Abstract 290: Haemogenic Endocardium Contribute To Definitive Hematopoiesis During Cardiogenesis

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Haruko Nakano ◽  
Xiaoqian Liu ◽  
Armin Arshi ◽  
Ben van Handel ◽  
Rajkumar Sasidharan ◽  
...  
Keyword(s):  
De Novo ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Circulatory System ◽  
Lineage Tracing ◽  
Mouse Heart ◽  
Heart Tube ◽  
Cardiac Progenitors ◽  
Definitive Hematopoiesis

The circulatory system is the first functional organ system that develops during mammalian life. Accumulating evidences suggest that cardiac and endocardial cells can arise from a single common progenitor cell during mammalian cardiogenesis. Notably, these early cardiac progenitors express multiple hematopoietic transcription factors, consistent with previous reports. Indeed, a close relationship among cardiac, endocardial and hematopoietic lineages has been suggested in fly, zebrafish, and embryonic stem cell in vitro differentiation models. However, it is unclear when, where and how this hematopoietic gene program is in operation during in vivo mammalian cardiogenesis. Hematopoietic colony assay suggests that mouse heart explants generate myeloids and erythroids in the absence of circulation, suggesting that the heart tube is a de novo site for the definitive hematopoiesis. Lineage tracing revealed that putative cardiac-derived Nkx2-5+/Isl1+ endocardial cells give rise to CD41+ hematopoietic progenitors that contribute to definitive hematopoiesis in vivo and ex vivo during embryogenesis earlier than in the AGM region. Furthermore, Nkx2-5 and Isl1 are both required for the hemogenic activity of the endocardium. Together, identification of Nkx2-5/Isl1-dependent hemogenic endocardial cells (1) adds hematopoietic component in the cardiogenesis lineage tree, (2) changes the long-held dogma that AGM is the only major source of definitive hematopoiesis in the embryo proper, and (3) represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway during the development of circulatory system.

scholarly journals Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Boon-Seng Soh ◽  
Shi-Yan Ng ◽  
Hao Wu ◽  
Kristina Buac ◽  
Joo-Hye C. Park ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Fate ◽  
Embryonic Stem ◽  
Circulatory System ◽  
Cardiac Progenitors ◽  
Cardiac Tissues ◽  
Endothelin 1 ◽  
Key Factor

Abstract Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human–mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1+ vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo.

Abstract 18845: Induced Cardiac Progenitor Cells Differentiate Into Cardiac Lineage Cells in vivo and Improve Survival in Mice post Myocardial Infarction

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Pratik A Lalit ◽  
Max R Salick ◽  
Daryl O Nelson ◽  
Jayne M Squirrell ◽  
Christina M Shafer ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Disease Modeling ◽  
Ex Vivo ◽  
Cardiac Progenitor Cells ◽  
Heart Tube ◽  
Whole Embryo Culture ◽  
Cardiac Actin ◽  
Cardiac Lineage

Several studies have reported reprogramming of fibroblasts (Fibs) to induced cardiomyocytes, and we have recently reprogrammed mouse Fibs to induced cardiac progenitor cells (iCPCs), which may be more favorable for cardiac repair because of their expandability and multipotency. Adult cardiac (AC), lung and tail-tip Fibs from an Nkx2.5-EYFP reporter mouse were reprogrammed using a combination of five defined factors into iCPCs. Transcriptome and immunocytochemistry analysis revealed that iCPCs were cardiac mesoderm-restricted progenitors that expressed CPC markers including Nkx2.5, Gata4, Irx4, Tbx5, Cxcr4, Flk1 etc. iCPCs could be extensively expanded (over 30 passages) while maintaining multipotency to differentiate in vitro into cardiac lineage cells including cardiomyocytes (CMs), smooth muscle cells and endothelial cells. iCPC derived CMs upon co-culture with mESC-derived CMs formed intercellular gap junctions, exhibited calcium transients, and contractions. The purpose of this study was to determine the in vivo potency of iCPCs. Given that the Nkx2.5-EYFP reporter identifies embryonic CPCs, we first tested the embryonic potency of iCPCs using an ex vivo whole embryo culture model injecting cells into the cardiac crescent (CC) of E8.5 mouse embryos and culturing for 24 to 48 hours. GFP labeled AC Fibs were first tested and live imaging revealed that after 24 hours these cells were rejected from the embryo proper and localized to the ecto-placental cone. In contrast, iCPCs reprogrammed from AC Fibs when injected into the CC localized to the developing heart tube and differentiated into MLC2v, αMHC and cardiac actin expressing CMs. Further we injected iCPCs into infarcted adult mouse hearts and determined their regenerative potential after 1-4 wks. The iCPCs significantly improved survival (p<0.01 Mantel-Cox test) in treated animals (75%) as compared to control (11%). Immunohistochemistry revealed that injected iCPCs localized to the scar area and differentiated into cardiac lineage cells including CMs (cardiac actin). These results indicate that lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for cardiac regenerative therapy as well as drug discovery and disease modeling.

