Abstract 98: Embryonic Extracellular Matrix and Cell Fate Determination

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Stefan M Kren ◽  
Christopher S Chapman ◽  
Andrew Loza ◽  
Daniel J Garry ◽  
Mary G Garry
Keyword(s):  
Extracellular Matrix ◽  
Cell Fate ◽  
Cellular Proliferation ◽  
Cultured Cells ◽  
Culture Media ◽  
Fluorescent Microscopy ◽  
Embryonic Stem ◽  
Vascular Perfusion ◽  
Murine Embryos

Background - The determination of cell fate during development is governed by intrinsic factors, but also by interaction with the milieu in which they reside. The extracellular matrix (ECM) from rapidly developing tissue should form a rich signaling environment for cellular proliferation and differentiation. Hypothesis - Murine embryonic ECM can be prepared by detergent decellularization that is morphologically preserved, biocompatible for cell culture, and at E13.5 substantial enough to permit vascular catheterization and recellularization by perfusion. Methods and Results - To test the contribution of embryonic extracellular matrix (ECM) to the determination of cell fate, we undertook isolation of ECM from developing murine embryos. Triton X-100 and SDS detergent decellularization were used to isolate ECM from E10.5 and E13.5 embryos. Acellularity was confirmed by pico-green DNA assay (98.7 % ± 0.95 of DNA removed compared to control), as well as the lack of visible nuclei by H & E and DAPI histology. The matrix scaffolds were washed thoroughly with PBS and culture media to return them to a biocompatible state. Murine embryonic stem cells (mESC) modified to express EGFP were cultured on the exterior or the interior of the ECM scaffolds. mESCs seeded on the exterior of the E10.5 scaffolds or perfused through the E13.5 umbilical vasculature were highly adherent and proliferative during the 17 day culture period as evidenced by fluorescent microscopy. Perfused mESCs exhibited engrafted in the heart, liver, and vascular conduit E13.5 matrix 2 days post-infusion. Histology confirmed the attachment and morphologic alteration of the cultured cells on the exterior of the E10.5 ECM and presence of the perfused cells in the E13.5 embryo matrix interior. Conclusion - Biocompatible, acellular morphologically preserved embryonic ECM can be extracted from E10.5 and E13.5 murine embryos. By E13.5 the structural integrity of the acellular matrix can sustain vascular perfusion for delivery of mESCs to internal organoid structures. These ECM preparations support the proliferation and maintenance of mESCs externally and internally.

scholarly journals Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides

2017 ◽  
Vol 37 (7) ◽  
pp. 647-660 ◽  
Author(s):  
Saradaprasan Muduli ◽  
Li-Hua Chen ◽  
Meng-Pei Li ◽  
Zhao-wen Heish ◽  
Cheng-Hui Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Poly Vinyl Alcohol ◽  
Hematopoietic Stem ◽  
Stem Cell Culture

Abstract The physical characteristics of cell culture materials, such as their elasticity, affect stem cell fate with respect to cell proliferation and differentiation. We systematically investigated the morphologies and characteristics of several stem cell types, including human amniotic-derived stem cells, human hematopoietic stem cells, human induced pluripotent stem (iPS) cells, and embryonic stem (ES) cells on poly(vinyl alcohol) (PVA) hydrogels immobilized with and without extracellular matrix-derived oligopeptide. Human ES cells did not adhere well to soft PVA hydrogels immobilized with oligovitronectin, whereas they did adhere well to PVA hydrogel dishes with elasticities greater than 15 kPa. These results indicate that biomaterials such as PVA hydrogels should be designed to possess minimum elasticity to facilitate human ES cell attachment. PVA hydrogels immobilized with and without extracellular matrix-derived oligopeptides are excellent candidates of cell culture biomaterials for investigations into how cell culture biomaterial elasticity affects stem cell culture and differentiation.

scholarly journals Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3629 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Adrianna Jensen ◽  
Ralitsa T. Loewen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Trabecular Meshwork ◽  
Degrees Of Freedom ◽  
Culture Media ◽  
Ex Vivo ◽  
Fluorescent Microscopy ◽  
Anterior Segment ◽  
Perfusion Culture ◽  
Control Group ◽  
Freeze Thaw

