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Background: Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are effective in the treatment of skin photoaging; however, their low yield and functional decline with passage progression limit their clinical application. Cell‐derived nanovesicles (CNVs) are potential alternatives that can address the limitations of EVs derived from MSCs and are conducive to clinical transformations. Hair follicle mesenchymal stem cells (HFMSCs), a type of MSCs, have demonstrated the function of repairing skin tissues; nevertheless, the efficacy of CNVs from HFMSCs (HFMSC‐CNVs) in the treatment of skin photoaging remains unclear. Therefore, ultraviolet radiation B (UVB)‐induced photoaging nude mice and human dermal fibroblasts (HDFs) were used as experimental models to investigate the therapeutic effects of HFMSC–CNVs in photoaging models. Methods: HFMSC–CNVs were successfully prepared using the mechanical extrusion method. UVB‐induced nude mice and HDFs were used as experimental models of photoaging. Multiple approaches, including hematoxylin–eosin and Masson staining, immunohistochemistry, immunofluorescence, detection of reactive oxygen species (ROS), flow cytometry, western blotting, and other experimental methods, were combined to investigate the possible effects and mechanisms of HFMSC–CNVs in the treatment of skin photoaging. Results: In the nude mouse model of skin photoaging, treatment with HFMSC–CNVs reduced UVB‐induced skin wrinkles (p < 0.05) and subcutaneous capillary dilation, alleviated epidermis thickening (p < 0.001), and dermal thinning (p < 0.001). Furthermore, HFMSC–CNVs upregulated proliferating cell nuclear antigen (PCNA) expression (p < 0.05) and decreased the levels of ROS, β‐galactosidase (β‐Gal), and CD86 (p < 0.01). In vitro experiments, treatment with HFMSC–CNVs enhanced the cellular activity of UVB‐exposed HDFs (p < 0.05), and reduced ROS levels and the percentage of senescent cells (p < 0.001), and alleviated cell cycle arrest (p < 0.001). HFMSC–CNVs upregulated the expression of Collagen I (Col I), SMAD2/3, transforming growth factor beta (TGF‐β), catalase (CAT), glutathione peroxidase‐1 (GPX‐1), and superoxide dismutase‐1 (SOD‐1) (p < 0.05) and downregulated the expression of cycle suppressor protein (p53), cell cycle suppressor protein (p21), and matrix metalloproteinase 3 (MMP3) (p < 0.05). Conclusion: Conclusively, the anti‐photoaging properties of HFMSC–CNVs were confirmed both in vivo and in vitro. HFMSC–CNVs exert anti‐photoaging effects by alleviating cell cycle arrest, decreasing cellular senescence and macrophage infiltration, promoting cell proliferation and extracellular matrix (ECM) production, and reducing oxidative stress by increasing the activity of antioxidant enzymes. [ABSTRACT FROM AUTHOR]

Hair follicle mesenchymal stem cells (HFMSCs) are slow cycling label retaining cells with self-regeneration ability, high proliferative potential, and multi-directional differentiation. HFMSCs can induce and promote the regeneration of the epidermis, hair follicles and sebaceous glands. HFMSCs have important advantages in regenerative medicine because of their abundance, wide availability, expansion in vitro, no genetic manipulation, no tumor formation and no ethical restriction. The synergy and antagonism between Wnt and other signaling pathways such as Shh, Notch and BMP signaling pathways play a crucial role in the regulation of stem cell homeostasis, epidermal development and hair follicle regeneration. The dysregulation of these pathways may result in hair loss diseases or skin tumors. This paper summarizes the classification and some specific biomarkers of HFMSCs, and discusses the induction of HFMSCs and the biomolecular pathways that affect hair regeneration, so as to shed light on the treatment strategies of hair diseases.

