ISL1 cardiovascular progenitor cells for cardiac repair after myocardial infarction

Abstract

Cardiovascular progenitor cells (CPCs) expressing the ISL1-LIM-homeodomain transcription factor contribute developmentally to cardiomyocytes in all 4 chambers of the heart. Here, we show that ISL1-CPCs can be applied to myocardial regeneration following injury. We used a rapid 3D methylcellulose approach to form murine and human ISL1-CPC spheroids that engrafted after myocardial infarction in murine hearts, where they differentiated into cardiomyocytes and endothelial cells, integrating into the myocardium and forming new blood vessels. ISL1-CPC spheroid-treated mice exhibited reduced infarct area and increased blood vessel formation compared with control animals. Moreover, left ventricular (LV) contractile function was significantly better in mice transplanted with ISL1-CPCs 4 weeks after injury than that in control animals. These results provide proof-of-concept of a cardiac repair strategy employing ISL1-CPCs that, based on our previous lineage-tracing studies, are committed to forming heart tissue, in combination with a robust methylcellulose spheroid-based delivery approach.

Figure 1. Cardiac differentiation of mouse ISL1 cardiovascular progenitor cell–derived spheroids (mISL1-CPC spheroids).

(A) Test for spheroid formation using 96-U wells with (top left) or without (top right) methylcellulose, hanging drops (bottom left), or forced aggregation in 96-V wells (bottom right). Mouse ISL1-CPC spheroids were only formed in 96-U wells, in the presence of methylcellulose (top left, black arrow). Scale bars: 100 μm. (B) Mouse ISL1-CPCs were isolated by FACS and plated as monolayer culture (single cells) or spheroids on Matrigel-coated plates for 1 week. Cells were fixed and stained for cardiac troponin T (cTnT; green). Scale bar: 200 μm. Scale bar for inset pictures: 50 μm. (C) Staining for cTnT (green) in ISL1-CPC spheroids plated on the Matrigel-coated 4-well chamber slide for 6 days. Pictures represent ×20 magnifications (Scale bar: 50 μm) on the left and ×90 magnification (Scale bar: 20 μm) on the right. Note in the ×90 picture the presence of sarcomeric structures. (D) One week after plating on Matrigel, mISL1-CPC single cells or spheroids were subjected to FACS analysis to quantify the percentage of cardiomyocytes marked by the specific cardiac marker cTnT and the early cardiomyocyte marker smooth muscle α-actin (SMA). Quantifications of SMA⁺ cells and cardiomyocytes (cTnT⁺/SMA⁺) from n = 4 independent experiments (unpaired 2-tailed Student’s t test). **P < 0.01. See also Supplemental Figures 1 and 2 and Movies 1 and 2.

Figure 2. Mouse ISL1-CPC spheroid–derived cells in infarcted hearts.

(A) Bioluminescent images in a representative live animal injected with mISL1-CPC spheroids and recorded at 3 different time points: left (72 hours); middle (2 weeks); right (4 weeks). The far right: Graph representing the bioluminescence signal quantified in live animals injected with mISL1-CPC spheroids, n = 7. Statistical analysis performed with 1-way repeated measurements ANOVA with Greenhouse-Geisser correction (P = 0.35). (B) Bioluminescent image of a heart explanted 4 weeks after artery ligation and injection with mISL1-CPC spheroids. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. In A and B, the numbers in the scale bars were introduced manually to enlarge the font size. (C and D) Histological sections of explanted hearts 4 weeks after surgery, showing that mISL1-CPC spheroids differentiated into cardiomyocytes (C) and endothelial cells (D). Note the integration of donor endothelial cells in host blood vessels. Pictures in C represent ×10 (left), ×20 (second and third from left), and ×60 (fourth and fifth from left) magnification. Pictures in D represent ×10 (left), ×20 (second from left), and ×60 (third and fourth from left) magnification. White rectangles in ×10 and ×20 pictures represent the area magnified in ×20 and ×60, respectively. Y-chromosome FISH staining (red), cTnT (green), and CD31 (cyan). White arrowheads indicate the location in the ×60 pictures of Y-FISH⁺/cTnT⁺ and Y-FISH⁺/CD31⁺ cells in C and D, respectively. Scale bars: 100 μm in ×10, 50 μm in ×20, and 20 μm in ×60 pictures. (E) Quantification of double-positive Y-FISH/cTnT, Y-FISH/CD31, and Y-FISH/SMA cells in 4 infarcted hearts 4 weeks after surgery/cell injection. Seven slides, containing coronal sections, were analyzed per heart in the anatomical region where the bioluminescence was detected. See also Supplemental Figures 3 and 4.

Figure 3. Left ventricular heart function in infarcted mice implanted with murine ISL1-CPC spheroids.

