Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair

Abstract

In this report we describe a human pluripotent stem cell-derived vascular progenitor (MesoT) cell of the mesothelium lineage. MesoT cells are multipotent and generate smooth muscle cells, endothelial cells, and pericytes and self-assemble into vessel-like networks in vitro. MesoT cells transplanted into mechanically damaged neonatal mouse heart migrate into the injured tissue and contribute to nascent coronary vessels in the repair zone. When seeded onto decellularized vascular scaffolds, MesoT cells differentiate into the major vascular lineages and self-assemble into vasculature capable of supporting peripheral blood flow following transplantation. These findings demonstrate in vivo functionality and the potential utility of MesoT cells in vascular engineering applications.

Keywords: mesothelium; regenerative medicine; stem cells; tissue engineering; vascular progenitor.

Figure 1.. hPSC-Derived MesoT Displays Molecular Characteristics of Primary Mesothelium

(A) Sources of cells used for RNA-seq analysis. hPSC-derived MesoT cells and tdTomato+ mesothelium isolated from mouse embryonic gut, liver, heart, and lungs (embryonic day 15.5 [E15.5]) were compared to RNA-seq data in the public domain. (B) Addition of Wnt3a, BMP4, and retinoic acid (RA) to SplM (ISL1⁺, NKX2.5⁺, ZO1⁺) efficiently generates MesoT (WT1⁺, αSMA⁺, VIM⁺) with a mesenchymal phenotype. Scale bars, 50 μm. (C) qRT-PCR data showing fold-change of transcript levels for markers of SplM (ISL1, NKX2.5, and GATA4) and MesoT (WT1, TBX18, and TCF21) following directed differentiation of human embryonic stem cells (hESCs, WA09). TaqMan assays for each transcript were performed in technical triplicate and fold-change shown relative to untreated hESCs (WA09) after normalization with 18S RNA. (D) Left: hierarchical clustering (Euclidean distance, complete linkage) of all human (black box) and mouse (red box) RNA-seq samples according to mouse and human orthologs. Data from both species were log transformed and scaled to mean = 0 and standard deviation = 1 prior to clustering. Right: zoomed-in section of the highlighted portion of the array tree dendrogram (left) showing that hPSC-derived MesoT cells are closely related to human (h) and adult (a) epicardium (m, mouse mesothelium; f, fetal mesothelium). Replicate numbers from independent experiments are indicated. Error bars ± standard deviation. See also Figures S1 and S2 and Table S4.

Figure 2.. Epigenetic and Transcript Profiles of MesoT Are Similar to Vascular Cell Types

(A) Hierarchical clustering (Euclidean distance, complete linkage) of human tissue and hESC-derived samples according to beta values for the 1,846 cytosines comprising module 9 of the DNA methylation profile. Array tree dendrograms and the distribution of beta values for these cytosines are presented in heatmap form (top) and as box and whisker plots (bottom). (B) Cartoon depicting the epigenetic landscape at primed and activated enhancers as MesoT cells transition to a vascular fate. Top portion depicts vascular genes “primed” in MesoT with the presence of K4me1 on histone H3 at enhancer sites. Bottom portion depicts the primed enhancers for vascular genes being activated by addition of K27ac as they differentiate to smooth muscle cells (SMCs) or endothelial cells (ECs). (C) Enhancer and gene ontology discovery pipeline for data in (D) (Figures S3B–S3D). (D) Heatmaps of H3K4me1 (red) and H3K27ac (blue) ChIP-seq data for enhancers linked to upregulated genes in primary smooth muscle and ECs, compared to MesoT cells. (E) Principal component analysis of the top 50% of highly expressed genes in hESC-derived mesothelium cells(MesoT and MLC), primary fetal tissue (liver, heart, brain, spinal cord, and hindbrain), and primary cells (Endo and SMC). MesoT and MesoT (FCS) (self-renewing) cells cluster tightly with vascular cell types (Endo and SMC) and hESC-derived mesothelium (MLC). See also Figure S3 and Tables S1 and S4.

Figure 3.. MesoT Cells Efficiently Differentiate to Smooth Muscle and ECs

(A) MesoT cells treated with PDGF-BB (50 ng/ml) in CDM (–Activin A) for 12 days were probed with antibodies for alpha smooth muscle actin (αSMA), calponin, and myosin heavy chain 11 (MYH11). Scale bars, 100 μm (left) and 50 μm (right). (B) MesoT cells grown in CDM (–A) supplemented with PDGF-BB or VEGF-A₁₆₅ and SB431542 were quantified based on expression of lineage specific markers for SMCs and ECs. All experiments in biological triplicate. (C) 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO)-labeled SMCs as shown in (A) were treated with 100 μM carbachol or 50 mM KCl to stimulate functional contraction. Scale bar, 200 μm. (D) SMC surface area was measured after treatment (C). Contraction is shown as the % change in cell surface area for individual cells. Each treatment group was compared to corresponding control time point to determine statistical significance. b = 20. (E and F) MesoT cells treated with CDM (–A), VEGF-A₁₆₅, and SB431542 for 12 days were fixed and probed with antibodies for CD31, vWF, and DAPI (E) or characterized by flow cytometry with VE-cadherin (blue) or isotype control (red) (F). Scale bar, 50 μm. (G) Trans-endothelial electrical resistance (TEER) was measured after culturing MesoT cells in CDM (–A) supplemented with VEGF-A₁₆₅ alone (+VEGF) or with SB431542 (+VEGF +SB) after 14, 21, and 28 days and compared against primary dermal microvascular endothelium (1° ECs). n = 3. (H) Barrier integrity was tested by measuring FITC-dextran (40 kDa) perfusion from the apical to basolateral side. Control represents the absence of cells. No statistical significance was determined when comparing cells to 1° ECs. (I) Schematic of the bioreactor culture system used in (G) and (H). (J) Immunofluorescence of cell monolayer as in (I) showing expression of tight junction marker ZO1 (top) and H&E staining (bottom). Scale bars, 100 μm. (K) Transmission electron microscopy image of MesoT-derived endothelium. Red arrows depict tight cell junctions. Inset (top) is depicted on bottom. Scale bars, 500 nm and 200 nm, respectively. **p = 0.0027 for two-way ANOVA. ****p < 0.0001 for one-tailed t test. Error bars ± SEM. See also Figure S4.