Objective The trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might be a viable therapeutic approach. Decellularized TM scaffolds have previously been produced by ablating cells with suicide gene therapy or saponin, which risks incomplete cell removal or dissolution of the extracellular matrix, respectively. We hypothesized that improved trabecular meshwork cell ablation would result from freeze-thaw cycles compared to chemical treatment. Materials and Methods We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion within two hours of sacrifice. Intraocular pressure (IOP) was recorded continuously by a pressure transducer system. After 72 h of IOP stabilization, eight eyes were assigned to freeze-thaw (F) ablation (−80 °C × 2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The TM was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture media for 180 h. TM histology was assessed by hematoxylin and eosin staining. TM viability was evaluated by a calcein AM/propidium iodide (PI) assay. The TM extracellular matrix was stained with Picro Sirius Red. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with five degrees of freedom. Results F and S experienced a similar IOP reduction of 30% from baseline (P = 0.64). IOP reduction of about 30% occurred in F within 24 h and in S within 48 h. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis and Picro Sirius staining confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to many PI-labeled, dead TM cells and calcein-labeled viable TM cells in S. Conclusion We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM extracellular matrix in an organotypic perfusion culture.

Abstract 359: The Production of Acellular Cardiac Extracellular Matrix from P3 Murine Heart

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Stefan M Kren ◽  
Daniel J Garry ◽  
Mary G Garry
Keyword(s):  
Extracellular Matrix ◽  
Cardiac Tissue ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Culture Media ◽  
Heart Tissue ◽  
Cell Nuclei ◽  
Dapi Staining ◽  
Neonatal Heart ◽  
The Matrix

Introduction: Understanding the role of extracellular matrix (ECM) in the creation of the cellular microenvironment during tissue formation and regeneration could be vital in extending this capability to injured adult tissue. To date the only confirmed mammalian heart tissue regeneration/regrowth has been in neonatal murine heart, with the regenerative capacity ceasing after day P3. By focusing on ECM from P3 heart tissue we hope to elucidate its contribution to regenerative plasticity and transfer this capacity to injured adult myocardium. Hypothesis: Detergent decellularization protocols used in adult tissues can be modified to function on a neonatal scale, serving to remove the cellular components of the neonatal heart, leaving structurally intact ECM to serve as a scaffold for the generation of cardiac tissue equivalents. Materials and Methods: Murine P3 hearts were perfused with 1% SDS in water at 20mm Hg for 12 hrs. Following detergent decellularization, perfusion with water, 1% Triton X-100, PBS and culture media restored biocompatibility to the isolated ECM. P1 and P7 primary cardiomyocytes expressing the mCherry red fluorescent protein reporter under control of the alpha myosin promoter were isolated by enzymatic disassociation and cultured in the heart matrix in a perfusion based bioreactor. Results: The decellularized ECM demonstrated removal of 97% of native DNA when compared to control by pico-green dsDNA binding assay. Histologic analysis demonstrated an absence of cell nuclei by H & E and DAPI staining. The preservation of the matrix structure and the maintenance of matrix immunoreactivity (collagen IV) were also demonstrated histologically. Following infusion of P1 or P7 mCherry positive cells, contractile behavior of the recellularized heart constructs was observed, and markers of cardiac linage (alpha-actinin in mCherry positive cells) were present. Conclusions: Neonatal heart matrix can be effectively decellularized. With appropriate modification of perfusion parameters, pediatric ECM structure can be preserved. This isolated matrix can serve as a scaffold for growth and maintenance of immature and mature cardiomyocytes, supporting continued contractility of cultured cells.

