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In vivo multimodal microscopy for detecting bone-marrow-derived cell contribution to skin regeneration.

ABSTRACT: Bone-marrow (BM)-derived cells have been shown to be capable of aiding skin regeneration in vivo by differentiating into keratinocytes. However, the conditions under which this occurs are not fully understood. Characterizing innate mechanisms of skin regeneration by stem cells in vivo is important for the area of stem cell biology. In this study, we investigate the use of novel in vivo imaging technology for characterizing the contribution of BM-derived cells to regeneration of the epidermis in mouse skin in vivo. In vivo imaging provides the ability to non-invasively observe the spatial positions and morphology of the BM-derived cells. Using a GFP BM-transplanted mouse model and in vivo multimodal microscopy, BM-derived cells can be observed in the skin. Our in vivo imaging method was used to search for the presence and identify the 3D spatial distribution of BM-derived cells in the epidermis of the skin under normal conditions, following wound healing, and after syngeneic skin grafting. We did not observe any evidence of BM-derived keratinocytes under these conditions, but we did observe BM-derived dendritic cells in the skin grafts. In vivo multimodal imaging has great potential for characterizing the conditions under which BM-derived cells contribute to skin regeneration.

SUBMITTER: Graf BW

PROVIDER: S-EPMC4058964 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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In vivo multimodal microscopy for detecting bone-marrow-derived cell contribution to skin regeneration.

Graf Benedikt W BW Bower Andrew J AJ Chaney Eric J EJ Marjanovic Marina M Adie Steven G SG De Lisio Michael M Valero Maria C MC Boppart Marni D MD Boppart Stephen A SA

Journal of biophotonics 20130208 1-2

Bone-marrow (BM)-derived cells have been shown to be capable of aiding skin regeneration in vivo by differentiating into keratinocytes. However, the conditions under which this occurs are not fully understood. Characterizing innate mechanisms of skin regeneration by stem cells in vivo is important for the area of stem cell biology. In this study, we investigate the use of novel in vivo imaging technology for characterizing the contribution of BM-derived cells to regeneration of the epidermis in ...[more]

PMID: 23401460

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Project description:Current wound healing models generally employ full-thickness or irregular split wounds. Consequently, assessing the type of healing at varying wound depths and determining the deepest level at which wounds can regenerate has been a challenge. We describe a wound model that allows assessment of the healing process over a continuous gradient of wound depth, from epidermal to full-thickness dermal loss. Further, we investigate whether green fluorescent protein-labeled bone marrow mesenchymal stem cells (BM-MSCs/GFP) transplantation could regenerate deeper wounds that might otherwise lead to scar formation. A wound gradient was created on the back of 120 Sprague Dawley rats, which were randomized into the BM-MSCs/GFP and control group. These were further subdivided into 6 groups where terminal biopsies of the healing wounds were taken at days 1, 3, 5, 7, 14, and 21 post-operatively. At each observed time point, the experimental animals were anesthetized and photographed, and depending on the group, the animals euthanized and skin taken for rapid freezing, haemotoxylin and eosin staining, and vascular endothelial growth factor (VEGF) immunohistochemistry. We found the deepest layer to regenerate in the control group was at the level of the infundibulum apex, while in the BM-MSCs/GFP group this was deeper, at the opening site of sebaceous duct at hair follicle in which had the appearance of normal skin and less wound contraction than the control group (P value less than .05). The expression of VEGF in BM-MSCs/GFP group was higher than that in control group (P value less than .05). The number of vessels increased from 2.5 ± 0.2/phf of control group to 5.0 ± 0.3/phf of BM-MSCs/GFP (P value less than .05). The progressively deepening wound model we described can identify the type of wound repair at increasing depths. Further, topical transplantation of BM-MSCs/GFP significantly improved regeneration of deeper wounds from infundibulum apex (maximum depth of control group regeneration) to the opening site of sebaceous duct at hair follicle level.

Project description:BACKGROUND: Definite identification of the cell types and the mechanism relevant to cardiomyogenesis is essential for effective cardiac regenerative medicine. We aimed to identify the cell populations that can generate cardiomyocytes and to clarify whether generation of donor-marker(+) cardiomyocytes requires cell fusion between BM-derived cells and recipient cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: Purified BM stem/progenitor cells from green fluorescence protein (GFP) mice were transplanted into C57BL/6 mice or cyan fluorescence protein (CFP)-transgenic mice. Purified human hematopoietic stem cells (HSCs) from cord blood were transplanted into immune-compromised NOD/SCID/IL2r?(null) mice. GFP(+) cells in the cardiac tissue were analyzed for the antigenecity of a cardiomyocyte by confocal microscopy following immunofluorescence staining. GFP(+) donor-derived cells, GFP(+)CFP(+) fused cells, and CFP(+) recipient-derived cells were distinguished by linear unmixing analysis. Hearts of xenogeneic recipients were evaluated for the expression of human cardiomyocyte genes by real-time quantitative polymerase chain reaction. In C57BL/6 recipients, Lin(-/low)CD45(+) hematopoietic cells generated greater number of GFP(+) cardiomyocytes than Lin(-/low)CD45(-) mesenchymal cells (37.0+/-23.9 vs 0.00+/-0.00 GFP(+) cardiomyocytes per a recipient, P?=?0.0095). The number of transplanted purified HSCs (Lin(-/low)Sca-1(+) or Lin(-)Sca-1(+)c-Kit(+) or CD34(-)Lin(-)Sca-1(+)c-Kit(+)) showed correlation to the number of GFP(+) cardiomyocytes (P<0.05 in each cell fraction), and the incidence of GFP(+) cardiomyocytes per injected cell dose was greatest in CD34(-)Lin(-)Sca-1(+)c-Kit(+) recipients. Of the hematopoietic progenitors, total myeloid progenitors generated greater number of GFP(+) cardiomyocytes than common lymphoid progenitors (12.8+/-10.7 vs 0.67+/-1.00 GFP(+) cardiomyocytes per a recipient, P?=?0.0021). In CFP recipients, all GFP(+) cardiomyocytes examined coexpressed CFP. Human troponin C and myosin heavy chain 6 transcripts were detected in the cardiac tissue of some of the xenogeneic recipients. CONCLUSIONS/SIGNIFICANCE: Our results indicate that HSCs resulted in the generation of cardiomyocytes via myeloid intermediates by fusion-dependent mechanism. The use of myeloid derivatives as donor cells could potentially allow more effective cell-based therapy for cardiac repair.

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In vivo multimodal microscopy for detecting bone-marrow-derived cell contribution to skin regeneration.

Publications

In vivo multimodal microscopy for detecting bone-marrow-derived cell contribution to skin regeneration.

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