Oxidative stress accelerates intestinal tumorigenesis by enhancing 8-oxoguanine-mediated mutagenesis in MUTYH-deficient mice.

Oxidative stress-induced DNA damage and its repair systems are related to cancer etiology; however, the molecular basis triggering tumorigenesis is not well understood. Here, we aimed to explore the causal relationship between oxidative stress, somatic mutations in pre-tumor-initiated normal tissues, and tumor incidence in the small intestines of MUTYH-proficient and MUTYH-deficient mice. MUTYH is a base excision repair enzyme associated with human colorectal cancer. Mice were administered different concentrations of potassium bromate (KBrO3; an oxidizing agent)-containing water for 4 wk for mutagenesis studies or 16 wk for tumorigenesis studies. All Mutyh -/- mice treated with >0.1% KBrO3 developed multiple tumors, and the average tumor number increased dose dependently. Somatic mutation analysis of Mutyh -/-/rpsL transgenic mice revealed that G:C  > T:A transversion was the only mutation type correlated positively with KBrO3 dose and tumor incidence. These mutations preferentially occurred at 5'G in GG and GAA sequences in rpsL This characteristic mutation pattern was also observed in the genomic region of Mutyh -/- tumors using whole-exome sequencing. It closely corresponded to signature 18 and SBS36, typically caused by 8-oxo-guanine (8-oxoG). 8-oxoG-induced mutations were sequence context dependent, yielding a biased amino acid change leading to missense and stop-gain mutations. These mutations frequently occurred in critical amino acid codons of known cancer drivers, Apc or Ctnnb1, known for activating Wnt signal pathway. Our results indicate that oxidative stress contributes to increased tumor incidence by elevating the likelihood of gaining driver mutations by increasing 8-oxoG-mediated mutagenesis, particularly under MUTYH-deficient conditions.

Development of adolescent childhood cancer survivors' psychosocial issues scale.

BACKGROUND: Follow-up care for adolescent childhood cancer survivors (ACCS) after they return to school requires an understanding of their psychosocial issues. Therefore, this study developed the adolescent childhood cancer survivors' psychosocial issues scale (ACCSPIS) and evaluated its reliability and validity. METHODS: In the development phase, pediatric oncology clinical professionals created the 24 item questionnaire of ACCS's psychosocial issues. In the feasibility phase, a survey was administered to 165 ACCS aged 12-18 years after discharge from hospital in Japan, and 57 completed questionnaires were analyzed. The survey items were psychosocial issues, attributes, K6 scale, and impact of event scale-revised (IES-R) scale. Factor analysis was conducted for psychosocial issues. Regarding reliability, Cronbach's α coefficients and item-total correlation coefficients were calculated. Regarding validity, Spearman's rank correlation coefficients between ACCSPIS and K6 and IES-R were calculated, and confirmatory factor analysis was conducted. RESULTS: Four factors comprising 15 items were extracted: "appearance changes due to treatment effects," "anxiety about marriage and the future," "change in appearance due to treatment", and "psychological distress due to interpersonal relationships and information about the disease." The model fit was good, with a total ACCSPIS α coefficient of 0.901 and α coefficients for the subscales ranging from 0.651 to 0.914. The K6 and IES-R were significantly associated with the total ACCSPIS, and item-total correlations were satisfactory. CONCLUSIONS: The reliability and validity of ACCSPIS were generally confirmed. This scale could be useful to measure psychosocial issues in ACCS aged 12-18 years after their return to school.

Discovery of 7-Azanorbornane-Based Dual Agonists for the Delta and Kappa Opioid Receptors through an In Situ Screening Protocol.

In medicinal chemistry, the copper-catalyzed click reaction is used to prepare ligand candidates. This reaction is so clean that the bioactivities of the products can be determined without purification. Despite the advantages of this in situ screening protocol, the applicability of this method for transmembrane proteins has not been validated due to the incompatibility with copper catalysts. To address this point, we performed ligand screening for the µ, δ, and κ opioid receptors using this protocol. As we had previously reported the 7-azanorbornane skeleton as a privileged scaffold for the G protein-coupled receptors, we performed the click reactions between various 7-substituted 2-ethynyl-7-azanorbornanes and azides. Screening assays were performed without purification using the CellKeyTM system, and the putative hit compounds were re-synthesized and re-evaluated. Although the "hit" compounds for the µ and the δ receptors were totally inactive after purifications, three of the four "hits" for the κ receptor were true agonists for this receptor and also showed activities for the δ receptor. Although false positive/negative results exist as in other screening projects for soluble proteins, this in situ method is effective in identifying novel ligands for transmembrane proteins.

