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a Cardiovascular Surgery and Cardiology Department, Hôpital Haut-Lévêque, Pessac 33604, France
b Medical University of Victor Segalen Bordeaux 2, Bordeaux, France
c Inserm U828, 33600 Pessac, France
Received 15 January 2008; received in revised form 11 March 2008; accepted 14 March 2008.
* Corresponding author. Address: Cardiovascular Surgery Department, Haut-Lévêque Hospital, Avenue de Magellan, 33600 Pessac, France. Tel.: +33 5 57 89 19 79; fax: +33 5 56 36 89 79. (Email: laurent.barandon{at}chu-bordeaux.fr).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionReferences |
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Key Words: Mesenchymal stem cell • Bone marrow cells • Progenitor cells • Tissue regeneration • Engraftment • Angiogenesis
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionReferences |
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We have previously developed a 3D scaffold method for cardiac tissue engineering in a mouse model of myocardial infarction, in which a muscle patch containing bone marrow cell (BMC) embedded into a 3D collagen matrix is maintained vis-à-vis the myocardial infarct area. In this model, we showed that BMC were able to migrate from the muscle patch into the infarct zone, to increase capillary density and restore cardiac function. The beneficial effect of this cell therapy on ischemic myocardium was likely due to a paracrine release of pro-angiogenic factors and cytokines, thereby facilitating cell survival, cell recruitment and angiogenesis [4].
Bone marrow-derived subpopulations, i.e., endothelial progenitor cells (EPC) or mesenchymal stromal cells (MSC), have shown promise in regenerative medicine [5,6]. MSC have indeed demonstrated their ability to regenerate vessels and damaged myocardium in several experimental studies [7–9]. EPC are present in the systemic circulation, increase in response to tissue ischemia, home to and incorporate into sites of neovascularization [10]. Moreover, mobilization and recruitment of EPC contributes to vasculogenesis in vivo [11]. EPC and MSC appear to be attractive cells in therapy because of their role as source of cytokines and growth factors but also because of their supposed cardiomyocyte and endothelial transdifferentiation potential [12,13]. As stem cells reside in highly regulated microenvironments called niches [14,15], a recent scenario for stem cell therapy envisioned the use of a mixture of bone marrow cells with enriched purified stem cells to recreate a niche in the treated tissue.
In view of these findings, we sought to assess the fate and outcome of purified and expanded EPC and MSC on vascular and cardiac regeneration into the infarct myocardium following engraftment in a 3D collagen patch model. In addition, as environment has a crucial role in the commitment of stem cells, we compared the functional benefit of mixing unfractionated BMC with either purified progenitor cell populations on angiogenesis and myogenesis within the ischemic heart.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionReferences |
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2.1.2 Endothelial progenitor cells (EPC)
1 x 106/ml mononuclear cells without any further cell subpopulation enrichment procedure were plated on different culture dishes coated with fibronectin (Sigma) in endothelial basal medium (EBM2) (Clonetics Inc., San Diego, California, USA) supplemented with endothelial growth medium Single Quots and 20% FCS, and incubated at 37 °C in a 5% CO2 humidified environment. After 24 h, unattached cells and debris were removed by washing with medium. Culture medium was changed every 5 days, and cells were characterized and used after 12 days of the primary culture.
2.1.3 Mesenchymal stem cells (MSC)
Mononuclear cells were resuspended and seeded at a density of 1 x 106
cells/cm2 in petri dishes and cultured in McCoy's medium (Invitrogen) with 10% BFS and 10% horse serum. Five to seven days later the nonadherent hematopoietic cells were discarded. The adherent bone MSCs were maintained in a humidified incubator at 37 °C with 5% CO2 in the absence of any exogenous growth factor or anchoring materials such as fibronectin or collagen. The attached cells grew and developed colonies in 15–30 days, when the colonies were subcloned and re-seeded. Five selected clones were passed more than eight times prior to their characterization and further use. These clones displayed all the same phenotypic characteristics.
2.2 Phenotype characterization of EPC and MSC
To detect the uptake of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine-labeled acetylated LDL (DiI-acLDL), cells were incubated with DiI-acLDL (2.5 µg/ml) (molecular probe) at 37 °C for 1 h.
