Figure 2. Vascular cells reprogrammed from young vs. old donors show gene expression and functional differences.

Heat-map representing DE genes between reprogrammed vascular cells from young (N = 3) vs. old (N = 3) donors (iSMCs (A), iVECs (B)). Z score = ± 1.5. DE genes with log₂FC > 1 and FDR < 0.05. Quantification of BMP2 (C) and PALD1 (D) expression in human skin biopsies from young vs. old donors (N = 2 donors per condition; N = 10 tissue sections per condition; Student’s t-test with p<0.001 (***) and p<0.05 (*)). Representative images of BMP2 (E) and PALD1 (F) from skin biopsies obtained from young vs. old donors. Scale bars: 50 µm. Quantification of vascular permeability using young vs. old reprogrammed cells (G–J). Schematic showing the generation of an endothelial monolayer covering the interface with a 3D matrix embedding SMCs. Vascular permeability was quantified by measuring the variation of 70 kDa dextran fluorescent intensity across the interface (G). Representative images of dextran flow (red) through the endothelial monolayer in presence of young vs. old reprogrammed cells. Scale bar: 100 µm (H). Quantification of vascular permeability in presence of young vs. old iVECs (I, at least N = 40 independent measurements per condition in three biological replicates; ANOVA with Holm-Sidak test; comparison with primary VECs (** is p<0.01 and *** is p<0.001); comparison with young iVECs (♦♦♦is p<0.001); comparison with old iVECs (••• is p<0.001)) or young vs. old iSMCs (J, at least N = 40 independent measurements per condition in three biological replicates; ANOVA with Holm-Sidak test; * is p<0.05, ** is p<0.01 and *** is p<0.001). Quantification of vascular permeability in presence of PALD1 KD (K) or BMP2 KD (L) in primary VECs and SMCs, respectively (at least N = 60 independent measurements per condition in three biological replicates for each KD experiment; Student's t-test; *** is p<0.001).

Figure 2—figure supplement 1. Heat-maps representing key cell identity genes for both iSM cells and iVECs derived from young and old donors.

Fibroblasts were included as control. Z score = ± 1. All these genes were not DE comparing young and old donors.

Figure 2—figure supplement 2. BMP2 expression by iSMCs reprogrammed from young (19 y.o.) vs. old (67 y.o.) donor.

Representative image showing young (mCherry MYOCD) and old (EGFP MYOCD) iSMCs. Experiments were also performed with the reciprocal tagging. At least N = 25 independent measurements in three biological replicates. Student’s t-test, p<0.01 (**). Scale bar: 50 µm.

Figure 2—figure supplement 3. Representative images of GSTM1 expression by endothelial cells within human skin biopsies from young vs.old donors (N = 2 donors per condition, 10 tissue sections per condition; Student’s t-test with p<0.01 (**)).

Scale bars: 100 µm.

Figure 2—figure supplement 4. FPKM values for PALD1and GSTM1 from RNAseq performed on primary mouse endothelial cells from bone marrow, brain and skin of young (3 months) vs. old (18 months) mice.

Biological processes upregulated in old vs. young mouse VECs (pool of bone marrow, brain and skin VECs). It is interesting to observe the upregulation in old mouse VECs of biological processes associated with immune system activation and inflammation, consistent with the general concept that aging is associated with chronic inflammation. Cells were derived from N = 3 animals per age group and then pooled for RNAseq.

Figure 2—figure supplement 5. Representative images and quantification of Y658 VE-Cadherin (white) in iVECs derived from young and old donors.

The higher amount of Y658 staining in old iVECs (Student’s t-test with p<0.01 (**) from N = 3 biological replicates) suggests that cell-cell junctions might be compromised in reprogrammed cells derived from old donors. Nuclei: blue. Scale bars: 25 µm.

Figure 2—figure supplement 6. Representative images of microfluidic devices seeded with iSMCs reprogrammed from young or old fibroblasts.

VECs stained with VE-cadherin (white) cover the lateral channel and the interface with the 3D matrix embedding iSMCs (red, αSMA). iSMCs from young donors migrate towards the endothelial layer. Nuclei: blue. Scale bar: 50 µm.

Figure 2—figure supplement 7. Validation of KD of PALD1 and BMP2 in VECs and SMCs, respectively (N = 2 biological replicates).

Note that BMP2 KD was validated using qPCR due to technical problems with any of the tested anti-BMP2 antibodies (Western Blot). The use of these antibodies resulted in multiple aspecific bands.