Biallelic truncating variants in VGLL2 cause syngnathia in humans.

BACKGROUND: Syngnathia is an ultrarare craniofacial malformation characterised by an inability to open the mouth due to congenital fusion of the upper and lower jaws. The genetic causes of isolated bony syngnathia are unknown. METHODS: We used whole exome and Sanger sequencing and microsatellite analysis in six patients (from four families) presenting with syngnathia. We used CRISPR/Cas9 genome editing to generate vgll2a and vgll4l germline mutant zebrafish, and performed craniofacial cartilage analysis in homozygous mutants. RESULTS: We identified homozygous truncating variants in vestigial-like family member 2 (VGLL2) in all six patients. Two alleles were identified: one in families of Turkish origin and the other in families of Moroccan origin, suggesting a founder effect for each. A shared haplotype was confirmed for the Turkish patients. The VGLL family of genes encode cofactors of TEAD transcriptional regulators. Vgll2 is regionally expressed in the pharyngeal arches of model vertebrate embryos, and morpholino-based knockdown of vgll2a in zebrafish has been reported to cause defects in development of pharyngeal arch cartilages. However, we did not observe craniofacial anomalies in vgll2a or vgll4l homozygous mutant zebrafish nor in fish with double knockout of vgll2a and vgll4l. In Vgll2 -/- mice, which are known to present a skeletal muscle phenotype, we did not identify defects of the craniofacial skeleton. CONCLUSION: Our results suggest that although loss of VGLL2 leads to a striking jaw phenotype in humans, other vertebrates may have the capacity to compensate for its absence during craniofacial development.

Longitudinal health-related quality of life analysis in childhood cancer survivors after proton beam therapy.

PURPOSE: Whilst proton beam therapy (PBT) for children with cancer is expected to reduce their comorbidities, to date only a limited number of studies have been published. To analyze the long-term comorbidity and health-related quality of life (HRQoL) of childhood cancer survivors (CCSs) after PBT, we conducted a questionnaire-based study. METHODS: Questionnaires were sent to CCSs who underwent PBT at the University of Tsukuba Hospital during the period from 1984 to 2020. Scores from 41 CCSs who did not undergo PBT (noPBT-CCSs) and from the general population were used for comparison. RESULTS: In total, 110 individuals who underwent PBT participated in the study. Among them, 40 individuals were longitudinally analyzed. The range of change in the scores was significantly greater in the CCSs whose initial scores were low. Although the comorbidity levels were more severe, HRQoL tended to be better in the PBT-CCSs than in the noPBT-CCSs with central nervous system (CNS) or solid tumors, respectively. When compared with the general population, the psychosocial health summary scores and its components were not different in the noPBT-CNS-CCSs. On the other hand, the psychosocial health summary scores and/or at least one of the scores of emotional, social, and school functioning were significantly higher in the other CCSs groups. CONCLUSIONS: The HRQoL scores of CCSs with low initial scores can be greatly changed over time. Appropriate psychosocial support for this population is warranted. PBT may avoid reduction in HRQoL in terms of the psychosocial functioning of CCSs with CNS tumors.

DNA polymerase delta Exo domain stabilizes mononucleotide microsatellites in human cells.

In prokaryotes and yeasts, DNA polymerase proofreading (PPR) and DNA mismatch repair (MMR) cooperatively counteracts replication errors leading to repeat sequence destabilization (i.e. insertions/deletions of repeat units). However, PPR has not thus far been regarded as a mechanism stabilizing repeat sequences in higher eukaryotic cells. In a human cancer cell line, DLD-1, which carries mutations in both MSH6 and the Exo domain of POLD1, we previously observed that mononucleotide microsatellites were markedly destabilized whereas being stable in the simple MMR-defective backgrounds. In this study, we introduced the Exo domain mutation found in DLD-1 cells into MSH2-null HeLa cell clones, using CRISPR/Cas9 system. In the established Exo-/MMR-mutated HeLa clones, mononucleotide repeat sequences were remarkably destabilized as in DLD-1 cells. In contrast, dinucleotide microsatellites were readily destabilized in the parental MMR-deficient backgrounds, and the instability was not notably increased in the genome-edited HeLa clones. Here, we show an involvement of the Exo domain functions of DNA polymerase delta in mononucleotide repeat stabilization in human cells, which also suggests a possible role division between DNA polymerase and MMR in repeat maintenance in the human genome.

