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  • Mesenchymal stem cells promote human melanocytes proliferation and resistance to apoptosis through PTEN pathway in vitiligo
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-15
    Lifei Zhu; Xi Lin; Lin Zhi; Yushan Fang; Keming Lin; Kai Li; Liangcai Wu

    Vitiligo is an acquired chronic and recurrent skin disease that causes a depigmentation disorder, resulting in selective destruction of melanocytes (MC). However, the mechanism that leads to melanocyte dysfunction and death remains unclear. We performed RNA sequencing, immunohistochemistry, and immunoblotting to characterize the patterns of phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway activation in vitiligo. We also cocultured primary melanocytes with mesenchymal stem cells (MSCs) in a Transwell system to explore how MSCs inhibit the PTEN/PI3K/AKT pathway in melanocytes. We identified that vitiligo normal-lesional junction skin presented with high expression of PTEN, which led to the inhibition of AKT phosphorylation (p-AKT) at S-473. Furthermore, PTEN overexpression led to oxidative stress-induced apoptosis in melanocytes. Coculturing with MSCs enhanced the cell proliferation of human melanocytes and repressed PTEN expression, which inhibited oxidative stress-induced apoptosis. We report that vitiligo patients present with high PTEN expression, which may play a role in the impairment of melanocytes. Furthermore, our study provides evidence that MSCs target the PTEN/PI3K/AKT pathway to regulate cell proliferation and apoptosis in human melanocytes, indicating that MSCs may serve as a promising therapy for vitiligo.

    更新日期:2020-01-15
  • Management of retinitis pigmentosa by Wharton’s jelly derived mesenchymal stem cells: preliminary clinical results
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-13
    Emin ÖZMERT; Umut ARSLAN

    The aim of this study is to determine if umbilical cord Wharton’s jelly derived mesenchymal stem cells implanted in sub-tenon space have beneficial effects on visual functions in retinitis pigmentosa patients by reactivating the degenerated photoreceptors in dormant phase. This prospective, open-label, phase-3 clinical trial was conducted between April of 2019 and October of 2019 at Ankara University Faculty of Medicine, Department of Ophthalmology. 32 RP patients (34 eyes) were included in the study. The patients were followed for 6 months after the Wharton’s jelly derived mesenchymal stem cell administration, and evaluated with consecutive examinations. All patients underwent a complete routine ophthalmic examination, and best corrected visual acuity, optical coherens tomography angiography, visual field, multifocal and full-field electroretinography were performed. The quantitative results were obtained from a comparison of the pre-injection and final examination (6th month) values. The mean best corrected visual acuity was 70.5 letters prior to Wharton’s jelly derived mesenchymal stem cell application and 80.6 letters at the 6th month (p = 0.01). The mean visual field median deviation value was 27.3 dB before the treatment and 24.7 dB at the 6th month (p = 0.01). The mean outer retinal thickness was 100.3 μm before the treatment and 119.1 μm at 6th month (p = 0.01). In the multifocal electroretinography results, P1 amplitudes improved in ring1 from 24.8 to 39.8 nv/deg2 (p = 0.01), in ring2 from 6.8 to 13.6 nv/deg2 (p = 0.01), and in ring3 from 3.1 to 5.7 nv/deg2 (p = 0.02). P1 implicit times improved in ring1 from 44.2 to 32.4 ms (p = 0.01), in ring2 from 45.2 to 33.2 ms (p = 0.02), and in ring3 from 41.9 to 32.4 ms (p = 0.01). The mean amplitude improved in 16 Tds from 2.4 to 5.0 nv/deg2 (p = 0.01) and in 32 Tds from 2.4 to 4.8 nv/deg2 (p = 0.01) in the full-field flicker electroretinography results. Full field flicker electroretinography mean implicit time also improved in 16 Tds from 43.3 to 37.9 ms (p = 0.01). No ocular or systemic adverse events related to the two types of surgical methods and/or Wharton’s jelly derived mesenchymal stem cells itself were observed during the follow-up period. RP is a genetic disorder that can result in blindness with outer retinal degeneration. Regardless of the type of genetic mutation, sub-tenon Wharton’s jelly derived mesenchymal stem cell administration appears to be an effective and safe option. There are no serious adverse events or ophthalmic / systemic side effects for 6 months follow-up. Although the long-term adverse effects are still unknown, as an extraocular approach, subtenon implantation of the stem cells seems to be a reasonable way to avoid the devastating side effects of intravitreal/submacular injection. Further studies that include long-term follow-up are needed to determine the duration of efficacy and the frequency of application. SHGM56733164. Redistered 28 January 2019 https://shgm.saglik.gov.tr/organ-ve-doku-nakli-koordinatorlugu/56733164/203 E.507.

    更新日期:2020-01-14
  • Mesenchymal stem cells alleviate liver injury induced by chronic-binge ethanol feeding in mice via release of TSG6 and suppression of STAT3 activation
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-13
    Yue-Meng Wan; Zhi-qiang Li; Qiong Zhou; Chang Liu; Men-Jie Wang; Hui-Xin Wu; Yun-Zhen Mu; Yue-Feng He; Yuan Zhang; Xi-Nan Wu; Yu-Hua Li; Zhi-Yuan Xu; Hua-Mei Wu; Ying Xu; Jin-Hui Yang; Xiao-Fang Wang

    Mesenchymal stem cells (MSCs) are a population of pluripotent cells that might be used for treatment of liver disease. However, the efficacy of MSCs for mice with alcoholic hepatitis (AH) and its underlying mechanism remains unclear. MSCs were isolated from the bone marrow (BM) of 4–6-week-old male C57BL/6 N mice. AH was induced in female mice by chronic-binge ethanol feeding for 10 days. The mice were given intraperitoneal injections of MSCs with or without transfection or AG490, recombinant mouse tumor necrosis factor (TNF)-α-stimulated gene/protein 6 (rmTSG-6), or saline at day 10. Blood samples and hepatic tissues were collected at day 11. Various assays such as biochemistry, histology, and flow cytometry were performed. MSCs reduced AH in mice, decreasing liver/body weight ratio, liver injury, blood and hepatic lipids, malondialdehyde, interleukin (IL)-6, and TNF-ɑ, but increasing glutathione, IL-10, and TSG-6, compared to control mice. Few MSCs engrafted into the inflamed liver. Knockdown of TSG-6 in MSCs significantly attenuated their effects, and injection of rmTSG-6 achieved similar effects to MSCs. The signal transducer and activator of transcription 3 (STAT3) was activated in mice with AH, and MSCs and rmTSG-6 inhibited the STAT3 activation. Injection of MSCs plus AG490 obtained more alleviation of liver injury than MSCs alone. BM-MSCs injected into mice with AH do not engraft the liver, but they secrete TSG-6 to reduce liver injury and to inhibit STAT3 activation.

    更新日期:2020-01-13
  • hUCMSC-extracellular vesicles downregulated hepatic stellate cell activation and reduced liver injury in S. japonicum-infected mice
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-09
    Liyang Dong; Yanan Pu; Xiaojun Chen; Xin Qi; Lina Zhang; Lei Xu; Wei Li; Yongbin Ma; Sha Zhou; Jifeng Zhu; Yalin Li; Xuefeng Wang; Chuan Su

    Accumulating evidence shows that mesenchymal stem cells (MSCs) have the potential as a cellular therapy avenue for schistosome-induced liver injury. Extracellular vesicles (EVs) are membranous vesicles released by almost all cell types, and EVs produced by MSCs (MSC-EVs) exert therapeutic effects in several disease models. However, the potential of MSC-EVs in schistosomiasis treatment remains unclear. Using survival analysis, HE and Masson’s trichrome staining, immunohistochemical, western blot analysis, real-time PCR, and EdU proliferation, we investigated the effects of human umbilical cord MSC-derived EVs (hUCMSC-EVs) on the survival and liver injury in the S. japonicum-infected mice and explored the underlying mechanism. Here, we found that like hUCMSCs, hUCMSC-EVs significantly ameliorated liver injury and improved the survival of schistosome-infected mice. Indeed, the hUCMSC-EV-mediated alleviation of liver injury is associated with decreased expression of α-smooth muscle actin (α-SMA), collagen 1, and collagen 3. More importantly, we showed that hUCMSC-EVs directly suppressed the proliferation of LX2 (human hepatic stellate cell) in vitro. In addition, hUCMSC-EVs significantly downregulated the activation of LX2 after transforming growth factor-β1 (TGF-β1) treatment. Our results provided the first evidence that hUCMSC-EVs reduced liver injury in S. japonicum-infected mice, potentially creating new avenues for the treatment of liver damage in schistosomiasis.

