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Stage-dependent differential gene expression profiles of cranial neural crest-like cells derived from mouse-induced pluripotent stem cells.

ABSTRACT: Cranial neural crest cells are multipotent cells that migrate into the pharyngeal arches of the vertebrate embryo and differentiate into various craniofacial organ derivatives. Therefore, migrating cranial neural crest cells are considered one of the most attractive candidate cell sources in regenerative medicine. We generated cranial neural crest like cell (cNCCs) using mouse-induced pluripotent stem cells cultured in neural crest-inducing medium for 14 days. Subsequently, we conducted RNA sequencing experiments to analyze gene expression profiles of cNCCs at different time points after induction. cNCCs expressed several neural crest specifier genes; however, some previously reported specifier genes such as paired box 3 and Forkhead box D3, which are essential for embryonic neural crest development, were not expressed. Moreover, ETS proto-oncogene 1, transcription factor and sex-determining region Y-box 10 were only expressed after 14 days of induction. Finally, cNCCs expressed multiple protocadherins and a disintegrin and metalloproteinase with thrombospondin motifs enzymes, which may be crucial for their migration.

SUBMITTER: Odashima A

PROVIDER: S-EPMC7033077 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Stage-dependent differential gene expression profiles of cranial neural crest-like cells derived from mouse-induced pluripotent stem cells.

Odashima Ayano A Onodera Shoko S Saito Akiko A Ogihara Yuuki Y Ichinohe Tatsuya T Azuma Toshifumi T

Medical molecular morphology 20190711 1

Cranial neural crest cells are multipotent cells that migrate into the pharyngeal arches of the vertebrate embryo and differentiate into various craniofacial organ derivatives. Therefore, migrating cranial neural crest cells are considered one of the most attractive candidate cell sources in regenerative medicine. We generated cranial neural crest like cell (cNCCs) using mouse-induced pluripotent stem cells cultured in neural crest-inducing medium for 14 days. Subsequently, we conducted RNA sequ ...[more]

PMID: 31297611

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Project description:Replacement of lost cranial bone (partly mesodermal and partly neural crest-derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow-derived mesenchymal stromal cells (mesoderm-derived BM-MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell-mesenchymal progenitor cells (iNCC-MPCs) improves implant-to-bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC-MPCs. BM-MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (μCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC-MPC-Luc2 vs BM-MSC-Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, μCT analysis showed enhanced structural parameters in the iNCC-MPC-Luc2 group and increased bone volume in the BM-MSC-Luc2 group compared to controls. Histology demonstrated improved integration of iNCC-MPC-Luc2 allografts compared to BM-MSC-Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft-host interphase in cell-seeded groups. The iNCC-MPC-Luc2 group also demonstrated improved biomechanical properties compared to BM-MSC-Luc2 implants and cell-free controls. Our results show an improved integration of iNCC-MPC-Luc2-coated allografts compared to BM-MSC-Luc2 and controls, suggesting the use of iNCC-MPCs as potential cell source for cranial bone repair.

Project description:Cranial bone loss presents a major clinical challenge and new regenerative approaches to address craniofacial reconstruction are in great demand. Induced pluripotent stem cell (iPSC) differentiation is a powerful tool to generate mesenchymal stromal cells (MSCs). Prior research demonstrated the potential of bone marrow-derived MSCs (BM-MSCs) and iPSC-derived mesenchymal progenitor cells via the neural crest (NCC-MPCs) or mesodermal lineages (iMSCs) to be promising cell source for bone regeneration. Overexpression of human recombinant bone morphogenetic protein (BMP)6 efficiently stimulates bone formation. The study aimed to evaluate the potential of iPSC-derived cells via neural crest or mesoderm overexpressing BMP6 and embedded in 3D printable bio-ink to generate viable bone graft alternatives for cranial reconstruction. Cell viability, osteogenic potential of cells, and bio-ink (Ink-Bone or GelXa) combinations were investigated in vitro using bioluminescent imaging. The osteogenic potential of bio-ink-cell constructs were evaluated in osteogenic media or nucleofected with BMP6 using qRT-PCR and in vitro μCT. For in vivo testing, two 2 mm circular defects were created in the frontal and parietal bones of NOD/SCID mice and treated with Ink-Bone, Ink-Bone + BM-MSC-BMP6, Ink-Bone + iMSC-BMP6, Ink-Bone + iNCC-MPC-BMP6, or left untreated. For follow-up, µCT was performed at weeks 0, 4, and 8 weeks. At the time of sacrifice (week 8), histological and immunofluorescent analyses were performed. Both bio-inks supported cell survival and promoted osteogenic differentiation of iNCC-MPCs and BM-MSCs in vitro. At 4 weeks, cell viability of both BM-MSCs and iNCC-MPCs were increased in Ink-Bone compared to GelXA. The combination of Ink-Bone with iNCC-MPC-BMP6 resulted in an increased bone volume in the frontal bone compared to the other groups at 4 weeks post-surgery. At 8 weeks, both iNCC-MPC-BMP6 and iMSC-MSC-BMP6 resulted in an increased bone volume and partial bone bridging between the implant and host bone compared to the other groups. The results of this study show the potential of NCC-MPC-incorporated bio-ink to regenerate frontal cranial defects. Therefore, this bio-ink-cell combination should be further investigated for its therapeutic potential in large animal models with larger cranial defects, allowing for 3D printing of the cell-incorporated material.

Dataset Information

Stage-dependent differential gene expression profiles of cranial neural crest-like cells derived from mouse-induced pluripotent stem cells.

Publications

Stage-dependent differential gene expression profiles of cranial neural crest-like cells derived from mouse-induced pluripotent stem cells.

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