Induction of vascular progenitor cells from endothelial cells stimulates coronary collateral growth

Abstract

Rationale: A well-developed coronary collateral circulation improves the morbidity and mortality of patients following an acute coronary occlusion. Although regenerative medicine has great potential in stimulating vascular growth in the heart, to date there have been mixed results, and the ideal cell type for this therapy has not been resolved.

Objective: To generate induced vascular progenitor cells (iVPCs) from endothelial cells, which can differentiate into vascular smooth muscle cells (VSMCs) or endothelial cells (ECs), and test their capability to stimulate coronary collateral growth.

Methods and results: We reprogrammed rat ECs with the transcription factors Oct4, Klf4, Sox2, and c-Myc. A population of reprogrammed cells was derived that expressed pluripotent markers Oct4, SSEA-1, Rex1, and AP and hemangioblast markers CD133, Flk1, and c-kit. These cells were designated iVPCs because they remained committed to vascular lineage and could differentiate into vascular ECs and VSMCs in vitro. The iVPCs demonstrated better in vitro angiogenic potential (tube network on 2-dimensional culture, tube formation in growth factor reduced Matrigel) than native ECs. The risk of teratoma formation in iVPCs is also reduced in comparison with fully reprogrammed induced pluripotent stem cells (iPSCs). When iVPCs were implanted into myocardium, they engrafted into blood vessels and increased coronary collateral flow (microspheres) and improved cardiac function (echocardiography) better than iPSCs, mesenchymal stem cells, native ECs, and sham treatments.

Conclusions: We conclude that iVPCs, generated by partially reprogramming ECs, are an ideal cell type for cell-based therapy designed to stimulate coronary collateral growth.

Figure 1

Generation of iVPCs from rat ECs transduced with transcription factors OKSM. Top panel is the schematic diagram of the protocol for reprogramming. (A) Confluent rat EC morphology before reprogramming. After transduction ECs showed ES-like morphology at day 4 (B), formed ES-like colonies at day 14 and retained the morphology until 16 days (C), and changed to ball-like iVPCs at day 21 (D). Most iVPCs retained stable morphology during expansion (E). iVPCs expressed ES cell marker SSEA-1 (F),Oct4 (G), Alkaline Phosphatase (H) by immunostaining. iVPCs showed only 0.39% EC marker PECAM by FACS analysis (I). iVPCs at passage 16 showed a normal 42xy Karyotype (J). Scale bar equals 100μm.

Figure 2

Gene expression profile of iVPC. RT–PCR analysis of endogenous gene expression in iVPCs and iPSCs (A) and exogenous expression of transcription factors Oct4, Klf4, Sox2, c-Myc in iVPCs (B). q RT-PCR analysis of E-cadherin (C) and cell lineage markers RUNX2 (D), GATA4 (E), and GATA6 (F) in iVPCs, ES cells and iPSCs. FACS analysis of iVPCs and iPSCs of pluripotent cell markers Oct4 and SSEA-1; hemangioblast progenitor markers CD133, Flk1, and c-kit (G).

Figure 3

DNA methylation status (bisulfite sequencing) of the Nanog (A) and Oct4 (B) promoter regions of endothelial cells (ECs), iPSCs from fully reprogrammed fibroblasts, and 4 randomly selected clones of induced vascular progenitor cells (iVPCs). Open and closed lollipops indicate unmethylated and methylated CpGs, respectively. The top panel shows the promoter region of Nanog and Oct4 relative to the translation start site. The bottom numbers indicate the methylation percentage of CpG in the region. C and D show the statistical summary for A and B.

Figure 4

Angiogenic potential of iVPCs. (A) iVPCs spontaneously formed a “tube”-like network on feeder cells. In GFR Matrigel, ECs (B) and iVPCs (C) formed tube-like network in medium with serum and VEGF (50ng/mL). In the absence of serum and VEGF, ECs did not form tubes in the GFR Matrigel (D). In contrast, iVPCs formed an extensive tube-like network under same conditions (E). When exposed to shear stress for 72 hr, ECs (F) and differentiated iVPCs (G) aligned parallel to shear stress. iVPCs could differentiate into ECs with induction of VEGF (50ng/mL) shown in RT-PCR analysis (H) and immunostained with the EC marker VWF (J, with VWF stained in green and DAPI stained in blue. iVPCs could differentiate into VSMCs with induction of PDGF (50ng/mL) shown in RT-PCR analysis (I) and immunostained with the VSMC marker α-SMA (K, α-SMA stained in red and DAPI stained in blue). But iVPCs could not differentiate into cardiomyocytes either with BMP4 (10ng/mL), Activin A (10ng/mL) and bFGF (10ng/mL) or with 3 μM 5-azacytidine shown by negative staining of α-Sarcomeric Actin (L). Scale bar equals 100μm in A-J, and scale bar in L equals to 10μm.

