|
|
Blood, Vol. 109, Issue 6, 2634-2642, March 15, 2007
In vitro and in vivo arterial differentiation of human multipotent adult progenitor cells
Blood Aranguren et al.
109: 2634
Supplemental materials for: Aranguren et al, Vol 109, Issue 6, 2634-3462
Files in this Data Supplement:
- Figure S1. Characterization of hMAPCs (PDF, 1.33 MB) -
(A) FACS phenotype of hMAPCs at 50 population doublings (PDs). Plots show isotype control IgG staining profile (black line) versus specific antibody staining profile (red line). A representative phenotype of 1 of the 8 clones is shown. (B) RT-PCR blot showing the expression profile of pluripotency markers in hMAPCs at 50 PDs. C+ indicates total RNA (Becton Dickinson); C-, H2O. (C-F) Immunofluorescent staining for pluripotency markers on hMAPCs at 50 PDs. The hMAPCs show expression of SSEA-4 (C), OCT3/4 (D), and nanog (E) but not SSEA-1 (F). A representative expression profile of 1 of the 8 clones is shown. (G) Smooth muscle differentiation from hMAPCs. The hMAPCs treated with TGF- 1 for 6 days up-regulated expression of SMC markers, shown by Q-RT-PCR (presented as percent expression in comparison with SMCs derived from umbilical artery) and immunofluorescence ( -actin, green). (H) Hepatocyte differentiation from hMAPCs. Expression of hepatocyte markers was up-regulated after 7 to 28 days in the presence of HGF and FGF-4, shown by Q-RT-PCR (presented as fold increase compared with day 0) and immunofluorescence (albumin, red). (I) Neuronal differentiation from hMAPCs. Expression of neuronal markers was up-regulated after 7 to 28 days in the presence of bFGF (week 1), Shh, FGF-8 (week 2), and BDNF (week 3),2 as shown by Q-RT-PCR (presented as fold increase compared with day 0), phase contrast imaging, and immunofluorescence (3-tubulin, green). Q-RT-PCR results are expressed as mean (± SEM) of 3 different experiments in triplicate, and immunofluorescence panels are representative of more than 3 experiments. DAPI (blue) in panels C and F-I was used for nuclear staining. (G-I) *P < .05; **P < .01 versus day 0. Magnifications ×40 (C-F), ×20 (G-H), and ×40 (I).
- Figure S2. Human AC133+ cells and hMAPCs differentiate into functional ECs (PDF, 289 KB) -
(A-B) Flow cytometric analysis of hAC133+ cells before (A) and 14 days after the start of the differentiation process (B). After 14 days of differentiation, hAC133+ cells down-regulated AC133 and hematopoietic markers (CD45 and CD34), while EC markers (including CD31, CD105, and v3) were up-regulated. A representative experiment of more than 5 experiments is shown. (C-G) Immunofluorescence of hAC133+ derived ECs. After 14 days of differentiation, most hAC133+ cell–derived cells stained positive for EC markers, including vWF (C), Tie-1 (D), Tie-2 (E), Flt-1 (F), and KDR (G). (H-I) Functionality of hAC133+-derived ECs. The hAC133+ cell–derived ECs were functional as shown by their ability to take up AcLDL (H) and to form vascular tubes in Matrigel (I). A representative experiment of more than 5 experiments is shown. (J-K) Flow cytometric analysis of hMAPCs before (J) and 21 days after the start of the differentiation process (K). After 21 days of differentiation, most hMAPCs down-regulated hematopoietic markers (like CD45) and up-regulated several EC markers (including CD105 and v3). (L-P) Immunofluorescence of hMAPC-derived ECs. After 14 days of differentiation, hMAPC-derived ECs expressed several EC markers, including vWF (L), Tie-1 (M), Tie-2 (N), Flt-1 (O), and KDR (P). A representative experiment of more than 5 experiments is shown. (Q-R) Functionality of hMAPC-derived ECs. The hMAPC-derived ECs were functional as shown by their ability to take up AcLDL (Q) and to form vascular tubes in Matrigel (R). A representative experiment of more than 5 experiments is shown. FACS plots in panels A, B, J, and K show isotype control IgG staining profile (black line) versus specific antibody staining profile (red line). Percentages of positive cells are shown. In panels C-H and L-Q, DAPI was used for nuclear staining. Magnifications ×40 (C-H,L-Q) and ×10 (I,R).