MSC and EPC were characterized using immunostaining. Cells from cultures were rinsed twice with PBS and fixed immediately with 2% PFA for 10 min, permeabilized (0.2% Triton X-100, 2 min) and saturated with 5% of BSA (Sigma). Endothelial or EPC markers were used: CD31 (Pharmingen), CD34 (Immunotech Inc.), and VE-cadherin (Santa Cruz Biotechnology Inc.), leukocytes markers: CD45 (30-F11, Pharmingen), CD4 (W3/25, Serotec), CD3 (Becton Dickinson), mesenchymal stem cell markers Sca1 (D7, Becton Dickinson), cKit (Santa Cruz), Thy1 (sc9163, Santa Cruz), Endoglin (sc18893, Santa Cruz), yoyo-1 (Molecular Probes) for nucleus staining. Biotinylated secondary antibodies used were: anti-rabbit IgG or anti-mouse IgG (Amersham), or anti-goat IgG (Jackson ImmunoResearch Laboratories Inc). Streptavidin–FITC complex or Alexa 568 was used for immunofluorescence analysis (Molecular Probes). All primary and secondary antibodies were diluted in PBS plus 1% BSA.
Total RNAs were prepared from cultured cells in guanidinium thiocyanate buffer, and reverse transcription-polymerase chain reactions (RT-PCR) were carried out as previously described [16]. Negative controls without RT were prepared in parallel for each RNA sample. The list of primers used to characterize each cell type is shown in Table 1 .
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2.4.1 Study treatments
Mice were randomly assigned to five treatment groups (Fig. 1A). One group underwent sham treatment (i.e., rethoracotomy and immediate closure) 30 days after MI induction. The other groups received rethoracotomy and placement of a muscle patch coated with EPC or MSC ± BMC adjunction 30 days after MI induction (Fig. 1A). All groups were also injected with BrdU 24 h before sacrifice in order to measure the rate of cell proliferation. All mice that survived for 15 days were sacrificed.
2.4.2 Isolation and placement of muscle patches
The muscle patches were obtained as previously described [4]. Briefly, 30 days after MI, a 1 cm x 1 cm patch of abdominal muscle was harvested and cupped so as to fit the left ventricular geometry and form a tank in which cells were deposited and secured (Fig. 1C and D).
2.4.3 Deposition of stem cells on muscle patch
We used 5 x 106 BMC, 5 x 105
eGFP/MSC and 5 x 105
eGFP/EPC in their corresponding groups. These stem cells were mixed into a growth factor-deprived liquid matrix rich in collagen type 1 (200 µl, 200 mg/ml, Becton Dickinson) [4]. This mixture was then deposited within the muscle patch and the coated muscle patch was positioned on the exposed epicardium directly above the MI zone.
2.5 Hemodynamic analysis
Hemodynamic analysis was performed as previously described using a 1.4F Millar transducer (Millar Instruments, Houston, TX) [4].
2.6 Histological analysis
2.6.1 Morphometric analysis
Infarct size was determined in a minimum of six mice in each treatment group as described previously [17].
2.6.2 Immunochemistry and confocal analysis
Mice were sacrificed by lethal injection of potassium chloride. Their hearts were fixed by pressure perfusion with a 4% paraformaldehyde solution and cut perpendicularly to the longitudinal axis of the LV in the middle of the patch. These two sections were embedded in OCT compound and stored at –80 °C. To detect cell repopulation in the scar, tissue sections were incubated with popo (popoTM-3 iodide, Molecular Probes). To detect angiogenic activity, endothelial cells were stained with CD31 antibody (Pharmingen). To determine the extent of transplanted cell proliferation, specimens were immunostained with a BrdU antibody (Harlan). A minimum of 30 random digital photographs was taken at x40 magnification for each mouse specimen analyzed. Positively stained eGFP cells or CD31-positive capillaries and BrdU positive cells were manually counted by a blinded observer with the help of Sigma Scan Plot software.
To evaluate cell differentiation, a CD31 antibody (BMA) was used for endothelial cell detection, a smooth muscle 
-actin antibody for smooth muscle cell detection, and an anti--cardiac sarcomeric actin (Abcam, 5C5) for myocyte detection. Slides were analyzed under a confocal microscope searching for eGFP/rhodamine double staining.
The number of transplanted cells, capillary density, and cell proliferation per square millimeter were determined and recorded.
2.7 Statistical analysis
All data were expressed as mean ± SD. All analyses were performed using appropriate software (Statview 5.1). Comparisons of continuous variables between two groups were made using one-way ANOVA and, when a statistically significant difference was observed, a two-sided paired t test. A value of p
< 0.05 was considered statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionReferences |
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3.1 Phenotype of EPC and MSCs
We used both EPC and MSC to test their vascular or cardiogenic potential in vitro and in vivo. First, the expression of genes of primitive and fully differentiated endothelial and smooth muscle cells was analyzed in both cell lines.