Decreased YAP activity reduces proliferative ability in human induced pluripotent stem cell of duchenne muscular dystrophy derived cardiomyocytes.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied by dilated cardiomyopathy. Recently, abnormality of yes-associated protein (YAP) has been reported as the pathogenesis of muscle degeneration of DMD; however YAP activity remains unclear in dystrophic heart of DMD. Herein, we investigated YAP activity using disease-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) in DMD. DMD-iPSCs were generated from DMD patient with exon 48-54 deletion in DMD, and genome-edited (Ed)-DMD-iPSCs with in-frame (Ed-DMD-iPSCs) were created using CRISPR/Cas9. Nuclear translocation of YAP [nuclear (N)/cytoplasmic (C) ratio] was significantly lower in DMD-iPSC-CMs than in Ed-DMD-iPSC-CMs. In addition, Ki67 expression, indicating proliferative ability, was significantly lower in DMD-iPSC-CMs than Ed-DMD-iPSC-CMs. Therefore, immunofluorescent staining showed that actin stress fibers associated with YAP activity by mechanotransduction were disorganized in DMD-iPSC-CMs. Lysophosphatidic acid (LPA), a known lipid mediator on induction of actin polymerization, significantly increased YAP activity and actin dynamics in DMD-iPSC-CMs using live cell imaging. These results suggested that altered YAP activity due to impaired actin dynamics reduced proliferative ability in DMD-iPSC-CMs. Hence, decreased YAP activity in dystrophic heart may contribute to DMD-cardiomyopathy pathogenesis.

Cognitive Functions of Pediatric Brain Tumor Survivors Treated With Proton Beam Therapy: A Case Series.

Pediatric brain tumor survivors who received proton beam therapy at the University of Tsukuba Hospital from 2004 to 2011 were retrospectively evaluated for cognitive function. Five patients were included. The median age of diagnosis was 5.4 years (range: 1.5 to 12.5 y) and the median follow-up time was 5.8 years (range: 3.1 to 8.1 y). IQ scores at follow-up were decreased in 2 of 5 patients; 1 underwent whole-brain irradiation and the other was examined just after surgical removal of recurrent tumors. Local proton beam therapy may preserve cognitive function in survivors of pediatric brain tumors.

Geometrical Patterning and Constituent Cell Heterogeneity Facilitate Electrical Conduction Disturbances in a Human Induced Pluripotent Stem Cell-Based Platform: An In vitro Disease Model of Atrial Arrhythmias.

Ectopic foci from pulmonary veins (PVs) comprise the main trigger associated with the initiation of atrial fibrillation (AF). An abrupt anatomical narrow-to-wide transition, modeled as in vitro geometrical patterning with similar configuration in the present study, is located at the junction of PVs and the left atrium (LA). Complex cellular composition, i.e., constituent cell heterogeneity, is also observed in PVs and the PVs-LA junction. High frequency triggers accompanied with anatomical irregularity and constituent cell heterogeneity provoke impaired conduction, a prerequisite for AF genesis. However, few experiments investigating the effects of these factors on electrophysiological properties using human-based cardiomyocytes (CMs) with atrial properties have been reported. The aim of the current study was to estimate whether geometrical patterning and constituent cell heterogeneity under high frequency stimuli undergo conduction disturbance utilizing an in vitro two-dimensional (2D) monolayer preparation consisting of atrial-like CMs derived from human induced pluripotent stem cells (hiPSCs) and atrial fibroblasts (Fbs). We induced hiPSCs into atrial-like CMs using a directed cardiac differentiation protocol with the addition of all-trans retinoic acid (ATRA). The atrial-like hiPSC-derived CMs (hiPSC-CMs) and atrial Fbs were transferred in defined ratios (CMs/Fbs: 100%/0% or 70%/30%) on manually fabricated plates with or without geometrical patterning imitating the PVs-LA junction. High frequency field stimulation emulating repetitive ectopic foci originated in PVs were delivered, and the electrical propagation was assessed by optical mapping. We generated high purity CMs with or without the ATRA application. ATRA-treated hiPSC-CMs exhibited significantly higher atrial-specific properties by immunofluorescence staining, gene expression patterns, and optical action potential parameters than those of ATRA-untreated hiPSC-CMs. Electrical stimuli at a higher frequency preferentially induced impaired electrical conduction on atrial-like hiPSC-CMs monolayer preparations with an abrupt geometrical transition than on those with uniform geometry. Additionally, the application of human atrial Fbs to the geometrically patterned atrial-like hiPSC-CMs tended to further deteriorate the integrity of electrical conduction compared with those using the atrial-like hiPSC-CM alone preparations. Thus, geometrical narrow-to-wide patterning under high frequency stimuli preferentially jeopardized electrical conduction within in vitro atrial-like hiPSC-CM monolayers. Constituent cell heterogeneity represented by atrial Fbs also contributed to the further deterioration of conduction stability.

Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing.

Repeat destabilisation is variously associated with human disease. In neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. However, several discrepancies have been pointed out. Firstly, the MSI+ phenotype is not uniform in human neoplasms. Established classification utilises the frequency of microsatellite changes, i.e. MSI-H (high) and -L (low), the former regarded as an authentic MMR-defective phenotype. In addition, we have observed the qualitatively distinct modes of MSI, i.e. Type A and Type B. One discrepancy we previously pointed out is that tumours occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumours (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). In the present study, MSH2 mutations reported in Lynch syndrome (LS) kindred have been introduced into HeLa cells using the CRISPR/Cas9 system. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. Nevertheless, microsatellites were not markedly destabilised as in MSI-H tumours occurring in LS patients, and all the observed alterations were uniformly Type A, which confirms the results in mice. Our findings suggest added complexities to the molecular mechanisms underlying repeat destabilisation in human genome.

Cited4 is related to cardiogenic induction and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis.

Cardiac progenitor cells have a limited proliferative capacity. The CREB-binding protein/p300-interacting transactivator, with the Glu/Asp-rich carboxy-terminal domain (Cited) gene family, regulates gene transcription. Increased expression of the Cited4 gene in an adult mouse is associated with exercise-induced cardiomyocyte hypertrophy and proliferation. However, the expression patterns and functional roles of the Cited4 gene during cardiogenesis are largely unknown. Therefore, in the present study, we investigated the expression patterns and functional roles of the Cited4 gene during in vitro cardiogenesis. Using embryoid bodies formed from mouse embryonic stem cells, we evaluated the expression patterns of the Cited4 gene by quantitative reverse transcriptase-polymerase chain reaction. Cited4 gene expression levels increased and decreased during the early and late phases of cardiogenesis, respectively. Moreover, Cited4 gene levels were significantly high in the cardiac progenitor cell population. A functional assay of the Cited4 gene in cardiac progenitor cells using flow cytometry indicated that overexpression of the Cited4 gene significantly increased the cardiac progenitor cell population compared with the control and knockdown groups. A cell proliferation assay, with 5-ethynyl-2'-deoxyuridine incorporation and Ki67 expression during the late phase of cardiogenesis, indicated that the number of troponin T-positive embryonic stem cell-direived cardiomyocytes with proliferative capacity was significantly greater in the overexpression group than in the control and knockdown groups. Our study results suggest that the Cited4 gene is related to cardiac differentiation and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis. Therefore, manipulation of Cited4 gene expression may be of great interest for cardiac regeneration.

Vestigial-like 2 contributes to normal muscle fiber type distribution in mice.

Skeletal muscle is composed of heterogeneous populations of myofibers that are classified as slow- and fast-twitch fibers. The muscle fiber-type is regulated in a coordinated fashion by multiple genes, including transcriptional factors and microRNAs (miRNAs). However, players involved in this regulation are not fully elucidated. One of the members of the Vestigial-like factors, Vgll2, is thought to play a pivotal role in TEA domain (TEAD) transcription factor-mediated muscle-specific gene expression because of its restricted expression in skeletal muscles of adult mice. Here, we generated Vgll2 null mice and investigated Vgll2 function in adult skeletal muscles. These mice presented an increased number of fast-twitch type IIb fibers and exhibited a down-regulation of slow type I myosin heavy chain (MyHC) gene, Myh7, which resulted in exercise intolerance. In accordance with the decrease in Myh7, down-regulation of miR-208b, encoded within Myh7 gene and up-regulation of targets of miR-208b, Sox6, Sp3, and Purß, were observed in Vgll2 deficient mice. Moreover, we detected the physical interaction between Vgll2 and TEAD1/4 in neonatal skeletal muscles. These results suggest that Vgll2 may be both directly and indirectly involved in the programing of slow muscle fibers through the formation of the Vgll2-TEAD complex.

