PBX1 Attenuates Hair Follicle-Derived Mesenchymal Stem Cell Senescence and Apoptosis by Alleviating Reactive Oxygen Species-Mediated DNA Damage Instead of Enhancing DNA Damage Repair.

Tissues and organs undergo structural deterioration and functional decline during aging. DNA damage is considered a major cause of stem cell senescence. Although stem cells develop sophisticated DNA repair systems, when the intrinsic and extrinsic insults exceed the DNA repair capacity, cellular senescence, and age-related diseases inevitably occur. Therefore, the prevention and alleviation of DNA damage is an alternative to DNA repair in attenuating stem cell senescence and preventing age-related diseases. Pre-B-cell leukaemia homeobox 1 (PBX1) participates in maintaining the pluripotency of human embryonic and haematopoietic stem cells. Our recent studies showed that PBX1 promotes hair follicle-derived mesenchymal stem cell (HF-MSC) proliferation, decreases cellular senescence and apoptosis, and enhances induced pluripotent stem cell generation. Whether PBX1 attenuates HF-MSC senescence and apoptosis by alleviating DNA damage or by enhancing DNA repair remains unknown. In this study, we aimed to determine the effects of PBX1 on the intrinsic ROS or extrinsic H2O2-induced cellular senescence of HF-MSCs. To this end, we generated HF-MSCs overexpressing either PBX1, or poly (ADP-ribose) polymerase 1, or both. Our results showed that PBX1 overexpression attenuates HF-MSC senescence and apoptosis by alleviating reactive oxygen species (ROS)-mediated DNA damage instead of enhancing DNA repair. This is the first study to report that PBX1 attenuates stem cell senescence and apoptosis by alleviating DNA damage. It provides new insight into the mechanism of stem cell senescence and lays the foundation for the development of strategies for age-related disease prevention and treatment, and in particular, hair follicle repair and regeneration.

Melanocytes derived from mouse hair follicles: A novel study model to assess pigmentation disorders.

Melanocytes are the major cells responsible for skin and fair pigmentation in vertebrates. They localize to hair follicles(HFs) and the epidermis during embryonic development. A reduced number or dysfunction of melanocytes results in pigmentation disorders.Thus, methods for isolation, culture, and identification of melanocytes in mouse hair follicles provide an experimental basis for thestudy of of pigmentation disorders. In the current work, we harvested the melanocytes from the anagen phase dorsal skin of C57BL/6 mice.After its separation from the skin, the dermis was digested, and the HFs were released. HFs were then also digested, and the cells released from HFs were cultured in melanocyte growth medium. Immunofluorescence and immunohistochemistry staining were used to localize the distribution of melanocytes in HFs . Reverse transcription polymerase chain reaction was performed to detect the expression of specific melanocyte marker genes. Immunofluorescence, immunohistochemistry, flow cytometry, and western blot were carried out to detect the expression of marker proteins in cells. 3,4-Dihydroxy-L-phenylalanine (L-DOPA) staining was used to detect the pigmentation functionality of melaonocytes. Based on our results, we conclude that mature and functional melanocytes can be successfully obtained from theHFs, providing a cell model to study pigmentation disorders. The current findings provide novel insights for the treatment of pigmentation disorders by autologous cell transplantation. Further, we believe that issues related to skin damage, insufficient numbers of autologous cells, and autoimmune problems can be resolved in future though the use of functional melanocytes.

Human hair follicle-derived mesenchymal stem cells: Isolation, expansion, and differentiation.

Hair follicles are easily accessible skin appendages that protect against cold and potential injuries. Hair follicles contain various pools of stem cells, such as epithelial, melanocyte, and mesenchymal stem cells (MSCs) that continuously self-renew, differentiate, regulate hair growth, and maintain skin homeostasis. Recently, MSCs derived from the dermal papilla or dermal sheath of the human hair follicle have received attention because of their accessibility and broad differentiation potential. In this review, we describe the applications of human hair follicle-derived MSCs (hHF-MSCs) in tissue engineering and regenerative medicine. We have described protocols for isolating hHF-MSCs from human hair follicles and their culture condition in detail. We also summarize strategies for maintaining hHF-MSCs in a highly proliferative but undifferentiated state after repeated in vitro passages, including supplementation of growth factors, 3D suspension culture technology, and 3D aggregates of MSCs. In addition, we report the potential of hHF-MSCs in obtaining induced smooth muscle cells and tissue-engineered blood vessels, regenerated hair follicles, induced red blood cells, and induced pluripotent stem cells. In summary, the abundance, convenient accessibility, and broad differentiation potential make hHF-MSCs an ideal seed cell source of regenerative medical and cell therapy.

