Figure 1.
Reprogramming of disease-specific CPCs.
(A) Schematic presentation of CPC isolation and iPS generation. (B) Retroviral transduction was verified by tagged-PCR and endogenous genes (C) are shown. (D) Retroviral silencing was confirmed during reprogramming after 3 months of infection. Trans- and endogenous-gene expressions are shown.
Figure 2.
Characterization of disease-specific iPS cells.
Representative patient-specific iPS clones at passage 10 (A, iPS30: HLHS; B, iPS65: TAPVC, representing BV). Colonies were stained with transcription factors typically expressed in iPS cells. Bar, 200 µm. (C and D) Bisulfite sequencing analysis of OCT4 and NANOG promoter regions during reprogramming is shown. Closed and open circles represent methylated and unmethylated CpG dinucleotides, respectively.
Figure 3.
Patient-specific CPCs were fully reprogrammed.
(A) Representative images of alkaline phosphatase staining are shown for iPS cells generated from HLHS (iPS30) and TAPVC representing BV (iPS65) patients. Bar, 200 µm. (B) Chromosomal abnormalities were not found in both iPS clones at 10 weeks by the G-banding method. (C) Heat map (right) and hierarchical cluster analysis (left) of global gene expression from patient-specific CPCs and iPS clones are shown. A commercially available 201B7 clone (Riken) was used as control human iPS cells.
Figure 4.
Patient-derived iPS cells differentiated into all three germ layer origins in vivo.
Gross morphology and hematoxylin and eosin staining of patient-specific iPS cell-derived teratomas are shown. Teratomas were found in the testes of NOD/SCID mice 10 to 12 weeks after transplantation. Histological sections of identified cells represent all three germ layers. Bar, 50 µm.
Figure 5.
HLHS-derived iPS cells could give rise to cardiomyocytes.
(A) Both HLHS- and BV-derived iPS cells could generate cardiac troponin-T (TNNT2)-positive cardiomyocytes (green) 3 weeks after lineage induction. Nuclei were shown by DAPI (blue). Bar, 30 µm. (B) Time course of TNNT2 expression in disease-specific iPS cells. Data were normalized using β2-microglobulin and human heart tissue for comparisons. *, p<0.05 vs. control and differentiated BV-derived iPS cells at 3 weeks. †, p<0.05 vs. before cardiac lineage induction (0 weeks) in each group.
Figure 6.
HLHS-iPS cell-derived cardiomyocytes showed decreased cardiac transcripts.
mRNA expressions in control 201B7 iPS cells and one BV- and two HLHS-derived iPS cell lines during cardiac lineage induction at respective time points were determined by quantitative RT-PCR. All data were obtained from more than five independent experiments with three different clonal derivatives and normalized using β2-microglobulin and human heart tissue for comparisons. *, p<0.05 vs. differentiated 201B7 and BV-derived iPS cells at corresponding time points. †, p<0.05 vs. 201B7 at corresponding time points.
Figure 7.
Synergistic restoration of target promoters by NKX2-5, HAND1, and NOTCH in HLHS-derived CPCs and iPS cells.
Transcriptional activation of SRE promoter luciferase construct by combinatorial transfection of NKX2-5, HAND1, and NOTCH1 in HLHS- and BV-derived CPCs (A) or iPS cells (B). Co-transfection of TNNT2 luciferase reporter with NKX2-5, HAND1, and NOTCH1 in CPCs (C) or iPS cells (D) is shown. NPPA luciferase construct was co-transfected with NKX2-5, HAND1, and NOTCH1 alone or in combination into CPCs (E) or iPS cells (F). (G-I) BV-derived CPCs were transfected with either control or shRNAs specific to inhibit NKX2-5, HAND1, and NOTCH1 expression. Results were normalized using an internal control (SEAP or hRluc) and obtained from more than triplicate sets of experiments. *, p<0.05 vs. the same HLHS sample without transfection of the gene of interest. †, p<0.05 vs. BV sample transfected with control vector alone. ‡, p<0.05 vs. both HLHS samples with the same treatment. §, p<0.01 vs. BV sample transfected with control vector alone.
Figure 8.
HLHS-iPS cell-derived cardiomyocytes showed suppressed H3K4 methylation and H3 acetylation, but increased H3K27 methylation.
Undifferentiated CPCs (A) and iPS cells (B) and differentiated (cardiac-lineage induction for 3 weeks) iPS cells (C) were analyzed by ChIP assay. N.D., not detected; *, p<0.05 vs. differentiated BV-derived iPS cells. Data are expressed as the percentage of input DNA.
Figure 9.
Orchestrated gene regulatory network in the development of HLHS.
(A) Core transcriptional factors expressed in cardiac progenitor cells serve as targets in response to inductive signals to initiate cardiogenesis. NKX2-5 is predominantly expressed in the primary heart field and controls progenitor cell proliferation. Genes regulate atrioventricular (AV) canal and valve development. Reduced expression may contribute to mitral- and aortic-valve stenosis/atresia often seen in HLHS. NOCTH modulates left heart outflow tract development and the resultant obstruction may cause secondary ventricle hypoplasia. HAND1/2 specify left and right ventricular chamber morphogenesis, and the absence of these genes may lead to a hypoplastic ventricle. (B–D) Schematic diagrams of SRE, TNNT2, and NPPA transcriptional activation. HLHS-derived CPCs and iPS cells showed significantly reduced luciferase activities compared with BV-derived cells. Co-transfection analysis of reporter constructs with NKX2-5, HAND1, and NOTCH1, proposed core transcriptional factors, could synergistically restore the transcriptional activation in these reporters equivalent to the levels in BV-derived cells. (E) Major chromatin features in differentiated HLHS- and BV-derived iPS cells are shown. Upon cardiomyocyte differentiation, HLHS-derived iPS cells failed to enrich the active histone marks such as H3K4me2 and acH3, whereas repressive histone marks such as H3K27me3 increased, resulting in compact chromatin that lost enhancer marks and gained repressor marks on the NKX2-5 promoter.