Comorbidity and quality of life in childhood cancer survivors treated with proton beam therapy.

BACKGROUND: The rate of childhood cancer survival has recently reached >80%. Various adverse events among childhood cancer survivors (CCS) have been reported. Proton beams are able to avoid unnecessary irradiation to normal/vital organs. We conducted a quality of life (QOL) study for CCS who were treated with proton beam therapy (PBT). METHODS: We included those patients treated with PBT to the brain, head, or neck and who were ≤15 years old at the University of Tsukuba Hospital between 1983 and 2011. Clinical information was collected from medical records. Questionnaires including the Pediatric Quality of Life Inventory (PedsQL) 4.0 Generic Core Scales (which assess health-related quality of life) were sent to the families/patients. RESULTS: Sixty patients were included. Median age at treatment was 6.2 years. The number of patients with status alive/dead/unknown was 32/24/4. Median follow-up period was 63.0 months (range, 48-340 months) for survivors. Questionnaires were sent to 25 families/patients and 19 were returned. PedsQL was assessed for 17 patients. Eleven of 32 living patients had at least one comorbidity grade 3/4. Average QOL score was above that for Japanese schoolchildren and adolescents. There was no correlation with comorbidity, and only longer time from treatment was correlated with a higher PedsQL score (P = 0.006). CONCLUSION: CCS who were treated with multimodal treatment using PBT had a higher QOL score. Higher score was related to longer time since treatment, regardless of comorbidity.

Electrophysiological properties of prion-positive cardiac progenitors derived from murine embryonic stem cells.

BACKGROUND: The prion protein (PrP) has been reported to serve as a surface maker for isolation of cardiomyogenic progenitors from murine embryonic stem (ES) cells. Although PrP-positive cells exhibited automaticity, their electrophysiological characteristics remain unresolved. The aim of the present study was therefore to investigate the electrophysiological properties of PrP-positive cells in comparison with those of HCN4p-or Nkx2.5-positive cells. METHODS AND RESULTS: Differentiation of AB1, HCN5p-EGFP and hcgp7 ES cells into cardiac progenitors was induced by embryoid body (EB) formation. EBs were dissociated and cells expressing PrP, HCN4-EGFP and/or Nkx2.5-GFP were collected via flow cytometry. Sorted cells were subjected to reverse transcriptase-polymerase chain reaction, immunostaining and patch-clamp experiments. PrP-positive cells expressed mRNA of undifferentiation markers, first and second heart field markers, and cardiac-specific genes and ion channels, indicating their commitment to cardiomyogenic progenitors. PrP-positive cells with automaticity showed positive and negative chronotropic responses to isoproterenol and carbamylcholine, respectively. Hyperpolarization-activated cation current (I(f)) was barely detectable, whereas Na(+) and L-type Ca(2+) channel currents were frequently observed. Their spontaneous activity was slowed by inhibition of sarcoplasmic reticulum Ca(2+) uptake and release but not by blocking I(f). The maximum diastolic potential of their spontaneous firings was more depolarized than that of Nkx2.5-GFP-positive cells. CONCLUSIONS: PrP-positive cells contained cardiac progenitors that separated from the lineage of sinoatrial node cells. PrP can be used as a marker to enrich nascent cardiac progenitors.

Different distribution of Cav3.2 and Cav3.1 transcripts encoding T-type Ca(2+) channels in the embryonic heart of mice.

We investigated the distribution of T-type Ca(2+) channel mRNAs in the mouse embryonic heart. Cav3.2, but not Cav3.1, was expressed in the E8.5 embryonic heart along with cardiac progenitor markers (Nkx2.5, Tbx5, Isl-1) and contractile proteins (alpha and beta MHC). In the E10.5 heart, the distribution of Cav3.1 mRNA was confirmed in the AV-canal and overlapped with that of MinK or Tbx2. Cav3.2 mRNA was observed not only in the AV-canal but also in the outflow tract, along with MinK and Isl-1, indicating the expression of Cav3.2 in the secondary heart field. Thus, Cav3.2 may contribute to the development of the outflow tract from the secondary heart field in the embryonic heart, whereas Cav3.1 may be involved in the development of the cardiac conduction-system together with Cav3.2.

RNA-binding motif protein 24 regulates myogenin expression and promotes myogenic differentiation.

