Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.

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Multipotent fetal-derived Cdx2 cells from placenta regenerate the heart

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811827116 ◽

2019 ◽

pp. 201811827 ◽

Cited By ~ 1

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.

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MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00271.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. H2038-H2049 ◽

Cited By ~ 79

Author(s):

Carley Glass ◽

Dinender K. Singla

Keyword(s):

Cell Differentiation ◽

Cardiac Myocyte ◽

Heart Function ◽

Es Cells ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Border Zone ◽

Es Cell ◽

Infarcted Heart ◽

Myocyte Differentiation

microRNAs (miRs) have emerged as critical modulators of various physiological processes including stem cell differentiation. Indeed, miR-1 has been reported to play an integral role in the regulation of cardiac muscle progenitor cell differentiation. However, whether overexpression of miR-1 in embryonic stem (ES) cells (miR-1-ES cells) will enhance cardiac myocyte differentiation following transplantation into the infarcted myocardium is unknown. In the present study, myocardial infarction (MI) was produced in C57BL/6 mice by left anterior descending artery ligation. miR-1-ES cells, ES cells, or culture medium (control) was transplanted into the border zone of the infarcted heart, and 2 wk post-MI, cardiac myocyte differentiation, adverse ventricular remodeling, and cardiac function were assessed. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared with ES cells. Assessment of apoptosis revealed that overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-AKT and inhibition of caspase-3, phosphatase and tensin homolog, and superoxide production. A significant reduction in interstitial and vascular fibrosis was quantified in miR-1-ES cell and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1-ES cell and ES cell groups was observed. Finally, mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls ( P < 0.05). Our data suggest miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI, ultimately giving rise to enhanced cardiac repair, regeneration, and function.

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A Rapid and Highly Efficient Genotyping Pipeline for Screening Gene Targeted Mouse Embryonic Stem Cells

10.21203/rs.3.rs-748320/v1 ◽

2021 ◽

Author(s):

Roger Caothien ◽

Charles Yu ◽

Lucinda Tam ◽

Robert Newman ◽

Brian Nakao ◽

...

Keyword(s):

Animal Models ◽

Gene Targeting ◽

Fluorescent Protein ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Rapid Screening ◽

Es Cell ◽

Screening Process ◽

Targeting Vector

Abstract Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. The addition of magnetic activated cell sorting (MACS) technology and multiplex droplet digital PCR (ddPCR) to the ES cell screening process can achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. This improved workflow reduces the time needed to generate preclinical animal models. Faster access to animal models for therapeutic target identification and experimental validation can accelerate the development of therapies for human disease.

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Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7419-7426.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7419-7426 ◽

Cited By ~ 207

Author(s):

Sara R. Cherry ◽

D. Biniszkiewicz ◽

L. van Parijs ◽

D. Baltimore ◽

R. Jaenisch

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Transcriptional Repression ◽

Fluorescent Protein ◽

De Novo ◽

Es Cells ◽

Embryonic Stem ◽

Hematopoietic Stem

ABSTRACT Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

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Herbal Extract from Codonopsis pilosula (Franch.) Nannf. Enhances Cardiogenic Differentiation and Improves the Function of Infarcted Rat Hearts

Life ◽

10.3390/life11050422 ◽

2021 ◽

Vol 11 (5) ◽

pp. 422

Author(s):

Jieh-Neng Wang ◽

Chung-Dann Kan ◽

Lain-Tze Lee ◽

Lynn L. H. Huang ◽

Ya-Li Hsiao ◽

...

Keyword(s):