Stem cell senescence and depletion are major causes of aging and aging-related diseases. The NAD (Nicotinamide adenine dinucleotide) – SIRT1 (Silent Information Regulator 1) – PARP1 (Poly (ADP-ribose) polymerase-1) axis has gained interest owing to its significant role in regulating stem cell senescence and organismal aging. A recent study from our lab showed that pre-B-cell leukemia transcription factor1 (PBX1) overexpression attenuates hair follicle-derived mesenchymal stem cells (HF-MSCs) senescence and apoptosis by regulating ROS-mediated DNA damage via PARP1 downregulation; thus, suggesting that PARP1 downregulation is a common manifestation of the roles of both PBX1 and SIRT1 in HF-MSCs senescence attenuation, and implying a potential link between PBX1 and SIRT1. To this end, HF-MSCs overexpressing PBX1, overexpressing both PBX1 and PARP1, downregulating SIRT1, and overexpressing PBX1 as well as downregulating SIRT1 were generated, and senescence, apoptosis, DNA damage, and repair biomarkers were analyzed. Our results showed that (1) PBX1 overexpression alleviated HF-MSCs senescence and apoptosis accompanied by SIRT1 upregulation, PARP1 downregulation, and increased intracellular NAD and ATP levels. (2) SIRT1 knockdown enhanced cellular senescence and apoptosis, accompanied by increased ROS accumulation, DNA damage aggravation, and decreased intracellular NAD and ATP levels. (3) PBX1 overexpression rescued HF-MSCs senescence and apoptosis induced by SIRT1 knockdown. (4) PBX1 rescued PARP1 overexpression-mediated ATP and NAD depletion, accompanied by increased SIRT1 expression. Collectively, our results revealed that a positive interaction feedback loop exists between PBX1 and SIRT1. To the best of our knowledge we are the first to report that there is a PBX1-SIRT1-PARP1 axis that plays a critical role in alleviating HF-MSCs senescence and apoptosis. We provide a new perspective on the mechanisms underlying stem cell senescence as well as age-related disease prevention and treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Hair follicle mesenchymal stem cells (HF-MSCs) have great potential for cell therapy. Traditional method to isolate whisker HF-MSC is time-consuming and few in cell numbers. How to quickly and conveniently obtain a large number of HF-MSC for experimental research is a problem worth exploring. Methods Two-step Ficoll Density Gradient Sedimentation (FDGS) was performed to isolate pelage HF-MSC from adult mice. The characteristic of the isolated cells was identified and compared with whisker HF-MSC by immunofluorescence staining, flow cytometry, three-lineage differentiation and hair follicle reconstruction. Pelage HF-MSC and exosomes were injected into the dorsal skin of mice as well as hair follicle organ culture to explore its role in promoting hair growth. The cells and exosomes distribution were located by immunofluorescence staining. Results Isolated pelage HF-MSC expressed similar markers (ALP, Versican, NCAM, Nestin), showed similar growth pattern, possessed similar mesenchymal stem cells function and hair follicle induction ability as whisker HF-MSC. A large number of cells can be obtained with fewer mice compared to traditional method. Injected pelage HF-MSC promoted hair growth by secreting exosomes. Conclusion A large number of Pelage HF-MSC can be isolated by FDGS, which can promote hair growth by secreting exosomes which may target the dermal papilla and hair matrix region of host hair follicle.

Background: Small intestinal ischemia-reperfusion (IR) injury is a common intestinal disease with high morbidity and mortality. Mesenchymal stem cells (MSCs) have been increasingly used in various intestinal diseases. This study aimed to evaluate the therapeutic effect of hair follicle MSCs (HFMSCs) on small intestinal IR injury.Methods: We divided Sprague–Dawley rats into three groups: the sham group, IR group and IR + HFMSCs group. A small intestinal IR injury rat model was established by clamping of the superior mesenteric artery (SMA) for 30 min and reperfusion for 2 h. HFMSCs were cultured in vitro and injected into the rats through the tail vein. Seven days after treatment, the intrinsic homing and differentiation characteristics of the HFMSCs were observed by immunofluorescence and immunohistochemical staining, and the paracrine mechanism of HFMSCs was assessed by Western blotting and enzyme-linked immunosorbent assay (ELISA).Results: A small intestinal IR injury model was successfully established. HFMSCs could home to damaged sites, express proliferating cell nuclear antigen (PCNA) and intestinal stem cell (ISC) markers, and promote small intestinal ISC marker expression. The expression levels of angiopoietin-1 (ANG1), vascular endothelial growth factor (VEGF) and insulin growth factor-1 (IGF1) in the IR + HFMSCs group were higher than those in the IR group. HFMSCs could prevent IR-induced apoptosis by increasing B-cell lymphoma-2 (Bcl-2) expression and decreasing Bcl-2 homologous antagonist/killer (Bax) expression. Oxidative stress level detection showed that the malondialdehyde (MDA) content was decreased, while the superoxide dismutase (SOD) content was increased in the IR + HFMSCs group compared to the IR group. An elevated diamine oxidase (DAO) level reflected the potential protective effect of HFMSCs on the intestinal mucosal barrier.Conclusion: HFMSCs are beneficial to alleviate small intestinal IR injury through intrinsic homing to the small intestine and by differentiating into ISCs, via a paracrine mechanism to promote angiogenesis, reduce apoptosis, regulate the oxidative stress response, and protect intestinal mucosal function potentially. Therefore, this study suggests that HFMSCs serve as a new option for the treatment of small intestinal IR injury.