The surface markers of EPC were determined by immunostaining and flow cytometry analyses as previously reported. EPC were co-stained with acetylated-LDL (Fig. 2A). EPC expressed VEGF Receptor 2, CD31 and CD34 (data not shown). Real-time PCR amplification showed that EPC featured expression of genes reflective of endothelial lineages (VE-cadherin, Flk-1, Flt-1, angiopoietin-1 and 2, Tie-1 and 2) (Fig. 2A). MSC expressed moderate to high level of Sca1, cKit, Thy1 while Endoglin, CD31, CD34, CD3 and CD45 were negative (data not shown and Fig. 2A). To track engrafted cells in vivo, both EPC and MSC were infected at a low level with a lentiviral vector expressing GFP that did not alter in vitro growth rate and viability. Almost 100% of the cells expressed GFP after a single round of infection.
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3.3 MSC but not EPC can migrate from the implanted patch to the infarcted area
We then investigated the capacity of the progenitors to penetrate and migrate into the ischemic and scar area. In MI hearts covered with a patch containing MSC, 46.6 ± 3.1 eGFP-positive MSC/mm2 were detected in the MI area, representing about 1.5% of the total cells of the scar. Most of eGFP-positive cells were detected in the ischemic border zone while none were found in uninjured zones. In contrast, only sporadic eGFP-positive EPC were detected in the MI area in patch plus EPC-treated mice (Table 2
and Fig. 3
). These findings suggested that, in this model of epicardial cell deposition, only MSC but not EPC could be successfully mobilized to migrate to the MI area.
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3.5 Mobilization of progenitor cells did not contribute to the regeneration of vascular endothelium, smooth muscle or cardiac tissue
Here, we analyzed whether mobilized progenitor cells can differentiate in situ to form capillary network or to regenerate cardiomyocytes using immunofluorescent techniques. In the myocardial area, a few of eGFP-positive MSC cells (less than one per MI heart) were positive for CD31 or -actin staining using confocal microscopy analysis (Fig. 4
), suggesting that transdifferentiation or fusion between transplanted and resident cells were very rare events. Moreover, no transdifferentiation into cardiomyocytes was observed as eGFP-positive MSC cells remained negative for -sarcomeric actin (Fig. 4).
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An eGFP-MSC mix with BMC adjunction led to a higher quantity of MSC in the scar as compared with eGFP-MSC alone (p < 0.01; Table 2). A mean of 286 ± 11.5 eGFP-positive MSC/mm2 was detected in the necrotic area, representing 7% of the cells into MI area, in the group MSC plus BMC (Table 2, Fig. 3). In contrast, BMC adjunction to EPC in the patch did not induce EPC migration in the scar. We next tested whether BMC could provide a microenvironment that would contribute to MSC commitment. We failed to detect any progenitor cell that co-expressed eGFP and either endothelial or cardiac markers indicating that BMC environment did not favor MSC incorporation into neovascular structure or into cardiac tissue.
Noteworthy, BMC adjunction modified infarct scar tissue since the total cell number in the scar was significantly higher in the BMC plus EPC or MSC groups than in the progenitor cells alone groups (Table 2). One of the consequences was a significant increase in scar thickness, probably caused by an increase in cell density due to BMC invasion. However, cell proliferation and capillary density were not significantly different between the progenitors groups with or without BMC (Table 2).