Abstract 343: Embryonic Extracellular Matrix and Cardiomyocyte Maturation

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Stefan M Kren ◽  
Daniel J Garry ◽  
Mary G Garry
Keyword(s):  
Cell Culture ◽  
Extracellular Matrix ◽  
Structural Integrity ◽  
Vascular Perfusion ◽  
Acellular Matrix ◽  
Intrinsic Factors ◽  
Contraction Velocity ◽  
Relaxation Velocity ◽  
Murine Embryos ◽  
Contractile Performance

Background: The progression of cell maturation is governed by intrinsic factors, but also by interaction with the milieu in which they reside. The extracellular matrix (ECM) from rapidly developing tissue should form a rich signaling environment for cellular development. Hypothesis: Murine embryonic ECM can be prepared by detergent decellularization that is morphologically preserved, biocompatible for cell culture, and at E13.5, substantial enough to permit vascular catheterization and recellularization by perfusion. When used as a scaffold for cell culture, it can be shown to have a salutatory effect on the morphologic and physiologic maturation of post-natal cardiomyocytes (CM). Methods and Results: To test the contribution of embryonic ECM to the performance of cardiomyocytes in culture, we undertook the isolation of ECM from developing murine embryos. Using a detergent decellularization protocol, we established an acellular scaffold that was shown to be free of native cells and returned to a biocompatible state, to serve as a scaffold for cell culture. Primary P7 mCherry expressing cardiomyocytes were isolated and introduced by perfusion into the embryonic ECM heart. The same cells were also plated, and the performance of the two culture modalities was compared at 3 and 28 days. Histologic comparison demonstrated the maintenance of the rod cellular morphology. Analysis of cellular contractile performance by video microscopy demonstrated improved contractile performance of CMs when cultured on ECM and paced at 1 Hz (3 Day plated contraction velocity/relaxation velocity (μm/sec) 1.18±0.3/0.91±0.2 and ECM contraction velocity/relaxation velocity (μm/sec) 6.65±1.0/4.52±0.7). At 28 days plated contraction velocity/relaxation velocity was (μm/sec) 2.71±0.2/1.59±0.1 and ECM contraction velocity/relaxation velocity was (μm/sec) 8.83±4.2/6.20±2.6). Conclusion: Biocompatible, acellular morphologically preserved embryonic ECM can be extracted from E13.5 murine embryos. By E13.5 the structural integrity of the acellular matrix can sustain vascular perfusion for delivery of P7 cardiomyocytes to internal organoid structures. These ECM preparations support the maturation and physiologic performance of cardiomyocytes.

scholarly journals JMJD3: a critical epigenetic regulator in stem cell fate

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yuanjie Ding ◽  
Yuanchun Yao ◽  
Xingmu Gong ◽  
Qi Zhuo ◽  
Jinhua Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Tumor Development ◽  
Embryonic Stem ◽  
Multipotent Stem Cells ◽  
Stem Cell Fate ◽  
Epigenetic Regulator ◽  
Induced Pluripotent

AbstractThe Jumonji domain-containing protein-3 (JMJD3) is a histone demethylase that regulates the trimethylation of histone H3 on lysine 27 (H3K27me3). H3K27me3 is an important epigenetic event associated with transcriptional silencing. JMJD3 has been studied extensively in immune diseases, cancer, and tumor development. There is a comprehensive epigenetic transformation during the transition of embryonic stem cells (ESCs) into specialized cells or the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). Recent studies have illustrated that JMJD3 plays a major role in cell fate determination of pluripotent and multipotent stem cells (MSCs). JMJD3 has been found to enhance self-renewal ability and reduce the differentiation capacity of ESCs and MSCs. In this review, we will focus on the recent advances of JMJD3 function in stem cell fate.

scholarly journals Vitamin C in Stem Cell Biology: Impact on Extracellular Matrix Homeostasis and Epigenetics

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Cristina D'Aniello ◽  
Federica Cermola ◽  
Eduardo Jorge Patriarca ◽  
Gabriella Minchiotti
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Vitamin C ◽  
Cell Fate ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Redox Status ◽  
Stem Cell Biology ◽  
Prolyl Hydroxylases