Background: Hair follicle mesenchymal stem cells (HF-MSCs) have great potential for cell therapy. Traditional method to isolate whisker HF-MSC is time-consuming and few in cell numbers. How to quickly and conveniently obtain a large number of HF-MSC for experimental research is a problem worth exploring. Methods: Two-step Ficoll Density Gradient Sedimentation (FDGS) was performed to isolate pelage HF-MSC from adult mice. The characteristic of the isolated cells was identified and compared with whisker HF-MSC by immunofluorescence staining, flow cytometry, three-lineage differentiation and hair follicle reconstruction. Pelage HF-MSC and exosomes were injected into the dorsal skin of mice as well as hair follicle organ culture to explore its role in promoting hair growth. The cells and exosomes distribution were located by immunofluorescence staining. Results: Isolated pelage HF-MSC expressed similar markers (ALP, Versican, NCAM, Nestin), showed similar growth pattern, possessed similar mesenchymal stem cells function and hair follicle induction ability as whisker HF-MSC. A large number of cells can be obtained with fewer mice compared to traditional method. Injected pelage HF-MSC promoted hair growth by secreting exosomes. Conclusion: A large number of Pelage HF-MSC can be isolated by FDGS, which can promote hair growth by secreting exosomes which may target the dermal papilla and hair matrix region of host hair follicle. [ABSTRACT FROM AUTHOR]

Liver cirrhosis (LC) involves multiple systems throughout the body, and patients with LC often die of multiple organ failure. However, few drugs are useful to treat LC. Hair follicle mesenchymal stem cells (HF-MSCs) are derived from the dermal papilla and the bulge area of hair follicles and are pluripotent stem cells in the mesoderm with broad prospects in regenerative medicine. As an emerging seed cell type widely used in skin wound healing and plastic surgery, HF-MSCs show considerable prospects in the treatment of LC due to their proliferation and multidirectional differentiation capabilities. We established an LC model in C57BL/6J mice by administering carbon tetrachloride (CCl4) and injected HF-MSCs through the tail vein to explore the therapeutic effects and potential mechanisms of HF-MSCs on LC. Here, we found that HF-MSCs improved liver function and ameliorated the liver pathology of LC. Notably, PKH67-labeled HF-MSCs were detected in the injured liver and expressed the hepatocyte-specific markers cytokeratin 18 (CK18) and albumin (ALB). In addition, in contrast to that in the LC group, the α-SMA expression showed a decreasing trend in the treatment group in vitro and in vivo, indicating that the pathological activation of hepatic stellate cells (HSCs) was inhibited by HF-MSC treatment. Moreover, the levels of transforming growth factor β (TGF-β1) and p-Smad3, a signaling molecule downstream of TGF-β1, were increased in mice with LC, while HF-MSC treatment reversed these changes in vivo and in vitro. Based on these findings, HF-MSCs may reverse LC by blocking the TGF-β/Smad pathway and inhibiting the pathological activation of HSCs, which may provide evidence for the application of HF-MSCs to treat LC. [ABSTRACT FROM AUTHOR]

