Parthenogenetic stem cells for tissue-engineered heart repair

Abstract

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.

Figure 1. Basic characterization of PSCs.

(A) Undifferentiated PSCs cultured on MEFs formed ESC-like colonies with alkaline phosphatase activity (red – inset). Scale bar: 100 μm. (B) Immunofluorescence labeling of POU5F1, NANOG, and FUT4 (also known as SSEA1) in undifferentiated PSC colonies. Scale bars: 20 μm. (C) Growth kinetics of ESC line R1 and PSC lines A3, A6, B2, and B3 (n = 3 per group and time point; data represent means ± SEM; cell-doubling time: 16–17 hours). (D) PCA of global gene expression profiles of pluripotent cells (PSCs, ESCs, iPSCs, and gPSCs) and somatic cells (MEFs and neural stem cells [NSCs]). The respective Gene Expression Omnibus accession numbers are: GSE11274 (includes MEF and gPSCs); GSE10806 (includes NSC and iPSCs); and GSE30868 (includes ESCs [R1], PSCs [A3] and PSC-derived EBs [A3 EB culture day 15]). Each microarray experiment is represented by a dot, which is positioned in 2D space according to its similarity or degree of variance to all samples analyzed. PC1 and PC2 show variances of 47% and 20%, respectively. (E) Heat map of 80 annotated imprinted genes expressed in ESCs (R1 passage 25; n = 3) and PSCs (A3 passage 25; n = 3). Each gene is represented by a single row of colored boxes. Red represents transcript levels above median; blue represents transcript levels below median. (F) Comparison of global gene expression in ESCs and PSCs with key reprogramming factors highlighted. (G) qPCR of selected stemness factors (n = 4 per group; data represent means ± SEM).

Figure 2. Characteristic genetic and epigenetic differences in ESCs and PSCs.

(A) Overview of chromosome 5 genotype demonstrating the expected variable allelic disparity in PSC lines (A3, A6, B2, B3). Color codes: C57BL/6J allele, blue; DBA/2J allele, yellow; alternative allele, green. (B) Bisulfite genomic sequencing of DMRs of imprinted genes in PSC line A3. Each row represents a cloned allele and each circle a CpG site (black circles represent methylated and white circles represent unmethylated CpG sites). (C) Differentially expressed imprinted genes in ESCs (R1; n = 3) versus PSCs (A3; n = 3). Each gene is represented by a single row of colored boxes. Color codes: yellow represents transcript levels above median; blue represents transcript levels below median.

Figure 3. Multilineage potential of PSCs in vitro and in vivo.

(A) RT-PCR analyses of lineage-specific transcripts in differentiating PSCs (PSC line B2). MEFs served as controls. Culture day 7; i.e., 2 days of hanging drop followed by 5 days in suspension culture. (+): Subsequent adhesion culture days. M, 100 bp DNA marker (full uncut gels are shown in the Supplemental Material). (B) EB culture day 22 (PSC line B3): pancytokeratin (pKRT), cytokeratin-18 (KRT18), α-fetoprotein (AFP), neurofilament protein M (NEFM), nebulin (NEB), GATA4. Respective lineage markers (red); nuclei (blue). Scale bars: 20 μm. (C and D) Comparison of teratoma size and composition 3 weeks after s.c. injection of ESCs and PSCs in SCID mice. Teratoma volume was quantified by ultrasonic biomicroscopy (representative image in C; data represent means ± SEM). H&E staining revealed multilineage potential of PSCs, including the capacity to generate cross-striated muscle (inset in D shows magnification of smaller boxed area). Scale bars: 50 μm.

Figure 4. H2-haploidentical PSCs as preferred allografts.

(A) Allele contribution on chromosome 17 (MHC locus is between 18.4 and 20.3 cM) in A3, A6, B2, and B3 PSC lines. Allele color codes: C57BL/6J, blue; DBA/2J, yellow; alternative alleles, green; no PCR product detected, white. (B) Schematic of transplant conditions (upper left); representative teratoma histologies (bottom left); teratoma formation after grafting of H2-haploidentical A3 PSC grafts in indicated recipient mice; n = 10 per group (upper right); teratoma formation after grafting of H2-heterozygous A6 PSC grafts in indicated recipient mice; n = 10 per group (bottom right).

Figure 5. Mesodermal differentiation and cardiogenicity.

qPCR for pluripotency (Pou5f1), (cardio)mesoderm (T, Kdr, Isl1), and cardiac (Nkx2-5, Myh6) markers in undifferentiated (UD) cells and differentiating EBs in (A) PSC (n = 4–5 per group) and (B) ESC (n = 3–4 per group) cultures (data represent means ± SEM). Decrease in Nkx2-5 and Mhy6 in ESCs on culture day 7 + 6 was the consequence of nonmyocyte overgrowth. (C) Enumeration of beating EBs at the indicated culture days (n = 2–3 per time point; data represent means). (D) Quantity of EGFP-positive cardiomyocytes (CMs) per input stem cell (SC; 400 ESCs or PSCs per EB; cells from 5 single EBs were pooled for analytic FACS; n = 5–6) on culture day 5 + 10 (box plots indicate median with interquartile range; whiskers indicate minimal and maximal values). PSC line A3 and ESC lines ES4 and ES7 were generated from the same αMHC-EGFP mouse strain.