Abstract 294: Hematopoietic Cells Are Required For Proper Development Of Coronary Vasculature

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Gentian Lluri ◽  
Xiaoqian Liu ◽  
Atsushi N
Keyword(s):  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Coronary Vessels ◽  
Explant Culture ◽  
Hematopoietic Cell ◽  
Mouse Heart ◽  
Blood Island ◽  
Epicardial Cells ◽  
Whole Mount

Objective: We examined whether the hematopoietic cells induce the coronary artery formation using genetically modified mouse models of hematopoietic ablation in vivo and ex vivo . Methods: As a model of for hematopoietic cell deficient animals, we used Runx1 (a transcription factor required for definitive hematopoiesis) knockout embryos and Vav1-cre; R26-DTA embryos, which ablates 2/3 of CD45+ hematopoietic cells. The coronary growth and the hematopoietic cells were evaluated in whole-mount, section and ex vivo explant culture. Results: The developing coronary endothelial cells form blood-island-like structure at around E12.5 in the subepicardial region. Interestingly, however, the histological analyses suggest that the first Ter119+ and CD45+ blood cells appear in the subendocardial area at E10.5, even before the formation of coronary channels. These initial hematopoietic cells in the heart are not likely derived from the epicardium, as the sorted epicardial cells yielded no hematopoietic cell in colony formation assay. These observations raised a question whether these heart-resident hematopoietic cells rather play an inductive role during coronary formation. To examine this possibility, we analyzed two hematopoietic ablation models. Both Runx1 knockout embryos and Vav1-cre; R26-DTA embryos revealed disorganized, hypoplastic microvasculature of coronary vessels on section and whole-mount stainings. Furthermore, coronary explant experiments showed that the mouse heart explants from Runx1 knockout embryos and Vav1-cre; R26-DTA embryos exhibited impaired coronary formation ex vivo. Conclusion: Hematopoietic cells are not merely transported via coronary vessels, but substantially involved in the induction of the coronary vessels during cardiogenesis.

scholarly journals Multipotent fetal-derived Cdx2 cells from placenta regenerate the heart

2019 ◽  
pp. 201811827 ◽  
Author(s):  
Sangeetha Vadakke-Madathil ◽  
Gina LaRocca ◽  
Koen Raedschelders ◽  
Jesse Yoon ◽  
Sarah J. Parker ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Contractile Function ◽  
Es Cells ◽  
Embryonic Stem ◽  
Lineage Tracing ◽  
Cardiac Differentiation ◽  
Cell Based Therapy ◽  
Allogeneic Cell Therapy

The extremely limited regenerative potential of adult mammalian hearts has prompted the need for novel cell-based therapies that can restore contractile function in heart disease. We have previously shown the regenerative potential of mixed fetal cells that were naturally found migrating to the injured maternal heart. Exploiting this intrinsic mechanism led to the current hypothesis that Caudal-type homeobox-2 (Cdx2) cells in placenta may represent a novel cell type for cardiac regeneration. Using a lineage-tracing strategy, we specifically labeled fetal-derived Cdx2 cells with enhanced green fluorescent protein (eGFP). Cdx2-eGFP cells from end-gestation placenta were assayed for cardiac differentiation in vitro and in vivo using a mouse model of myocardial infarction. We observed that these cells differentiated into spontaneously beating cardiomyocytes (CMs) and vascular cells in vitro, indicating multipotentiality. When administered via tail vein to infarcted wild-type male mice, they selectively and robustly homed to the heart and differentiated to CMs and blood vessels, resulting in significant improvement in contractility as noted by MRI. Proteomics and immune transcriptomics studies of Cdx2-eGFP cells compared with embryonic stem (ES) cells reveal that they appear to retain “stem”-related functions of ES cells but exhibit unique signatures supporting roles in homing and survival, with an ability to evade immune surveillance, which is critical for cell-based therapy. Cdx2-eGFP cells may potentially represent a therapeutic advance in allogeneic cell therapy for cardiac repair.