(A) Fractional shortening (M-Mode) measured along the mid-left ventricular short axis, and ejection fraction (B-Mode) and end diastolic volume along the mid-part of parasternal long axis of mice subjected to ligation of the left coronary artery. No injury (n = 5), no treatment (n = 7 mice), Matrigel injection (n = 6 mice), mISL1-CPC spheroid injection (n = 7 mice). Three measurements were averaged per animal for each time point to obtain the M-Mode data. Samples were analyzed by 2-way ANOVA and pair comparisons between groups using unpaired 2-tailed Student’s t test (*P < 0.05; **P < 0.01), as well as pair comparisons for time points (48 hours vs. 4 weeks) within the same group using paired 2-tailed Student’s t test (^†P < 0.05; ^††P ≤ 0.01; ^†††P < 0.001). (B) Representative ultrasound images in systole and diastole recorded along the mid-left ventricular parasternal long axis (B-Mode) 4 weeks after surgery. Ao, aorta; Apx, apex; LA, left atrium; LV, left ventricle.See also Supplemental Figure 5, A and C.

Figure 4. Human ISL1-CPC spheroid formation.

(A and B) Day 6 human ISL1-CPC staining for ISL1 (green) and NKX2.5 (red) and quantification (B). Quantifications presented as mean ± SEM are: ISL1⁺ (94.53 ± 4.42); NKX2.5⁺ (98.04 ± 1.44); ISL1⁺/NKX2.5⁺ (94.35 ± 4.46). Scale bars: 50 μm in ×20 pictures and 20 μm in ×90 insets. Four random fields per experiment, containing a total of 9,000 cells were quantified. n = 3. (C) Test for human ISL1-CPC methylcellulose–induced spheroid formation in 96-U well plates with (right panel; +Y27632) and without 5 μM ROCK inhibitor Y27632 (left panel; control). Human ISL1-CPC spheroid formation was detected in 96-U well plates with methylcellulose in the presence of Y-27632, 12 hours after plating. Scale bars: 100 μm. See also Supplemental Figure 5D.

Figure 5. Human ISL1-CPC adhesion and cardiac differentiation.

(A) Human ISL1-CPC single cells or ISL1-CPC spheroids were plated on Matrigel-coated plates at a density of 1 × 10⁵ cells/well (24-well plate). Human ISL1-CPC single cells but not spheroids required 5 μM Y-27632, which was removed 24 hours after cell plating. While hISL1-CPC spheroids exhibited robust cell survival (green), hISL1-CPC single cell culture showed significant and progressive cell death 48 and 72 hours after seeding (red). Green: calcein staining for live cells. Red: ethidium homodimer staining for dead cells. Scale bars: 100 μm. (B and C) Human ISL1-CPC spheroids were seeded on Matrigel-coated wells and stained for cTnT (green, B) and CD31 (red, C) 10 days after plating. Scale bars: 50 μm for first, second, and third panels from left in B and 20 μm for fourth panel from left in B and panel C. The white rectangle in the third panel from left indicates the area shown in the magnified image displayed in the fourth panel. A magnified inset picture for cTnT is presented to show the sarcomeres in the fourth panel from left in B. See also Movie 3.

Figure 6. ROCK inhibition by Y-27632 induces RAC1 activation during human ISL1-CPC spheroid formation.

(A) RAC1 pull-down with PBD-PAK beads showing active RAC1 (RAC-GTP) in hISL1-CPCs treated with Y27632 during spheroid formation. Cell lysate before pull-down was used to detect total RAC1 and α tubulin (loading control). n = 3 (unpaired 2-tailed Student’s t test, **P < 0.01). (B) Human ISL1-CPC spheroid formation is abolished in the presence of RAC1 inhibitor (50 μM NSC23766) or JNK inhibitor (10 μM SP600125) but not P38 inhibitor (10 μM BIRB796) or ERK1/2 inhibitor (10 μM U0126). Arrows point to formed spheroids. Scale bars: 100 μm. (C) Staining for hypophospho β-catenin (green) and phalloidin (cyan) in hISL1-CPCs treated with vehicle control (top panels), Y27632 (middle panels), or Y27632 + NSC23766 (bottom panels) 6 hours after cell plating. Note the rounded morphology in cells treated with control or Y27632 + NSC23766 vs. the spread morphology in cells treated with Y27632 only. Scale bars: 20 μm.

Figure 7. Colocalization of RAC1 and IQGAP1 and cadherin expression during human ISL1-CPC spheroid formation.

(A) Staining for RAC1, IQGAP1, and phalloidin in control- or Y27632-treated hISL1-CPCs 2 hours after cell plating during spheroid formation. Note the colocalization of RAC1, IQGAP1, and actin in Y27632-treated cells in the cell-cell contact regions. Arrowheads show the absence of RAC1 in the membrane of cells treated with vehicle control. Scale bars: 20 μm. (B) Spheroid formation 48 hours after hISL1-CPCs plating in the presence of Y27632 or Y27632 + 2.5 mM EGTA. Scale bar: 100 μm. (C) Cadherin gene expression by qPCR. DSG1; Desmoglein 1. n = 4 (Mann-Whitney U test). *P < 0.05. (D) Staining for N-cadherin, hypophospho β-catenin, and phalloidin in control- or Y27632-treated hISL1-CPCs 8 hours after cell plating during spheroid formation. Scale bars: 20 μm.