Figure 6. Cardiomyocyte differentiation from PSCs in vitro and in vivo.

(A) FACS of GFP-positive PCMs (culture day 7 + 15); nontransgenic ESCs served as the control cell population (right panel illustrates a PCM 24 hours after plating; also refer to Supplemental Video 1). (B) Morphology of FACS-purified PCMs (EGP-positive) assessed by confocal laser scanning microscopy. (C) Distinct APs in FACS-purified PCMs. (D) Epifluorescence images and histochemical staining of atrial, atrioventricular (AV) nodal (acetylcholinesterase-positive/connexin43-negative), and ventricular regions in chimeric adult mouse heart (generated by blastocyst injection of αMHC-EGFP PSCs, A3 line). (E) Heart section from a chimeric mouse after injection of αMHC-EGFP PSCs (A3 line) into αMHC-nLacZ blastocysts. Light microscopy after X-gal staining (left panel); epifluorescence image of the same section showing PSC-derived EGFP-positive cardiomyocytes (middle panel); merged image did not show cells double-positive for EGFP and nLacZ, ruling out overt fusion events (right panel). Scale bars: (A) 10 μm, (B and D) 20 μm, (E) 50 μm.

Figure 7. Retention and functional integration of PCMs after intramyocardial injection.

(A and B) Immunofluorescent labeling of α-actinin (red, A) and connexin43 (red, B) in adult ventricular mouse heart tissue 3 weeks after injection of PCMs (EGFP, green; nuclei, blue). (C–F) Two-photon laser scanning microscopy of intracellular Ca²⁺ transients in adult mouse hearts after injection of PCMs: 2D-scan (C) and line-scan (D) images of stimulated (3 Hz) and spontaneous Ca²⁺ transients. Arrow 1, EGFP-positive cell; arrow 2, EGFP-negative cell; the dotted line indicates the location of the line scan. Bands of increased rhod-2 fluorescence intensity reflect AP-induced Ca²⁺ transients. (E) Plots of rhod-2 and GFP line-scan data in the EGFP-expressing cardiomyocyte 1 and the GFP-negative (native) cardiomyocyte 2 as a function of time. (F) Superimposed tracings of AP-evoked changes in rhod-2 fluorescence as a function of time from cardiomyocytes 1 (green) and 2 (red). For each cell, the relative changes in fluorescence were normalized such that 0 represents the prestimulus fluorescence intensity (F0), and 1 represents the peak fluorescence intensity. Additional immunofluorescence analyses confirmed the maturity of engrafted PCMs: A3 line; EGFP-positive; (G) α-actinin (red); (H) actin (red); (I) troponin (red); nuclei (blue; DAPI). Scale bars: (A) 100 μm, (B, C, G–I) 20 μm.

Figure 8. Tissue-engineered myocardium from PSC derivatives.

(A) Light microscopic (top panel) and epifluorescence images (bottom panel) of an EHM (culture day 8; also refer to Supplemental Video 2). (B) Confocal laser scanning microscopy of a cardiomyocyte bundle in EHM. EGFP (green); α-actinin (red); nuclei (blue, DAPI). (C) Transcript abundance of Myh6 and Myh7 (normalized to Casq2) in native myocardium (fetal, neonatal, adult), EB cultures (ESC and PSC), and EHM (n = 3–4 per group; data represent means ± SEM). (D) Concentration response curve under increasing extracellular [Ca²⁺] for EHM (n = 8) and native mouse myocardium (ventricular wedge; n = 4). Data represent means ± SEM (also refer to Supplemental Table 2). Single isometric contractions of EHM and native heart muscle (inset) at low (0.4 mmol/l) and high (2.4 mmol/l) extracellular [Ca²⁺]. Scale bars: (A) 1 mm, (B) 50 μm.

Figure 9. Proof-of-concept for the therapeutic application of MHC-matched EHM grafts.

(A) F-EHM graft sutured onto a beating heart. (B) EHM graft on infarcted myocardium (PCMs, EGFP-positive; heart was explanted directly after EHM engraftment for demonstration). (C) Anterior wall thickness in diastole (AWThd) and (D) anterior wall thickening fraction (AWThF) measured by echocardiography 2 weeks after MI and the respective surgical intervention. Sham, no MI, and no graft; control (Ctrl), MI with no graft; F-EHM, MI with formaldehyde-fixed (nonviable) EHM; EHM, MI with viable EHM graft (n = 7–8). Data represent means ± SEM; *P < 0.05 versus control (also refer to Supplemental Table 3). (E) Heart explants with F-EHM and EHM (EGFP-positive) grafts; EGFP signal (lower panels) indicates retention of viable cardiomyocytes. (F) Anisotropically arranged PCMs (α-actinin and GFP) in EHM graft. (G) PCMs surrounded by CD31-positive capillaries. Scale bars: (A, B, and E) 2 mm; (F and G) 20 μm.