Stem Leydig cell (SLC) transplantation could provide a new strategy for treating the testosterone deficiency. Our previous study demonstrated that CD51 (also called integrin αv) might be a putative cell surface marker for SLCs, but the physiological function and efficacy of CD51+ SLCs treatment remain unclear. Here, we explore the potential therapeutic benefits of CD51+ SLCs transplantation and whether these transplanted cells can be regulated by the hypothalamic-pituitary-gonadal (HPG) axis. CD51+ cells were isolated from the testes of 12-weeks-old C57BL/6 mice, and we showed that such cells expressed SLC markers and that they were capable of self-renewal, extensive proliferation, and differentiation into multiple mesenchymal cell lineages and LCs in vitro. As a specific cytotoxin that eliminates Leydig cells (LCs) in adult rats, ethane dimethanesulfonate (EDS) was used to ablate LCs before the SLC transplantation. After being transplanted into the testes of EDS-treated rats, the CD51+ cells differentiated into mature LCs, and the recipient rats showed a partial recovery of testosterone production and spermatogenesis. Notably, a testosterone analysis revealed a circadian rhythm of testosterone secretion in cell-transplanted rats, and these testosterone secretions could be suppressed by decapeptyl (a luteinizing hormone-releasing hormone agonist), suggesting that the transplanted cells might be regulated by the HPG axis. This study is the first to demonstrate that CD51+ SLCs can restore the neuroendocrine regulation of testicular function by physiologically recovering the expected episodic changes in diurnal testosterone serum levels and that SLC transplantation may provide a new tool for the studies of testosterone deficiency treatment.

Mesenchymal stromal cells (MSCs) show potential for treating cardiovascular diseases, but their therapeutic efficacy exhibits significant heterogeneity depending on the tissue of origin. This study sought to identify an optimal source of MSCs for cardiovascular disease therapy. We demonstrated that Nestin was a suitable marker for cardiac MSCs (Nes⁺cMSCs), which were identified by their self-renewal ability, tri-lineage differentiation potential, and expression of MSC markers. Furthermore, compared with bone marrow-derived MSCs (Nes⁺bmMSCs) or saline-treated myocardial infarction (MI) controls, intramyocardial injection of Nes⁺cMSCs significantly improved cardiac function and decreased infarct size after acute MI (AMI) through paracrine actions, rather than transdifferentiation into cardiac cells in infarcted heart. We further revealed that Nes⁺cMSC treatment notably reduced pan-macrophage infiltration while inducing macrophages toward an anti-inflammatory M2 phenotype in ischemic myocardium. Interestingly, Periostin, which was highly expressed in Nes⁺cMSCs, could promote the polarization of M2-subtype macrophages, and knockdown or neutralization of Periostin remarkably reduced the therapeutic effects of Nes⁺cMSCs by decreasing M2 macrophages at lesion sites. Thus, the present work systemically shows that Nes⁺cMSCs have greater efficacy than do Nes⁺bmMSCs for cardiac healing after AMI, and that this occurs at least partly through Periostin-mediated M2 macrophage polarization.

