Murine Dermal Fibroblast Isolation by FACS

Walmsley, Graham G.; Maan, Zeshaan N.; Hu, Michael S.; Atashroo, David; Whittam, Alexander J.; Duscher, Dominik; Tevlin, Ruth; Marecic, Owen; Lorenz, H. Peter; Gurtner, Geoffrey C.; Longaker, Michael T.

doi:10.3791/53430

Cited by 20 publications

(26 citation statements)

References 31 publications

(15 reference statements)

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“…The final, and perhaps most important, consideration is the decision of whether or not to employ cell sorting. At a minimum, MACS or FACS can be beneficial to isolate live cells and potentially enrich for a population of interest (for example, a mouse wound fibroblast population can be enriched by lineage gating against CD45, CD326, CD324, CD31, Ter119, and Tie2) [ 55 ]. However, these techniques can severely traumatize cells, particularly those coming from an injury specimen that may be stressed and have high metabolic demand at baseline.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing

et al. 2020

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Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.

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Section: Discussionmentioning

confidence: 99%

Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing

et al. 2020

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“…Therefore, in vitro cultivation of asFbs is an indispensable procedure to study the repair of depressed scars. Combined digestion methods using trypsin and collagenase for the in vitro culture of asFbs are effective laboratory methods, and asFbs cell cultures are highly proliferative, easily adaptable and stable ( 24 – 27 ). The successful culture of asFbs has provided a reliable source of cells for the repair of human depressed scars ( 22 ).…”

Section: Discussionmentioning

confidence: 99%

Role of rat autologous skin fibroblasts and mechanism underlying the repair of depressed scars

Zhao

Liu

Shi

et al. 2016

Experimental and Therapeutic Medicine

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The aim of the present study was to provide reliable experimental evidence for the application of autologous skin fibroblasts (asFbs) in the repair of depressed scars. In the experiments, depressed trauma was induced in male Wistar rats, and fibroblasts were separated from the removed skin tissues to culture in medium. In vitro cultured asFbs were injected into the depressed scar sites of rats, and the repair function of asFbs in the depressed scars was then examined at the cellular and whole-animal levels. The expression levels of type I and type III collagen in the dermal layer of the skin injected with asFb cells were significantly higher, as compared with those of the control, and type I collagen expression was significantly higher compared with Type III. Re-injection of asFbs into the dermal layer of depressed scars can markedly improve their repair. These results may prove useful for skin repair in clinical settings.

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“…Analysis of murine cells isolated from explanted grafts was performed according to published protocols for flow cytometry on murine tissue ( 61 ). In brief, hSTSG and ADM were explanted on day 14 from the parabiosis and non-parabiosis implantation models, then micro-dissected and incubated in serum-free Dulbecco’s Modified Eagle Medium (DMEM) with 240 U of collagenase IV/ml for 1h at 37°C in a rotating oven.…”

Section: Methodsmentioning

confidence: 99%

Xenogeneic Skin Transplantation Promotes Angiogenesis and Tissue Regeneration Through Vitamin D-Activated Trem2+ Macrophages

Henn

Chen

Fehlmann

et al. 2021

Preprint

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Skin allo- and xenotransplantation are the standard treatment for major burns when donor sites for autografts are not available and have been shown to significantly accelerate wound healing. Although the cellular elements of foreign grafts are rejected, the extracellular matrix components integrate into the wound and may underlie their beneficial effects on wound healing. The molecular mechanisms defining the relationship between the immune response to foreign grafts and their impact on wound healing have not been fully elucidated. Here, we investigated changes in collagen architecture after xenogeneic implantation of clinically available human biologic scaffolds. We show that collagen deposition in response to the implantation of human split-thickness skin grafts (hSTSG) containing live cells recapitulates normal skin architecture, whereas human acellular dermal matrix (ADM) grafts led to highly aligned collagen deposition, characteristic of fibrosis and scar. Using single-cell RNA-sequencing, we show that macrophage differentiation in response to hSTSG is driven by vitamin D (VD) signaling toward Trem2+ subpopulations with an enrichment of pro-angiogenic and anti-fibrotic transcriptomic programs. We subsequently induced this regenerative subpopulation in vitro by treating bone marrow-derived cells with vitamin D3 and found that hydrogel delivery of Trem2+ macrophages significantly accelerated wound closure in a human-like murine excisional wound model. Our study identifies the preclinical therapeutic potential of Trem2+ macrophages to mitigate fibrosis and promote wound healing and provides a novel effective strategy to develop advanced cell therapies for complex wounds.

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Murine Dermal Fibroblast Isolation by FACS

Cited by 20 publications

References 31 publications

Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing

Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing

Role of rat autologous skin fibroblasts and mechanism underlying the repair of depressed scars

Xenogeneic Skin Transplantation Promotes Angiogenesis and Tissue Regeneration Through Vitamin D-Activated Trem2+ Macrophages

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