matrigel plugs. A semithin section (B, top panel), an ultrathin section (B, bottom panel), and a detail of iron particles (insets in lower panel of B) are shown. (C-F) Immunohistochemical staining of 3 μm paraffin cross-sections through Matrigel plugs for human-specific CD31 (C) and human-specific VE-cadherin (D) (both indicating their EC identity) and human-specific Hey-2 (E) and human-specific EphrinB1 (F) (both indicating their arterial EC identity). Arrows indicate red blood cells in vessel lumen. (G-H) Double confocal immunofluorescence staining of 40 μm cryopreserved cross-sections through Matrigel plugs with human endothelial-specific lectin UEA (green) and Hey-2 (red) (G) or UEA (green) and Ephrin B1 (red) (H). Topro (blue) was used for nuclear staining. (I) High-resolution live in vivo imaging of a Matrigel plug containing VEGF165 and hMAPCs labeled with CFSE 10 days after subcutaneous implantation and 30 minutes after intravenous injection of UEA lectin. Note colocalization (yellow; indicated by arrowheads) of CFSE-labeled cells (green) and UEA lectin (red) area, indicating that the vessels containing CFSE-labeled cells were connected to the host vascular system. (J) Double confocal immunofluorescence staining of 40 μm cryopreserved cross-sections through Matrigel plugs with human endothelial-specific lectin UEA (green) and α-actin (red), showing hMAPC-ECs (arrowheads) coated by α-actin–positive SMCs. Topro (blue) was used for nuclear staining. “L” in panels B and D-F indicates the vessel lumen. Magnifications ×63 (E-F), ×40 (C-D,J), and ×20 (G-I). Scale bars in panel B: 10 μm (semithin); 2.5 μm (ultrathin); 1 μm (upper inset); 0.5 μm (lower inset).