Figure 4.. MesoT Cells Are Multipotent Vascular Progenitor Cells

(A) Population doubling of MesoT (FBS) cells. Time = 0 is when cells are first passed into serum containing media. Simple linear regression analysis (blue dashes) applied to data shows a tightly fitted regression line with a coefficient of determination (R²) = 0.9774 and slope of 0.8282+/−0.0563. Experiment n = 3 performed in technical triplicate. (B) Cell cycle analysis of self-renewing MesoT (FBS) cells at passages 4 and 9 (p4 and p9) using Life Technologies Click-iT Plus EdU Alexa Fluor flow cytometry assay kit. n = 4 for p4 and n = 3 for p9, all in technical triplicate. (C and D) Representative two-dimensional flow plots for each passage showing gating strategy to determine percentage of cells in each cell cycle phase for (B). (E) Clonal assay strategy to determine multipotency of self-renewing MesoT (FBS). (F) Fluorescence-activated cell sorting (FACS) gating strategy to obtain single cells for clonal analysis. Triple positive single cells (CD44⁺/CD73⁺/CD105⁺) were sorted onto a 96-well plate for amplification and downstream lineage analysis. Cyan are isotype controls. (G) After amplification, 14 individual clones were selected for downstream lineage analysis. MesoT (FBS) cells were treated with 2% FBS +VEGF or +PDGF-BB for endothelial or smooth muscle cell differentiation, respectively. Cells were fixed and probed with antibodies against vWF (endothelium) or MYH11 (SMC). > 45 cells in 3 separate images per clone were quantified using ImageJ to determine percentage of cells that give rise to each lineage. (H) MesoT in CDM (–Activin A) treated with VEGF generate mixtures of ECs (vWF⁺) and SMCs (calponin⁺). Scale bar, 50 μm. (I and J) Cells as in (H) were cultured on Matrigel for 12 days. Bright-field images (I) of resulting vessel structures were probed with antibodies for αSMA and vWF and the nuclei counter stained with DAPI (J). Scale bars, 50 μm. Error bars ± SEM. See also Figure S4.

Figure 5.. MesoT Cells Incorporate into Newly Formed Blood Vessels in a Neonatal Mouse Heart Injury Model

(A) Surgery on P0.5 mouse pups that includes ventricle apex resection. (B) After resection, the damaged heart was overlaid with a 2 μL suspension of 1 × 10⁶ DiO-labeled MesoT cells followed by suturing of the rib cage and chest wall. Micron bar, 1 mm. (C) Cryo-section (10 μm) of a repaired mouse heart, 30 days post-injury. Tissue was probed with anti-bodies for αSMA, CD31, human Golgi antigen, and DAPI. Scale bars, 50 μm. (D) Repair zone showing a submesothelial blood vessel (bv) comprised of human μSMA⁺ cells and human CD31⁺ cells. Scale bars: top left, 20 μm; other panels, 10 μm.(E) Magnified images of panels shown in (E) showing incorporation of human endothelial and smooth muscle cells into nascent vessels. Scale bar, 10 μm. See also Figure S5.

Figure 6.. Reperfusion of a Decellularized Biological Scaffold with MesoT Vascular Progenitor Cells Repopulates the Vascular Network and Forms Functional Invested Vessels when Transplanted In Vivo

(A-C) Image of a decellularized rat jejunum (A) injected with phenol red to contrast the vasculature before cell perfusion (B and C). (D) MesoT cells were perfused through the arterial and venous cannulas with (+VEGF) media. MTT assay imaging depicts reseeded vasculature with metabolically active cells after 28 days. Right is blowup of inset on left. Red lines mark representative vessels of various sizes. Micron bar, 1 mm. (E) Vasculature derived by seeding MesoT cells was stained with human nuclear antigen (hNA) and DAPI. (F–H) Vessels of small (F), medium (G), and large (H) diameter as in (D) were stained with antibodies against CD31 or αSMA. Scale bars, 100 μm. (I) Vascular barrier integrity testing by perfusion with FITC-dextran. Left: bright-field image before perfusion. Middle: intravital microscopy image of FITC-dextran retention after repeated perfusion and washing of the vascular network. Right: uptake of acetylated low-density lipoprotein (LDL, red). Nuclei were visualized with NucBlue Live ReadyProbes. Scale bars, 100 μm. (J) Light sheet microscopy image of vessel networks after fixation and staining with CD31 antibody. Scale bar, 400 μm. (K) Reseeded vascular constructs were transplanted into 8-week-old immunodeficient female rats and anastomosed (white arrow) with the host circulatory system. (L) Gross anatomical image of a transplanted graft after harvesting, showing the presence of host oxygenated blood and the absence of occlusion or leakage. (M–O) Harvested grafts stained with antibodies for CD31 (red) and αSMA (green). Small (M), medium (N), and (O) large blood vessels are shown. Scale bars, 100 μm. See also Figure S6 and Videos S1 and S2.