Angiotensin II overflow from canine skeletal muscle in vivo: importance of plasma angiotensin I

1994 ◽  
Vol 266 (5) ◽  
pp. R1664-R1669 ◽  
Author(s):  
J. H. Schwieler ◽  
J. Nussberger ◽  
T. Kahan ◽  
P. Hjemdahl
Keyword(s):  
De Novo ◽  
Ex Vivo ◽  
Plasma Renin ◽  
Gracilis Muscle ◽  
Ang Ii ◽  
Concentration Difference ◽  
Beta Adrenoceptor ◽  
Arterial Blood ◽  
Catheter System

The overflows (i.e., veno-arterial concentration differences multiplied by plasma flow) of angiotensin-(1-10) decapeptide (ANG I) and angiotensin-(1-8) octapeptide (ANG II) from blood-perfused canine gracilis muscle in situ were studied. Special precautions were taken to minimized ex vivo generation and/or degradation of angiotensins in the sampled blood. ANG I was found to be generated in the catheter system supplying the gracilis muscle with arterial blood, but plasma renin activity and ANG II levels were uninfluenced by the catheter system. A positive venoarterial concentration difference over the muscle itself was found for ANG II but not for ANG I under basal conditions. Isoprenaline elicited vasodilatation, reduced ANG I overflow, and tended to increase ANG II overflow, whereas beta-adrenoceptor blockade by propranolol had no effect on these variables. In conclusion, we found no evidence for a local de novo synthesis of ANG II from the gracilis muscle vasculature in vivo. The net overflow of ANG II was most likely caused by local conversion in the tissue of ANG I artifactually generated in the arterial catheter system. beta-Adrenoceptor stimulation enhanced the local conversion of ANG I to ANG II, probably by exposing a greater endothelial surface containing angiotensin-converting enzyme activity.

Profiling substrate fluxes in the isolated working mouse heart using 13C-labeled substrates: focusing on the origin and fate of pyruvate and citrate carbons

2004 ◽  
Vol 286 (4) ◽  
pp. H1461-H1470 ◽  
Author(s):  
Maya Khairallah ◽  
François Labarthe ◽  
Bertrand Bouchard ◽  
Gawiyou Danialou ◽  
Basil J. Petrof ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Ex Vivo ◽  
Heart Function ◽  
Acid Oxidation ◽  
Mouse Heart ◽  
Relative Contribution ◽  
Cardiac Functions ◽  
Lactate And Pyruvate

The availability of genetically modified mice requires the development of methods to assess heart function and metabolism in the intact beating organ. With the use of radioactive substrates and ex vivo perfusion of the mouse heart in the working mode, previous studies have documented glucose and fatty acid oxidation pathways. This study was aimed at characterizing the metabolism of other potentially important exogenous carbohydrate sources, namely, lactate and pyruvate. This was achieved by using 13C-labeling methods. The mouse heart perfusion setup and buffer composition were optimized to reproduce conditions close to the in vivo milieu in terms of workload, cardiac functions, and substrate-hormone supply to the heart (11 mM glucose, 0.8 nM insulin, 50 μM carnitine, 1.5 mM lactate, 0.2 mM pyruvate, 5 nM epinephrine, 0.7 mM oleate, and 3% albumin). The use of three differentially 13C-labeled carbohydrates and a 13C-labeled long-chain fatty acid allowed the quantitative assessment of the metabolic origin and fate of tissue pyruvate as well as the relative contribution of substrates feeding acetyl-CoA (pyruvate and fatty acids) and oxaloacetate (pyruvate) for mitochondrial citrate synthesis. Beyond concurring with the notion that the mouse heart preferentially uses fatty acids for energy production (63.5 ± 3.9%) and regulates its fuel selection according to the Randle cycle, our study reports for the first time in the mouse heart the following findings. First, exogenous lactate is the major carbohydrate contributing to pyruvate formation (42.0 ± 2.3%). Second, lactate and pyruvate are constantly being taken up and released by the heart, supporting the concept of compartmentation of lactate and glucose metabolism. Finally, mitochondrial anaplerotic pyruvate carboxylation and citrate efflux represent 4.9 ± 1.8 and 0.8 ± 0.1%, respectively, of the citric acid cycle flux and are modulated by substrate supply. The described 13C-labeling strategy combined with an experimental setup that enables continuous monitoring of physiological parameters offers a unique model to clarify the link between metabolic alterations, cardiac dysfunction, and disease development.

scholarly journals Inhibition of Autism-Related Crm1 Disrupts Mitosis and Induces Apoptosis of the Cortical Neural Progenitors

2020 ◽  
Vol 30 (7) ◽  
pp. 3960-3976
Author(s):  
Xue Li ◽  
Yue Feng ◽  
Meifang Yan ◽  
Xiaomeng Tu ◽  
Bin Xie ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
De Novo ◽  
Ex Vivo ◽  
Brain Slices ◽  
Neural Progenitors ◽  
Embryonic Brain ◽  
Mitotic Slippage ◽  
Developing Mouse Brain