Transcription factors and signaling molecules are well-known regulators of stem cell identity and behavior; however, increasing evidence indicates that environmental cues contribute to this complex network of stimuli, acting as crucial determinants of stem cell fate.L-Ascorbic acid (vitamin C (VitC)) has gained growing interest for its multiple functions and mechanisms of action, contributing to the homeostasis of normal tissues and organs as well as to tissue regeneration. Here, we review the main functions of VitC and its effects on stem cells, focusing on its activity as cofactor of Fe+2/αKG dioxygenases, which regulate the epigenetic signatures, the redox status, and the extracellular matrix (ECM) composition, depending on the enzymes’ subcellular localization. Acting as cofactor of collagen prolyl hydroxylases in the endoplasmic reticulum, VitC regulates ECM/collagen homeostasis and plays a key role in the differentiation of mesenchymal stem cells towards osteoblasts, chondrocytes, and tendons. In the nucleus, VitC enhances the activity of DNA and histone demethylases, improving somatic cell reprogramming and pushing embryonic stem cell towards the naive pluripotent state. The broad spectrum of actions of VitC highlights its relevance for stem cell biology in both physiology and disease.

scholarly journals Intrinsic Mechanical Cues and Their Impact on Stem Cells and Embryogenesis

2021 ◽  
Vol 9 ◽  
Author(s):  
Jonna Petzold ◽  
Eileen Gentleman
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Cell Surface ◽  
Cell Fate ◽  
Cell Biology ◽  
Soft Tissues ◽  
Embryonic Stem ◽  
Focal Adhesions ◽  
Cell Fate Decisions ◽  
Applied Forces

Although understanding how soluble cues direct cellular processes revolutionised the study of cell biology in the second half of the 20th century, over the last two decades, new insights into how mechanical cues similarly impact cell fate decisions has gained momentum. During development, extrinsic cues such as fluid flow, shear stress and compressive forces are essential for normal embryogenesis to proceed. Indeed, both adult and embryonic stem cells can respond to applied forces, but they can also detect intrinsic mechanical cues from their surrounding environment, such as the stiffness of the extracellular matrix, which impacts differentiation and morphogenesis. Cells can detect changes in their mechanical environment using cell surface receptors such as integrins and focal adhesions. Moreover, dynamic rearrangements of the cytoskeleton have been identified as a key means by which forces are transmitted from the extracellular matrix to the cell and vice versa. Although we have some understanding of the downstream mechanisms whereby mechanical cues are translated into changes in cell behaviour, many of the signalling pathways remain to be defined. This review discusses the importance of intrinsic mechanical cues on adult cell fate decisions, the emerging roles of cell surface mechano-sensors and the cytoskeleton in enabling cells to sense its microenvironment, and the role of intracellular signalling in translating mechanical cues into transcriptional outputs. In addition, the contribution of mechanical cues to fundamental processes during embryogenesis such as apical constriction and convergent extension is discussed. The continued development of tools to measure the biomechanical properties of soft tissues in vivo is likely to uncover currently underestimated contributions of these cues to adult stem cell fate decisions and embryogenesis, and may inform on regenerative strategies for tissue repair.

Macrophage Inhibition of Collagen-Induced Platelet Aggregation

1979 ◽  
Vol 42 (04) ◽  
pp. 1207-1216 ◽  
Author(s):  
Berit Mørland
Keyword(s):  
Enzyme Activity ◽  
Platelet Aggregation ◽  
Peritoneal Macrophage ◽  
Cultured Cells ◽  
Culture Media ◽  
Human Platelets ◽  
Mouse Peritoneal Macrophage ◽  
Collagenase Activity ◽  
Partial Inhibition ◽  
Macrophage Phagocytosis

SummaryCollagen was incubated with cells or media fractions of mouse peritoneal macrophage cultures, and its aggregating effect on human platelets was tested. Incubation with lysates of cultured cells completely abolished the normal collagen-induced platelet aggregation, while incubation with media fractions only caused partial inhibition. The latter inhibition was more pronounced after macrophage phagocytosis of latex particles, while endocytosis of endotoxin had no effect.Corresponding macrophage cultures were also tested for specific collagenase activity, using 14C-glycine labelled collagen as substrate. Collagenase activity was found in the culture media fractions only, and the enzyme activity could be enhanced by endocytosis of latex as well as endotoxin.It appears that the effect of macrophage lysates and media on collagen-platelet interaction cannot be ascribed only to secretion of collagenase from macrophages.

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