3D spheroids versus 2D-cultured human adipose stem cells to generate smooth muscle cells in an internal anal sphincter-targeting cryoinjured mouse model.

BACKGROUND: The efficacy of cell implantation via 3D-spheroids to treat basal tone in fecal incontinence remains unclear. To address this, in this study, we aimed to identify cell differentiation and assess the development of a contractile phenotype corresponding to smooth muscle cells (SMCs) following implantation of 3D-spheroid and 2D-cultured human adipose stem cells (hASCs) in an in vivo internal anal sphincter (IAS)-targeted mouse model. METHODS: We developed an IAS-targeted in vivo model via rapid freezing (at - 196 °C) of the dorsal layers of the region of interest (ROI) of the IAS ring posterior quarter, between the submucosal and muscular layers, following submucosal dissection (n = 60 rats). After implantation of tetramethylindocarbocyanine perchlorate (Dil)-stained 3D and 2D-cells into randomly allocated cryoinjured rats, the entire sphincter ring or only the cryoinjured ROI was harvested. Expression of SMC markers, RhoA/ROCKII and its downstream molecules, and fibrosis markers was analyzed. Dil, α-smooth muscle actin (α-SMA), and RhoA signals were used for cell tracking. RESULTS: In vitro, 3D-spheroids exhibited higher levels of SMC markers and RhoA/ROCKII-downstream molecules than 2D-hASCs. The IAS-targeted cryoinjured model exhibited substantial loss of SMC layers of the squamous epithelium lining of the anal canal, as well as reduced expression of SMC markers and RhoA-related downstream molecules. In vivo, 3D-spheroid implantation induced SMC markers and contractile molecules weakly at 1 week. At 2 weeks, the mRNA expression of aSma, Sm22a, Smoothelin, RhoA, Mypt1, Mlc20, Cpi17, and Pp1cd increased, whereas that of fibrosis markers reduced significantly in the 3D-spheroid implanted group compared to those in the sham, non-implanted, and 2D-hASC implanted groups. Protein levels of RhoA, p-MYPT1, and p-MLC20 were higher in the 3D-spheroid-implanted group than in the other groups. At 2 weeks, in the implanted groups, the cryoinjured tissues (which exhibited Dil, α-SMA, and RhoA signals) were restored, while they remained defective in the sham and non-implanted groups. CONCLUSIONS: These findings demonstrate that, compared to 2D-cultured hASCs, 3D-spheroids more effectively induce a contractile phenotype that is initially weak but subsequently improves, inducing expression of RhoA/ROCKII-downstream molecules and SMC differentiation associated with IAS basal tone.

Differentiation of Adipose-Derived Stem Cells into Smooth Muscle Cells in an Internal Anal Sphincter-Targeting Anal Incontinence Rat Model.

OBJECTIVE: Studies on development of an anal incontinence (AI) model targeting smooth muscle cells (SMCs) of the internal anal sphincter (IAS) have not been reported. The differentiation of implanted human adipose-derived stem cells (hADScs) into SMCs in an IAS-targeting AI model has also not been demonstrated. We aimed to develop an IAS-targeting AI animal model and to determine the differentiation of hADScs into SMCs in an established model. MATERIALS AND METHODS: The IAS-targeting AI model was developed by inducing cryoinjury at the inner side of the muscular layer via posterior intersphincteric dissection in Sprague-Dawley rats. Dil-stained hADScs were implanted at the IAS injury site. Multiple markers for SMCs were used to confirm molecular changes before and after cell implantation. Analyses were performed using H&E, immunofluorescence, Masson's trichrome staining, and quantitative RT-PCR. RESULTS: Impaired smooth muscle layers accompanying other intact layers were identified in the cryoinjury group. Specific SMC markers, including SM22α, calponin, caldesmon, SMMHC, smoothelin, and SDF-1 were significantly decreased in the cryoinjured group compared with levels in the control group. However, CoL1A1 was increased significantly in the cryoinjured group. In the hADSc-treated group, higher levels of SMMHC, smoothelin, SM22α, and α-SMA were observed at two weeks after implantation than at one week after implantation. Cell tracking revealed that Dil-stained cells were located at the site of augmented SMCs. CONCLUSIONS: This study first demonstrated that implanted hADSc restored impaired SMCs at the injury site, showing stem cell fate corresponding to the established IAS-specific AI model.

CXXC5 Mediates DHT-Induced Androgenetic Alopecia via PGD2.