MSC-derived exosomes attenuate cell death through suppressing AIF nucleus translocation and enhance cutaneous wound healing.

BACKGROUND: Skin wounding is very common and may be slow to heal. Increasing evidence shows that exosomes derived from mesenchymal stem cells (MSCs) dramatically enhance skin wound healing in a paracrine manner. However, the mechanism underlying this phenomenon has not yet been elucidated. Thus, the objective of the present study was to identify the signaling pathways and paracrine factors by which MSC-derived exosomes promote de novo skin tissue regeneration in response to wound healing. METHODS: In vitro and in vivo skin wound healing models were created by treating immortalized human keratinocytes (HaCaT) with hydrogen peroxide (H2O2) and excising full-thickness mouse skin, respectively. Exosomes were extracted from human umbilical cord Wharton's jelly MSCs (hucMSC-Ex) by ultracentrifugation of cell culture supernatant. RESULTS: The hucMSC-Ex treatment significantly increased HaCaT cell proliferation and migration in a time- and dose-dependent manner, suppressed HaCaT apoptosis induced with H2O2 by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and upregulating poly ADP ribose polymerase 1 (PARP-1) and poly (ADP-ribose) (PAR). The animal experiments showed that relative to hucMSCs, hucMSC-Ex attenuated full-thickness skin wounding by enhancing epidermal re-epithelialization and dermal angiogenesis. CONCLUSIONS: These findings indicated that direct administration of hucMSC-Ex may effectively treat cutaneous wounding and could be of great value in clinical settings.

Internalization of the TAT-PBX1 fusion protein significantly enhances the proliferation of human hair follicle-derived mesenchymal stem cells and delays their senescence.

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 ß-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.

NANOG Attenuates Hair Follicle-Derived Mesenchymal Stem Cell Senescence by Upregulating PBX1 and Activating AKT Signaling.

Stem cells derived from elderly donors or harvested by repeated subculture exhibit a marked decrease in proliferative capacity and multipotency, which not only compromises their therapeutic potential but also raises safety concerns for regenerative medicine. NANOG-a well-known core transcription factor-plays an important role in maintaining the self-renewal and pluripotency of stem cells. Unfortunately, the mechanism that NANOG delays mesenchymal stem cell (MSC) senescence is not well-known until now. In our study, we showed that both ectopic NANOG expression and PBX1 overexpression (i) significantly upregulated phosphorylated AKT (p-AKT) and PARP1; (ii) promoted cell proliferation, cell cycle progression, and osteogenesis; (iii) reduced the number of senescence-associated-ß-galactosidase- (SA-ß-gal-) positive cells; and (iv) downregulated the expression of p16, p53, and p21. Western blotting and dual-luciferase activity assays showed that ectopic NANOG expression significantly upregulated PBX1 expression and increased PBX1 promoter activity. In contrast, PBX1 knockdown by RNA interference in hair follicle- (HF-) derived MSCs that were ectopically expressing NANOG resulted in the significant downregulation of p-AKT and the upregulation of p16 and p21. Moreover, blocking AKT with the PI3K/AKT inhibitor LY294002 or knocking down AKT via RNA interference significantly decreased PBX1 expression, while increasing p16 and p21 expression and the number of SA-ß-gal-positive cells. In conclusion, our findings show that NANOG delays HF-MSC senescence by upregulating PBX1 and activating AKT signaling and that a feedback loop likely exists between PBX1 and AKT signaling.

PBX homeobox 1 enhances hair follicle mesenchymal stem cell proliferation and reprogramming through activation of the AKT/glycogen synthase kinase signaling pathway and suppression of apoptosis.

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) 3ß 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 ß-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) 3ß 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/GSK3ß 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/GSK3ß pathway during the early stages of reprogramming.