Novel 3D culture system with similarities to the human heart for studies of the cardiac stem cell niche

The gastrointestinal epithelium is characterized by a high turnover of cells and intestinal stem cells predominantly reside at the bottom of crypts and their progeny serve to maintain normal intestinal homeostasis. Accumulating evidence demonstrates the pivotal role of a niche surrounding intestinal stem cells in crypts, which consists of cellular and soluble components and creates an environment constantly influencing the fate of stem cells. Here we describe different 3D culture systems to culture gastrointestinal epithelium that should enable us to study the stem cell nichein vitroin the future: organoid culture and multilayered systems such as organotypic cell culture and culture of intestinal tissue fragmentsex vivo. These methods mimic thein vivosituationin vitroby creating 3D culture conditions that reflect the physiological situation of intestinal crypts. Modifications of the composition of the culture media as well as coculturing epithelial organoids with previously described cellular components such as myofibroblasts, collagen, and neurons show the impact of the methods applied to investigate niche interactionsin vitro. We further present a novel method to isolate labeled nerves from the enteric nervous system using Dclk1-CreGFP mice.

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Abstract 259: Role of Connexin 43 in Human Bone Marrow Derived Mesenchymal Stem Cell Cardiac Integration and Cardiac Stem cell Niche Formation.

Circulation Research ◽

10.1161/res.113.suppl_1.a259 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Cristina Sanina ◽

Claudia Rodrigues ◽

Michael Bellio ◽

Ivonne Schulman ◽

Wayne Balkan ◽

...

Keyword(s):

Stem Cell ◽

Gap Junctions ◽

Stem Cell Niche ◽

Connexin 43 ◽

Three Dimensional ◽

Human Mscs ◽

Cardiac Stem Cell ◽

Niche Formation ◽

Cell Niche

Introduction: Bone marrow-derived mesenchymal stem cells (MSCs) and cardiac progenitor cells (CPCs) have been used successfully as a cell-based approach for cardiac regeneration after myocardial infarction. While MSCs and CPCs are effective, in part due to differentiation, paracrine effect, and integration into the heart, the mechanism underlying these effects remains controversial. Objective: We hypothesized that functional connexin 43 (Cx43) gap junctions are crucial for MSC and CPCs interaction and integration into cardiac tissue and cardiac stem cell niche formation. Methods and Results: Human MSCs were co-cultured with neonatal rat ventricular cardiomyocytes (NRVMs). The ability of MSCs to form gap junctions was modulated using lentiviral constructs to either knockdown (Cx43KD) or overexpress (Cx43OE) Cx43. Co-culture of Cx43OE or control MSCs with NRVMs led to the formation of beating, three-dimensional tubes whereas Cx43KD MSCs failed to form tubes (n=5, p<0.05). Furthermore, we replicated the cardiac stem cell niche by combining human CPCs and MSCs in an in vitro model (the same lentiviral constructs were used). As a result, Cx43 expressing MSCs and CPCs formed organotypic three-dimensional structures similar to human MSCs and NRVMs, whereas the lack of Cx43 significantly affected the structure formation (n=6, p<0.05). Cx43KD significantly increased proliferation (n=5, p<0.05) and changed culture phenotype (n=4, p<0.05) of MSCs or CPCs cultured separately. The angiogenic tube length, assessed by a Matrigel assay in vitro, was significantly reduced in MSCs and HUVEC Cx43 KD groups compared to control (n=5, p<0.05). The change/onset of endothelial (KDR, PECAM-1, VE-cadherin), and cardiomyocyte (Nkx2.5, Gata4, Troponin I, Na+ channel, K+ channel, Ca2+ channel, Na+-Ca2+ exchanger) gene expression was investigated in co-culture of MSCs and CPCs. Only voltage ion channel gene expression was upregulated in control and Cx43OE groups by co-culture (n=5, p<0.05), but not affected in Cx43KD group. Conclusion: These findings reveal that cell−cell contact mediated by Cx43 gap junctions enables MSCs to interact with CPCs, integrate and reconstitute cardiac stem cell niches.

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