Generation and characterization of two immortalized dermal fibroblast cell lines from the spiny mouse (Acomys)

Abstract

The spiny mouse (Acomys) is gaining popularity as a research organism due to its phenomenal regenerative capabilities. Acomys recovers from injuries to several organs without fibrosis. For example, Acomys heals full thickness skin injuries with rapid re-epithelialization of the wound and regeneration of hair follicles, sebaceous glands, erector pili muscles, adipocytes, and dermis without scarring. Understanding mechanisms of Acomys regeneration may uncover potential therapeutics for wound healing in humans. However, access to Acomys colonies is limited and primary fibroblasts can only be maintained in culture for a limited time. To address these obstacles, we generated immortalized Acomys dermal fibroblast cell lines using two methods: transfection with the SV40 large T antigen and spontaneous immortalization. The two cell lines (AcoSV40 and AcoSI-1) maintained the morphological and functional characteristics of primary Acomys fibroblasts, including maintenance of key fibroblast markers and ECM deposition. The availability of these cells will lower the barrier to working with Acomys as a model research organism, increasing the pace at which new discoveries to promote regeneration in humans can be made.

Fig 1. Immortalized AcoSV40 and AcoSI-1 fibroblasts demonstrate logarithmic growth over at least 30 passages.

(a) AcoSV40 fibroblasts maintain logarithmic growth across 30 passages and 3 freeze/thaw cycles. (b) pAFs subjected to continuous subculture spontaneously immortalize after about 40 days. (c) AcoSI-1 fibroblasts maintain logarithmic growth across 30 passages and 3 freeze/thaw cycles.

Fig 2. AcoSV40 and AcoSI-1 fibroblasts maintain morphological characteristics and fibroblast markers (vimentin and αSMA) of pAFs.

(a) Phase images of passage 1 pAFs, AcoSV40, and AcoSI-1 fibroblast demonstrate similar morphology; scale = 100um. (b) pAFs, AcoSV40, and AcoSI-1 fibroblasts express vimentin at similar levels, but αSMA is weekly present in AcoSV40 and absent in AcoSI-1 fibroblasts under standard culture conditions. We demonstrated that under serum starvation, AcoSV40 and AcoSI-1 can (c) upregulate αSMA and (d) co-localize it to stress fibers; scale = 100μm.

Fig 3. Traction forces of pAFs, AcoSV40, and AcoSI-1 fibroblasts are similar, and differences in strain energy correlate with differences in cell area.

Cells were seeded on 8kPa polyacrylamide gels to evaluate root-mean-square of traction (rmst), max rmst, strain energy, and area. pAFs have higher maximum contraction stresses than AcoSI-1 cells, but similar average stresses to both immortalized lines. While pAFs have higher strain energy (i.e., do more work) than the immortalized fibroblasts, it is likely a function of their larger area. *p<0.05 as assessed by ANOVA with Bonferroni test.

Fig 4. Immortalized lines AcoSV40 and AcoSI-1 share most proteins with pAFs.

The comparison of AcoSV40 and pAF CDMs via mass spectrometry resulted in no significant difference in deposited proteins. (a) AcoSI-1 CDMs shared 604 out of 686 proteins identified with pAFs and enriched biological processes within the dissimilar proteins were unrelated to ECM organization. (b) In comparison, NIH3T3 fibroblasts share 641 out of 853 identified proteins with Mus primary fibroblasts, some of which are related to ECM organization. GO biological processes were determined by running the proteins that were exclusive to Primary Acomys, AcoSI-1, Primary Mus, and NIH3T3 fibroblasts through the PANTHER classification system. For brevity, only the top eight enriched biological processes were reported in the NIH3T3 exclusive proteins, all of which had a fold enrichment change of over 50.

Fig 5. AcoSV40 cells are female while all AcoSI cells are male.

(a) Forward and reverse primers specific to Acomys SrY. (b) PCR products for Y chromosome in all 4 cell lines compared to a DNA sample obtained from the blood of an Acomys adult male.