The organoid-initiating cells in mouse pancreas and liver are phenotypically and functionally similar.

Pancreatic Lgr5 expression has been associated with organoid-forming epithelial progenitor populations but the identity of the organoid-initiating epithelial cell subpopulation has remained elusive. Injury causes the emergence of an Lgr5(+) organoid-forming epithelial progenitor population in the adult mouse liver and pancreas. Here, we define the origin of organoid-initiating cells from mouse pancreas and liver prior to Lgr5 activation. This clonogenic population was defined as MIC1-1C3(+)/CD133(+)/CD26(-) in both tissues and the frequency of organoid initiation within this population was approximately 5% in each case. The transcriptomes of these populations overlapped extensively and showed enrichment of epithelial progenitor-associated regulatory genes such as Sox9 and FoxJ1. Surprisingly, pancreatic organoid cells also had the capacity to generate hepatocyte-like cells upon transplantation to Fah(-/-) mice, indicating a differentiation capacity similar to hepatic organoids. Although spontaneous endocrine differentiation of pancreatic progenitors was not observed in culture, adenoviral delivery of fate-specifying factors Pdx1, Neurog3 and MafA induced insulin expression without glucagon or somatostatin. Pancreatic organoid cultures therefore preserve many key attributes of progenitor cells while allowing unlimited expansion, facilitating the study of fate determination.

Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors.

Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the ancient cell-fate regulator Numb (Nb) and its homologue Numblike (Nbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, flow cytometric and functional analyses, we find that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Nb- and Nbl-deficient CPCs by lineage-specific mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These findings demonstrate that Nb and Nbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and that the PA2 serves as a microenvironment for their expansion.DOI: http://dx.doi.org/10.7554/eLife.02164.001.

Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Direct contact with endoderm-like cells efficiently induces cardiac progenitors from mouse and human pluripotent stem cells.

RATIONALE: Pluripotent stem cell-derived cardiac progenitor cells (CPCs) have emerged as a powerful tool to study cardiogenesis in vitro and a potential cell source for cardiac regenerative medicine. However, available methods to induce CPCs are not efficient or require high-cost cytokines with extensive optimization due to cell line variations. OBJECTIVE: Based on our in-vivo observation that early endodermal cells maintain contact with nascent pre-cardiac mesoderm, we hypothesized that direct physical contact with endoderm promotes induction of CPCs from pluripotent cells. METHOD AND RESULT: To test the hypothesis, we cocultured mouse embryonic stem (ES) cells with the endodermal cell line End2 by co-aggregation or End2-conditioned medium. Co-aggregation resulted in strong induction of Flk1(+) PDGFRa(+) CPCs in a dose-dependent manner, but the conditioned medium did not, indicating that direct contact is necessary for this process. To determine if direct contact with End2 cells also promotes the induction of committed cardiac progenitors, we utilized several mouse ES and induced pluripotent (iPS) cell lines expressing fluorescent proteins under regulation of the CPC lineage markers Nkx2.5 or Isl1. In agreement with earlier data, co-aggregation with End2 cells potently induces both Nkx2.5(+) and Isl1(+) CPCs, leading to a sheet of beating cardiomyocytes. Furthermore, co-aggregation with End2 cells greatly promotes the induction of KDR(+) PDGFRa(+) CPCs from human ES cells. CONCLUSIONS: Our co-aggregation method provides an efficient, simple and cost-effective way to induce CPCs from mouse and human pluripotent cells.

Non-canonical Notch signaling: emerging role and mechanism.

Notch is an ancient transmembrane receptor with crucial roles in cell-fate choices. Although the 'canonical' Notch pathway and its core members are well established - involving ligand-induced cleavage of Notch for transcriptional regulation - it has been unclear whether Notch can also function independently of ligand and transcription ('non-canonically') through a common mechanism. Recent studies suggest that Notch can non-canonically exert its biological functions by post-translationally targeting Wnt/ß-catenin signaling, an important cellular and developmental regulator. The non-canonical Notch pathway appears to be highly conserved from flies to mammals. Here, we discuss the emerging conserved mechanism and role of ligand/transcription-independent Notch signaling in cell and developmental biology.

Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart.