Bone-derived mesenchymal stem cell (BMSC) transplantation has been reported to be effective for the treatment of ischemic heart disease, but whether BMSCs are the optimal cell type remains under debate. Increasing numbers of studies have shown that Nestin, an intermediate filament protein, is a potential marker for MSCs, which raises the question of whether Nestin+ cells in BMSCs may play a more crucial role in myocardial repair. Nestin+ cells were isolated using flow cytometry by gating for CD45− Ter119− CD31− cells from the compact bone of Nestin-GFP transgenic mice, expressing GFP driven by the Nestin promoter. Colony-forming and proliferative curve assays were conducted to determine the proliferative capacity of these cells, while qRT-PCR was used to analyze the mRNA levels of relative chemokines and growth factors. Cardiac endothelial cell (CEC) recruitment was assessed via a transwell assay. Moreover, permanent ligation of the left anterior descending (LAD) coronary artery was performed to establish an acute myocardial infarction (AMI) mouse model. After cell transplantation, conventional echocardiography was conducted 1 and 4 weeks post-MI, and hearts were harvested for hematoxylin-and-eosin (HE) staining and immunofluorescence staining 1 week post-MI. Further evaluation of paracrine factor levels and administration of a neutralizing antibody (TIMP-1, TIMP-2, and CXCL12) or a CXCR4 antagonist (AMD3100) in MI hearts were performed to elucidate the mechanism involved in the chemotactic effect of Nestin+ BMSCs in vivo. Compared with Nestin− BMSCs, a greater proliferative capacity of Nestin+ BMSCs was observed, which further exhibited moderately high expression of chemokines instead of growth factors. More CEC recruitment in the Nestin+ BMSC-cocultured group was observed in vitro, while this effect was obviously abolished after treatment with neutralizing antibodies against TIMP-1, TIMP-2, or CXCL12, and more importantly, blocking the CXCL12/CXCR4 axis with a AMD3100 significantly reduced the chemotactic effect of Nestin+ BMSCs. After transplantation into mice exposed to myocardial infarction (MI), Nestin+ BMSC-treated mice showed significantly improved survival and left ventricular function compared with Nestin− BMSC-treated mice. Moreover, endogenous CECs were markedly increased, and chemokine levels were significantly higher, in the infarcted border zone with Nestin+ BMSC treatment. Meanwhile, neutralization of each TIMP-1, TIMP-2, or CXCL12 in vivo could reduce the left ventricular function at 1 and 4 weeks post-MI; importantly, the combined use of these three neutralizing antibodies could make a higher significance on cardiac function. Finally, blocking the CXCL12/CXCR4 axis with AMD3100 significantly reduced the left ventricular function and greatly inhibited Nestin+ BMSC-induced CEC chemotaxis in vivo. These results suggest that Nestin+ BMSC transplantation can improve cardiac function in an AMI model by recruiting resident CECs to the infarcted border region via the CXCL12/CXCR4 chemokine pathway. And we demonstrated that Nestin+BMSC-secreted TIMP-1/2 enhances CXCL12(SDF1α)/CXCR4 axis-driven migration of endogenous Sca-1+ endothelial cells in ischemic heart post-AMI. Taken together, our results show that Nestin is a useful marker for the identification of functional BMSCs and indicate that Nestin+ BMSCs could be a better therapeutic candidate for cardiac repair.

Male infertility has become an important health problem that is primarily caused by testicular dysfunction with abnormal spermatogenesis. In this study, we demonstrated that the neuropeptide, substance P (SP), is essential for spermatogonia proliferation in a seminiferous tubule culture system. In addition, SP (5 nmol/kg) treatment markedly restored spermatogenesis, improved sperm quality, and increased the number of ZBTB16⁺ or LIN28⁺ undifferentiated spermatogonia as well as STRA8⁺ differentiated spermatogonia in a busulfan-induced non-obstructive azoospermic mouse model. Furthermore, 100 nM SP treatment in vitro significantly stimulated the proliferation of GC-1 spg cells (a spermatogonia cell line) via activation of the Erk1/2 signaling pathway. Moreover, the sperm quality and the number of spermatogonia were significantly reduced after treatment with RP67580, a selective NK-1 receptor antagonist, suggesting that SP-NK1R signaling plays an important role in spermatogenesis. Taken together, these results suggest that SP may be a potential therapeutic agent for male infertility by accelerating the restoration of spermatogenesis.

Corni Fructus has traditionally been used as herbal medicine for the treatment of tuberculosis, asthma, hepatitis, and chronic nephritis in Korea, Japan, and China. This research was carried out to evaluate the proliferative-inhibitory effect of CF extracts against cancer cells and to identify the new pro-substance from medicinal plants. Among these herbal extracts extracted from KCF (Korean Corni Fructus), JCF (Japanese Corni Fructus) and CCF (Chinese Corni Fructus), KCF extracts strongly induced anti-proliferation of cancer cells in a dose-dependent manner compared with other extracts. Moreover, after treatment with CM/F3 (fraction 3 obtained from KCF extracts) for 24 h, A549 cells were evaluated by several indicators such as cell viability, LDH release, DNA fragmentation, nuclear condensation, and apoptotic proteins in vitro. CM/F3 showed the tumor-selective growth inhibitory activity in a dose- and time-dependent manner in A549 cells. Consistently, CM/F3 effectively induced the activation of bax, cytochrome-c, caspase-3, -8, -9, p53, and p21 causing apoptosis, and caused the suppression of Cdk2, pRb, and E2F1 related to cell arrest in A549 cells. These results demonstrate that CM/F3 caused not only anti-proliferation but also cell death involving cell arrest through interaction between apoptotic proteins and the upregulation of p53 in A549 cells.