Tracking of CM-DiI⁺/MPIO⁺ human ATMSCs and BMMSCs on three-dimensional reconstruction MRI and Micro CT Scanning.

<p>Exemplary three-dimensional reconstruction analysis confirmed labeled cell clusters of human CM-DiI⁺/MPIO⁺ ATMSCs after intramyocardial transplantation in the anterior-lateral and septal ventricular wall (<b>A and B</b>; <i>white arrows; anterior-lateral and septal view</i>). In addition, labeled cells were also detectable in the costal/para-vertebral area (<b>A and B</b>; <i>white *</i>) and in the Sinus phrenico-costalis (<b>A and B</b>; <i>white #</i>) indicative that the cells had been distributed in the fetal thorax. Abdominal 3D reconstruction analysis showed labeled cell clusters of CM-DiI⁺/MPIO⁺ human BMMSCs following intra-peritoneal injection within and around the liver (<b>C</b>; <i>white arrows; anterior-inferior view</i>) as well as in the anterior the Gerota's fascia (<b>D</b>; <i>white arrows; lateral view</i>). The intra-myocardial presence of CM-DiI⁺/MPIO⁺ human ATMSCs could be further confirmed showing several labeled cell clusters in the fetal myocardium on 2-dimensional as well as 3-dimensional high-resolution Micro CT analysis (<b>E</b>). <i>Scale Bar: 2 cm</i>.</p

Detection of CM-DiI⁺/MPIO⁺ human ATMSCs in the pre-immune fetal sheep myocardium via immunohistochemistry.

<p>Exemplary image series post intramyocardial transplantation of human ATMSCs into the fetal myocardium. Morphologically CM-DiI⁺/MPIO⁺ human ATMSCs could be easily identified within the fetal heart tissue suggesting to be in good shape and viable. The cells appeared to be integrated within the fetal myocardium and could be found as clusters as well as in the interstitial and intravascular spaces (<b>A–I</b>; <i>black arrows</i>). The cells stained positive for human specific Major Histo Compatibility Complex 1 (MHC-1) clearly confirming the human origin (<b>A and B</b>; <i>black arrows</i>) and also stained positive for anti-FITC detecting the Dragon Green fluorochrome labelled MPIOs within the human cells (<b>C and D</b>; <i>black arrows</i>). In addition, double staining for ALU Sequence and anti-FITC further confirmed the presence of the injected CM-DiI⁺/MPIO⁺ human ATMSCs within healthy heart tissue (<b>E and F</b>; <i>black arrows</i>) as well as in infarcted myocardium (<b>G–I</b>; <i>black arrows) Scale Bar: 100 um (A–C, E, G, H), 50 um (D, F, I)</i>.</p

Labeling of human BMMSCs and ATMSCs with superparamagnetic microspheres (MPIOs; co-labeled with Dragon-green fluorochromes) and CM-Dil.

<p>Successful labeling of human BMMSCs and ATMSCs was demonstrated on Prussian Blue staining before (<b>A and B</b>) and after MPIO labeling (<b>C and D</b>) as well as on immunofluorescence clearly showing the presence of MPIOs (<b>E–G</b>). After labeling, CM-Dil⁺/MPIO⁺ cells displayed excellent cell labeling efficiency in excess of 95% on FACS analysis (<b>H</b>). <i>Scale Bar: 100 um (A and C), 50 um (B, D–G)</i>.</p

Distribution of CM-DiI⁺/MPIO⁺ human mesenchymal stem cells in ovine fetal tissue by Flow Cytometry.

<p>Distribution of CM-DiI⁺/MPIO⁺ human mesenchymal stem cells in ovine fetal tissue by Flow Cytometry.</p

Preoperative & Procedural Data.

*<p> <i>the two animals that died had received human BMMSCs.</i></p><p>Animal Weight (gr) / Size (cm): 331 ± 81 / 20 ± 2. Overall Survival (%): 10 (77%).</p

Concept of intra-uterine induction of myocardial infarction and intra-myocardial stem cell delivery into the pre-immune fetal sheep.

<p>The uterus was exteriorized through a maternal midline laparotomy. Following digital palpation of the fetus, the uterus was opened through a 10 cm incision. The fetal chest was opened via a left-sided mini-thoracotomy <i>(4^th intercostal space)</i> (<b>A</b>). After sharp dissection of the pericardium, the heart was positioned for an optimal access of the anterior wall and the apex (<b>A and D</b>). After evaluation of the myocardial vasculature, the left anterior descending coronary artery (LAD) and the diagonal branches (DB) were identified (<b>E</b>). To achieve a sufficient myocardial infarction involving the anterior wall and the apex, the LAD (± appropriate diagonal branches) were suture ligated using a 7/0 suture (<b>B and F</b>). Sufficient ligation was confirmed by instant changes of the regional wall movement and the colour in the anterior-apical area (<b>B and F</b>). After ligation of the coronary vessels, the 5–6 target zones for stem cell delivery were defined. Following careful exposure of the fetal heart, the cells were slowly injected into the fetal myocardium (<b>C and G</b>). <i>Scale Bar: 5 cm (D–G)</i>.</p

Assessment of cell fate and early bio-distribution of injected of human ATMSCs and BMMSCs via Flow Cytometry and PCR analysis.