The formation of muscle fibers involves sequential expression of many proteins that regulate key steps during myoblast-to-myotube transition. Myogenin is a major player in the initiation and maintenance of myogenic differentiation in a mouse myoblast cell line, C2C12. RNA-binding proteins bind to specific target RNA sequences and regulate gene expression in a post-transcriptional manner. This study demonstrates that RNA-binding motif protein 24 (Rbm24) interacts with the 3'-untranslated region of myogenin mRNA and affects its half-life in C2C12 myogenesis. Knockdown of Rbm24 expression by RNA interference significantly decreased myogenin expression associated with the inhibition of myogenesis. In contrast, the overexpression of Rbm24 by stable transfection of a plasmid increased myogenin expression and had a positive effect on myogenic differentiation. Ectopic expression of myogenin was also able to restore myogenic differentiation in Rbm24-knockdown cells. Together, our results suggest that Rbm24 binds to myogenin mRNA and regulates its stability in C2C12 cells. Rbm24 plays a crucial role in myogenic differentiation at least in part through a myogenin-dependent post-transcriptional regulatory pathway.

Differentiation of pharyngeal endoderm from mouse embryonic stem cell.

Embryonic stem cells are considered to be a good in vitro tool to study the induction of various cell types including cardiomyocytes; however, induction of the pharyngeal endoderm (PE), the underlying heart-forming region, in vivo has been scarcely reported. In the present study, we found that many PE-related genes, such as Paxl, Pax9, Sixl, and Tbxl, were up-regulated in cardiomyocyte-rich embryoid bodies (EBs). The third pouch-related genes including Hoxa3, Foxn1, and Aire, which are crucial for thymus development and function, were also detected in later stages. Nkx2.5, a cardiac transcription factor gene, is known to be transiently expressed in the PE. By crossing Nkx2.5-Cre mice with Cre-dependent EGFP reporter mice, we found that Nkx2.5(+) lineage exclusively contributed to thymic epithelial cell development, followed by thymus development. Gene expression analysis using Nkx2.5-EGFP ES cells also revealed that PE-related mRNAs were specifically enriched in the transiently appearing E-cadherin(+)Nkx2.5(+) cell fraction. Interestingly, the EB-derived cells were found capable of supporting T-cell differentiation to CD4 or CD8 double-positive cells in a reaggregation organ culture in vitro. Our results suggest that EBs contain cells that resemble third pharyngeal pouch endoderm and confer a thymus-like microenvironment.

Nongenetic method for purifying stem cell-derived cardiomyocytes.

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.

The cellular prion protein identifies bipotential cardiomyogenic progenitors.

RATIONALE: The paucity of specific surface markers for cardiomyocytes and their progenitors has impeded the development of embryonic or pluripotent stem cell-based transplantation therapy. Identification of relevant surface markers may also enhance our understanding of the mechanisms underlying differentiation. OBJECTIVE: Here, we show that cellular prion protein (PrP) serves as an effective surface marker for isolating nascent cardiomyocytes as well as cardiomyogenic progenitors. METHODS AND RESULTS: Embryonic stem (or embryo-derived) cells were analyzed using flow cytometry to detect surface expression of PrP and intracellular myosin heavy chain (Myhc) proteins. Sorted cells were then analyzed for their differentiation potential. CONCLUSIONS: PrP+ cells from beating embryoid bodies (EBs) frequently included nascent Myhc+ cardiomyocytes. Cultured PrP+ cells further differentiated, giving rise to cardiac troponin I+ definitive cardiomyocytes with either an atrial or a ventricular identity. These cells were electrophysiologically functional and able to survive in vivo after transplantation. Combining PrP with a second marker, platelet-derived growth factor receptor (PDGFR)alpha, enabled us to identify an earlier cardiomyogenic population from prebeating EBs, the PrP+PDGFRalpha+ (PRa) cells. The Myhc- PRa cells expressed cardiac transcription factors, such as Nkx2.5, T-box transcription factor 5, and Isl1 (islet LIM homeobox 1), although they were not completely committed. In mouse embryos, PRa cells in cardiac crescent at the 1 to 2 somite stage were Myhc+, whereas they were Myhc- at headfold stages. PRa cells clonally expanded in methlycellulose cultures. Furthermore, single Myhc- PRa cell-derived colonies contained both cardiac and smooth muscle cells. Thus, PrP demarcates a population of bipotential cardiomyogenic progenitor cells that can differentiate into cardiac or smooth muscle cells.