A naturally derived cardiac extracellular matrix enhances cardiac progenitor cell behavior in vitro

Limitations associated with cardiac progenitor cell (CPC) therapy of myocardial infarction (MI) including poor engraftment, cell death and incomplete cardiac differentiation have hindered the efficacy of treatment in pre-clinical trials. Given that the extracellular environment plays an important role in regulating cell function and that it is significantly remodeled following MI, it is critical to understand how these changes impact the therapeutic potential of CPCs. In this study, we investigated how the alterations to the extracellular matrix (ECM) following MI impacted the regenerative potential of CPCs in vitro. Hearts were decellularized with 1% SDS prior to MI and 1 and 4 weeks post-MI (Fig A) and the composition of the left ventricle or scar was characterized through LC-MS/MS. While Periostin and Collagen I increased post-MI, Laminin decreased (Fig B). c-kit+ CPCs isolated from rat hearts 1 week post-MI were cultured on tissue culture plastic (TCP) coated with pepsin-solubilized ECM. Our results demonstrated that the healthy matrix promoted the expression of pro-angiogenic growth factors, while maintaining the cells in an undifferentiated state (Fig D,E). Alternatively, 1 week ECM promoted cell adherence (Fig C) and the expression of pro-survival growth factors (Fig D) and GATA-4 (Fig E). Cells cultured on 4 week ECM demonstrated significant differentiation towards vascular lineages through their expression of smooth muscle (TAGLN) and endothelial (VWF) markers. By characterizing how the changing ECM composition following MI impacts CPC fate, we may be able to develop therapeutic strategies that modulate cell fate/ function in vivo following implantation.

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Abstract 356: Krueppel-Like Factor 15 Regulates Wnt/β-Catenin Transcription and Controls Cardiac Progenitor Cell Fate in the Postnatal Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a356 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Claudia Noack ◽

Maria P Zafiriou ◽

Anke Renger ◽

Hans J Schaeffer ◽

Martin W Bergmann ◽

...

Keyword(s):

Cell Fate ◽

Transcriptional Activation ◽

Progenitor Cell ◽

Cellular Localization ◽

Target Genes ◽

Critical Role ◽

Isolated Cells ◽

Cardiac Progenitor Cell

Wnt/β-catenin signaling controls adult heart remodeling partly by regulating cardiac progenitor cell (CPC) differentiation. We now identified and characterized a novel cardiac interaction of the transcription factor Krueppel-like factor 15 (KLF15) with the Wnt/β-catenin signaling on adult CPCs. In vitro mutation, reporter gene assays and co-localization studies revealed that KLF15 requires two distinct domains for nuclear localization and for repression of β-catenin-mediated transcription. KLF15 had no effect on β-catenin stability or cellular localization, but interacted with its co-factor TCF4, which is required for activation of β-catenin target gene expression. Moreover, increased TCF4 ubiquitination was induced by KLF15. In line with this finding we found KLF15 to interact with the Nemo-like kinase, which was shown to phosphorylate and target TCF4 for degradation. In vivo analyses of adult Klf15 functional knock-out (KO) vs. wild-type (WT) mice showed a cardiac β-catenin-mediated transcriptional activation and reduced TCF4 degradation along with cardiac dysfunction assessed by echocardiography (n=10). FACS analysis of the CPC enriched-population of KO vs. WT mice revealed a significant reduction of cardiogenic-committed precursors identified as Sca1+/αMHC+ (0.8±0.2% vs. 1.8±0.1%) and Tbx5+ (3.5±0.3% vs. 5.2±0.5%). In contrast, endothelial Sca1+/CD31+ cells were significantly higher in KO mice (11.3±0.4% vs. 8.6±0.4%; n≥9). In addition, Sca1+ isolated cells of Klf15 KO showed increased RNA expression of endothelial markers von Willebrand Factor, CD105, and Flk1 along with upregulation of β-catenin target genes. CPCs co-cultured on adult fibroblasts resulted in increased endothelial Flk1 cells and reduction of αMHC and Hand1 cardiogenic cells in KO vs. WT CPCs (n=9). Treating these co-cultures with Quercetin, an inhibitor of nuclear β-catenin, resulted in partial rescue of the observed phenotype. This study uncovers a critical role of KLF15 for the maintenance of cardiac tissue homeostasis. Via inhibition of β-catenin transcription, KLF15 controls cardiomyogenic cell fate similar to embryonic cardiogenesis. This knowledge may provide a tool for activation of endogenous CPCs in the postnatal heart.

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Decellularized Extracellular Matrix for Cancer Research

Materials ◽

10.3390/ma12081311 ◽

2019 ◽

Vol 12 (8) ◽

pp. 1311 ◽

Cited By ~ 14

Author(s):

Takashi Hoshiba

Keyword(s):

Cancer Research ◽

Extracellular Matrix ◽

Cancer Cell ◽

Cancer Progression ◽

Intracellular Signaling ◽

Genetic Mutation ◽

Cell Behavior ◽

Characterization Methods ◽

Decellularized Extracellular Matrix

Genetic mutation and alterations of intracellular signaling have been focused on to understand the mechanisms of oncogenesis and cancer progression. Currently, it is pointed out to consider cancer as tissues. The extracellular microenvironment, including the extracellular matrix (ECM), is important for the regulation of cancer cell behavior. To comprehensively investigate ECM roles in the regulation of cancer cell behavior, decellularized ECM (dECM) is now used as an in vitro ECM model. In this review, I classify dECM with respect to its sources and summarize the preparation and characterization methods for dECM. Additionally, the examples of cancer research using the dECM were introduced. Finally, future perspectives of cancer studies with dECM are described in the conclusions.