Accurate nuclear identification is crucial for distinguishing the role of cardiac myocytes in intrinsic and experimentally induced regenerative growth of the myocardium. Conventional histologic analysis of myocyte nuclei relies on the optical sectioning capabilities of confocal microscopy in conjunction with immunofluorescent labeling of cytoplasmic proteins such as troponin T, and dyes that bind to double-strand DNA to identify nuclei. Using heart sections from transgenic mice in which the cardiomyocyte-restricted alpha-cardiac myosin heavy chain promoter targeted the expression of nuclear localized beta-galactosidase reporter in >99% of myocytes, we systematically compared the fidelity of conventional myocyte nuclear identification using confocal microscopy, with and without the aid of a membrane marker. The values obtained with these assays were then compared with those obtained with anti-beta-galactosidase immune reactivity in the same samples. In addition, we also studied the accuracy of anti-GATA4 immunoreactivity for myocyte nuclear identification. Our results demonstrate that, although these strategies are capable of identifying myocyte nuclei, the level of interobserver agreement and margin of error can compromise accurate identification of rare events, such as cardiomyocyte apoptosis and proliferation. Thus these data indicate that morphometric approaches based on segmentation are justified only if the margin of error for measuring the event in question has been predetermined and deemed to be small and uniform. We also illustrate the value of a transgene-based approach to overcome these intrinsic limitations of identifying myocyte nuclei. This latter approach should prove quite useful when measuring rare events.

Randomized, controlled trial of intramuscular or intracoronary injection of autologous bone marrow cells into scarred myocardium during CABG versus CABG alone.

BACKGROUND: Studies of the transplantation of autologous bone marrow cells (BMCs) in patients with chronic ischemic heart disease have assessed effects on viable, peri-infarct tissue. We conducted a single-blinded, randomized, controlled study to investigate whether intramuscular or intracoronary administration of BMCs into nonviable scarred myocardium during CABG improves contractile function of scar segments compared with CABG alone. METHODS: Elective CABG patients (n = 63), with established myocardial scars diagnosed as akinetic or dyskinetic segments by dobutamine stress echocardiography and confirmed at surgery, were randomly assigned CABG alone (control) or CABG with intramuscular or intracoronary administration of BMCs. The BMCs, which were obtained at the time of surgery, were injected into the mid-depth of the scar in the intramuscular group or via the graft conduit supplying the scar in the intracoronary group. Contractile function was assessed in scar segments by dobutamine stress echocardiography before and 6 months after treatment. RESULTS: The proportion of patients showing improved wall motion in at least one scar segment after BMC treatment was not different to that observed in the control group (P = 0.092). Quantitatively, systolic fractional thickening in scar segments did not improve with BMC administration. Furthermore, BMCs did not improve scar transmurality, infarct volume, left ventricular volume, or ejection fraction. CONCLUSION: Injection of autologous BMCs directly into the scar or into the artery supplying the scar is safe but does not improve contractility of nonviable scarred myocardium, reduce scar size, or improve left ventricular function more than CABG alone.

Lineage tracing of cardiac explant derived cells.

AIMS: Cultured cardiac explants produce a heterogeneous population of cells including a distinctive population of refractile cells described here as small round cardiac explant derived cells (EDCs). The aim of this study was to explore the source, morphology and cardiogenic potential of EDCs. METHODS: Transgenic MLC2v-Cre/ZEG, and actin-eGFP mice were used for lineage-tracing of EDCs in vitro and in vivo. C57B16 mice were used as cell transplant recipients of EDCs from transgenic hearts, as well as for the general characterisation of EDCs. The activation of cardiac-specific markers were analysed by: immunohistochemistry with bright field and immunofluorescent microscopy, electron microscopy, PCR and RT-PCR. Functional engraftment of transplanted cells was further investigated with calcium transient studies. RESULTS: Production of EDCs was highly dependent on the retention of blood-derived cells or factors in the cultured explants. These cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo. However, EDCs failed to differentiate into functional cardiac myocytes in vivo as demonstrated by the absence of stimulation-evoked intracellular calcium transients following transplantation into the peri-infarct zone. CONCLUSIONS: This study highlights that positive identification based upon one parameter alone such as morphology or immunofluorescene is not adequate to identify the source, fate and function of adult cardiac explant derived cells.