Figure 8. Human ISL1-CPC spheroid–derived cells in infarcted hearts.

(A) Bioluminescence (BLI) imaging recorded in 1 mouse at 3 different time points after left anterior coronary artery ligation and human ISL1-CPC spheroid injection. Graph shows BLI quantification at 72 hours, 2 weeks, and 4 weeks after cell delivery. n = 8. Statistical analysis performed with 1-way repeated measurements ANOVA with Greenhouse-Geisser correction (P = 0.14). (B) Explanted heart 4 weeks after surgery and human ISL1-CPC spheroid injection showing BLI in the peri-infarct area. LV, left ventricle; RV, right ventricle. In A and B, the numbers in the scale bars were introduced manually to enlarge the font size. (C and D) Histological analysis of a heart, 4 weeks after MI. (C) Human ISL1-CPC spheroids differentiated into CMs in the midst of the scar. The white rectangle indicates the area shown in the magnified image displayed below. Scale bars: 50 μm (top panels) and 20 μm (bottom panels). (D) Human ISL1-CPC spheroids differentiated into functional blood vessels. The images marked by the numbers in the middle and bottom panels represent the magnified area demarcated by the rectangles in the top panels. HLA, human leukocyte antigen, which marks human ISL1-CPC–derived cells. Scale bars: 50 μm (top panels) and 20 μm (middle and bottom panels). (E) M-Mode fractional shortening recorded along the mid-left ventricular short axis, and B-Mode ejection fraction recorded along the parasternal long axis of mice subjected to ligation of the left coronary artery. Matrigel injection and human ISL1-CPC spheroid injection (n = 8 mice). Note that Matrigel-injected animals included six mice used in mouse ISL1-CPC studies. Three measurements were averaged per animal for each time point to obtain the M-Mode data. Samples were analyzed by 2-way ANOVA and pair comparisons using unpaired 2-tailed Student’s t test between groups (*P < 0.05), as well as pair comparisons for time points (2–3 days vs. 4 weeks) within the same group using paired 2-tailed Student’s t test (^††P < 0.01; ^†††P < 0.001). See also Supplemental Figure 5, B and C, and Figures 6 and 7.

Figure 9. Induction of YAP activation in host cardiomyocytes by human ISL1-CPC spheroids.

(A) Heart section of a representative mouse injected with Matrigel (top panels) or human ISL1-CPC spheroids (bottom panels) showing nuclear (active) YAP in host cardiomyocytes. Scale bar: 20 μm. (B) Graph representing host cardiomyocytes with active YAP. Two sections, in the mid-part of the heart and close to ligation, were quantified per heart. n = 3 hearts per experimental condition (unpaired 2-tailed Student’s t test). *P < 0.05. See also Supplemental Figure 8, A and B.

Figure 10. Human ISL1-CPC spheroids produced growth factors with cardio-protective properties.

(A) qPCR showing an upregulation of the expression of Neuregulin 1β (NRG1β), Vascular endothelial growth factor C (VEGF-C), and Angiopoietin 1 (ANGPT1) in hISL1-CPC spheroids. n = 4 (Mann-Whitney U test). *P < 0.05. (B) Representative Western blots and graph showing increased levels of active YAP (lower p-YAP/Total YAP ratio) in neonatal rat cardiomyocytes cultured under hypoxia and serum starvation in the presence of hISL1-CPC conditioned medium (CM). YAP activation was abolished when cardiomyocytes were treated with the ERBB inhibitor Lapatinib. n = 3 (unpaired 2-tailed Student’s t test, *P < 0.05, **P < 0.01). (C and D) NRG1β induces YAP activation (nuclear translocation) in neonatal rat cardiomyocytes under hypoxia and serum deprivation. Arrows indicate cardiomyocytes with nuclear YAP. Scale bars: 20 μm. Graph representing quantification of cardiomyocytes containing nuclear YAP normalized by total number of cardiomyocytes. n = 3 (unpaired 2-tailed Student’s t test, **P < 0.01). (E) Representative Western Blots and graph quantification showing NRG1-mediated activation of AKT in neonatal rat cardiomyocytes under hypoxia and glucose and serum deprivation. n = 4 (unpaired 2-tailed Student’s t test).See also Supplemental Figure 8, C–F.

Figure 11. Human ISL1-CPC spheroids produced growth factors with antifibrotic properties.

(A) SMA staining in human adult ventricular cardiac fibroblasts, 48 hours after treatment with the indicated growth factors. Scale bars: 50 μm. Graph showing reduced numbers of human cardiac fibroblasts with SMA⁺ filament bundle formation, 48 hours after treatment with NRG1β or ANGPT1. n = 4 (paired 2-tailed Student’s t test, *P < 0.05, **P < 0.01). (B) Western blots and graph quantification showing that NRG1β and ANGPT1 reduced collagen 1a1 protein and that NRG1β reduced SMA in human adult ventricular cardiac fibroblasts. n = 4 (paired 2-tailed Student’s t test, *P < 0.05).