Dataset Information

Fibroblast reticular cells engineer a blastema extracellular network during digit tip regeneration in mice.

ABSTRACT: The regeneration blastema which forms following amputation of the mouse digit tip is composed of undifferentiated cells bound together by an organized network of fibers. A monoclonal antibody (ER-TR7) that identifies extracellular matrix (ECM) fibers produced by fibroblast reticular cells during lymphoid organogenesis was used to characterize the ECM of the digit, the blastema, and the regenerate. Digit fibroblast reticular cells produce an ER-TR7⁺ ECM network associated with different tissues and represent a subset of loose connective tissue fibroblasts. During blastema formation there is an upregulation of matrix production that returns to its pre-existing level and anatomical pattern in the endpoint regenerate. Co-localization studies demonstrate a strong spatial correlation between the ER-TR7 antigen and collagen type III (COL3) in histological sections. ER-TR7 and COL3 are co-induced in cultured digit fibroblasts following treatment with tumor necrosis factor alpha and a lymphotoxin beta receptor agonist. These results provide an initial characterization of the ECM during digit regeneration and identify a subpopulation of fibroblasts involved in producing the blastema provisional matrix that is remodeled during the regeneration response.

SUBMITTER: Marrero L

PROVIDER: S-EPMC5469731 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Fibroblast reticular cells engineer a blastema extracellular network during digit tip regeneration in mice.

Marrero Luis L Simkin Jennifer J Sammarco Mimi M Muneoka Ken K

Regeneration (Oxford, England) 20170401 2

The regeneration blastema which forms following amputation of the mouse digit tip is composed of undifferentiated cells bound together by an organized network of fibers. A monoclonal antibody (ER-TR7) that identifies extracellular matrix (ECM) fibers produced by fibroblast reticular cells during lymphoid organogenesis was used to characterize the ECM of the digit, the blastema, and the regenerate. Digit fibroblast reticular cells produce an ER-TR7<sup>+</sup> ECM network associated with differen ...[more]

PMID: 28616246

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Project description:Purpose: Repair of distal phalanx amputation is widely studied and thought to occur through a regenerative process. Single-cell RNA sequencing (scRNA) has greatly improved the ability to characterize the cells that are involved in regeneration and abnormal healing of bone and soft tissue. Given the disorganized architecture of digit tip “regenerated” bone and its common progenitor cell to the pathologic process of heterotopic ossification, we hypothesized that digit tip repair after injury follows a program of aberrant wound repair rather than true regeneration of normal bone tissue. Methods: ScRNA data of mouse digit tip amputation (DTA) and embryonic limb development (EMB) was downloaded from NBCI GEO data base ascension number GSE135985. Additionally, scRNA data from heterotopic ossification (HO) was downloaded from GSE126060. HO, DTA, and EMB datasets were clustered individually. HO was merged and aligned with EMB and DT in separate analyses. Cells expressing the mesenchymal progenitor cell (MPC) marker Pdgfra, which we have shown to differentiate into HO and others have shown to differentiate into bone after DTA, were identified and analyzed in the HO-DTA and HO-EMB datasets. Monocle 2, Seurat 3.1.1, and Pearson correlation analysis were used for analysis and visualization. Immunostaining for markers present in DTA (ACAN, FBN-2, LTBP2) was performed on sections collected from a murine model of traumatic HO in Pdgfra-CreER;TdTomato mice induced with tamoxifen one-week prior to HO-forming injury. Results: MPC clusters from DTA and HO and from EMB and HO were aligned and projected using UMAP to compare transcription profiles. Across the timepoints, MPCs in the DTA and HO occupy similar regions of the UMAP plot, while MPCs in EMB and HO occupy largely independent regions, with only overlap at the latest time points. Using Monocle 2, cells were placed on a virtual timeline based on similarities in transcription. MPCs from DTA and HO meet at the same location on the trajectory following injury. In the EMB to HO comparison, cells only occupy similar regions of the trajectory at post-natal day 3 suggesting that bone is formed by different pathways. To understand correlations between time points, transcriptomes were also compared by Pearson analysis. MPCs following HO and DTA injuries occupy similar regions of the plot while MPCs in EMB and HO only occupy similar regions of the correlation plot at P3. Markers previously identified to be upregulated in MPCs in DTA were also upregulated in MPCs in HO by both scRNA and immunofluorescent histology. Conclusions: Cells responsible for bone repair following DTA show similar transcriptional profiles as MPCs in traumatic HO and dissimilar to MPCs in EMB. Furthermore, MPCs in both digit tip repair and HO follow trajectories that do not overlap with embryonic limb development. Thus, we demonstrate for the first time, that the process following DTA is not truly regeneration and follows programs of aberrant repair as seen in HO.

Project description:ObjectivesLimb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells (BM-MSCs) as an alternative accessible cell source to BlCs for regeneration of appendages.Materials and methodsIn this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and lineage-specific staining were used to assess their ability to differentiate into skeletal cell lineages. The colony forming ability, proliferation, alkaline phosphatase (ALP) activity, calcium content, and osteogenic gene expression were evaluated in both BMMSCs and BlCs cultures at days 7, 14, and 21.ResultsqRT-PCR analysis revealed that the cells from both sources readily differentiated into mesodermal lineages. There was significantly higher colony forming ability in BM-MSCs compared to BlCs (P<0.05). Alizarin red staining (ARS), calcium, and the ALP assay showed the same degree of mineral deposition in both BlCs and BM-MSCs. Gene expression levels of osteblastic markers indicated similar bone differentiation capacity for both BlCs and BM-MSCs at all time-points.ConclusionsCharacteristics of BlCs in vitro appear to be similar to BM-MSCs. Therefore, they could be considered as a substitute for BlCs for a regenerative approach with potential use in future clinical settings for regenerating human appendages.

Dataset Information

Fibroblast reticular cells engineer a blastema extracellular network during digit tip regeneration in mice.

Publications

Fibroblast reticular cells engineer a blastema extracellular network during digit tip regeneration in mice.

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