Inhibition of growth of Asian keloid cells with human umbilical cord Wharton's jelly stem cell-conditioned medium

Abstract

Background: Keloid formation occurs in Caucasian, African, and Asian populations and is a severe psychosocial burden on patients. There is no permanent treatment for this problem as its pathogenesis is not properly understood. Furthermore, differences in keloid behavior between ethnic groups are not known. It has been hypothesized that keloids behave like benign tumors because of their uncontrolled growth. The present study evaluated the tumoricidal properties of human Wharton's jelly stem cell-conditioned medium (hWJSC-CM) on fresh Asian keloid cells (AKCs).

Methods: Human Wharton's jelly stem cells (hWJSCs) and AKCs were isolated based on our previous methods. hWJSCs and human skin fibroblasts (HSF) (controls) were used to collect hWJSC-CM and HSF-conditioned medium (HSF-CM). AKCs were treated with hWJSC-CM and HSF-CM in vitro and in vivo in a human keloid xenograft SCID mouse model. The inhibitory effect of hWJSC-CM on AKCs was tested in vitro using various assays and in vivo for attenuation/abrogation of AKC tumors created in a xenograft mouse model.

Results: qRT-PCR analysis showed that the genes FN1, MMP1, and VCAN were significantly upregulated in AKCs and ANXA1, ASPN, IGFBP7, LGALS1, and PTN downregulated. AKCs exposed to hWJSC-CM in vitro showed significant decreases in cell viability and proliferation, increases in Annexin V-FITC+ cell numbers, interruptions of the cell cycle at Sub-G1 and G2/M phases, altered CD marker expression, downregulated anti-apoptotic-related genes, and upregulated pro-apoptotic and autophagy-related genes compared to controls. When AKCs were administered together with hWJSC-CM into immunodeficient mice there were no keloid tumors formed in 7 mice (n = 10) compared to the untreated control mice. When hWJSC-CM was injected directly into keloid tumors created in mice there were significant reductions in keloid tumor volumes and weights in 30 days.

Conclusions: hWJSC-CM inhibited the growth of AKCs in vitro and in xenograft mice, and it may be a potential novel treatment for keloids in the human. The specific molecule(s) in hWJSC-CM that induce the anti-keloid effect need to be identified, characterized, and tested separately in larger preclinical and clinical studies.

Keywords: Cell inhibition; Characterization; Human Wharton’s jelly stem cell-conditioned medium; Keloid; Keloid tumor volume and weight; SCID mice.

Fig. 1

a qRT-PCR analysis of keloid-related genes (A2M, FN1, MMP1, VCAN, C5orf13, HIF1a, SERPINH1, ACAN3, TNFAIP6, INHBA, DCN, FMOD, TGF-β1, TGF-β3, ANXA1, ASPN, IGFBP7, LGALS1, and PTN). Confocal analysis of b paraffin section of keloid tissue and c AKCs adherent cells. All values are represented as mean ± SEM of at least three independent experiments. p < 0.05 was considered as statistically significant

Fig. 2

a Phase-contrast inverted optical images of AKCs grown in keloid medium (control), HSF-CM and hWJSC-CM. b Viable AKCs cell counts assessed by Trypan blue staining for AKCs grown in keloid medium (control), HSF-CM and hWJSC-CM. Assesment of c cell viability (MTT) and d cell proliferation (BrdU) of AKCs grown in keloid medium (control), HSF-CM and hWJSC-CM at day 3. All values are represented as mean ± SEM of at least three independent experiments. p < 0.05 was considered as statistically significant

Fig. 3

a Flow cytometry analysis of MSC CD marker profile for AKCs cultured in keloid medium (control), HSF-CM, and hWJSC-CM. b Cell cycle analysis of AKCs cultured in keloid medium (control), HSF-CM, and hWJSC-CM. All values are represented as mean ± SEM of at least three independent experiments. p < 0.05 was considered as statistically significant

Fig. 4

a Annexin-V analysis of AKCs grown in keloid medium (control), HSF-CM, and hWJSC-CM. b qRT-PCR analysis of pro-apoptotic, anti-apoptotic, and autophagy-related genes (BAX, SURVIVIN, BECLIN-1, ATG5, and ATG7) for AKCs grown in keloid medium (control), HSF-CM, and hWJSC-CM. All values are represented as mean ± SEM of at least three independent experiments. p < 0.05 was considered as statistically significant

Fig. 5

a, b qRT-PCR analysis of keloid and matrix assembly-related genes (A2M, FN1, MMP1, VCAN, C5orf13, HIF1a, TNFAIP6, INHBA, ACAN3, ANXA1, ASPN, SERPINH1, COL1, IGFBP7, PTN, DCN, FMOD, TGF-β1, and TGF-β3) for AKCs cultured in keloid medium (control), HSF-CM, and hWJSC-CM. All values are represented as mean ± SEM of at least three independent experiments. p < 0.05 was considered as statistically significant

Fig. 6

a Combined protocol (The administration of AKCs together with hWJSC-CM into SCID mice) of keloid xenograft SCID mice. a(a, c, and e) AKCs + HA, AKCs + HA + HSF-CM, and AKCs + HA + hWJSC-CM were injected into subcutaneous sites (both hind limbs) per animal (n = 10). a(b, d, and f) Human nuclear antigen (HNA) staining of keloid tumor from AKCs + HA, AKCs + HA + HSF-CM, and AKCs + HA + hWJSC-CM. b Evaluation of keloid tumor volume and c tumor weight. All values are represented as mean ± SEM of at least three independent experiments. p < 0.05 was considered as statistically significant

Fig. 7

a Separate protocol (Keloid tumors were first created in SCID mice about the size of 0.3–6 cm in diameter and respective treatments administered through intra-tumorally) of keloid xenograft SCID mice. a(a) AKCs + HA were injected into subcutaneous sites to induce keloid tumors. a(b, c) Human nuclear antigen (HNA) staining of keloid tumor from AKCs + HA on day 7. b(a–c) PBS, HSF-CM, and hWJSC-CM treatments were administered intra-tumorally to each animal (n = 9). c Evaluation of keloid tumor volume and d tumor weight. All values are represented as mean ± SEM of at least three independent experiments. p < 0.05 was considered as statistically significant