1. Volume 23, Issue 3, Pages 510-522 (March 2015)
Sourcing of an Alternative Pericyte-Like Cell Type from Peripheral Blood in Clinically Relevant Numbers for Therapeutic Angiogenic Applications 
Anna Blocki, Yingting Wang, Maria Koch, Anna Goralczyk, Sebastian Beyer, Nikita Agarwal, Michelle Lee, Shehzahdi Moonshi, Jean- Yves Dewavrin, Priscilla Peh, Herbert Schwarz, Kishore Bhakoo, Michael Raghunath 
Molecular Therapy 
Volume 23, Issue 3, Pages (March 2015)
DOI: /mt
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
MMC induces BDAC proliferation and enhances growth factor signaling in monocyte-derived cultures. Spindle-shaped cells were generated from PBMCs without (−MMC), or in the presence of macromolecules (+Fc) during the first day of culture. Increased proliferation of adherent spindle-shaped cells under +Fc is evident (a) in phase contrast (PhC) images at day 5 (b) adherent cytometry of DAPI stained nuclei of adherent cells resulting from cell culture of 106 PBMC at 6 hours (0.25 days), 1 day, 2 days and 5 days and (c) Quantitation of cell doublings during 5 days of culture. (d–e) MTS assay of spindle-shaped cells normalized to cell number on day 1 and 5, respectively. NADH/NADPH levels are displayed as percentages relative to –MMC controls. (f–g) G6PDH activity assay normalized to cell number on day 1 and 5, respectively. (h) M1 polarization of THP-1 derived macrophages under MMC (+Fc) or without macromolecules (−MMC). Read-out for successful polarization is TNF-α production. *P < 0.05; **P < All experiments were done at least in triplicates with comparable results.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
BDACs express a unique set of markers and are distinguishable from other angiogenic cells. (a) Flow cytometry analysis for hematopoietic, endothelial, and mesenchymal markers. Results are displayed as average ± SD from three independent blood samples. (b) Immunocytochemistry for vWF in BDAC and HUVEC cultures. (c) SDS-Page of pepsin-digested conditioned media by Pl-Prc, IMR-90s (fibroblasts), and BDACs. Collagen I standards (Col I Std) served as molecular weight standards. Bands from the same gel are displayed. (d) RT-PCR of collagen α1 and vWF mRNAs in Pl-Prc, IMR-90s, HUVEC and BDACs. (e) Immunocytochemistry for Tie-2, VEGFR-1, CD31, and antigen recognized by PM-2k antibody clone in HUVECs, BDAC, Pl-Prc, and bmMSCs. (f) Immunocytochemistry for PDGFR-β, NG2, α-SMA and desmin in cell types from e. All experiments were repeated at least three times with comparable results.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Although monocyte-derived, BDACs differ from standard macrophages. (a–c) BDACs and macrophages derived from the same blood samples were polarized into either M1 or M2. (a) Graph shows ELISA results for M1 cytokine TNFα measured in supernatant. (b) ELISA for M2 cytokine IL10 in conditioned media. (c) Immunocytochemistry for CD206 expression in cell types from a) and b). (d–f) Phagocytosis assay. (d) Quantification of cells up-taking fluorescent microbeads by flow cytometry. Data are presented as average of three independent blood samples. HUVEC served as negative controls. (e) Exemplary flow cytometry results with (f) corresponding images of cells before harvesting (Phase contrast images (PhC) overlaid with green fluorescence are displayed). Scale bar 100 μm.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
BDACs colocalize and stabilize endothelial tubular networks on matrigel. (a) Stills of live-cell imaging of coculture of BDAC (green label) with HUVEC on matrigel over 24 hours (Supplementary Video S1). (b) 40× magnification of BDAC (green label)-HUVEC coculture on matrigel. Phase contrast (PhC) and green fluorescence are displayed. Bar: 50 μm. (c) Culture of HUVECs alone or HUVECs together with BDAC on matrigel in starving media at 4 hours and 16 hours. Bar: 500 μm. (d) Quantification of cumulative tube length per field of view (FOV) at 16 hours of cultures in c. All experiments were repeated at least three times with comparable results.*P < 0.02.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
BDACs colocalize with endothelial sprouts and enhance endothelial sprouting in a cell-dose dependent manner in vitro. (a) Spheroids containing HUVEC and BDAC (green label) in a ratio of 2:1 were embedded into a collagen I gel and allowed to sprout for 3 days. Bar: 50 μm. (b) HUVECs spheroids were embedded in a collagen I gel with various BDAC concentrations suspended. Graphs show measured cumulative sprout length/spheroid and representative phase contrast (PhC) pictures are shown below. Bar: 100 μm. (c) Close-ups of sprouts for HUVEC control condition and in the presence of 2.7 x 105 BDAC suspended per ml of collagen I. All experiments were repeated at least three times with comparable results.*P < 0.01; **P <
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
BDACs are proangiogenic in vivo. U87 human glioma cells were injected subcutaneously into nude mice, either alone (U87 control) or with BDACs (U87 + BDAC) (n = 5). Animals were sacrificed after 8 days and tumors analyzed. (a) Immunohistochemical analysis of CD31 staining. Graph: Total fluorescent area per tumor section was quantified. Representative images are displayed below. (b) Immunohistochemical analysis of vWF staining. Graph: Total fluorescent area per tumor section was quantified. Representative images are displayed below. (c) Images of labeled BDACs (red) and CD31 stained blood vessels (green staining) at various locations within a tumor. DAPI was used to visualize nuclei staining (blue). Arrows: BDACs in perivascular location. Bar: 100 μm *P < 0.05.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
BDACs secrete proangiogenic factors involved in early stages of angiogenesis, of which secreted MMP9 is a pivotal effector in the enhancement of endothelial sprouting in vitro. (a) Proteome array for various pro- and antiangiogenic factors. Only strong signals are labeled on the membrane. Quantifications of signals can be found in Supplementary Figure S9. (b) Gelatin and collagen zymography of gel samples from spheroid sprouting assay (from Figure 5b), from HUVECs and BDACs suspended as single cells in collagen I gels and from supernatant of monolayer cultures of HUVEC and BDACs. Western blot (WB) for MMP-9. (c) Inhibition of MMP9 by small molecules in spheroids sprouting assay. (d) Representative images of spheroids from c. All experiments were repeated at least three times with comparable results.*P <
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 8
BDACs accelerate and enhance revascularization in a murine critical limb ischemia model. Affected limbs in a murine hind limb ischemia model were treated with intramuscular injected BDACs or the vehicle only. Revascularization was monitored by MRI angiography. (a) Reconstructed MRI images with the region of interest (ROI) indicated by red box. (b) Quantification of vessel volume in ROI. (c) Representative pictures of affected limbs in both treatment groups. (d) Ischemic scoring table. (e) Semiquantitative ischemia scoring accordingly to d. (f,g) H&E staining of the murine gastrocnemicus muscle of the calf in the vehicle- and BDAC-treated group respectively. Arrows indicate neutrophil infiltration, arrowheads indicate fibrosis and stars (*) indicate muscle degeneration with adipose replacement.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions