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Transplanting hematopoietic and peripheral blood-derived stem/progenitor cells can have beneficial effects in slowing the effects of heart failure. We investigated whether human bone marrow CD133 + -derived cells (BM-CD133 + cells) might be used for cell therapy of heart injury in combination with tissue engineering. We examined these cells for: 1) their in vitro capacity to be converted into cardiomyocytes (CMs), and 2) their potential for in vivo differentiation when delivered to a tissue-engineered type I collagen patch placed on injured hearts (group II). To ensure a microvascular network ready for use by the transplanted cells, cardiac injury and patching were scheduled 2 weeks before cell injection. The cardiovascular potential of the BM-CD133 + cells was compared with that of a direct injection (group I) of the same cells in heart tissue damaged according to the same schedule as for group II. While a small fraction (2 ± 0.5%) of BM-CD133 + cells cocultured with rat CMs switched in vitro to a CM-like cell phenotype, in vivo—and in both groups of nude rats transplanted with BM-CD133 + —there was no evidence of any CM differentiation (as detected by cardiac troponin I expression), but there were signs instead of new capillaries and small arterioles. While capillaries prevailed over arterioles in group II, the opposite occurred in group I. The transplanted cells further contributed to the formation of new microvessels induced by the patch (group II) but the number of vessels did not appear superior to the one developed after directly injecting the BM-CD133 + cells into the injured heart. Although chimeric human–rat microvessels were consistently found in the hearts of both groups I and II, they represented a minority (1.5–2.3%) compared with those of rat origin. Smooth muscle myosin isoform expression suggested that the arterioles achieved complete differentiation irrespective of the presence or absence of the collagen patch. These findings suggest that: 1) BM-CD133 + cells display a limited propensity for in vitro conversion to CMs; 2) the preliminarily vascularized bioscaffold did not confer a selective homing and differentiation advantage for the phenotypic conversion of BM-CD133 + cells into CMs; and 3) combined patching and cell transplantation is suitable for angiogenesis and arteriogenesis, but it does not produce better results, in terms of endothelial and smooth muscle cell differentiation, than the “traditional” method of cell injection into the myocardium.

Long-term engraftment and phenotype correction has been difficult to achieve in humans after in utero stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained during gestation from the amniotic fluid with minimal risk for the fetus and the mother. Using a sheep model, we explored the possibility of using amniotic fluid mesenchymal stem cells (AFMSCs) for autologous in utero stem cell/gene therapy. We collected amniotic fluid (AF) under ultrasound-guided amniocentesis in early gestation pregnant sheep ( n = 9, 58 days of gestation, term = 145 days). AFMSCs were isolated and expanded in all sampled fetal sheep. Those cells were transduced using an HIV vector encoding enhanced green fluorescent protein (GFP) with 63.2% (range 38.3-96.2%) transduction efficiency rate. After expansion, transduced AFMSCs were injected into the peritoneal cavity of each donor fetal sheep at 76 days under ultrasound guidance. One ewe miscarried twin fetuses after amniocentesis. Intraperitoneal injection was successful in the remaining 7 fetal sheep giving a 78% survival for the full procedure. Tissues were sampled at postmortem examination 2 weeks later. PCR analysis detected GFP-positive cells in fetal tissues including liver, heart, placenta, membrane, umbilical cord, adrenal gland, and muscle. GFP protein was detected in these tissues by Western blotting and further confirmed by cytofluorimetric and immunofluorescence analyses. This is the first demonstration of autologous stem cell transplantation in the fetus using AFMSCs. Autologous cells derived from AF showed widespread organ migration and could offer an alternative way to ameliorate prenatal congenital disease.

Long-term engraftment and phenotype correction has been difficult to achieve in humans after in utero stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained during gestation from the amniotic fluid with minimal risk for the fetus and the mother. Using a sheep model, we explored the possibility of using amniotic fluid mesenchymal stem cells (AFMSCs) for autologous in utero stem cell/gene therapy. We collected amniotic fluid (AF) under ultrasound-guided amniocentesis in early gestation pregnant sheep (n = 9, 58 days of gestation, term = 145 days). AFMSCs were isolated and expanded in all sampled fetal sheep. Those cells were transduced using an HIV vector encoding enhanced green fluorescent protein (GFP) with 63.2% (range 38.3-96.2%) transduction efficiency rate. After expansion, transduced AFMSCs were injected into the peritoneal cavity of each donor fetal sheep at 76 days under ultrasound guidance. One ewe miscarried twin fetuses after amniocentesis. Intraperitoneal injection was successful in the remaining 7 fetal sheep giving a 78% survival for the full procedure. Tissues were sampled at postmortem examination 2 weeks later. PCR analysis detected GFP-positive cells in fetal tissues including liver, heart, placenta, membrane, umbilical cord, adrenal gland, and muscle. GFP protein was detected in these tissues by Western blotting and further confirmed by cytofluorimetric and immunofluorescence analyses. This is the first demonstration of autologous stem cell transplantation in the fetus using AFMSCs. Autologous cells derived from AF showed widespread organ migration and could offer an alternative way to ameliorate prenatal congenital disease.

