MOESM2 of Human stem cells express pannexins

Additional file 2: Figure S1. Relative expression of germ layer-specific marker mRNAs in the differentiated stem cell lines (A) hCBiPS2, (B) HSC-1285, and (C) HES-3. The following symbols indicate a significant difference in relative mRNA expression with p < 0.05: # against each other marker; Δ against WNT1, TTR, AFP, and NKX2-5; □ against WNT1, TTR, NKX2-5, and o against WNT1, AFP, NKX2-5, and MYL7

Transplantation of purified iPSC-derived cardiomyocytes in myocardial infarction

<div><p>Background</p><p>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.</p><p>Methods and results</p><p>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. <i>In vitro</i> and <i>in vivo</i> 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.</p><p>Conclusions</p><p>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.</p></div

Genetically purified iPSC-derived CMs form mature grafts <i>in vivo</i>.

<p><b>A+B:</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. <b>C+D:</b> 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.</p

Cardiac bodies and cardiomyocytes derived from murine iPSCs.

<p><b>A:</b> CBs after antibiotic selection (dd14, brightfield view); inset: CBs after CFDA SE tracer staining (dd14). Scale bars: 100μm. <b>B:</b> CBs are positive for cTNT and show CMs with sarcomeric striations (inset, arrow). Scale bar: 50μm. <b>C+D:</b> 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. <b>E+F:</b> Reseeded CMs exhibit a mature sarcomeric intracellular organisation. Staining for cTnT and α-sarcomeric actinin shows Z-lines (arrows). Scale bars: 50μm. <b>G:</b> 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.</p

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

<p>Hemodynamic evaluation by magnetic resonance imaging (MRI; POD 27; A+B) and conductance catheter analysis (CC; POD 28; C-F). <b>A</b>: 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>B:</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 <b>C:</b> LV-EF (%) as measured by CC on POD 28: Sham²⁸ = 59±1; PBS²⁸ = 18±1; iPSC-CM²⁸ = 33±3 <b>D:</b> End-diastolic volume (EDV [μl]) as measured by CC on POD 28: Sham²⁸ = 13±1; PBS²⁸ = 30±1; iPSC-CM²⁸ = 21±2 <b>E:</b> 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 <b>F:</b> Preload adjusted maximal power (mWatts/μl²) as measured by CC on POD 28: Sham²⁸ = 205±23; PBS²⁸ = 20±3; iPSC-CM²⁸ = 62±8. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; ****<i>P</i><0.0001 (for group comparison); ##<i>P</i><0.01; ###<i>P</i><0.001; ####<i>P</i><0.0001 (for paired longitudinal comparison).</p

Graftsize after intramyocardial transplantation of iPSC derived CMs.

<p>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. *** <i>P</i><0.001.</p

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

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

Myocardial microsphere concentrations and venous drainage of microspheres.

<p><b>A</b> Comparision of myocardial microsphere concentrations after injection <i>ex vivo</i> (EVMI) and <i>in vivo</i> (IVMI). <b>B</b> Myocardial fluorescence analysis unveils venous drainage (arrows) from the injection zone (*) to the right atrium (**).</p

Distribution of microspheres after injection in vivo (IVMI).

<p>The IVMI-group showed distribution to both lungs (right lobe: 1 – left lobe: 3) 10 minutes after injection into the heart (2). Additionally, the homogenization (Organ – Filter – Homogenate) process involved filter retention of microspheres leading to lower microsphere counts in homogenate dilutions.</p

Ex vivo microsphere injection (EVMI).

<p><b>A</b> Macroscopic fluorescence imaging of a murine heart following injection of 5×10⁵ microspheres. The injection zone (blue circle) shows low (dark red) to high (yellow) microsphere concentrations. For proper fluorescence analysis, background fluorescence (dotted blue circle) must be assessed on every image. <b>B</b> Histological assessment of a murine heart after microsphere injection <b>C</b> Augmentation of the microsphere distribution passage inside the left ventricle (LV).</p