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

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

Co-aggregation with endoderm-like (End2) cells potently induces early cardiac progenitor cells from mouse ES cells.

<p><b>A,</b> Time course of early CPC markers, Flk1 and Pdgfra, during mouse ES cell differentiation with and without End2 cells. <b>B,</b> Expression profiles of Flk1 and Pdgfra. End2 cells promote CPC induction in a dose-dependent manner. <b>C,</b> mES cells colonies formed in 2D co-culture with End2 cells. <b>D,</b> Expression profiles of Flk1 and Pdgfra. Early CPCs were not efficiently induced in 2D co-culture.</p

Early endoderm is in physical contact with nascent cardiovascular progenitors.

<p><b>A–C</b>, <i>Mesp1^Cre; Rosa^RFP</i> embryos at E7 (A), E8 (B), and E9 (C). <b>D, E</b>, Transverse section of E7 (D) or E8 (E) <i>Mesp1^Cre; Rosa^RFP</i> embryo stained with RFP (red), Troma1 (green), and DAPI (blue). The cutting plane is indicated by a dotted line in (A) or (B). <b>F,</b> Endoderm-like (End2) cells stained with Troma1 and DAPI. DAPI was used to counterstain the nuclei. Scale bars, 75 (A), 200 (B), 250 (C) µm. a, anterior; p, posterior; cc, cardiac crescent; h, head; ht, heart; da, dorsal aorta.</p

Co-aggregation with End2 cells efficiently induces Isl1⁺/Nkx2.5⁺ cardiac progenitors from mouse pluripotent cells.

<p><b>A–D,</b> Gene expression profiles during ESC differentiation with and without End2 cells (n = 3, mean±SD). Mesendoderm marker, <i>T</i> (A), pre-cardiac mesoderm marker, <i>Mesp1</i> (B), cardiac progenitor cell/cardiomyocyte marker, <i>Nkx2.5</i> (C), cardiac progenitor cell marker, <i>Isl1</i> (D). <b>E,</b> Ratio of GFP⁺ cells for day 4–7 of differentiation from ES<i>^Nkx2.^5-GFP</i> cells. <b>F,</b> Ratio of RFP⁺ cells for day 4–7 of differentiation from ES<i>^{Isl1-Cre; Rosa-RFP}</i> cells. <b>G,</b> Ratio of YFP⁺ cells for day 4–7 of differentiation from iPS<i>^{Isl1-Cre; Rosa-YFP}</i> cells. (n = 3, mean±SD, **; p < 0.01, ***; p < 0.001, Two-way anova and Bonferroni post-tests (E–F) and unpaired t-test (G)) <b>H,</b> Gene expression profiles of RFP^+/− cells isolated at day 6 and RFP⁺ cells differentiated for additional 4 days after sorting (Day6+4), normalized to Gapdh. <b>I–J</b> immunostaining of cells at 4 days after sorting of YFP⁺ cells at day 6. Cardiac troponin T (red) and DAPI (blue) (I). Smooth Muscle Actin (SMA, red), Vimentin (green) and DAPI (blue) (J). Scale Bars, 400 µm.</p

Co-aggregation with End2 cells efficiently induces cardiac progenitor cells from human ES cells.

<p><b>A–C</b>, Gene expression profiles during human ES cells differentiation with or without End2 (n = 4, mean±SD). Undifferentiated cell marker, <i>OCT3/4</i> (A), mesendoderm marker, <i>T</i> (B), pre-cardiac mesoderm marker, <i>MESP1</i> (C). <b>D</b>, Plots of pre-cardiac mesoderm. KDR⁺ PDGFRa⁺ cells were only observed in EBs with End2 cells. <b>E</b>, Gene signature of KDR⁺ PDGFRa⁺ cells and KDR⁻ PDGFRa⁻ cells at day 5 of differentiation. Cardiac progenitor markers (<i>TBX5</i>, <i>GATA4</i>, <i>NKX2.5</i> and <i>ISL1</i>) and vascular progenitor marker (<i>ETV2</i>) were significantly upregulated in KDR⁺ PDGFRa⁺ cells. Early cardiomyocyte markers (<i>ACTC1</i> and <i>MYH6</i>) were also significantly upregulated. (n = 5, mean + SD, *; <i>p</i> < 0.05, **; <i>p</i> < 0.01, unpaired <i>t</i>-test).</p

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