Transplantation of purified iPSC-derived cardiomyocytes in myocardial infarction

Abstract

Background: Induced pluripotent stem cells (iPSC) can be differentiated into cardiomyocytes and represent a possible autologous cell source for myocardial repair. We analyzed the engraftment and functional effects of murine iPSC-derived cardiomyocytes (iPSC-CMs) in a murine model of myocardial infarction.

Methods and results: To maximize cardiomyocyte yield and purity a genetic purification protocol was applied. Murine iPSCs were genetically modified to express a Zeocin™ resistance gene under control of the cardiac-specific α-myosin heavy chain (α-MHC, MYH6) promoter. Thus, CM selection was performed during in vitro differentiation. iPSC-CM aggregates ("cardiac bodies", CBs) were transplanted on day 14 after LAD ligation into the hearts of previously LAD-ligated mice (800 CBs/animal; 2-3x106 CMs). Animals were treated with placebo (PBS, n = 14) or iPSC-CMs (n = 35). Myocardial remodeling and function were evaluated by magnetic resonance imaging (MRI), conductance catheter (CC) analysis and histological morphometry. In vitro and in vivo differentiation was investigated. Follow up was 28 days (including histological assessment and functional analysis). iPSC-CM purity was >99%. Transplanted iPSC-CMs formed mature grafts within the myocardium, expressed cardiac markers and exhibited sarcomeric structures. Intramyocardial transplantation of iPSC-CMs significantly improved myocardial remodeling and left ventricular function 28 days after LAD-ligation.

Conclusions: We conclude that iPSCs can effectively be differentiated into cardiomyocytes and genetically enriched to high purity. iPSC derived cardiomyocytes engraft within the myocardium of LAD-ligated mice and contribute to improve left ventricular function.

Fig 1. Cardiac bodies and cardiomyocytes derived from murine iPSCs.

A: CBs after antibiotic selection (dd14, brightfield view); inset: CBs after CFDA SE tracer staining (dd14). Scale bars: 100μm. B: CBs are positive for cTNT and show CMs with sarcomeric striations (inset, arrow). Scale bar: 50μm. C+D: CBs at dd14 are positive either for MLC2A or MLC2V indicating spontaneous differentiation into both an atrial and ventricular phenotype; negative CBs are marked with *, respectively. Scale bars: 50μm. E+F: Reseeded CMs exhibit a mature sarcomeric intracellular organisation. Staining for cTnT and α-sarcomeric actinin shows Z-lines (arrows). Scale bars: 50μm. G: Relative amount of reseeded CMs expressing cardiac markers α-sarcomeric actinin (99.1±1.5%), cTnT (99.3±0.4%), Titin (99.4±0.3%), MLC2A (47.3±1.9%) and MLC2V (52.1±1.8%); N = 8.

Fig 2. Genetically purified iPSC-derived CMs form mature grafts in vivo.

A+B: CFDA SE cell tracer positive iPSC-CM grafts 7 days after intramyocardial transplantation: Adjacent to the host myocardium iPSC-CMs align in a parallel, longitudinal fashion and exhibit sarcomeric structures (arrows). Within central portions of broader grafts (approximately > 200 μm) they maintain a small, round shape (* in A). In the infarct penumbra iPSC-CMs lie in close proximity to host CMs (arrowheads in B1), occasionally with direct cell contact (arrowheads in the bottom right corner of B1). Inside the infarct area iPSC-CMs are typically surrounded by infiltrating host cells (arrowheads in B2). Tissue disruption during histological preparation (* in B1+2) indicates loose cell adhesion within iPSC-CM grafts. C+D: CFDA SE cell tracer positive iPSC-CM graft 28 days after intramyocardial transplantation: The cell tracer remains visible 28 days after engraftment, but iPSC-CMs develop an amorphic appearance. Sarcomeric structures are not observed. Vacuoles form during histological preparation (* in C). A+D: brightfield overlay. Scale bars: 400μm.

Fig 3. Graftsize after intramyocardial transplantation of iPSC derived CMs.

Intramyocardial grafts were detected by their CFDA SE fluorescence on POD7 and POD28. Graft size (μl): after 7 days: 7.6±2.5; after 28 days: 0.78±0.21. *** P<0.001.

Fig 4. Intramyocardial transplantation of iPSC derived CMs improves ventricular remodeling and function after myocardial infarction.

Hemodynamic evaluation by magnetic resonance imaging (MRI; POD 27; A+B) and conductance catheter analysis (CC; POD 28; C-F). A: Left ventricular ejection fraction (LV-EF [%]) as measured by MRI: On POD 2: Sham²⁸ = 56±5; PBS²⁸ = 39±5; iPSC-CM²⁸ = 47±3. On POD 27: Sham²⁸ = 60±4; PBS²⁸ = 19±2; iPSC-CM²⁸ = 34±4 B: End-diastolic volume (EDV [μl]) as measured by MRI: On POD 2: Sham²⁸ = 34±3; PBS²⁸ = 38±5; iPSC-CM²⁸ = 37±2. On POD 27: Sham²⁸ = 40±3; PBS²⁸ = 99±9; iPSC-CM²⁸ = 73±6 C: LV-EF (%) as measured by CC on POD 28: Sham²⁸ = 59±1; PBS²⁸ = 18±1; iPSC-CM²⁸ = 33±3 D: End-diastolic volume (EDV [μl]) as measured by CC on POD 28: Sham²⁸ = 13±1; PBS²⁸ = 30±1; iPSC-CM²⁸ = 21±2 E: maximum Pressure increase (ΔP/dt max. [mmHg/sec]) as measured by CC on POD 28: Sham²⁸ = 5863±351; PBS²⁸ = 2893±207; iPSC-CM²⁸ = 4135±232 F: Preload adjusted maximal power (mWatts/μl²) as measured by CC on POD 28: Sham²⁸ = 205±23; PBS²⁸ = 20±3; iPSC-CM²⁸ = 62±8. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (for group comparison); ##P<0.01; ###P<0.001; ####P<0.0001 (for paired longitudinal comparison).

Fig 5. Intramyocardial transplantation of iPSC derived CMs alleviates adverse myocardial remodeling and increases the amount of viable myocardium.

A: Infarct size (%): after 2 days (MRI): PBS²⁸ = 34±3; iPSC-CM²⁸ = 36±4; after 28 days (Masson’s): PBS²⁸ = 46±3; iPSC-CM²⁸ = 25±4 B: LV wall thickness after 28 days (Masson’s; μm): iPSC-CM⁷ = 990±57; iPSC-CM²⁸ = 669±64; PBS²⁸ = 328±12 C: Expansion index after 28 days (Masson’s): iPSC-CM⁷ = 1.1±0.1; iPSC-CM²⁸ = 2.9±0.5; PBS²⁸ = 4.5±0.4 D: Viable myocardium (Masson’s; % of infarct area): iPSC-CM⁷ = 32±2; iPSC-CM²⁸ = 46±1; PBS²⁸ = 15±1. iPSC-CM²⁸ vs. PBS²⁸: * P<0.05, ** P<0.01, **** P<0.0001. iPSC-CM⁷ vs. iPSC-CM²⁸: # P<0.05, ##P<0.01; ###P<0.001; ####P<0.0001.