Cardiac Function ◽

Fluorescent Protein ◽

Es Cells ◽

Embryonic Stem ◽

Herbal Extract ◽

Egfp Expression ◽

Herbal Extracts ◽

Es Cell ◽

Codonopsis Pilosula ◽

Cardiogenic Differentiation

Background: The roots of Codonopsis pilosula (Franch.) Nannf. have been used in traditional Chinese medicine for treating cardiovascular disease. In the current study, we aimed to discover herbal extracts from C. pilosula that are capable of improving cardiac function of infarcted hearts to develop a potential therapeutic approach. Methods: A mouse embryonic stem (ES) cell-based model with an enhanced green fluorescent protein (eGFP) reporter driven by a cardiomyocyte-specific promoter, the α-myosin heavy chain, was constructed to evaluate the cardiogenic activity of herbal extracts. Then, herbal extracts from C. pilosula with cardiogenic activity based on an increase in eGFP expression during ES cell differentiation were further tested in a rat myocardial infarction model with left anterior descending artery (LAD) ligation. Cardiac function assessments were performed using echocardiography, 1, 3, and 6 weeks post LAD ligation. Results: The herbal extract 417W from C. pilosula was capable of enhancing cardiogenic differentiation in mouse ES cells in vitro. Echocardiography results in the LAD-ligated rat model revealed significant improvements in the infarcted hearts at least 6 weeks after 417W treatment that were determined based on left ventricle fractional shortening (FS), fractional area contraction (FAC), and ejection fraction (EF). Conclusions: The herbal extract 417W can enhance the cardiogenic differentiation of ES cells and improve the cardiac function of infarcted hearts.

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Monkey Embryonic Stem Cell Lines Expressing Green Fluorescent Protein

Cell Transplantation ◽

10.3727/000000002783985350 ◽

2002 ◽

Vol 11 (7) ◽

pp. 631-635 ◽

Cited By ~ 27

Author(s):

Tatsuyuki Takada ◽

Yutaka Suzuki ◽

Yasushi Kondo ◽

Nae Kadota ◽

Kinji Kobayashi ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Transplantation ◽

Cell Lines ◽

Fluorescent Protein ◽

Es Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Es Cell ◽

Green Fluorescent

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.

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309 DEVELOPMENT OF A CULTURE SYSTEM CAPABLE OF LONG-TERM MAINTENANCE OF BUFFALO (BUBALUS BUBALIS) EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab309 ◽

2011 ◽

Vol 23 (1) ◽

pp. 251 ◽

Cited By ~ 1

Author(s):

R. Sharma ◽

A. George ◽

N. M. Kamble ◽

K. P. Singh ◽

S. K. Panda ◽

...

Keyword(s):

Colony Size ◽

Inner Cell Mass ◽

Es Cells ◽

Formation Rate ◽

Cell Mass ◽

Embryonic Stem ◽

Bubalus Bubalis ◽

Es Cell

The present study was aimed at developing a system for long-term culture of buffalo embryonic stem (ES) cells, which, to our knowledge, have not been maintained beyond passage 10 in reports available to date, primarily because of lack of information on their specific requirements during in vitro culture. Inner cell mass (n = 181) cells, mechanically isolated from in vitro produced day 8 blastocysts, were cultured on mitomycin-C-treated buffalo fetal fibroblast feeder layers in stem cell medium (SCM), which consisted of Knockout-DMEM® + 15% Knockout serum replacer® + 1% minimal essential medium nonessential amino acids + 50 μg mL–1 of gentamicin, supplemented with 1000 IU mL–1 of leukemia inhibitory factor (LIF) and fibroblast growth factor-2 (FGF-2) at different concentrations. The medium was changed every 24 h. The primary colony formation rate, which was similar for 5, 10, 20, and 40 ng mL–1 of FGF-2 (63.7 ± 5.2, 65.7 ± 6.5, 57.0 ± 10.5, and 62.8 ± 13.30, respectively), was significantly higher (P ≤ 0.05) than that of controls (22.4 ± 5.5). In Experiment 2, ES-cell-like cell colonies at passages 6 through 7 (n = 441) were cultured for 5 to 6 days to examine the effects of media supplements. The percentage of colonies that survived was significantly higher (P ≤ 0.05) when these were cultured in SCM+LIF+5 ng mL–1 of FGF-2 (93.1 ± 1.8) than when these were cultured in SCM alone (73.5 ± 9.0) or in SCM supplemented with FGF-2 (88.8 ± 5.4) or LIF (85.8 ± 3.7). Following examination of the colony size at 0 and 120 h of culture, the increase in colony size was found to be nearly 4- (P ≤ 0.01) and 2-fold higher (P ≤ 0.05) with SCM+LIF+5 ng mL–1 of FGF-2 (41.9 ± 3.4) and SCM+FGF-2 (21.0 ± 3.0), respectively, than with SCM alone (10.8 ± 2.6) or with SCM+LIF (9.3 ± 3.3). The ES cell colonies cultured in the presence of FGF-2 were compact and had defined edges, whereas those cultured in its absence were less compact, irregularly shaped, and had less defined edges. To confirm the role of FGF-2 in maintenance of buffalo ES cells, the cell colonies cultured in the presence of 5 ng mL–1 of FGF-2 (n = 487) were exposed to different concentrations (10, 20, or 30 μM) of SU5402, a FGF-2 receptor inhibitor, for 5 to 6 days. The percentage of cell colonies that were found to have differentiated was significantly higher (P ≤ 0.05) when these had been cultured in the presence of 30 (78.6 ± 4.2) or 20 μM (47.9 ± 1.0) than when these were cultured with 10 (24.5 ± 5.1) or 0 μM (28.6 ± 2.3) of SU5402. Following culture in SCM+LIF+FGF-2, buffalo ES cells, in which the expression of pluripotency markers such as OCT-4, NANOG, and SOX-2 was regularly confirmed, have been maintained for more than 80 passages for over an year’s time to date, indicating that a combination of LIF and FGF-2 is beneficial for the maintenance of buffalo ES cells. Supported by NAIP grant No. C4/C-2067 from ICAR, India.