<p>Flow cytometric analysis in an exemplary recipient after direct intra-myocardial injection (IMI) of human ATMSCs. CM-DiI⁺/MPIO⁺ human ATMSCs were primarily detected within the heart and within the spleen (<b>A and B</b>). In an animal that had received intra-peritoneal injection (IPI) of human BMMSCs, CM-DiI⁺/MPIO⁺ BMMSCs were primarily identified within the lymphatic organs, in particular within spleen, while no CM-DiI⁺/MPIO⁺ cells were found within the heart (<b>C and D</b>). Intra-myocardial presence of CM-DiI⁺/MPIO⁺ human ATMSCs was further confirmed via PCR analysis using human β-2 microglobulin (<b>E</b>), a component of the class I antigen complex. Negative controls from non-injected sheep hearts as well as human mesenchymal stem cells used as a positive control clearly confirmed the human specificity of the staining for human β-2 microglobulin within the fetal heart: Agarose gel analysis of human β-2 microglobulin <i>(left panel, A)</i> and β -actin <i>(right panel, B)</i> PCR products. Lane 1: molecular size marker (100 bp DNA ladder, Genecraft, Germany). Lane 2: human mesenchymal stem cells as a positive control. Lane 3: ovine fetal heart after human ATMSCs injection. Lane 4: tissue from sheep heart as a negative control for the human β-2 microglobulin sequence.</p

Assessment of intra-uterine induction of myocardial infarction in the pre-immune fetal sheep.

<p>Induction of acute myocardial infarction (MI) was confirmed by instant changes of the regional wall movement and the colour in the anterior-apical area (<b>A</b>; <i>purple discolouration; white arrow</i>). The area of myocardial infarction (MI) could be easily identified showing the typical areas of necrosis when compared to the surrounding border zone and the healthy myocardium. In detail, loss of the classical myocardial morphology, necrotic cell death with the loss of the nuclei and cardiac proteins as well as the complete loss of entire muscle fibres could be observed (<b>B</b>; <i>magnification x2.5</i>). MI was further confirmed on histology via positive staining for activated caspase-3 (<b>C and D</b>; <i>black arrows</i>) and TUNEL (<b>E</b>; <i>black arrows</i>) in fetal infarcts at 7 days suggesting programmed cardiomyocyte death within the infracted region. <i>Scale Bar: 500 um (B), 100 um (C–E)</i>.</p

Characterization of human BMMSCs and ATMSCs.

<p>Cell surface proteins of human BMMSCs and ATMSCs were evaluated by flow cytometric analysis (<b>panel A and B</b>; <i>blue population represents isotype control</i>). Positive expression of CD44, CD73, CD90, CD105, CD166 and none/weak expression of CD146 was observed for both MSC cell types. Human BMMSCs (<b>C–E</b>) and ATMSCs (<b>F–H</b>) demonstrated their differentiation potential to the adipogenic <i>(Oil Red O Staining, (C, F))</i>, osteogenic <i>(Alizarin Red S Staining, (D, G))</i> and chondrogenic lineages <i>(Toluidine Blue Staining, (E, H)). Scale Bar: 100 um</i>.</p

Tracking of CM-DiI⁺/MPIO⁺ human ATMSCs and BMMSCs on two-dimensional magnetic resonance imaging (MRI).

<p>CM-DiI⁺/MPIO⁺ human ATMSCs and BMMSCs could be detected after stem cell transplantation on high resolution 4.7 Tesla MRI. Exemplary image series of an animal that had received an intra-myocardial injection of CM-DiI⁺/MPIO⁺ human ATMSCs the labeled cell clusters were clearly visible as areas of strong focal signal loss in the anterior-lateral and septal ventricular wall corresponding to the injection sites (<b>A and B</b>; <i>white arrows; axial and sagittal view</i>; <b>and </b><b>G</b>; <i>black arrow</i>; <i>showing a non-injected myocardium as respective negative control</i>). Next, an exemplary image series of an animal that underwent intra-peritoneal injection of CM-DiI⁺/MPIO⁺ human BMMSCs displayed the distribution within the entire intra-peritoneal cavity (<b>C–E</b>; <i>white arrow heads</i>; <b>and H</b>; <i>black arrows</i>; <i>showing a non-injected intra-peritoneal cavity as respective negative control</i>) involving the liver (<b>C</b>; <i>white arrow heads; axial and view</i>), the kidneys, the Gerota's fascia (<b>D</b>; <i>white arrow heads; axial view</i>), diffuse cell clusters in the surrounding intra-peritoneal areas (<b>D</b>; <i>white arrow heads; axial view</i>) as well as between the intestines (<b>E</b>; <i>white arrow heads; sagittal view</i>). Comparing the dilution series (<b>F</b>) of MPIO labeled mesenchymal stem cells to the morphological images, the amount of cells in the myocardium could be estimated to approximately 1×10⁵–5×10⁵ cells and the cells found in the liver to 5×10⁵ cells and around the kidneys to 5×10⁵–1×10⁶ cells (<b>A–D and F</b>). <i>(Cell Dilution: 1×10⁶</i><i>[1]</i>, <i>5×10⁵</i><i>[2]</i>, <i>1×10⁵</i><i>[3]</i>, <i>5×10⁴</i><i>[4]</i>, <i>2×10⁴</i><i>[5]</i><i>. Scale Bar: 2.5 cm</i>.</p