Project name: scRNA-Seq datasets profiling peripheral leukocyte response when co-cultured with fibroblasts isolated from reindeer back skin or antler velvet.

Description: In adult mammals, skin wound healing has evolved to favor rapid repair through the formation of fibrotic scar. These dermal scars are dysfunctional and may lead to chronic disfigurement and disability, yet the biologic mechanisms that drive fibrosis and prevent tissue regeneration remain unknown. Here, we report that reindeer (Rangifer tarandus) antler velvet exhibits regenerative wound healing, whereas identical full-thickness injury in dorsal back skin of the same animal forms fibrotic scar. This regenerative capacity is retained even following ectopic transplantation of velvet to a scar-forming site, demonstrating that this latent regenerative capacity is innate to velvet cells and independent of local factors derived from the growing antler. Single cell RNA-sequencing of uninjured skin revealed a marked divergence in resting fibroblast transcriptional states and immunomodulatory function. Uninjured velvet fibroblast shared a striking resemblance with human fetal fibroblasts whereas uninjured back skin fibroblasts exhibited an overrepresentation of pro-inflammatory genes resembling adult human fibroblasts. Identical skin injury resulted in site-specific fibroblast polarization; back fibroblasts exacerbated the inflammatory response, whereas velvet fibroblasts adopted an immunosuppressive state and reverted back to a regeneration-competent ground state. Consequently, velvet wounds exhibited an accelerated adoption of anti-inflammatory immune states and an expedited resolution of immune response. This study demonstrates reindeer as a novel comparative mammalian model to study both adult skin regeneration (velvet) and scar formation (back skin) within the same animal. Our study underscores the importance of fibroblast heterogeneity in shaping local immune cell functions that ultimately polarize wound healing outcomes. Purposeful, acute modulation of fibroblast-mediated immune signaling represents an important therapeutic avenue to mitigate scar and improve wound healing.Overall design: Whole blood was collected 3 days post-wound from venous blood supply into EDTA tubes. Blood was spun at 300g for 10 mins and plasma was removed and stored at -80oC. Buffy coat was collected, and red blood cells were lysed with ACK buffer (1:10 ACK to blood cells) for 5 minutes and then spun at 300g for 5min and then supernatant. To liberate fibroblasts from reindeer back skin and velvet, either a 12mm circular biopsy punches (velvet) or an elliptical skin excision (back skin) was collected and dissociated to single cells by mincing the skin into 1–2 mm pieces using a pair of scalpels. Minced tissue was first submerged in dispase (2 mg/ml at 37°C for 10 minutes, Stem Cell Technologies) to remove epidermis and then in collagenase type IV (2 mg/ml at 37 for 1-3 hours with mechanical trituration at set intervals, Sigma) to digest the dermal extracellular matrix. Dissociated cell suspensions were passed through 100μm and then 70μm cell strainers, centrifuged at 1200rpm for 6 minutes to pellet the liberated cells, and resuspended in complete fibroblast culture media (DMEM + 10%FBS, 5mL of L-glutamine, penicillin-streptomycin). Fibroblasts were expanded for three days in a 37 °C 5% CO2 tissue culture incubator prior to initiating co-culture with circulating immune cells. Following co-culture incubation, immune cells cells were resuspended in 2% BSA in PBS at a final concentration prior to loading into the 10X controller. All samples were processed according to 10X Genomics ChromiumTM Single Cell 3’ Reagent Guidelines NextGEM Chemistry as per the manufacturer’s protocol. In brief, cells were partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. D11 primers, D12 and C12  primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using DTape station 1000. Sequencing was performed using Illumina NovaSeq with an S2 flow cell.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Other

Last updated: 2022-06-27