Figure 5

iVPCs co-localized with Isolectin B4 after implantation into rat ischemic myocardium. A diagram for surgical procedure was shown in A. The iVPCs were labeled with Td-tomato by lentiviral vector before injection (B). Fluorescent images show rat myocardium in control group and iVPC-implanted group after 10 days of the RI protocol (C). iVPCs were labeled with tdTomato, the native endothelium was labeled with FITC-isolectin B4, and DAPI reveals nuclei. Note the absence of tdTomato in the control images, but the presence of this fluorochrome and its overlap with FITC in the iVPCs implantation images. Scale bar equals 100μm in B and 10μm in C.

Figure 6

iVPCs engrafted into blood vessels after implantation into rat ischemic myocardium. Images are from immunostaining of rat myocardium in control group and iVPC- and iPSC-implanted group after 10 days of the RI protocol. DAPI staining reveals cell nuclei. (A) Immunostaining of rat myocardium with ES cell marker SSEA-1 (green) and endothelial cell marker VWF (red). In control group, there was no obvious SSEA-1 expression. The iVPC- and iPSC-implanted groups highly expressed SSEA-1 and co-localized with vascular ECs. In the bottom panel, iVPCs branched and split from one big vessel into several small vessels, which is one type of angiogenesis (intussusceptive angiogenesis). (B) Immunostaining of rat myocardium with ES cell marker SSEA-1 (green) and vascular smooth cell marker αSMA (red). In the control group, again there was no obvious SSEA-1 expression. In the iVPC-implanted group, iVPCs expressing SSEA-1 co-localized with VSMCs. However, no co-localization of iPSCs with VSMCs was observed. (C) Immunostaining of rat myocardium with cardiomyocytes marker α-sarcomeric actin. In the iVPC delievered group, there was no co-localization of iVPCs with cardiomyocytes, but in the iPSC-implanted group, co-localization of iPSCs with cardiomyocytes was observed. Scale bar equals 100μm in A and B and 10μm in C.

Figure 7

iVPCs improved coronary collateral flow and heart function in vivo after implantation into rat myocardium. Compared to control (n=10), iPSC (n=7), EC (n=6), and MSC (n=8) implantation groups, iVPCs (n=8) augmented coronary collateral blood flow measured by microspheres. Collateral flow is expressed as a ratio of flows to the collateral and normal zones (CZ/NZ) (A). Ejection fraction (EF%), fractional shortening (FS%) and left ventricular end systolic volume (LVESV) were measured by echocardiography at day 0 and day 10 of RI protocol, and before and after balloon inflation (to produce ischemia). The percentage changes of each parameter before and after balloon inflation were calculated at day 0 and day 10. The improvement in ejection fraction (B), fractional shortening (C) and systolic volume (D) was most striking in the iVPC-treated group (n=8) between day 0 and day 10, compared to the other groups (control, n=10, iPSC, n=9, EC, n=5, and MSC (n=9). These data suggest heart function of the iVPC group improved significantly.

Figure 8

Less Risk of Teratoma Formation from iVPCs than from iPSCs. A. Morphology of iPSC, iVPC and clone #9 (named as iVPC/iPSC because ES-cell-like fully reprogrammed cells appeared in the iVPCs). B. When seeded on low attachment dishes with differentiation medium, iPSC grew in suspension, aggregated and formed embryo bodies. In contrast, iVPCs attached to the plates and spread, and did not from embryo bodies. C. Kidneys and testis harvested after injection with iPSC, iVPCs and iVPCs/iPSCs. iVPCs did not form teratomas while iPSCs and iVPCs formed teratomas. D shows the probabilities of teratoma formation from iVPCs, iPSCs and iVPC/iPSC. 1 refers the teratoma formed and 0 refers no teratoma formed. The “N” refers to injection times. So for iPSCs, the probability of teratoma formation was 100% (4 out of 4). For iVPC, the probability of teratoma formation is 0% (0 out of 15). For iVPC/iPSC, the probability of teratoma formation is 100% (3 out of 3). Even if including iVPC/iPSC (clone#9), the probability of teratoma formation of iVPCs is 0.46% (3 out of 18 injections in the context of one clone out of 36 clones became iVPC/iPSC). Scale bar equals 100μm.