- Figure S3. VEGF165 induces high expression levels of EC markers in hMAPCs (PDF, 12.2 KB) -
Q-RT-PCR analysis of EC markers before and 14 or 21 days after differentiation of hMAPCs. The increase in mRNA expression varied between the different genes from a 5-fold (CD31) to a more than 600-fold increase (Flt-1) versus the undifferentiated state. The mRNA levels in all panels are expressed as fold increase compared with day 0 and were normalized using GAPDH as housekeeping gene. The mean and SEM of 3 different experiments is shown. *P < .05; **P < .01 versus day 0.
- Figure S4. Detailed analysis of hAC133+-derived CD45+ fraction (PDF, 20.7 KB) -
CD45+ cells present in hAC133+ cultures 14 days after differentiation in the presence of VEGF165 were analyzed by FACS with antibodies against (A) lymphoid markers (CD19 for B cells, CD3 for T cells, CD16 and CD56 for natural killer NK cells) and (B) myeloid (CD14, CD11c, CD11b, and CD13) cells. None of the CD45+ cells coexpressed markers associated with lymphoid cells. Instead they expressed markers associated with myeloid cells.
- Figure S5. Control immunofluorescence and in vitro Matrigel assays with endothelial-specific antibodies using HUVECs and HUAECs (PDF, 146 KB) -
(A-F) HUVECs were incubated with antibodies against KDR (A), Flt-1 (B), vWF (C), Tie-1 (D), Tie-2 (E), and EphB4 (F) and visualized with FITC-conjugated secondary antibody. (G-H) HUAECs were incubated with antibodies against Hey-2 (G) and EphrinB1 (H) and visualized with FITC-conjugated secondary antibody. Note the heterogeneity in morphology. (I) In vitro Matrigel assay: 50,000 HUVECs per milliliter were cultured for 24 hours on Matrigel, in which condition they started forming tubes. Magnifications ×200 (A-H) and (F) ×100.
- Figure S6. Effect of Notch and patched ligands on arteriovenous differentiation of hAC133+ and hMAPC-derived ECs (PDF, 37.3 KB) -
Q-RT-PCR analysis of arterial and venous markers in hAC133+ cells (black bars) and hMAPCs (gray bars) after 14 days of differentiation with different combinations of Notch and patched ligands (x-axis). The mRNA levels in all panels are expressed as mean percentage of a positive control (HUAECs for arterial and HUVECs for venous markers) and were normalized using GAPDH as housekeeping gene. The mean and SD of 3 different experiments is shown. The combination of VEGF165, Shh, and Dll-4 gives the best induction of Hey-2, paralleled by the best reduction in EphB4. No significant change in Hey-2 expression was induced in hAC133+ cells. *P < .05; **P < .01 versus VEGF165 alone.
- Figure S7. Matrigel engraftment of hMAPCs correlates with cell dose (PDF, 285 KB) -
Histologic analysis on 3 µm paraffin cross-sections through Matrigel plugs containing hMAPCs (revealed with human-specific antivimentin in green) and VEFG165 (A-C) or hMAPCs and VEGF165 + Shh + Dll-4 (D-F) showing increasing engraftment in Matrigels injected with increasing numbers of hMAPCs. DAPI (blue) was used as nuclear counterstain in all panels. Magnification ×100.
- Figure S8. In vivo arterial differentiation of hMAPCs in standard media (PDF, 72.7 KB) -
Immunostaining of 3 µm paraffin cross-sections through Matrigel plugs containing 2.5 × 106 hMAPC cells and VEGF165, stained with human-specific vimentin (A green) and human-specific EphrinB1 (B red), showing colocalization (C yellow indicated by arrowheads) of both markers, indicating the arterial EC identity of the hMAPC-ECs. Magnification ×200.
- Figure S9. Subcutaneous Matrigel assay with hAC133+ cells (PDF, 75.1 KB) -
Immunohistochemical analysis of 3 µm paraffin cross-sections through Matrigel plugs containing hAC133+ cells, stained with the human-specific lectin UEA (B) or human-specific CD31 (D), both indicating their EC identity. Positive control is human muscle (A,C). Despite the presence of hAC133-derived ECs (B,D, arrows), they do not associate with or organize into larger vascular structures. Magnification × 400.
|
|