    更新日期:2020-01-09
  • IGF-1 enhances BMSC viability, migration, and anti-apoptosis in myocardial infarction via secreted frizzled-related protein 2 pathway
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-09
    Mingzhuo Lin; Xinyue Liu; Haoxiao Zheng; Xiaohui Huang; Yu Wu; Anqing Huang; Hailan Zhu; Yunzhao Hu; Weiyi Mai; Yuli Huang

    Bone marrow mesenchymal stem cell (BMSC) transplantation represents a promising therapeutic strategy for ischemic heart disease. However, its effects are hampered by the poor viability of transplanted cells and the hostile microenvironment of the ischemic region. Insulin-like growth factor-1 (IGF-1) is an important paracrine growth factor of BMSC and plays an important role in the properties of BMSC. Here, we investigated whether overexpressing IGF-1 could enhance the BMSC viability, migration, anti-apoptosis, and protective effects of cardiomyocytes, and explore the underlying mechanisms’ focus on the role of the AKT/secreted frizzled-related protein 2 (SFRP2)/β-catenin pathway. We constructed BMSCs overexpressing insulin-like growth factor-1 (BMSCs-IGF-1) or empty vector (BMSCs-NC) using lentivirus, and evaluated cell survival, proliferation, and migration under normoxic and hypoxic conditions. Co-culture of rat cardiomyoblasts with BMSCs was performed to explore the paracrine effect of BMSCs-IGF-1 for rescuing cardiomyoblasts under hypoxia. Transplantation of BMSCs in acute myocardial infarction rats was used to explore the effect of BMSCs-IGF-1 therapy. BMSCs-IGF-1 exhibited a higher cell proliferation rate, migration capacity, and stemness, and were more resistant to apoptosis under hypoxia. Overexpression of IGF-1 upregulated the expression of total and nuclear β-catenin via the AKT-secreted frizzled-related protein 2 (SFRP2) pathway, which enhanced cell survival. Inhibition of AKT or SFRP2 knockdown by siRNA significantly antagonized the effect of IGF-1 and decreased the expression of β-catenin. The expression of β-catenin target genes, including cyclin D1 and c-Myc, were accordingly decreased. Moreover, BMSCs-IGF-1 could rescue cardiomyoblasts from hypoxia-induced apoptosis and preserve cell viability under hypoxia. Transplantation of BMSCs-IGF-1 into myocardial infarction rats greatly reduced infarct volume than BMSCs-NC, with significantly greater expression of SFRP2 and β-catenin. These results suggest that in BMSCs overexpressing IGF-1, SFRP2 is an important mediator for the enhancement of stem cell viability via activating, rather than antagonizing, the Wnt/β-catenin pathway.

    更新日期:2020-01-09
  • Mircrining the injured heart with stem cell-derived exosomes: an emerging strategy of cell-free therapy
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-09
    Khawaja Husnain Haider; Beatrice Aramini

    Bone marrow-derived mesenchymal stem cells (MSCs) have successfully progressed to phase III clinical trials successive to an intensive in vitro and pre-clinical assessment in experimental animal models of ischemic myocardial injury. With scanty evidence regarding their cardiogenic differentiation in the recipient patients’ hearts post-engraftment, paracrine secretion of bioactive molecules is being accepted as the most probable underlying mechanism to interpret the beneficial effects of cell therapy. Secretion of small non-coding microRNA (miR) constitutes an integral part of the paracrine activity of stem cells, and there is emerging interest in miRs’ delivery to the heart as part of cell-free therapy to exploit their integral role in various cellular processes. MSCs also release membrane vesicles of diverse sizes loaded with a wide array of miRs as part of their paracrine secretions primarily for intercellular communication and to shuttle genetic material. Exosomes can also be loaded with miRs of interest for delivery to the organs of interest including the heart, and hence, exosome-based cell-free therapy is being assessed for cell-free therapy as an alternative to cell-based therapy. This review of literature provides an update on cell-free therapy with primary focus on exosomes derived from BM-derived MSCs for myocardial repair.

    更新日期:2020-01-09
  • Neurovascular effects of umbilical cord blood-derived stem cells in growth-restricted newborn lambs
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-08
    Atul Malhotra; Margie Castillo-Melendez; Beth J. Allison; Amy E. Sutherland; Ilias Nitsos; Yen Pham; Courtney A. McDonald; Michael C. Fahey; Graeme R. Polglase; Graham Jenkin; Suzanne L. Miller

    Neonatal ventilation exacerbates brain injury in lambs with fetal growth restriction (FGR), characterized by neuroinflammation and reduced blood-brain barrier integrity, which is normally maintained by the neurovascular unit. We examined whether umbilical cord blood stem cell (UCBC) treatment stabilized the neurovascular unit and reduced brain injury in preterm ventilated FGR lambs. Surgery was performed in twin-bearing pregnant ewes at 88 days’ gestation to induce FGR in one fetus. At 127 days, FGR and appropriate for gestational age (AGA) lambs were delivered, carotid artery flow probes and umbilical lines inserted, lambs intubated and commenced on gentle ventilation. Allogeneic ovine UCBCs (25 × 106 cells/kg) were administered intravenously to lambs at 1 h of life. Lambs were ventilated for 24 h and then euthanized. FGR (n = 6) and FGR+UCBC (n = 6) lambs were growth restricted compared to AGA (n = 6) and AGA+UCBC (n = 6) lambs (combined weight, FGR 2.3 ± 0.4 vs. AGA 3.0 ± 0.3 kg; p = 0.0002). UCBC therapy did not alter mean arterial blood pressure or carotid blood flow but decreased cerebrovascular resistance in FGR+UCBC lambs. Circulating TNF-α cytokine levels were lower in FGR+UCBC vs. FGR lambs (p < 0.05). Brain histopathology showed decreased neuroinflammation and oxidative stress, increased endothelial cell proliferation, pericyte stability, and greater integrity of the neurovascular unit in FGR+UCBC vs. FGR lambs. Umbilical cord blood stem cell therapy mitigates perinatal brain injury due to FGR and ventilation, and the neuroprotective benefits may be mediated by stabilization of the neurovascular unit.

    更新日期:2020-01-08
  • Metformin alleviates muscle wasting post-thermal injury by increasing Pax7-positive muscle progenitor cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-08
    Yusef Yousuf; Andrea Datu; Ben Barnes; Saeid Amini-Nik; Marc G. Jeschke

    Profound skeletal muscle wasting and weakness is common after severe burn and persists for years after injury contributing to morbidity and mortality of burn patients. Currently, no ideal treatment exists to inhibit muscle catabolism. Metformin is an anti-diabetic agent that manages hyperglycemia but has also been shown to have a beneficial effect on stem cells after injury. We hypothesize that metformin administration will increase protein synthesis in the skeletal muscle by increasing the proliferation of muscle progenitor cells, thus mitigating muscle atrophy post-burn injury. To determine whether metformin can attenuate muscle catabolism following burn injury, we utilized a 30% total burn surface area (TBSA) full-thickness scald burn in mice and compared burn injuries with and without metformin treatment. We examined the gastrocnemius muscle at 7 and 14 days post-burn injury. At 7 days, burn injury significantly reduced myofiber cross-sectional area (CSA) compared to sham, p < 0.05. Metformin treatment significantly attenuated muscle catabolism and preserved muscle CSA at the sham size. To investigate metformin’s effect on satellite cells (muscle progenitors), we examined changes in Pax7, a transcription factor regulating the proliferation of muscle progenitors. Burned animals treated with metformin had a significant increase in Pax7 protein level and the number of Pax7-positive cells at 7 days post-burn, p < 0.05. Moreover, through BrdU proliferation assay, we show that metformin treatment increased the proliferation of satellite cells at 7 days post-burn injury, p < 0.05. In summary, metformin’s various metabolic effects and its modulation of stem cells make it an attractive alternative to mitigate burn-induced muscle wasting while also managing hyperglycemia.