Abstract Background PBX homeobox 1 (PBX1) is involved in the maintenance of the pluripotency of human embryonic and hematopoietic stem cells; however, the effects of PBX1 in the self-renewal and reprogramming of hair follicle mesenchymal stem cells (HF-MSCs) are unclear. The AKT/glycogen synthase kinase (GSK) 3β pathway regulates cell metabolism, proliferation, apoptosis, and reprogramming, and p16 and p21, which act downstream of this pathway, regulate cell proliferation, cell cycle, and apoptosis induced by reprogramming. Here, we aimed to elucidate the roles of PBX1 in regulating the proliferation and reprogramming of HF-MSCs. Methods A lentiviral vector designed to carry the PBX1 sequence or PBX1 short hairpin RNA sequence was used to overexpress or knock down PBX1. The roles of PBX1 in proliferation and apoptosis were investigated by flow cytometry. Real-time polymerase chain reaction was performed to evaluate pluripotent gene expression. Dual-luciferase reporter assays were performed to examine the transcriptional activity of the NANOG promoter. Western blotting was performed to identify the molecules downstream of PBX1 involved in proliferation and reprogramming. Caspase3 activity was detected to assess HF-MSC reprogramming. The phosphatidylinositol 3-kinase/AKT inhibitor LY294002 was used to inhibit the phosphorylation and activity of AKT. Results Overexpression of PBX1 in HF-MSCs increased the phosphorylation of AKT and nuclear translocation of β-catenin, resulting in the progression of the cell cycle from G0/G1 to S phase. Moreover, transfection with a combination of five transcription factors (SOMKP) in HF-MSCs enhanced the formation of alkaline phosphatase-stained colonies compared with that in HF-MSCs transfected with a combination of four transcription factors (SOMK). PBX1 upregulated Nanog transcription by activating the promoter and promoted the expression of endogenous SOX2 and OCT4. Furthermore, PBX1 expression activated the AKT/glycogen synthase kinase (GSK) 3β pathway and reduced apoptosis during the early stages of reprogramming. Inhibition of phospho-AKT or knockdown of PBX1 promoted mitochondrion-mediated apoptosis and reduced reprogramming efficiency. Conclusions PBX1 enhanced HF-MSC proliferation, and HF-MSCs induced pluripotent stem cells (iPSC) generation by activating the AKT/GSK3β signaling pathway. During the reprogramming of HF-MSCs into HF-iPSCs, PBX1 activated the NANOG promoter, upregulated NANOG, and inhibited mitochondrion-mediated apoptosis via the AKT/GSK3β pathway during the early stages of reprogramming.

Objectives: To express a TAT-PBX1 fusion protein using a prokaryotic expression system and to explore potential effects of TAT-PBX1 in the proliferation and senescence of human hair follicle-derived mesenchymal stem cells. Results: The TAT-PBX1 fusion was produced in inclusion bodies and heterogenously expressed in Rosetta (DE3) cells. Immunofluorescence staining showed that TAT-PBX1 fusion proteins were internalized by human hair follicle-derived mesenchymal stem cells. The growth rate of cells was increased after treatment with more than 5.0 μg/mL of TAT-PBX1. The rate of senescence-associated β-galactosidase positive cells was reduced in the 10.0 μg/mL TAT-PBX1 group (28%) than the 0 μg/mL control group (60%). Cells treated with the TAT-PBX1 fusion protein showed higher expression of p-AKT (1.22-fold that of the control), which indicates that TAT-PBX1 activated AKT pathway after cellular uptake. Conclusions: The TAT-PBX1 fusion protein increased the proliferation of hair follicle mesenchymal stem cells and delayed their senescence by activating the AKT pathway following internalization by cells. [ABSTRACT FROM AUTHOR]

Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming. [ABSTRACT FROM AUTHOR]

The use of human mesenchymal stem cells (hMSCs) in cell therapies has increased the demand for strategies that allow efficient cell scale-up. Preliminary data on the three-dimensional (3D) spinner culture describing the potential use of microcarriers for hMSCs culture scale-up have been reported. We exploited a rich source of autologous stem cells (human hair follicle) and demonstrated the robust in vitro long-term expansion of human hair follicle-derived mesenchymal stem cells (hHF-MSCs) by using CultiSpher-G microcarriers. We analyzed the feasibility of 3D culture by using hHF-MSCs/CultiSpher-G microcarrier constructs for its potential applicability in regenerative medicine by comparatively analyzing the performance of hHF-MSCs adhered to the CultiSpher-G microspheres in 3D spinner culture and those grown on the gelatin-coated plastic dishes (2D culture), using various assays. We showed that the hHF-MSCs seeded at various densities quickly adhered to and proliferated well on the microspheres, thus generating at least hundreds of millions of hHF-MSCs on 1 g of CultiSpher-G within 12 days. This resulted in a cumulative cell expansion of greater than 26-fold. Notably, the maximum and average proliferation rates in 3D culture were significantly greater than that of the 2D culture. However, the hHF-MSCs from both the cultures retained surface marker and nestin expression, proliferation capacity and differentiation potentials toward adipocytes, osteoblasts and smooth muscle cells and showed no significant differences as evidenced by Edu incorporation, cell cycle, colony formation, apoptosis, biochemical quantification and qPCR assays. [ABSTRACT FROM AUTHOR]