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Influence of injectable microparticle size on cardiac progenitor cell response

Journal of Applied Biomaterials & Functional Materials ◽

10.1177/2280800018782844 ◽

2018 ◽

Vol 16 (4) ◽

pp. 241-251 ◽

Cited By ~ 1

Author(s):

Elisabetta Rosellini ◽

Niccoletta Barbani ◽

Caterina Frati ◽

Denise Madeddu ◽

Diana Massai ◽

...

Keyword(s):

Cell Response ◽

Progenitor Cell ◽

Ex Vivo ◽

Physicochemical Characterization ◽

Hydrolytic Degradation ◽

Spherical Shape ◽

Oil Emulsion ◽

Cardiac Progenitor Cell ◽

Injectable Scaffolds

Introduction: Injectable scaffolds are emerging as a promising strategy in the field of myocardial tissue engineering. Among injectable scaffolds, microparticles have been poorly investigated. The goal of this study was the development of novel gelatin/gellan microparticles that could be used as an injectable scaffold to repair the infarcted myocardium. In particular, the effect of particle size on cardiac progenitor cell response was investigated. Methods: Particles were produced by a water-in-oil emulsion method. Phosphatidylcholine was used as a surfactant. Particles with different diameter ranges (125–300 µm and 350–450 µm) were fabricated using two different surfactant concentrations. Morphological, physicochemical, and functional characterizations were carried out. Cardiac progenitor cell adhesion and growth on microparticles were tested both in static and dynamic suspension culture conditions. Results: Morphological analysis of the produced particles showed a spherical shape and porous surface. The hydrophilicity of particle matrix and the presence of intermolecular interactions between gellan and gelatin were pointed out by the physicochemical characterization. A weight loss of 75 ± 5 % after 90 days of hydrolytic degradation was observed. Injectability through a narrow needle (26 G) and persistence of the microparticles at the injection site were preliminarily verified by ex vivo test. In vitro cell culture tests showed a preservation of rat cardiac progenitor biologic properties and indicated a preferential cell adherence to microparticles with a smaller size. Conclusion: Overall, the obtained results indicate that the produced gelatin/gellan microparticles could be potentially employed as injectable scaffolds for myocardial regeneration.

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Human foreskin-derived dermal stem/progenitor cell-conditioned medium combined with hyaluronic acid promotes extracellular matrix regeneration in diabetic wounds

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02116-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yu Xin ◽

Peng Xu ◽

Xiangsheng Wang ◽

Yunsheng Chen ◽

Zheng Zhang ◽

...

Keyword(s):

Extracellular Matrix ◽

Hyaluronic Acid ◽

Matrix Metalloproteinases ◽

Conditioned Medium ◽

Progenitor Cell ◽

Bioactive Molecules ◽

Published Data ◽

Collagen Iii ◽

Diabetic Wounds

Abstract Background Diabetic wounds remain a challenging clinical problem, which requires further treatment development. Published data showed that dermis-derived stem/progenitor cells (DSPCs) display superior wound healing in vitro. The beneficial effects of DSPCs are mediated through paracrine secretion, which can be obtained from conditioned medium (CM). Hyaluronic acid (HA) is especially suitable for skin regeneration and delivering bioactive molecules in CM. This study investigated the effect of human foreskin-derived dermal stem/progenitor cell (hFDSPC)-CM combined with HA on a diabetic mouse model and relevant mechanism in vitro. Methods hFDSPCs and human adipose-derived stem cells (hADSCs) were identified, and the respective CM was prepared. PBS, HA, hFDSPC-CM combined with HA, or hADSC-CM combined with HA was topically applied to mice. HE, CD31, CD68, CD86, and CD206 staining was performed to evaluate gross wound condition, angiogenesis, and inflammation, respectively. Masson and Picrosirius red staining was performed to evaluate collagen deposition and maturation. The effects of hFDSPC-CM and hADSC-CM on human keratinocyte cells (HaCaT) and fibroblasts were evaluated in vitro using CCK-8 and EdU assays to determine cell viability and proliferation, respectively. The scratch assay was performed to evaluate cell migration. Tube formation assay was performed on human umbilical vein endothelial cells (HUVECs) to confirm angiogenesis. Extracellular matrix (ECM) metabolic balance-related genes and proteins, such as collagen I (COL 1), collagen III (COL 3), fibronectin (FN), α-SMA, matrix metalloproteinases 1 (MMP-1), matrix metalloproteinases 3 (MMP-3), and transforming growth factor-beta 1 (TGF-β1), were analysed. Results hFDSPC-CM combined with HA showed superior wound closure rate over hADSC-CM. Histologically, the hFDSPC-CM combined with HA group showed significantly improved re-epithelialisation, angiogenesis, anti-inflammation, collagen regeneration, and maturation compared to hADSC-CM combined with HA group. In vitro assays revealed that hFDSPC-CM displayed significant advantages on cell proliferation, migration, and ECM regeneration through a TGF-β/Smad signalling pathway compared with hADSC-CM. Conclusions hFDSPC-CM combined with HA was superior for treating diabetic wounds. The underlying mechanism may promote proliferation and migration of epidermal cells with fibroblasts, thus leading to ECM deposition and remodelling. Reduced inflammation may be due to the above-mentioned mechanism.

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