    更新日期:2020-01-08
  • Stem cell-derived cell sheet transplantation for heart tissue repair in myocardial infarction
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-08
    Rui Guo; Masatoshi Morimatsu; Tian Feng; Feng Lan; Dehua Chang; Feng Wan; Yunpeng Ling

    Stem cell-derived sheet engineering has been developed as the next-generation treatment for myocardial infarction (MI) and offers attractive advantages in comparison with direct stem cell transplantation and scaffold tissue engineering. Furthermore, induced pluripotent stem cell-derived cell sheets have been indicated to possess higher potential for MI therapy than other stem cell-derived sheets because of their capacity to form vascularized networks for fabricating thickened human cardiac tissue and their long-term therapeutic effects after transplantation in MI. To date, stem cell sheet transplantation has exhibited a dramatic role in attenuating cardiac dysfunction and improving clinical manifestations of heart failure in MI. In this review, we retrospectively summarized the current applications and strategy of stem cell-derived cell sheet technology for heart tissue repair in MI.

    更新日期:2020-01-08
  • Establishment of long-term serum-free culture for lacrimal gland stem cells aiming at lacrimal gland repair
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-08
    Sa Xiao; Yan Zhang

    Aqueous-deficient dry eye disease (ADDED) resulting from dysfunction of the lacrimal gland (LG) is currently incurable. Although LG stem/progenitor cell-based therapy is considered to be a promising strategy for ADDED patients, the lack of a reliable serum-free culture method to obtain enough lacrimal gland stem cells (LGSCs) and the basic standard of LGSC transplantation are obstacles for further research. Adult mouse LGSCs were cultured in Matrigel-based 3D culture under serum-free culture condition, which contained EGF, FGF10, Wnt3A, and Y-27632. LGSCs were continuously passaged over 40 times every 7 days, and the morphology and cell numbers were recorded. LGSCs were induced to differentiate to ductal cells by reducing Matrigel rigidity, while fetal bovine serum was used for the induction of acinar cells. RT-PCR or qRT-PCR analysis, RNA-sequence analysis, H&E staining, and immunofluorescence were used for characterization and examining the differentiation of LGSCs. LGSCs were allotransplanted into diseased LGs to examine the ability of repairing the damage. The condition of eye orbits was recorded using a camera, the tear production was measured using phenol red-impregnated cotton threads, and the engraftments of LGSCs were examined by immunohistochemistry. We established an efficient 3D serum-free culture for adult mouse LGSCs, in which LGSCs could be continuously passaged for long-term expansion. LGSCs cultured from both the healthy and ADDED mouse LGs expressed stem/progenitor cell markers Krt14, Krt5, P63, and nestin, had the potential to differentiate into acinar or ductal-like cells in vitro and could engraft into diseased LGs and relieve symptoms of ADDED after orthotopic injection of LGSCs. We successfully established an efficient serum-free culture for adult mouse LGSCs aiming at LG repair for the first time. Our approach provides an excellent theoretical and technical reference for future clinical research for ADDED stem cell therapy.

    更新日期:2020-01-08
  • Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-07
    Sun Hwa Kim; Hae In Choi; Mi Ran Choi; Ga Yeong An; Bert Binas; Kyoung Hwa Jung; Young Gyu Chai

    Toll-like receptor 4 (TLR4) ligands such as lipopolysaccharide (LPS) activate immunomodulatory functions and the migration of human mesenchymal stromal cells (hMSCs). Here, we study the migration-related gene expression of LPS-stimulated hMSCs and the role and regulation of one of the upregulated genes, encoding the interferon-induced transmembrane protein 1 (IFITM1). Gene expression profiles were determined by whole-transcriptome analysis (RNA-seq) and quantitative real-time PCR (qRT-PCR). Bioinformatics approaches were used to perform network and pathway analyses. The cell migration-related genes were identified with an in vitro wound healing assay. RNA interference (RNAi) was used to suppress the IFITM1 gene expression. The IFITM1 gene enhancer was analyzed by chromatin immunoprecipitation (ChIP) sequencing, ChIP-to-PCR, luciferase reporter assays, and qRT-PCR for enhancer RNAs (eRNAs). RNA-seq confirmed IFITM1 as an LPS-stimulated gene, and RNAi demonstrated its importance for the LPS-stimulated migration. LPS treatment increased the eRNA expression in enhancer region R2 (2 kb upstream) of the IFITM1 gene and enriched R2 for H3K27ac. Bioinformatics implicated the transcription factors NF-κB and IRF1, ChIP assays revealed their binding to R2, and chemical inhibition of NF-κB and RNAi directed against IRF1 prevented R2 eRNA and IFITM1 gene expression. Increased expression of the IFITM1 gene is required for LPS-stimulated hMSC migration. We described several underlying changes in the IFITM1 gene enhancer, most notably the NF-κB-mediated activation of enhancer region R2.

    更新日期:2020-01-07
  • Endometrial stromal cells exhibit a distinct phenotypic and immunomodulatory profile
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-06
    Suzanna Queckbörner; Elisabeth Syk Lundberg; Kristina Gemzell-Danielsson; Lindsay C. Davies

    In Asherman’s syndrome (AS), intrauterine scarring and fibrotic adhesions lead to menstrual disorders, pregnancy loss, or infertility. A few clinical trials have piloted cell therapy to overcome AS. Understanding the role of the stromal compartment in endometrial regeneration remains poorly understood. We hypothesize that endometrial stromal cells (eSCs) represent a relevant cell population to establish novel cell-based therapeutics for endometrial disorders. The aim of this study was to characterize eSCs and evaluate their immune-cell interactions. eSCs were isolated from healthy donors, during the proliferative stage of the menstrual cycle. Cells were characterized for expression of mesenchymal stromal cell (MSC) markers and assessed for their tumorigenic potential. eSCs were co-cultured with interferon γ and tumor necrosis factor α, and cell surface expression of their respective receptors and human leukocyte antigen (HLA) I and II determined by flow cytometry. Secreted levels of key immunomodulatory factors were established. eSCs were cultured with activated peripheral blood mononuclear cells, and T cell differentiation and proliferation determined. eSCs demonstrated an MSC surface phenotype and exhibited multipotency. Expanded eSCs retained chromosomal stability and demonstrated no tumorigenicity. Upon stimulation, eSCs licensed to an anti-inflammatory phenotype with upregulated secretion of immunomodulatory factors. Stimulated eSCs did not express HLA class II. eSCs suppressed the proliferation and activation of CD4+ T cells, with the eSC secretome further downregulating central memory T cells and upregulating effector memory (EM) cells. Differential responsiveness to inflammation by eSCs, compared to other MSC sources, demonstrates the need to understand the specific functional effects of individual stromal cell sources. A lack of HLA class II and triggering of EM T cell differentiation strongly links to innate in vivo roles of eSCs in tissue repair and immune tolerance during pregnancy. We conclude that eSCs may be an ideal cell therapy candidate for endometrial disorders.

    更新日期:2020-01-06
  • Alteration of fatty acid oxidation by increased CPT1A on replicative senescence of placenta-derived mesenchymal stem cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Jin Seok; Hyun Sook Jung; Sohae Park; Jung Ok Lee; Chong Jai Kim; Gi Jin Kim

    Human placenta-derived mesenchymal stem cells (PD-MSCs) are powerful sources for cell therapy in regenerative medicine. However, a limited lifespan by senescence through mechanisms that are well unknown is the greatest obstacle. In the present study, we first demonstrated the characterization of replicative senescent PD-MSCs and their possible mitochondrial functional alterations. Human PD-MSCs were cultured to senescent cells for a long period of time. The cells of before passage number 8 were early cells and after passage number 14 were late cells. Also, immortalized cells of PD-MSCs (overexpressed hTERT gene into PD-MSCs) after passage number 14 were positive control of non-senescent cells. The characterization and mitochondria analysis of PD-MSCs were explored with long-term cultivation. Long-term cultivation of PD-MSCs exhibited increases of senescent markers such as SA-β-gal and p21 including apoptotic factor, and decreases of proliferation, differentiation potential, and survival factor. Mitochondrial dysfunction was also observed in membrane potential and metabolic flexibility with enlarged mitochondrial mass. Interestingly, we founded that fatty acid oxidation (FAO) is an important metabolism in PD-MSCs, and carnitine palmitoyltransferase1A (CPT1A) overexpressed in senescent PD-MSCs. The inhibition of CPT1A induced a change of energy metabolism and reversed senescence of PD-MSCs. These findings suggest that alteration of FAO by increased CPT1A plays an important role in mitochondrial dysfunction and senescence of PD-MSCs during long-term cultivation.

