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Bone morphogenetic protein-2/-4 upregulation promoted by endothelial cells in coculture enhances mouse embryoid body differentiation.

ABSTRACT: Endothelial cells (ECs) provide inductive signals for cell differentiation in vivo. However, it is unknown if these cells promote such differentiation in vitro and the signals involved. We investigated whether ECs are able to enhance the differentiation of the three germ layers and the underlying mechanisms. We established a coculture system of mouse embryoid bodies (EBs) and ECs. Then, we analyzed the expression of markers representative of the three germ layers, such as PDX-1, proinsulin, insulin1 (endoderm), nestin, neurofilament light (ectoderm), CD31, cardiotin, and cardiac troponin I (mesoderm) in EBs cultured alone (controls) or with ECs. A significant increase of these markers was observed in EBs cocultured with ECs compared to controls. The cocultured EBs also exhibited more robust vascular networks similar to those EBs treated with bone morphogenetic protein-2 or -4 (BMP-2 or -4). Therefore, the role of these peptides in the differentiation was investigated. We found a significant upregulation of BMP-2/-4 and BMP receptor 1A in EBs treated with EC conditioned medium (EC-CM) at early or middle stages of EB development. Recombinant human BMP-2 and BMP-4 exerted similar effects than EC-CM in the expression of BMPs or in the upregulation of the three germ layer specific markers. BMP-2/-4 antagonists, such as noggin and chordin-like-1, respectively inhibited the EC-CM inductive effects. These results demonstrate that ECs enhance the differentiation in vitro of cells that derived from the three germ layers and that BMP-2/-4 play a central role in this process.

SUBMITTER: Talavera-Adame D

PROVIDER: S-EPMC3868407 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Bone morphogenetic protein-2/-4 upregulation promoted by endothelial cells in coculture enhances mouse embryoid body differentiation.

Talavera-Adame Dodanim D Gupta Ankur A Kurtovic Silvia S Chaiboonma Kira L KL Arumugaswami Vaithilingaraja V Dafoe Donald C DC

Stem cells and development 20130924 24

Endothelial cells (ECs) provide inductive signals for cell differentiation in vivo. However, it is unknown if these cells promote such differentiation in vitro and the signals involved. We investigated whether ECs are able to enhance the differentiation of the three germ layers and the underlying mechanisms. We established a coculture system of mouse embryoid bodies (EBs) and ECs. Then, we analyzed the expression of markers representative of the three germ layers, such as PDX-1, proinsulin, insu ...[more]

PMID: 23924071

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Project description:Skeletal tissue regeneration following traumatic injury involves a complex cascade of growth factor signals that direct the differentiation of mesenchymal stem cells (MSCs) within the fracture. The necessity for controlled and localized expression of these factors has highlighted the role gene therapy may play as a promising treatment option for bone repair. However, the design of nanocarrier systems that negotiate efficient intracellular trafficking and nuclear delivery represents a significant challenge. Recent investigations have highlighted the roles histone tail sequences play in directing nuclear delivery and activating DNA transcription. We previously established the ability to recapitulate these natural histone tail activities within non-viral nanocarriers, improving gene transfer and expression by enabling effective navigation to the nucleus via retrograde vesicular trafficking. Herein, we demonstrate that histone-targeting leads to ∼4-fold enhancements in osteogenic bone morphogenetic protein-2 (BMP-2) expression by MSCs over 6 days, as compared with standard polymeric transfection reagents. This improved expression augmented chondrogenesis, an essential first step in fracture healing. Importantly, significant enhancements of cartilage-specific protein expression were triggered by histone-targeted gene transfer, as compared with the response to treatment with equivalent amounts of recombinant BMP-2 protein. In fact, an ∼100-fold increase in recombinant BMP-2 was required to achieve similar levels of chondrogenic gene and protein expression. The enhancements in differentiation achieved using histone-targeting were in part enabled by an increase in transcription factor expression, which functioned to drive MSC chondrogenesis. These novel findings demonstrate the utility of histone-targeted gene transfer strategies to enable substantial reductions in BMP-2 dosing for bone regenerative applications.Statement of significanceThis contribution addresses significant limitations in non-viral gene transfer for bone regenerative applications by exploiting a novel histone-targeting approach for cell-triggered delivery that induces osteogenic BMP-2 expression coincident with the initiation of bone repair. During repair, proliferating MSCs respond to a complex series of growth factor signals that direct their differentiation along cellular lineages essential to mature bone formation. Although these MSCs are ideal targets for enhanced transfection during cellular mitosis, few non-viral delivery approaches exist to enable maximization of this effect. Accordingly, this contribution seeks to utilize our histone-targeted nanocarrier design strategy to stimulate BMP-2 gene transfer in dividing MSCs. This gene-based approach leads to significantly augmented MSC chondrogenesis, an essential first step in bone tissue repair.

Dataset Information

Bone morphogenetic protein-2/-4 upregulation promoted by endothelial cells in coculture enhances mouse embryoid body differentiation.

Publications

Bone morphogenetic protein-2/-4 upregulation promoted by endothelial cells in coculture enhances mouse embryoid body differentiation.

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