Acute pancreatitis (AP) is commonly accompanied by intense pain and is associated with high mortality rates. However, the effectiveness of existing therapeutic approaches remains unsatisfactory. Stem cell therapy, which can promote the regeneration of damaged tissue and alleviate systemic inflammatory responses, has brought new possibility for patients suffering from AP. In particular, hair follicle-derived mesenchymal stem cells (HF-MSCs) are proposed as a suitable cell source for treating pancreatic diseases, but further research on their effectiveness, safety, and underlying mechanisms is warranted for clinical implementation. In this work, the therapeutic potential of HF-MSC transplantation was studied in an L-arginine-induced AP rat model. HF-MSCs were extracted from infant Sprague-Dawley (SD) rats, expanded in vitro , and detected by flow cytometry. HF-MSCs were labeled by PKH67 and transplanted into rats with AP via tail vein injection. Serum specimens were collected at 24 h, 48 h, and 72 h after transplantation, and the levels of amylase, lipase, and anti-inflammatory factors, namely interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), were analyzed. Pancreas samples were collected and assayed by immunofluorescence and immunohistochemistry 1 week after transplantation to monitor the differentiation of HF-MSCs and the functional recovery of the damaged pancreas. Intravenously delivered rat HF-MSCs spontaneously homed to the damaged pancreas and expressed pancreatic progenitor cell markers, relieved inflammation, and boosted pancreatic regeneration. These findings indicate that HF-MSC transplantation is a potentially effective treatment for AP., Competing Interests: None., (AJTR Copyright © 2021.)

Background: PBX homeobox 1 (PBX1) is involved in the maintenance of the pluripotency of human embryonic and hematopoietic stem cells; however, the effects of PBX1 in the self-renewal and reprogramming of hair follicle mesenchymal stem cells (HF-MSCs) are unclear. The AKT/glycogen synthase kinase (GSK) 3β pathway regulates cell metabolism, proliferation, apoptosis, and reprogramming, and p16 and p21, which act downstream of this pathway, regulate cell proliferation, cell cycle, and apoptosis induced by reprogramming. Here, we aimed to elucidate the roles of PBX1 in regulating the proliferation and reprogramming of HF-MSCs. Methods: A lentiviral vector designed to carry the PBX1 sequence or PBX1 short hairpin RNA sequence was used to overexpress or knock down PBX1. The roles of PBX1 in proliferation and apoptosis were investigated by flow cytometry. Real-time polymerase chain reaction was performed to evaluate pluripotent gene expression. Dual-luciferase reporter assays were performed to examine the transcriptional activity of the NANOG promoter. Western blotting was performed to identify the molecules downstream of PBX1 involved in proliferation and reprogramming. Caspase3 activity was detected to assess HF-MSC reprogramming. The phosphatidylinositol 3-kinase/AKT inhibitor LY294002 was used to inhibit the phosphorylation and activity of AKT. Results: Overexpression of PBX1 in HF-MSCs increased the phosphorylation of AKT and nuclear translocation of β-catenin, resulting in the progression of the cell cycle from G0/G1 to S phase. Moreover, transfection with a combination of five transcription factors (SOMKP) in HF-MSCs enhanced the formation of alkaline phosphatase-stained colonies compared with that in HF-MSCs transfected with a combination of four transcription factors (SOMK). PBX1 upregulated Nanog transcription by activating the promoter and promoted the expression of endogenous SOX2 and OCT4. Furthermore, PBX1 expression activated the AKT/glycogen synthase kinase (GSK) 3β pathway and reduced apoptosis during the early stages of reprogramming. Inhibition of phospho-AKT or knockdown of PBX1 promoted mitochondrion-mediated apoptosis and reduced reprogramming efficiency. Conclusions: PBX1 enhanced HF-MSC proliferation, and HF-MSCs induced pluripotent stem cells (iPSC) generation by activating the AKT/GSK3β signaling pathway. During the reprogramming of HF-MSCs into HF-iPSCs, PBX1 activated the NANOG promoter, upregulated NANOG, and inhibited mitochondrion-mediated apoptosis via the AKT/GSK3β pathway during the early stages of reprogramming. [ABSTRACT FROM AUTHOR]