    更新日期:2020-01-04
  • Effect of hCMSCs and liraglutide combination in ALI through cAMP/PKAc/β-catenin signaling pathway
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Yun Feng; Linlin Wang; Xiaoying Ma; Xiaotong Yang; Ocholi Don; Xiaoyan Chen; Jieming Qu; Yuanlin Song

    ALI/ARDS is the major cause of acute respiratory failure in critically ill patients. As human chorionic villi-derived MSCs (hCMSCs) could attenuate ALI in the airway injury model, and liraglutide, glucagon-like peptide 1 (GLP-1) agonist, possesses anti-inflammatory and proliferation promotion functions, we proposed to probe the potential combinatory effect of hCMSCs and liraglutide on ALI. We examined the time- and dose-dependent manner of GLP-1R, SPC, Ang-1, and FGF-10 with LPS via western blot and qRT-PCR. Western blot and chromatin immunoprecipitation assay detected the effects of liraglutide on GLP-1R, SPC, Ang-1, and FGF-10 through PKAc/β-catenin pathway and cAMP pathway. In the ALI animal model, we detected the effects of MSC and liraglutide combination on ALI symptoms by H&E staining, western blot, ELISA assays, calculating wet-to-dry ratio of the lung tissue, and counting neutrophils, leukocytes, and macrophages in mouse bronchoalveolar lavage fluid (BALF). The data demonstrated that LPS reduced hCMSC proliferation and GLP-1R, SPC, Ang-1, and FGF-10 levels in a dose- and time-dependent manner. Liraglutide significantly dampened the reduction of GLP-1R, SPC, Ang-1, and FGF-10 and reversed the effect of LPS on hCMSCs, which could be regulated by GLP-1R and its downstream cAMP/PKAc/β-catenin-TCF4 signaling. Combination of hCMSCs with liraglutide showed more therapeutic efficacy than liraglutide alone in reducing LPS-induced ALI in the animal model. These results reveal that the combination of hCMSCs and liraglutide might be an effective strategy for ALI treatment.

    更新日期:2020-01-04
  • Small extracellular vesicles derived from embryonic stem cells restore ovarian function of premature ovarian failure through PI3K/AKT signaling pathway
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Mengyu Liu; Yu Qiu; Zhuowei Xue; Ruoyu Wu; Jie Li; Xin Niu; Ji Yuan; Yang Wang; Qingkai Wu

    Premature ovarian failure (POF) has a great impact on reproductive endocrine function in females, and it is an important cause of infertility. Previous studies have demonstrated that small extracellular vesicles (sEVs) derived from stem cells play an important role in tissue regeneration. This study aimed to investigate the therapeutic effect of sEVs derived from embryonic stem cells (ESCs-sEVs) on damaged ovaries and explore the underlying molecular mechanisms. Mice POF models were established by injecting mice with cyclophosphamide and busulfan. Then, ESCs-sEVs were intravenously transplanted into POF mice. The plasma of mice was harvested at 1 and 2 weeks after treatment to analyze the levels of anti-Mullerian hormone (AMH), estradiol (E2), and follicle stimulating hormone (FSH) by ELISA. The morphology of ovaries and follicles was observed by H&E staining, and apoptosis of granulosa cells was detected by TUNEL. In vitro, EdU and CCK-8 tests were used to evaluate the proliferation of cultured granulosa cells stimulated by ESCs-sEVs. Western blotting was used to determine the expression of PI3K/AKT and apoptotic-related proteins. After transplantation of ESCs-sEVs, the levels of serum sex hormones recovered to normal levels. In addition, the number of follicles was significantly increased, and the number of apoptotic cells was decreased. The results in vitro revealed that ESCs-sEVs could significantly improve the proliferation rate of granulosa cells and increase the expression of phosphorylated PI3K and AKT. Meanwhile, the positive effect on proliferation and the negative effect on apoptosis observed in granulosa cells were obviously decreased when the PI3K/AKT signaling pathway was inhibited. Our findings suggested that ESCs-sEVs could improve ovarian function by regulating the PI3K/AKT signaling pathway, which could provide a promising clinical therapy for POF.

    更新日期:2020-01-04
  • Age-dependent impairment of adipose-derived stem cells isolated from horses
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Michalina Alicka; Katarzyna Kornicka-Garbowska; Katarzyna Kucharczyk; Martyna Kępska; Michael Rӧcken; Krzysztof Marycz

    Progressive loss of cell functionality caused by an age-related impairment in cell metabolism concerns not only mature specialized cells but also its progenitors, which significantly reduces their regenerative potential. Adipose-derived stem cells (ASCs) are most commonly used in veterinary medicine as an alternative treatment option in ligaments and cartilage injuries, especially in case of high-value sport horses. Therefore, the main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young (< 5), middle-aged (5–15), and old (> 15 years old). ASCs were isolated from three age-matched horse groups using an enzymatic method. Molecular changes were assessed using qRT-PCR, ELISA and western blot methods, flow cytometry-based system, and confocal and scanning electron microscopy. Our findings showed that ASCs derived from the middle-aged and old groups exhibited a typical senescence phenotype, such as increased percentage of G1/G0-arrested cells, binucleation, enhanced β-galactosidase activity, and accumulation of γH2AX foci, as well as a reduction in cell proliferation. Moreover, aged ASCs were characterized by increased gene expression of pro-inflammatory cytokines and miRNAs (interleukin 8 (IL-8), IL-1β, tumor necrosis factor α (TNF-α), miR-203b-5p, and miR-16-5p), as well as apoptosis markers (p21, p53, caspase-3, caspase-9). In addition, our study revealed that the protein level of mitofusin 1 (MFN1) markedly decreased with increasing age. Aged ASCs also displayed a reduction in mRNA levels of genes involved in stem cell homeostasis and homing, like TET-3, TET-3 (TET family), and C-X-C chemokine receptor type 4 (CXCR4), as well as protein expression of DNA methyltransferase (DNMT1) and octamer transcription factor 3/4 (Oct 3/4). Furthermore, we observed a higher splicing ratio of XBP1 (X-box binding protein 1) mRNA, indicating elevated inositol-requiring enzyme 1 (IRE-1) activity and, consequently, increased endoplasmic reticulum (ER) stress. We also observed reduced levels of glucose transporter 4 (GLUT-4) and insulin receptor (INSR) which indicated impaired insulin sensitivity. Obtained data suggest that ASCs derived from horses older than 5 years old exhibited several molecular alternations which markedly limit their regenerative capacity. The results provide valuable information that allows for a better understanding of the molecular events occurring in ASCs in the course of aging and may help to identify new potential drug targets to restore their regenerative potential.