Abstract De novo microdeletion of chromosome 2p15–16.1 presents clinically recognizable phenotypes that include mental retardation, autism, and microcephaly. Chromosomal maintenance 1 (CRM1) is a gene commonly missing in patients with 2p15–16.1 microdeletion and one of two genes found in the smallest deletion case. In this study, we investigate the role and mechanism of Crm1 in the developing mouse brain by inhibiting the protein or knocking down the gene in vivo. Inhibition of Crm1 reduces the proliferation and increases p53-dependent apoptosis of the cortical neural progenitors, thereby impeding the growth of embryonic cerebral cortex. Live imaging of mitosis in ex vivo embryonic brain slices reveals that inhibition of CRM1 arrests the cortical progenitors at metaphase. The arrested cells eventually slip into a pseudo-G1 phase without chromosome segregation. The mitotic slippage cells are marked by persistent expression of the spindle assembly checkpoint (SAC), repressing of which rescues the cells from apoptosis. Our study reveals that activating the SAC and inducing the mitotic slippage may lead to apoptosis of the cortical neural progenitors. The resulting cell death may well contribute to microcephaly associated with microdeletion of chromosome 2p15–16.1 involving CRM1.

GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4108-4118 ◽  
Author(s):  
Naruyoshi Suwabe ◽  
Satoru Takahashi ◽  
Toru Nakano ◽  
Masayuki Yamamoto
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Es Cell ◽  
Globin Mrna ◽  
Differentiated Cells ◽  
Vitro Analysis ◽  
Definitive Hematopoiesis

Abstract Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

Transdifferentiation of pancreatic ductal cells to endocrine β-cells

2008 ◽  
Vol 36 (3) ◽  
pp. 353-356 ◽  
Author(s):  
Susan Bonner-Weir ◽  
Akari Inada ◽  
Shigeru Yatoh ◽  
Wan-Chun Li ◽  
Tandy Aye ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Duct Cell ◽  
Cell Types ◽  
Lineage Tracing ◽  
Β Cells ◽  
Partial Pancreatectomy ◽  
Pancreatic Cell ◽  
Ductal Cells ◽  
Pancreatic Ductal Cells

The regenerative process in the pancreas is of particular interest, since diabetes, whether Type 1 or Type 2, results from an inadequate amount of insulin-producing β-cells. Islet neogenesis, or the formation of new islets, seen as budding of hormone-positive cells from the ductal epithelium, has long been considered to be one of the mechanisms of normal islet growth after birth and in regeneration, and suggested the presence of pancreatic stem cells. Results from the rat regeneration model of partial pancreatectomy led us to hypothesize that differentiated pancreatic ductal cells were the pancreatic progenitors after birth, and that with replication they regressed to a less differentiated phenotype and then could differentiate to form new acini and islets. There are numerous supportive results for this hypothesis of neogenesis, including the ability of purified primary human ducts to form insulin-positive cells budding from ducts. However, to rigorously test this hypothesis, we took a direct approach of genetically marking ductal cells using CAII (carbonic anhydrase II) as a duct-cell-specific promoter to drive Cre recombinase in lineage-tracing experiments using the Cre-Lox system. We show that CAII-expressing pancreatic cells act as progenitors that give rise to both new islets and acini after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor for all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes either in vivo or ex vivo.

scholarly journals Targeted Deletion of the Lipopolysaccharide (LPS)-binding Protein Gene Leads to Profound Suppression of LPS Responses Ex Vivo, whereas In Vivo Responses Remain Intact

1997 ◽  
Vol 186 (12) ◽  
pp. 2051-2056 ◽  
Author(s):  
Mark M. Wurfel ◽  
Brian G. Monks ◽  
Robin R. Ingalls ◽  
Russell L. Dedrick ◽  
Russell Delude ◽  
...  
Keyword(s):  
Lipid Transfer Protein ◽  
Ex Vivo ◽  
Binding Protein ◽  
Embryonic Stem ◽  
Cellular Responses ◽  
Tnf Α ◽  
Deficient Mice ◽  
Lps Binding

Gram-negative bacterial lipopolysaccharide (LPS) stimulates phagocytic leukocytes by interacting with the cell surface protein CD14. Cellular responses to LPS are markedly potentiated by the LPS-binding protein (LBP), a lipid-transfer protein that binds LPS aggregates and transfers LPS monomers to CD14. LBP also transfers LPS to lipoproteins, thereby promoting the neutralization of LPS. LBP present in normal plasma has been shown to enhance the LPS responsiveness of cells in vitro. The role of LBP in promoting LPS responsiveness in vivo was tested in LBP-deficient mice produced by gene targeting in embryonic stem cells. Whole blood from LBP-deficient animals was 1,000-fold less responsive to LPS as assessed by the release of tumor necrosis factor (TNF)-α. Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS. These activities were restored by the addition of exogenous recombinant murine LBP to the plasma. Despite these striking in vitro findings, no significant differences in TNF-α levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS. These data suggest the presence of an LBP-independent mechanism for responding to LPS. These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.

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