Transplanting hematopoietic and peripheral blood-derived stem/progenitor cells can have beneficial effects in slowing the effects of heart failure. We investigated whether human bone marrow CD133(+)-derived cells (BM-CD133(+) cells) might be used for cell therapy of heart injury in combination with tissue engineering. We examined these cells for: 1) their in vitro capacity to be converted into cardiomyocytes (CMs), and 2) their potential for in vivo differentiation when delivered to a tissue-engineered type I collagen patch placed on injured hearts (group II). To ensure a microvascular network ready for use by the transplanted cells, cardiac injury and patching were scheduled 2 weeks before cell injection. The cardiovascular potential of the BM-CD133(+) cells was compared with that of a direct injection (group I) of the same cells in heart tissue damaged according to the same schedule as for group II. While a small fraction (2 ± 0.5%) of BM-CD133(+)cells cocultured with rat CMs switched in vitro to a CM-like cell phenotype, in vivo-and in both groups of nude rats transplanted with BM-CD133(+)--there was no evidence of any CM differentiation (as detected by cardiac troponin I expression), but there were signs instead of new capillaries and small arterioles. While capillaries prevailed over arterioles in group II, the opposite occurred in group I. The transplanted cells further contributed to the formation of new microvessels induced by the patch (group II) but the number of vessels did not appear superior to the one developed after directly injecting the BM-CD133(+)cells into the injured heart. Although chimeric human-rat microvessels were consistently found in the hearts of both groups I and II, they represented a minority (1.5-2.3%) compared with those of rat origin. Smooth muscle myosin isoform expression suggested that the arterioles achieved complete differentiation irrespective of the presence or absence of the collagen patch. These findings suggest that: 1) BM-CD133(+) cells display a limited propensity for in vitro conversion to CMs; 2) the preliminarily vascularized bioscaffold did not confer a selective homing and differentiation advantage for the phenotypic conversion of BM-CD133(+) cells into CMs; and 3) combined patching and cell transplantation is suitable for angiogenesis and arteriogenesis, but it does not produce better results, in terms of endothelial and smooth muscle cell differentiation, than the "traditional" method of cell injection into the myocardium.

Efficacy of adult (bone marrow, BM) versus fetal (amniotic fluid, AF) mesenchymal stem cells (MSCs) to replenish damaged rat heart tissues with new cardiovascular cells has not yet been established. We investigated on the differentiation potential of these two rat MSC populations in vitro and in a model of acute necrotizing injury (ANI) induced by cryoinjury. Isolated BM-MSCs and AF-MSCs were characterized by flow cytometry and cytocentrifugation and their potential for osteogenic, adipogenic, and cardiovascular differentiation assayed in vitro using specific induction media. The left anterior ventricular wall of syngeneic Fisher 344 (n = 48) and athymic nude (rNu) rats (n = 6) was subjected to a limited, nontransmural epicardial ANI in the approximately one third of wall thickness without significant hemodynamic effects. The time window for in situ stem cell transplantation was established at day 7 postinjury. Fluorochrome (CMTMR)-labeled BM-MSCs (2 x 10(6)) or AF-MSCs (2 x 10(6)) were injected in syngeneic animals (n = 26) around the myocardial lesion via echocardiographic guidance. Reliability of CMTMR cell tracking in this context was ascertained by transplanting genetically labeled BM-MSCs or AF-MSCs, expressing the green fluorescent protein (GFP), in rNu rats with ANI. Comparison between the two methods of cell tracking 30 days after cell transplantation gave slightly different values (1420,58 +/- 129,65 cells/mm2 for CMTMR labeling and 1613.18 +/- 643.84 cells/mm2 for genetic labeling; p = NS). One day after transplantation about one half CMTMR-labeled AF-MSCs engrafted to the injured heart (778.61 +/- 156.28 cells/mm2) in comparison with BM-MSCs (1434.50 +/- 173.80 cells/mm2, p < 0.01). Conversely, 30 days after cell transplantation survived MSCs were similar: 1275.26 +/- 74.51/mm2 (AF-MSCs) versus 1420.58 +/- 129.65/mm2 for BM-MSCs (p = NS). Apparent survival gain of AF-MSCs between the two time periods was motivated by the cell proliferation rate calculated at day 30, which was lower for BM-MSCs (6.79 +/- 0.48) than AF-MSCs (10.83 +/- 3.50; p < 0.01), in the face of a similar apoptotic index (4.68 +/- 0.20 for BM-MSCs and 4.16 +/- 0.58 for AF-MSCs; p = NS). These cells were also studied for their expression of markers specific for endothelial cells (ECs), smooth muscle cells (SMCs), and cardiomyocytes (CMs) using von Willebrand factor (vWf), smooth muscle (SM) alpha-actin, and cardiac troponin T, respectively. Grafted BM-MSCs or AF-MSCs were found as single cell/small cell clusters or incorporated in the wall of microvessels. A larger number of ECs (227.27 +/- 18.91 vs. 150.36 +/- 24.08 cells/mm2, p < 0.01) and CMs (417.91 +/- 100.95 vs. 237.43 +/- 79.99 cells/mm2, p < 0.01) originated from AF-MSCs than from BM-MSCs. Almost no SMCs were seen with AF-MSCs, in comparison to BM-MSCs (98.03 +/- 40.84 cells/mm2), in concordance with lacking of arterioles, which, instead, were well expressed with BM-MSCs (71.30 +/- 55.66 blood vessels/mm2). The number of structurally organized capillaries was slightly different with the two MSCs (122.49 +/- 17.37/mm2 for AF-MSCs vs. 148.69 +/- 54.41/mm2 for BM-MSCs; p = NS). Collectively, these results suggest that, in the presence of the same postinjury microenvironment, the two MSC populations from different sources are able to activate distinct differentiation programs that potentially can bring about a myocardial-capillary or myocardial-capillary-arteriole reconstitution.