    更新日期:2020-01-04
  • Long non-coding RNA FER1L4 promotes osteogenic differentiation of human periodontal ligament stromal cells via miR-874-3p and vascular endothelial growth factor A
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Yiping Huang; Yineng Han; Runzhi Guo; Hao Liu; Xiaobei Li; Lingfei Jia; Yunfei Zheng; Weiran Li

    Periodontal ligament stromal cells (PDLSCs) are ideal cell sources for periodontal tissue repair and regeneration, but little is known about what determines their osteogenic capacity. Long non-coding RNAs (lncRNAs) are important regulatory molecules at both transcriptional and post-transcriptional levels. However, their roles in the osteogenic differentiation of PDLSCs are still largely unknown. The expression of lncRNA Fer-1-like family member 4 (FER1L4) during the osteogenic differentiation of PDLSCs was detected by quantitative reverse transcription polymerase chain reaction. Overexpression or knockdown of FER1L4 was used to confirm its regulation of osteogenesis in PDLSCs. Alkaline phosphatase and Alizarin red S staining were used to detect mineral deposition. Dual luciferase reporter assays were used to analyze the binding of miR-874-3p to FER1L4 and vascular endothelial growth factor A (VEGFA). Bone regeneration in critical-sized calvarial defects was assessed in nude mice. New bone formation was analyzed by micro-CT, hematoxylin and eosin staining, Masson’s trichrome staining, and immunohistochemical analyses. FER1L4 levels increased gradually during consecutive osteogenic induction of PDLSCs. Overexpression of FER1L4 promoted the osteogenic differentiation of PDLSCs, as revealed by alkaline phosphatase activity, Alizarin red S staining, and the expression of osteogenic markers, whereas FER1L4 knockdown inhibited these processes. Subsequently, we identified a predicted binding site for miR-874-3p on FER1L4 and confirmed a direct interaction between them. Wild-type FER1L4 reporter activity was significantly inhibited by miR-874-3p, whereas mutant FER1L4 reporter was not affected. MiR-874-3p inhibited osteogenic differentiation and reversed the promotion of osteogenesis in PDLSCs by FER1L4. Moreover, miR-874-3p targeted VEGFA, a crucial gene in osteogenic differentiation, whereas FER1L4 upregulated the expression of VEGFA. In vivo, overexpression of FER1L4 led to more bone formation compared to the control group, as demonstrated by micro-CT and the histologic analyses. FER1L4 positively regulates the osteogenic differentiation of PDLSCs via miR-874-3p and VEGFA. Our study provides a promising target for enhancing the osteogenic potential of PDLSCs and periodontal regeneration.

    更新日期:2020-01-04
  • Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Neety Sahu; Gaurav Budhiraja; Anuradha Subramanian

    Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.

    更新日期:2020-01-04
  • Pro-inflammatory cytokines induce cell death, inflammatory responses, and endoplasmic reticulum stress in human iPSC-derived beta cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Stéphane Demine; Andrea Alex Schiavo; Sandra Marín-Cañas; Piero Marchetti; Miriam Cnop; Decio L. Eizirik

    Adult human pancreatic beta cells are the “gold standard” for studies on diabetes pathogenesis, but their use is limited by insufficient availability and variable quality. An important effort has recently taken place to differentiate beta cells from human induced pluripotent stem cells (iPSCs) and validate their use for diabetes research. We presently used a 7-stage protocol to generate beta cells from human iPSC and evaluated whether these cells are responsive to the pro-inflammatory cytokines (IFNγ, IL-1β, or IFNα) that play a role in type 1 diabetes. The iPSC-derived islet-like cell clusters contained 40–50% beta and 10–15% alpha cells and expressed the receptors for IFNγ, IL-1β, or IFNα. Cells were exposed to either IFNγ (1000 U/mL) + IL-1β (50 U/mL) or IFNα alone (2000 U/mL) for 24/48 h. Apoptosis was quantified using Hoechst/propidium iodide staining or the RealTime Glo Apoptosis Kit (Promega). After treatment, CXCL10 secretion was quantified by ELISA. The expression of multiples genes (Ins, Gcg, Nkx2.2, Nkx6.1, Pdx1, Mafa, BiP, Chop, Atf3, CXCL10, CXCL9, CCL5, and HLA-ABC) was quantified by RT-qPCR. Phosphorylation state and total expression of STAT1/STAT2, as well as expression of PDL1 and of the ER chaperone BiP, were quantified by Western blotting. The co-localization of HLA-ABC or cleaved caspase-3 and Ins/Gcg expression was assessed by immunohistochemistry. The presence of HLA-ABC at the plasma membrane was measured by flow cytometry. IFNγ + IL-1β and IFNα induced apoptosis of the cells after 48 h of exposure. Cleaved caspase-3 co-localized mostly but not exclusively with Ins+ cells. Exposure to IFNγ + IL-1β induced a pro-inflammatory phenotype, including increased CXCL10, CXCL9, and CCL5 expression; CXCL10 secretion; and HLA-ABC expression. HLA overexpression was confirmed at the protein level by Western blotting and flow cytometry. Exposure to IFNγ + IL-1β (but not IFNα) also induced beta cell dedifferentiation and endoplasmic reticulum stress (increase in BiP, Chop, and Atf3 mRNA expression). Phosphorylation of STAT1 was stimulated already after 1 h by IFNγ + IL-1β and IFNα, while phosphorylation of STAT2 was only activated by IFNα at 1–4 h. PDL1 expression was increased by both IFNγ + IL-1β and IFNα. Our data show that human iPSC-derived beta cells respond to pro-inflammatory cytokines IL-1β + IFNγ and IFNα, by activating the same pathogenic processes as adult human primary beta cells. These cells thus represent a valuable tool for future research on the pathogenesis of type 1 diabetes.

    更新日期:2020-01-04
  • Long noncoding RNA HULC accelerates the growth of human liver cancer stem cells by upregulating CyclinD1 through miR675-PKM2 pathway via autophagy
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Chen Wang; Xiaoxue Jiang; Xiaonan Li; Shuting Song; Qiuyu Meng; Liyan Wang; Yanan Lu; Xiaoru Xin; Hu Pu; Xin Gui; Tianming Li; Dongdong Lu

    The functions of HULC have been demonstrated in several cancers. However, its mechanism has not been elucidated in human liver cancer stem cells. Liver cancer stem cells were isolated from Huh7 cells; gene infection and tumorigenesis test in vitro and in vivo were performed. We demonstrate that HULC promotes growth of liver cancer stem cells in vitro and in vivo. Mechanistically, HULC enhances the expression of Sirt1 dependent on miR675 and then induces the cellular autophagy through Sirt1. HULC enhances CyclinD1 and thereby increases pRB and inhibited P21 WAF1/CIP 1 via autophagy-miR675-PKM2 pathway in human liver cancer stem cells. Ultimately, our results demonstrate that CyclinD1 is required for the oncogenic functions of HULC in liver cancer stem cells. It reveals the key molecular signaling pathways for HULC and provides important basic information for finding effective tumor therapeutic targets based on HULC.

    更新日期:2020-01-04
  • Reproducibility of GMP-compliant production of therapeutic stressed peripheral blood mononuclear cell-derived secretomes, a novel class of biological medicinal products
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Maria Laggner; Alfred Gugerell; Christiane Bachmann; Helmut Hofbauer; Vera Vorstandlechner; Marcus Seibold; Ghazaleh Gouya Lechner; Anja Peterbauer; Sibylle Madlener; Svitlana Demyanets; Dirk Sorgenfrey; Tobias Ostler; Michael Erb; Michael Mildner; Hendrik Jan Ankersmit

    The recent concept of secretome-based tissue regeneration has profoundly altered the field of regenerative medicine and offers promising novel therapeutic options. In contrast to medicinal products with a single active substance, cell-derived secretomes comprise pleiotropic bioactive ingredients, representing a major obstacle for reproducible drug product efficacy and warranting patient safety. Good manufacturing practice (GMP)-compliant production guarantees high batch-to-batch consistency and reproducible efficacy of biological medicinal products, but different batches of cellular secretomes produced under GMP have not been compared yet, and suitable quality control parameters have not been established. To this end, we analyzed diverse biological and functional parameters of different batches produced under GMP of the secretome obtained from γ-irradiated peripheral blood mononuclear cells with proven tissue regenerative properties in infarcted myocardium, stroke, spinal cord injury, and skin wounds. We quantified key secretome ingredients, including cytokines, lipids, and extracellular vesicles, and functionally assessed potency in tube formation assay, ex vivo aortic ring sprouting assay, and cell-based protein and reporter gene assays. Furthermore, we determined secretome stability in different batches after 6 months of storage at various ambient temperatures. We observed that inter-batch differences in the bioactive components and secretome properties were small despite considerable differences in protein concentrations and potencies between individual donor secretomes. Stability tests showed that the analytical and functional properties of the secretomes remained stable when lyophilisates were stored at temperatures up to + 5 °C for 6 months. We are the first to demonstrate the consistent production of cell-derived, yet cell-free secretome as a biological medicinal product. The results from this study provide the basis for selecting appropriate quality control parameters for GMP-compliant production of therapeutic cell secretomes and pave the way for future clinical trials employing secretomes in tissue regenerative medicine.