The number of people suffering from hair loss is increasing, and hair loss occurs not only in older men but also in women and young people. Prostaglandin D2 (PGD2) is a well-known alopecia inducer. However, the mechanism by which PGD2 induces alopecia is poorly understood. In this study, we characterized CXXC5, a negative regulator of the Wnt/ß-catenin pathway, as a mediator for hair loss by PGD2. The hair loss by PGD2 was restored by Cxxc5 knock-out or treatment of protein transduction domain-Dishevelled binding motif (PTD-DBM), a peptide activating the Wnt/ß-catenin pathway via interference with the Dishevelled (Dvl) binding function of CXXC5. In addition, suppression of neogenic hair growth by PGD2 was also overcome by PTD-DBM treatment or Cxxc5 knock-out as shown by the wound-induced hair neogenesis (WIHN) model. Moreover, we found that CXXC5 also mediates DHT-induced hair loss via PGD2. DHT-induced hair loss was alleviated by inhibition of both GSK-3ß and CXXC5 functions. Overall, CXXC5 mediates the hair loss by the DHT-PGD2 axis through suppression of Wnt/ß-catenin signaling.

Pyruvate Kinase M2 Promotes Hair Regeneration by Connecting Metabolic and Wnt/ß-Catenin Signaling.

Hair follicle stem cells (HFSCs) utilize glycolytic metabolism during their activation and anagen induction. However, the role of pyruvate kinase M2 (PKM2), which catalyzes the final step of glycolysis, in hair regeneration has not been elucidated. In this study, we investigated the expression pattern and activity of PKM2 during the depilation-induced anagen progression in mice. We found that TEPP-46, a selective activator of PKM2, enhanced hair re-growth and proliferation of HFSCs. PKM2 expression was increased via up-regulation of Wnt/ß-catenin signaling, which is involved in hair regeneration. Moreover, a combined treatment with KY19382, a small molecule that activates Wnt/ß-catenin signaling, and TEPP-46 significantly enhanced hair re-growth and wound-induced hair follicle neogenesis (WIHN). These results indicate that simultaneous activation of the PKM2 and Wnt/ß-catenin signaling could be a potential strategy for treating alopecia patients.

Wnt/ß-catenin signaling activator restores hair regeneration suppressed by diabetes mellitus.

Diabetes mellitus is one of the most prevalent diseases in modern society. Many complicationssuch as hepatic cirrhosis, neuropathy, cardiac infarction, and so on are associated with diabetes. Although a relationship between diabetes and hair loss has been recently reported, the treatment of diabetic hair loss by Wnt/ß-catenin activators has not been achieved yet. In this study, we found that the depilation-induced anagen phase was delayed in both db/db mice and high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic mice. In diabetic mice, both hair regrowth and wound-induced hair follicle neogenesis (WIHN) were reduced because of suppression of Wnt/ß-catenin signaling and decreased proliferation of hair follicle cells. We identified that KY19382, a small molecule that activates Wnt/ß-catenin signaling, restored the capabilities of regrowth and WIHN in diabetic mice. The Wnt/ß-catenin signaling activator also increased the length of the human hair follicle which was decreased under high glucose culture conditions. Overall, the diabetic condition reduced both hair regrowth and regeneration with suppression of the Wnt/ß-catenin signaling pathway. Consequently, the usage of Wnt/ß-catenin signaling activators could be a potential strategy to treat diabetes-induced alopecia patients. [BMB Reports 2022; 55(11): 559-564].

KY19382, a novel activator of Wnt/ß-catenin signalling, promotes hair regrowth and hair follicle neogenesis.

BACKGROUND AND PURPOSE: The promotion of hair regeneration and growth heavily depends on the activation of Wnt/ß-catenin signalling in the hair follicle, including dermal papilla (DP). KY19382, one of the newly synthesized analogues of indirubin-3'-monoxime (I3O), was identified as a Wnt/ß-catenin signalling activator via inhibition of the interaction between CXXC-type zinc finger protein 5 (CXXC5) and dishevelled (Dvl). Given the close relationship between the Wnt/ß-catenin signalling and hair regeneration, we investigated the effect of KY19382 on hair regrowth and hair follicle neogenesis. EXPERIMENTAL APPROACH: In vitro hair induction effects of KY19382 were performed in human DP cells. The hair elongation effects of KY19382 were confirmed through the human hair follicle and vibrissa culture system. In vivo hair regeneration abilities of KY19382 were identified in three models: hair regrowth, wound-induced hair follicle neogenesis (WIHN) and hair patch assays using C57BL/6 mice. The hair regeneration abilities were analysed by immunoblotting, alkaline phosphatase (ALP) and immunohistochemical staining. KEY RESULTS: KY19382 activated Wnt/ß-catenin signalling and elevated expression of ALP and the proliferation marker PCNA in DP cells. KY19382 also increased hair length in ex vivo-cultured mouse vibrissa and human hair follicles and induced hair regrowth in mice. Moreover, KY19382 significantly promoted the generation of de novo hair follicles as shown by WIHN and hair patch assays. CONCLUSION AND IMPLICATIONS: These results indicate that KY19382 is a potential therapeutic drug that exhibits effective hair regeneration ability via activation of the Wnt/ß-catenin signalling for alopecia treatments.