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Hair loss, or alopecia, is associated with several psychosocial and medical comorbidities, and it remains an economic burden to individuals and the society. Alopecia is attributable to varied mechanisms and features a multifactorial predisposition, and the available conventional medical interventions have several limitations. Thus, several therapeutic strategies for alopecia in regenerative medicine are currently being explored, with increasing evidence suggesting that mesenchymal stem cell (MSC) implantation, MSC-derived secretome treatment, and blood-derived platelet-rich plasma therapies are potential treatment options. In this review, we searched the Cochrane Library, MEDLINE (PubMed), EMBASE, and Scopus using various combinations of terms, such as "stem cell," "alopecia," "hair loss," "Androgenetic alopecia," "male-pattern hair loss," "female-pattern hair loss," "regenerative hair growth," "cell therapy," "mesenchymal stem cells," "MSC-derived extracellular vesicles," "MSC-derived exosomes," and "platelet-rich plasma" and summarized the most promising regenerative treatments for alopecia. Moreover, further opportunities of improving efficacy and innovative strategies for promoting clinical application were discussed., Competing Interests: The authors have no conflicts of interest to declare for this work., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Introduction: Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs)., Methods: Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes ( ALB , AFP , α1-AT , CDH1 , CYP2E1 , CYP3A12 , and TAT ) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes., Results: The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4-7., Conclusion: These results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs., Competing Interests: The authors declare no conflict of interest., (© 2021 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Introduction: Multipotent mesenchymal stem cells (MSCs) are widespread in adult organisms and are implicated in tissue maintenance and repair, regulation of hematopoiesis, and immunologic responses. Human (h)MSCs have applications in tissue engineering, cell-based therapy, and medical devices but it is unclear how they respond to unfavorable conditions, such as hypoxia or inflammation after transplantation in vivo . Although endotoxin testing is required for evaluating the quality and safety of transplanted MSCs, no reports on their dose response to endotoxins are available to establish the limits for in vitro MSC culture systems. In the present study, we aimed to accurately quantify the risk of endotoxin contamination in cell culture systems to establish an acceptable endotoxin limit for the differentiation of hMSC osteoblasts and adipocytes., Methods: Three types of bone marrow-derived hMSCs (hMSC-1: 21-year-old, M/B; hMSC-2: 36-year-old, M/B; hMSC-3: 43-year-old, M/C) and adipose-derived stem cells (ADSCs; StemPro Human) were cultured in osteogenic or adipogenic differentiation media, respectively, from commercial kits, containing various concentrations of endotoxin (0.01-100 ng/ml). The degree of adipocyte and osteoblast differentiation was estimated by fluorescent staining of lipid droplets and hydroxyapatite, respectively. To clarify the molecular mechanism underlying the effect of endotoxin on hMSC differentiation, cellular proteins were extracted from cultured cells and subjected to liquid chromatograph-tandem mass spectrometry shotgun proteomics analysis., Results: Although endotoxin did not effect the adipocyte differentiation of hMSCs, osteoblast differentiation was enhanced by various endotoxin concentrations: over 1 ng/ml, for hMSC-1; 10 ng/ml, for hMSC-2; and 100 ng/ml, for hMSC-3. Proteomic analysis of hMSC-1 cells revealed up-regulation of many proteins related to bone formation. These results suggested that endotoxin enhances the osteoblast differentiation of MSCs depending on the cell type., Conclusions: Since endotoxins can affect various cellular functions, an endotoxin limit should be established for in vitro MSC cultures. Its no-observed-adverse-effect level was 0.1 ng/ml based on the effect on the hMSC osteoblast differentiation, but it may not necessarily be the limit for ADSCs.

Introduction: Multipotent mesenchymal stem cells (MSCs) are widespread in adult organisms and are implicated in tissue maintenance and repair, regulation of hematopoiesis, and immunologic responses. Human (h)MSCs have applications in tissue engineering, cell-based therapy, and medical devices but it is unclear how they respond to unfavorable conditions such as hypoxia or inflammation after in vivo transplantation. Although endotoxin testing is a requirement for evaluating the quality and safety of transplanted MSCs, there have been no reports on the dose response to endotoxins to establish limits for in vitro MSC culture systems. The present study aimed to accurately quantify the risk of endotoxin contamination in cell culture systems in order to establish the acceptable endotoxin limit for hMSC proliferation., Methods: Three types of bone marrow-derived hMSC (hMSC-1: 21 years, M/B; hMSC-2: 36 years, M/B; hMSC-3: 43 years, M/C) and adipose-derived stem cells (ADSCs; StemPro Human) were cultured in medium from commercial kits containing various concentrations of endotoxin (0.1-1000 ng/ml). The proliferative capacity of cells was estimated by cell counts using a hemocytometer. To clarify the molecular mechanism underlying the effect of endotoxin on hMSCs proliferation, cellular proteins were extracted from cultured cells and subjected to liquid chromatograph-tandem mass spectrometry shotgun proteomics analysis. The expression of Cu/Zn-type superoxide dismutase (SOD1) and Fe/Mn-type superoxide dismutase (SOD2) induced in hMSCs by endotoxin stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA), and the effect of SOD2 on hMSC proliferation was also estimated., Results: Although there was no change in cell morphology during the culture period, proliferative capacity increased with endotoxin concentration to over 0.1 ng/ml for ADSCs, 1 ng/ml for hMSC-1, and 100 ng/ml for hMSC-2; hMSC-3 proliferation was unaffected by the presence of endotoxin. A proteomic analysis of hMSC-1 revealed that various proteins related to the cell cycle, apoptosis, and host defense against infection were altered by endotoxin stimulation, whereas SOD2 expression was significantly and consistently upregulated during the culture period. The latter was also confirmed by ELISA. Moreover, recombinant SOD2 increased proliferative capacity in hMSC-1 cells in a manner similar to endotoxin. These results suggest that endotoxin protects MSCs from oxidative stress via upregulation of SOD2 to improve cell survival., Conclusions: Since endotoxins can affect various cellular functions, an endotoxin limit should be set for in vitro MSC cultures. The lowest observed adverse effect level was determined to be 0.1 ng/ml based on the effect on MSC proliferation.