    更新日期:2020-01-04
  • Aerobic and resistance training enhances endothelial progenitor cell function via upregulation of caveolin-1 in mice with type 2 diabetes
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Lu Zhai; Yuhua Liu; Wenpiao Zhao; Qingyun Chen; Tao Guo; Wei Wei; Zhuchun Luo; Yanfeng Huang; Cui Ma; Feng Huang; Xia Dai

    To explore the effect of aerobic training (AT), resistance training (RT) or a combination of AT and RT (AT+RT) on the function of endothelial progenitor cells (EPCs) in mice with type 2 diabetes and the potential effective mechanisms Eight-week-old db/db male mice were used as type 2 diabetic animal models in this study. Mice were randomly assigned to the control group (n = 5), AT group (n = 5), RT group (n = 5) and AT+RT group (n = 5). Mice in the control group remained sedentary with no specific training requirement. Mice were motivated to perform AT, RT or AT+RT by a gentle pat on their body for 3 or 4 days/week for 14 days. AT was performed by treadmill running, RT was performed by ladder climbing and AT+RT involved both AT and RT. Bone-derived EPCs were isolated after 14 days of the intervention. EPC expression of CD31, CD34, CD133, CD144 and VEGFR2 was detected by immunofluorescence staining. Fluorescence detection was performed on attached mononuclear cells to detect double-positive EPCs. We then explored the effect of caveolin-1 knockdown (lentiviral vector with caveolin-1-siRNA) on the proliferation and adherence of EPCs and the concentration of caveolin-1 and PI3K/AKT via western blot analyses. Compared to the mice in the control group, the mice in the AT, RT and AT+RT groups presented significant increases in proliferation and adherence after 14 days of intervention. AT+RT induced an increase in EPC adherence, which was greater than that of the control, RT and AT groups. Caveolin-1 knockdown inhibited the EPC proliferative and adherent abilities. The AT+RT group showed higher levels of caveolin-1 and p-AKT than the control group, but these changes were decreased by caveolin-1-siRNA transfection. Combined AT and RT is an effective way to improve EPC function through upregulation of caveolin-1 in mice with type 2 diabetes.

    更新日期:2020-01-04
  • Extracellular vesicles for acute kidney injury in preclinical rodent models: a meta-analysis
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Chao Liu; Jin Wang; Jie Hu; Bo Fu; Zhi Mao; Hengda Zhang; Guangyan Cai; Xiangmei Chen; Xuefeng Sun

    Extracellular vesicles (EVs), especially stem cell-derived EVs, have emerged as a potential novel therapy for acute kidney injury (AKI). However, their effects remain incompletely understood. Therefore, we performed this meta-analysis to systematically review the efficacy of EVs on AKI in preclinical rodent models. We searched PubMed, EMBASE, and the Web of Science up to March 2019 to identify studies that reported the treatment effects of EVs in a rodent AKI model. The primary outcome was serum creatinine (Scr) levels. The secondary outcomes were the blood urea nitrogen (BUN) levels, renal injury score, percentage of apoptotic cells, and interleukin (IL)-10 and tumour necrosis factor (TNF)-α levels. Two authors independently screened articles based on the inclusion and exclusion criteria. The meta-analysis was conducted using RevMan 5.3 and R software. Thirty-one studies (n = 552) satisfied the inclusion criteria. Pooled analyses demonstrated that the levels of Scr (SMD = − 3.71; 95% CI = − 4.32, − 3.10; P < 0.01), BUN (SMD = − 3.68; 95% CI = − 4.42, − 2.94; P < 0.01), and TNF-α (SMD = − 2.65; 95% CI = − 4.98, − 0.32; P < 0.01); the percentage of apoptotic cells (SMD = − 6.25; 95% CI = − 8.10, − 4.39; P < 0.01); and the injury score (SMD = − 3.90; 95% CI = − 5.26, − 2.53; P < 0.01) were significantly decreased in the EV group, and the level of IL-10 (SMD = 2.10; 95% CI = 1.18, 3.02; P < 0.01) was significantly increased. Meanwhile, no significant difference was found between stem cell-derived EVs and stem cells. The present meta-analysis confirmed that EV therapy could improve renal function and the inflammatory response status and reduce cell apoptosis in a preclinical rodent AKI model. This provides important clues for human clinical trials on EVs.

    更新日期:2020-01-04
  • Dopamine D1 receptor-mediated activation of the ERK signaling pathway is involved in the osteogenic differentiation of bone mesenchymal stem cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Chen-Xi Wang; Xi-Yuan Ge; Ming-Yue Wang; Ting Ma; Yu Zhang; Ye Lin

    Osteogenic differentiation of bone mesenchymal stem cells (BMSCs) is regulated by numerous signaling pathways. Dopamine (DA), a neurotransmitter, has previously been demonstrated to induce new bone formation by stimulating the receptors on BMSCs, but the essential mediators of DA-induced osteogenic signaling remain unclear. In this work, we evaluated the influence of both dopamine D1 and D2 receptor activation on BMSC osteogenic differentiation. Gene and protein expression of osteogenic-related markers were tested. The direct binding of transcriptional factor, Runx2, to those markers was also investigated. Additionally, cellular differentiation-associated signaling pathways were evaluated. We showed that the expression level of the D1 receptor on BMSCs increased during osteogenic differentiation. A D1 receptor agonist, similar to DA, induced the osteogenic differentiation of BMSCs, and this phenomenon was effectively inhibited by a D1 receptor antagonist or by D1 receptor knockdown. Furthermore, the suppression of protein kinase A (PKA), an important kinase downstream of the D1 receptor, successfully inhibited DA-induced BMSC osteogenic differentiation and decreased the phosphorylation of ERK1/2. Compared with P38, MAPK, and JNK, DA mainly induced the phosphorylation of ERK1/2 and led to the upregulation of Runx2 transcriptional activity, thus facilitating BMSC osteogenic differentiation. On the other hand, an ERK1/2 inhibitor could reverse these effects. Taken together, these results suggest that ERK signaling may play an essential role in coordinating the DA-induced osteogenic differentiation of BMSCs by D1 receptor activation.

    更新日期:2020-01-04
  • Influence of mesenchymal stem cell-derived extracellular vesicles in vitro and their role in ageing
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Juan Fafián-Labora; Miriam Morente-López; María José Sánchez-Dopico; Onno J. Arntz; Fons A. J. van de Loo; Javier De Toro; María C. Arufe

    This study assessed whether mesenchymal stem cell (MSC)-derived extracellular vesicles influenced ageing and pluripotency markers in cell cultures where they are added. MSC-derived extracellular vesicles from old and young rat bone marrows were isolated by ultracentrifugation and were characterised by western blotting, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). They were added to young and old MSC cultures. Real-time quantitative reverse transcription polymerase chain reactions and western blot analysis were performed to check the markers of ageing (vinculin and lamin A), pluripotency markers (Nanog and Oct4) and components of the mTOR signalling pathway (Rictor, Raptor, AKT and mTOR) in these cell populations. Subsequently, microRNA (miR)-188-3p expression was transiently inhibited in young MSCs to demonstrate the influence of mTOR2 on MSC ageing. Incubation with young MSC-derived extracellular vesicles decreased the levels of ageing markers and components of the mTOR pathway and increased the pluripotency markers from old MSC populations. By contrast, incubation of young MSCs with old MSC-derived extracellular vesicles generated the reverse effects. Inhibition of miR-188-3p expression in young MSCs produced extracellular vesicles that when incubated with old MSCs produced an increase in the levels of Rictor, as well as a decrease of phosphor-AKT, as indicated by a significant decrease in beta-galactosidase staining. MSC-derived extracellular vesicles affected the behaviour of MSC cultures, based on their composition, which could be modified in vitro. These experiments represented the basis for the development of new therapies against ageing-associated diseases using MSC-derived extracellular vesicles.