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ROCK inhibitor Y-27632 enhances the survivability of dissociated buffalo (Bubalus bubalis) embryonic stem cell-like cells

Reproduction Fertility and Development ◽

10.1071/rd11315 ◽

2013 ◽

Vol 25 (2) ◽

pp. 446 ◽

Cited By ~ 3

Author(s):

Ruchi Sharma ◽

Aman George ◽

Manmohan S. Chauhan ◽

Suresh Singla ◽

Radhey S. Manik ◽

...

Keyword(s):

Fluorescent Protein ◽

Es Cells ◽

Single Cells ◽

Formation Rate ◽

Embryonic Stem ◽

Rho Kinase ◽

Specific Inhibitor ◽

Embryoid Bodies ◽

Es Cell ◽

Rock Inhibitor

This study investigated the effects of supplementation of culture medium with 10 μM Y-27632, a specific inhibitor of Rho kinase activity, for 6 days on self-renewal of buffalo embryonic stem (ES) cell-like cells at Passage 50–80. Y-27632 increased mean colony area (P < 0.05) although it did not improve their survival. It decreased OCT4 expression (P < 0.05), increased NANOG expression (P < 0.05), but had no effect on SOX2 expression. It also increased expression of anti-apoptotic gene BCL-2 (P < 0.05) and decreased that of pro-apoptotic genes BAX and BID (P < 0.05). It increased plating efficiency of single-cell suspensions of ES cells (P < 0.05). Following vitrification, the presence of Y-27632 in the vitrification solution or thawing medium or both did not improve ES cell colony survival. However, following seeding of clumps of ES cells transfected with pAcGFP1N1 carrying green fluorescent protein (GFP), Y-27632 increased colony formation rate (P < 0.01). ES cell colonies that formed in all Y-27632-supplemented groups were confirmed for expression of pluripotency markers alkaline phosphatase, SSEA-4 and TRA-1–60, and for their ability to generate embryoid bodies containing cells that expressed markers of ectoderm, mesoderm and endoderm. In conclusion, Y-27632 improves survival of buffalo ES cells under unfavourable conditions such as enzymatic dissociation to single cells or antibiotic-assisted selection after transfection, without compromising their pluripotency.

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Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients

AJP Cell Physiology ◽

10.1152/ajpcell.00188.2019 ◽

2019 ◽

Vol 317 (4) ◽

pp. C725-C736

Author(s):

Gurbind Singh ◽

Divya Sridharan ◽

Mahmood Khan ◽

Polani B. Seshagiri

Keyword(s):

Cell Biology ◽

Fluorescent Protein ◽

Es Cells ◽

Monochromatic Light ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Medical Diagnostics ◽

Calcium Transients ◽

Egfp Expression ◽

Mitochondrial Activity

We earlier established the mouse embryonic stem (ES) cell “GS-2” line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420–495), green (~510–575), and red (~600–700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

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