Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.

We recently described a murine embryonic stem cell (ESC) line engineered to express the activated Notch 4 receptor in a tetracycline (doxcycline; Dox) regulated fashion (tet-notch4 ESCs). Notch 4 induction in Flk1(+) hematopoietic and vascular progenitors from this line respecified them to a cardiovascular fate. We reasoned that these cells would be ideal for evaluating the contribution of the cardiomyocyte and vascular lineages to the functional improvement noted following stem cell transplantation in infarcted hearts. Flk-1(+) Tet-notch4 cells from d 3 embryoid bodies exposed to doxycycline (Dox(+)) were compared to uninduced (Dox(-)) Flk-1(+) cells. Mice underwent transplantation of 5 x 10(5) Dox(+) cells, Dox(-)cells, or an equal volume of serum-free medium after surgically induced myocardial infarction. The mean ejection fraction was 59 + or - 15, 46 + or - 17, and 39 + or - 13% in the Dox(+), Dox(-), and serum-free medium groups, respectively (P<0.05 for the differences among all 3 groups). Immunohistochemistry of hearts injected with Dox(+) grafts expressed myocardial and vascular markers, whereas grafts of Dox(-) cells expressed primarily vascular markers. We conclude that cardiovascular progenitors are more effective than vascular progenitors in improving function after myocardial infarction. The transplantation of appropriate cell types is critical for maximizing the benefit of cardiovascular cell therapy.-Adler, E. D., Chen, V. C., Bystrup, A., Kaplan, A. D., Giovannone, S., Briley-Saebo, K., Young, W., Kattman, S., Mani, V., Laflamme, M., Zhu, W.-Z., Fayad, Z., Keller, G. The cardiomyocyte lineage is critical for optimization of stem cell therapy in a mouse model of myocardial infarction.

Positive selection is a process that ensures that peripheral T cells express TCR that are self-MHC restricted. This process occurs in the thymus and requires both self-MHC and self-peptides. We have recently established a TCR transgenic (TCRtrans+) mouse model using the C10.4 TCR restricted to the MHC class Ib molecule, H2-M3. Having defined H2-M3 as the positively selecting MHC molecule, the severely limited number of H2-M3 binding peptides allowed us to characterize a mitochondrial NADH dehydrogenase subunit 1-derived 9-mer peptide as the physiological ligand of positive selection. Here, we demonstrate that the NADH dehydrogenase subunit 1 self-peptide is seen by mature C10.4 TCRtrans+ T cells as a weak agonist and induces positive selection at a defined concentration range. We also found that the full-length cognate peptide, a strong agonist for mature C10.4 TCRtrans+ T cells, initiated positive selection, albeit at significantly lower concentrations. At increased peptide concentrations, and thus increased epitope densities, either peptide only induced the development of partially functional T cells. We conclude that successful positive selection only proceeded at a defined, yet fairly narrow window of avidity.

BACKGROUND: Intramyocardial transplantation of stem cells improves left ventricular ejection fraction (EF) in animal studies and preliminary clinical trials. The mechanism may involve either replacement of myocytes or improved vascular supply to existing myocytes. We recently identified an Embyronic Stem cell derived cardiovascular progenitor cell (ES-CPC) that is the common precursor of cardiomyocyte and vascular cell lineages. To determine whether myocyte transplantation improves myocardial function more than angiogenesis alone does, we compared the effect of ES-CPCs to hemangioblasts (vascular/hematopoetic progenitor cells) on EF in a mouse model of myocardial infarction. METHODS: ES-CPC and hemangioblasts were isolated from a doxycycline-responsive, Notch-inducible ES cell line containing Notch 4 cDNA under the control of a tetracycline-inducible promoter. Notch induction of mesoderm-derived ES cells resulted in a CPC phenotype, whereas non-induced cells developed into hemangioblasts. Mice underwent transplantation of 500,000 ES-CPC (n=20), hemangioblasts (n=16), or an equal volume of serum-free media (n=12) 30 minutes after surgically-induced myocardial infarction. All cell lines constitutively expressed green fluorescent protein (GFP). EF was assessed two weeks post-transplantation using 9.4 Tesla MRI. Mice were then euthanized and frozen heart sections were examined using fluorescent microscopy. RESULTS: The mean EF was 59Â ± 15, 46Â ± 17, and 39Â ± 13% in the ES-CPC, hemangioblast, and control groups, respectively (p<0.05 for the differences among all 3 groups; ANOVA). GFP + cells were detected in frozen sections of both the ES-CPC and hemangioblast groups. GFP + cells in ES-CPC treated hearts expressed markers associated with both cardiomyocyte and vascular phenotypes, whereas the GFP + cells in the hemangioblast group expressed markers associated with vascular phenotypes. CONCLUSIONS: Both hemangioblast and ES-CPC transplantation improves EF in a mouse model of myocardial infarction, but ES-CPC transplantation was more effective. This suggests that enhancement of myocardial function by transplantation of both cardiomyocyte and vascular phenotypes exceeds that with vascular phenotypes alone.