    更新日期:2020-01-04
  • Cellular therapy of corneal epithelial defect by adipose mesenchymal stem cell-derived epithelial progenitors
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2020-01-03
    Francisco Bandeira; Tze-Wei Goh; Melina Setiawan; Gary Hin-Fai Yam; Jodhbir S. Mehta

    Persistent epithelial defects (PED), associated with limbal stem cell deficiency (LSCD), require ocular surface reconstruction with a stable corneal epithelium (CE). This study investigated CE reformation using human adipose mesenchymal stem cells (ADSC), which derived epithelial progenitors via mesenchymal-epithelial transition (MET). STEMPRO human ADSC were cultured with specific inhibitors antagonizing glycogen synthase kinase-3 and transforming growth factor-β signaling, followed by culture under a defined progenitor cell targeted-epithelial differentiation condition to generate epithelial-like cells (MET-Epi), which were characterized for cell viability, mesenchymal, and epithelial phenotypes using immunofluorescence and flow cytometry. Tissue-engineered (TE) MET-Epi cells on fibrin gel were transplanted to corneal surface of the rat LSCD model caused by alkali injury. Epithelial healing, corneal edema, and haze grading, CE formation were assessed by fluorescein staining, slit lamp bio-microscopy, anterior segment optical coherence tomography, and immunohistochemistry. CD73high/CD90high/CD105high/CD166high/CD14negative/CD31negative human ADSC underwent MET, giving viable epithelial-like progenitors expressing δNp63, CDH1 (E-cadherin), epidermal growth factor receptor, integrin-β4, and cytokeratin (CK)-5, 9. Under defined epithelial differentiation culture, these progenitors generated MET-Epi cells expressing cell junction proteins ZO1 and occludin. When transplanted onto rat corneal surface with LSCD-induced PED, TE-MET-Epi achieved more efficient epithelial healing, suppressed corneal edema, and opacities, when compared to corneas without treatment or transplanted with TE-ADSC. CE markers (CK3, 12, and CDH1) were expressed on TE-MET-Epi-transplanted corneas but not in other control groups. Human ADSC-derived epithelial-like cells, via MET, recovered the CE from PED associated with LSCD. ADSC can be a viable adult stem cell source for potential autologous epithelial cell-based therapy for corneal surface disorders.

    更新日期:2020-01-04
  • Correction to: miR199a-3p regulates P53 by targeting CABLES1 in mouse cardiac c-kit+ cells to promote proliferation and inhibit apoptosis through a negative feedback loop
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-20
    Jingjin Liu; Yongshun Wang; Jinjin Cui; Meng Sun; Zhongyue Pu; Chao Wang; Wenjuan Du; Xinxin Liu; Jian Wu; Jingbo Hou; Shuo Zhang; Bo Yu

    After publication of our article [1] we became aware that there were errors in Fig. 5b and Fig. 6c, namely that the immunofluorescence of EDU-positive cells of the CABLES1 transfection group in Fig. 5b (panel 2) and the cell cycle distribution of the combination group (treatment with the antimiR199a-3p and shRNA-CABLES1) in Fig. 6c (panel 3) were incorrectly presented.

    更新日期:2019-12-21
  • Healthy, mtDNA-mutation free mesoangioblasts from mtDNA patients qualify for autologous therapy
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-21
    Florence van Tienen; Ruby Zelissen; Erika Timmer; Marike van Gisbergen; Patrick Lindsey; Mattia Quattrocelli; Maurilio Sampaolesi; Elvira Mulder-den Hartog; Irenaeus de Coo; Hubert Smeets

    Myopathy and exercise intolerance are prominent clinical features in carriers of a point-mutation or large-scale deletion in the mitochondrial DNA (mtDNA). In the majority of patients, the mtDNA mutation is heteroplasmic with varying mutation loads between tissues of an individual. Exercise-induced muscle regeneration has been shown to be beneficial in some mtDNA mutation carriers, but is often not feasible for this patient group. In this study, we performed in vitro analysis of mesoangioblasts from mtDNA mutation carriers to assess their potential to be used as source for autologous myogenic cell therapy. We assessed the heteroplasmy level of patient-derived mesoangioblasts, isolated from skeletal muscle of multiple carriers of different mtDNA point-mutations (n = 25). Mesoangioblast cultures with < 10% mtDNA mutation were further analyzed with respect to immunophenotype, proliferation capacity, in vitro myogenic differentiation potential, mitochondrial function, and mtDNA quantity. This study demonstrated that mesoangioblasts in half of the patients contained no or a very low mutation load (< 10%), despite a much higher mutation load in their skeletal muscle. Moreover, none of the large-scale mtDNA deletion carriers displayed the deletion in mesoangioblasts, despite high percentages in skeletal muscle. The mesoangioblasts with no or a very low mutation load (< 10%) displayed normal mitochondrial function, proliferative capacity, and myogenic differentiation capacity. Our data demonstrates that in half of the mtDNA mutation carriers, their mesoangioblasts are (nearly) mutation free and can potentially be used as source for autologous cell therapy for generation of new muscle fibers without mtDNA mutation and normal mitochondrial function.

    更新日期:2019-12-21
  • Menstrual blood-derived stem cells: toward therapeutic mechanisms, novel strategies, and future perspectives in the treatment of diseases
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-21
    Lijun Chen; Jingjing Qu; Tianli Cheng; Xin Chen; Charlie Xiang

    Menstrual blood-derived stem cells (MenSCs) have great potential in the treatment of various diseases. As a novel type of mesenchymal stem cells (MSCs), MenSCs have attracted more interest due to their therapeutic effects in both animal models and clinical trials. Here, we described the differentiation, immunomodulation, paracrine, homing, and engraftment mechanisms of MenSCs. These include differentiation into targeting cells, immunomodulation with various immune cells, the paracrine effect on secreting cytokines, and homing and engraftment into injured sites. To better conduct MenSC-based therapy, some novel hotspots were proposed such as CRISPR (clustered regularly interspaced short palindromic repeats)/cas9-mediated gene modification, exosomes for cell-free therapy, single-cell RNA sequence for precision medicine, engineered MenSC-based therapy for the delivery platform, and stem cell niches for improving microenvironment. Subsequently, current challenges were elaborated on, with regard to age of donor, dose of MenSCs, transplantation route, and monitoring time. The management of clinical research with respect to MenSC-based therapy in diseases will become more normative and strict. Thus, a more comprehensive horizon should be considered that includes a combination of traditional solutions and novel strategies. In summary, MenSC-based treatment has a great potential in treating diseases through diverse strategies, and more therapeutic mechanisms and novel strategies need to be elucidated for future regenerative medicine and clinical applications.

    更新日期:2019-12-21
  • Electromagnetic field treatment increases purinergic receptor P2X7 expression and activates its downstream Akt/GSK3β/β-catenin axis in mesenchymal stem cells under osteogenic induction
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-21
    Yingchi Zhang; Wenkai Li; Chaoxu Liu; Jiyuan Yan; Xuefeng Yuan; Wei Wang; Huaixi Wang; Hua Wu; Yong Yang

    Imbalance in bone formation and resorption is a crucial component of the pathological process leading to osteoporosis. Electromagnetic fields (EMFs) have been reported to be beneficial to osteogenesis, although the exact mechanism has not been fully clarified. Purinergic receptor P2X7 is expressed in osteoblasts and is reported to participate in the regulation of bone metabolism. To elucidate the link between EMFs and P2X7 expression and investigate its potential as a novel therapeutic target in osteoporosis. We investigated the effect of EMFs on P2X7 expression and downstream signaling in human bone marrow mesenchymal stem cells (h-MSCs). We also established an ovariectomized (OVX) osteoporosis rat model to evaluate the therapeutic efficacy of combining EMFs with P2X7 agonists. EMF treatment increased P2X7 expression in h-MSCs under conditions of osteogenic induction but not under regular culture conditions. P2X7 or PI3K/Akt inhibition partially inhibited the pro-osteogenic effect of EMF and lowered the EMF-stimulated activity of the Akt/GSK3β/β-catenin axis. No additive effect of this suppression was observed following simultaneous inhibition of P2X7 and PI3K/Akt. EMF treatment in the presence of a P2X7 agonist had a greater effect in increasing osteogenic marker expression than that of EMF treatment alone. In the OVX osteoporosis model, the therapeutic efficacy of combining EMFs with P2X7 agonists was superior to that of EMF treatment alone. EMF treatment increases P2X7 expression by h-MSCs during osteogenic differentiation, leading to activation of the Akt/GSK3β/β-catenin axis, which promotes the osteogenesis. Our findings also indicate that combined EMF and P2X7 agonist treatment may be an effective novel strategy for osteoporosis therapy.