3.7 MSC and EPC engraftment improved cardiac function
Infarct size as measured by the percentage of left ventricle necrosis was similar in all groups, control, and EPC or MSC-patch with or without BMC. This was not surprising as the cryolesion-induced MI was done 30 days before cell therapy was applied. Cardiac function as measured by dp/dt was significantly increased in all groups compared to the control group. However no significant differences were found between the four progenitor groups (Table 3
).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionReferences |
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4.1 Background and muscle patch cell delivery
In a precedent study, we showed the feasibility of improving cardiac function and remodeling irreversibly damaged myocardial tissue after MI by embedding BMCs in a collagen-rich matrix, applying the matrix directly to the damaged epicardium. The intramyocardial injection technique is limited by (1) the extensive mechanical trauma to cells (and consequent apoptosis) that can be caused by the syringes and needles that must be used, (2) the limited total number of cells in solution that can be injected, and (3) and the inhospitability of the scar environment to transplanted cells. Thus, in developing our epicardial deposition strategy, we have tried to offset these disadvantages by making it possible to (1) use unrestricted numbers of transplanted cells; (2) deposit cells in a trauma-free way that avoids the use of inappropriate devices and avoids immunogenic reactions; (3) place transplanted cells in a favorable environment (i.e., collagen) conducive to their survival, proliferation, and migration; and (4) use a homologous muscle patch as a reservoir in which to allow transplanted cells in the matrix to form a stronger, more adherent bond with the patch itself. We demonstrated that this combined strategy could increase capillary density, improve cardiac function and increase scar thickness. The effect on scar thickness could be related to an important cell migration, cell survival and proliferation rate. Moreover, we had found that autologous abdominal muscle patches could become easily integrated into, and in some cases even merge with, the epicardium. These advantages were offset somewhat by the tendency of the abdominal muscle patches to become thinner over time because of ischemic complications; however, this thinning was significantly less severe when the patch was coated with BMCs, presumably owing to the evident tropic and degradation-inhibiting effects of the BMCs. We were not able to demonstrate any inflammatory response in the different sites of fixation. This data showed that the paracrine effect, increasing capillary density in the scar, could not be related to a secretion of growth factor by the postoperative inflammatory response [4].
4.2 Progenitor cell differentiation
Our goal was to define the optimal cell type for myocardial revascularization in this epicardial deposition model. It has been demonstrated that, in some culture conditions, progenitor cells are able to transdifferentiate into vascular cells or cardiomyocytes [18,19]. We isolated and cultured MSC and EPC that presented the same characteristics and properties as previously reported [20,21]. To date, EPC and MSC have been used for cardiac regeneration [2,9,22]. Functional efficacy of such progenitor cells has been well documented, even if there are some controversies in their in vivo and in vitro differentiation abilities [23,24]. However, with more than 50 mice carefully examined, we found no evidence of vascular or myocardial regeneration from immunolabeled injected progenitor cells.
4.3 Modification of infarct scar
3D engraftment of EPC and MSC has improved cardiac function although scar area was not reduced by any of the cell types. Indeed, we chose a delayed timing of cell delivery to make the protocol more clinically relevant. In a clinical perspective, such a deleterious environment remains a major step for rationalizing the progenitor use. It is also possible that this delay of 1 month after MI onset for progenitor engraftment could be a limit in the repair benefit. Nevertheless, MSC, but not EPC, were able to migrate to the infarct scar but to a limited extent (1% of the scar cellularity), limiting their functional potential in situ.
However, independent of their migration capacities, both progenitor cells induced modifications of the infarcted myocardial tissue (increased in capillary density and cell proliferation) as compared to control mice. As we did not detect any transdifferentiation into endothelial or smooth muscle cells or cardiomyocytes, the angiogenic effect and cell proliferation in the scar were probably induced via a paracrine effect of the progenitors. In vitro characterization has shown that both MSC and EPC were able to secrete growth factors, angiogenic factors and some cytokines (laboratory unpublished data) [13,25].
4.4 Bone marrow cell environment benefit
We investigated whether the adjunction of BMC could enhance EPC or MSC numbers in the infarct area. Mix of MSC with unfractionated BMC gave rise to larger MSC fraction in the scar than that with MSC alone. EPC did not migrate in the scar even if BMC were added. This suggests that BMC create a specific and favorable environment that facilitate and/or preserve MSC repopulation into the scar. This further emphasizes that BMC environment is crucial to ensure a proper MSC engraftment or migration to the scar but may not allow their commitment toward a vascular or myogenic phenotype. We should however point out that the increase in MSC population did not account for a better capillary formation or cell proliferation.
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5. Conclusion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionReferences |
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Footnotes |
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This work was supported by a grant from the European vascular genomics network (EVGN, # 503254). L.B. is a recipient of a grant from the Groupe de Réflexion pour la Recherche Cardiovasculaire and from the Fondation pour la Recherche Médicale. |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionReferences |
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  H. Hamdi, A. Furuta, V. Bellamy, A. Bel, E. Puymirat, S. Peyrard, O. Agbulut, and P. Menasche Cell Delivery: Intramyocardial Injections or Epicardial Deposition? A Head-to-Head Comparison Ann. Thorac. Surg., April 1, 2009; 87(4): 1196 - 1203. [Abstract] [Full Text] [PDF]
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 C. Heilmann Editorial comment Myocardial regeneration by stem cells: still a black box. Eur. J. Cardiothorac. Surg., August 1, 2008; 34(2): 255 - 255. [Full Text] [PDF]
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