    更新日期:2019-12-21
  • Melatonin promotes the BMP9-induced osteogenic differentiation of mesenchymal stem cells by activating the AMPK/β-catenin signalling pathway
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-21
    Tianyuan Jiang; Chao Xia; Xiaoting Chen; Yan Hu; Yan Wang; Jin Wu; Shuyan Chen; Yanhong Gao

    Mesenchymal stem cells (MSCs) play a crucial role in maintaining the dynamic balance of bone metabolism. Melatonin may have a regulatory effect on bone metabolism by regulating the lineage commitment and differentiation signalling pathways of MSCs. Among the BMP families, the osteogenesis of BMP9 is considered to be one of the strongest in MSCs. Here, we explored whether melatonin and BMP9 act synergistically on MSC osteogenic differentiation. The C3H10T1/2 osteogenic differentiation function induced by melatonin synergizes with BMP9, as detected by the expression of osteogenic markers at different periods. The result was further confirmed by foetal limb explant culture and in vivo stem cell implantation experiments. The effects of the AMPK/β-catenin pathway on the osteogenic differentiation of C3H10T1/2 cells were evaluated by Western blotting. Melatonin combined with BMP9 significantly enhanced the expression of osteogenic markers at different periods in C3H10T1/2 cells, effectively enhancing BMP9-induced bone formation in cultured foetal explants and ectopic bone formation in vivo in stem cell transplantation experiments. Melatonin increases the expression of BMP9 in C3H10T1/2 cells and induces Smad1/5/8 translocation from the cytoplasm to the nucleus. In addition, melatonin and BMP9 synergistically promote AMPK and β-catenin phosphorylation, which can be largely eliminated by AMPK siRNA pretreatment. Melatonin and BMP9 in C3H10T1/2 cells synergistically promote osteogenic differentiation at least in part by activating the AMPK/β-catenin signalling pathway.

    更新日期:2019-12-21
  • Pre-aggregation of scalp progenitor dermal and epidermal stem cells activates the WNT pathway and promotes hair follicle formation in in vitro and in vivo systems
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-19
    Yiqun Su; Jie Wen; Junrong Zhu; Zhiwei Xie; Chang Liu; Chuan Ma; Qun Zhang; Xin Xu; Xunwei Wu

    Billions of dollars are invested annually by pharmaceutical companies in search of new options for treating hair loss conditions; nevertheless, the challenge remains. One major limitation to hair follicle research is the lack of effective and efficient drug screening systems using human cells. Organoids, three-dimensional in vitro structures derived from stem cells, provide new opportunities for studying organ development, tissue regeneration, and disease pathogenesis. The present study focuses on the formation of human hair follicle organoids. Scalp-derived dermal progenitor cells mixed with foreskin-derived epidermal stem cells at a 2:1 ratio aggregated in suspension to form hair follicle-like organoids, which were confirmed by immunostaining of hair follicle markers and by molecular dye labeling assays to analyze dermal and epidermal cell organization in those organoids. The hair-forming potential of organoids was examined using an in vivo transplantation assay. Pre-aggregation of dermal and epidermal cells enhanced hair follicle formation in vivo. In vitro pre-aggregation initiated the interactions of epidermal and dermal progenitor cells resulting in activation of the WNT pathway and the formation of pear-shape structures, named type I aggregates. Cell-tracing analysis showed that the dermal and epidermal cells self-assembled into distinct epidermal and dermal compartments. Histologically, the type I aggregates expressed early hair follicle markers, suggesting the hair peg-like phase of hair follicle morphogenesis. The addition of recombinant WNT3a protein to the medium enhanced the formation of these aggregates, and the Wnt effect could be blocked by the WNT inhibitor, IWP2. In summary, our system supports the rapid formation of a large number of hair follicle organoids (type I aggregates). This system provides a platform for studying epithelial-mesenchymal interactions, for assessing inductive hair stem cells and for screening compounds that support hair follicle regeneration.

    更新日期:2019-12-19
  • Microvesicles isolated from 5-azacytidine-and-resveratrol-treated mesenchymal stem cells for the treatment of suspensory ligament injury in horse—a case report
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-18
    Katarzyna Kornicka-Garbowska; Rafał Pędziwiatr; Paulina Woźniak; Katarzyna Kucharczyk; Krzysztof Marycz

    In athlete horses, suspensory ligament (SL) injuries are the most common cause of lameness. Healing of SL injury is still problematic, and even proper rehabilitation and pharmacological therapy do not guarantee returning to the initial performance level. In our previous studies, we have shown that a combination of 5-azacytidine (AZA) and resveratrol (RES) exerts beneficial, rejuvenating effects on metabolic syndrome derived adipose-derived stem cells (ASCs). Thus, in the presented research, we investigate whether not only rejuvenated ASC but also microvesicles (MVsAZA/RES) secreted by them possess enhanced regenerative properties in SL injury. In the presented study, a 6-year-old Dutch Warmblood gelding, working in jumping, was diagnosed with SL injury using ultrasonography, Doppler, real-time elastography and thermography. As a therapeutic strategy, the affected animal was treated with extracellular microvesicles derived from ASC treated with the combination of 5-azacytydine (AZA) and resveratrol (RES) (MVsAZA/RES). First, anti-apoptotic effects of MVsAZA/RES were tested in co-culture with metabolic syndrome derived ASC. The proliferation of cells and expression of pro-apoptotic genes were investigated. Then, MVsAZA/RES were injected directly into the injured SL of the Dutch Warmblood gelding. In vitro assays revealed that MVsAZA/RES enhance the proliferation of ASC and exert an anti-apoptotic effect. In the affected horse, the application of MVsAZA/RES resulted in increased lesion filling and improvement of angiogenesis and elasticity in injured tissue. As MVsAZA/RES mimic several of the biological actions exerted by ASC, they have become an alternative for stem cell-based therapies and can be effectively applied for the treatment of SL injury in horses.

    更新日期:2019-12-19
  • Combination therapies enhance immunoregulatory properties of MIAMI cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-18
    Fiorella Rossi; Hunter Noren; Leonor Sarria; Paul C. Schiller; Lubov Nathanson; Vladimir Beljanski

    Mesenchymal stromal cells (MSCs), adult stromal cells most commonly isolated from bone marrow (BM), are being increasingly utilized in various therapeutic applications including tissue repair via immunomodulation, which is recognized as one of their most relevant mechanism of action. The promise of MSC-based therapies is somewhat hindered by their apparent modest clinical benefits, highlighting the need for approaches that would increase the efficacy of such therapies. Manipulation of cellular stress-response mechanism(s) such as autophagy, a catabolic stress-response mechanism, with small molecules prior to or during MSC injection could improve MSCs’ therapeutic efficacy. Unfortunately, limited information exists on how manipulation of autophagy affects MSCs’ response to inflammation and subsequent immunoregulatory properties. In this study, we exposed BM-MSC precursor cells, “marrow-isolated adult multilineage inducible” (MIAMI) cells, to autophagy modulators tamoxifen (TX) or chloroquine (CQ), together with IFN-γ. Exposed cells then underwent RNA sequencing (RNAseq) to determine the effects of TX or CQ co-treatments on cellular response to IFN-γ at a molecular level. Furthermore, we evaluated their immunoregulatory capacity using activated CD4+ T cells by analyzing T cell activation marker CD25 and the percentage of proliferating T cells after co-culturing the cells with MIAMI cells treated or not with TX or CQ. RNAseq data indicate that the co-treatments alter both mRNA and protein levels of key genes responsible for MSCs’ immune-regulatory properties. Interestingly, TX and CQ also altered some of the microRNAs targeting such key genes. In addition, while IFN-γ treatment alone increased the surface expression of PD-L1 and secretion of IDO, this increase was further enhanced with TX. An improvement in MIAMI cells’ ability to decrease the activation and proliferation of T cells was also observed with TX, and to a lesser extent, CQ co-treatments. Altogether, this work suggests that both TX and CQ have a potential to enhance MIAMI cells’ immunoregulatory properties. However, this enhancement is more pronounced with TX co-treatment.

    更新日期:2019-12-19
  • Extracellular matrix derived from human urine-derived stem cells enhances the expansion, adhesion, spreading, and differentiation of human periodontal ligament stem cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-18
    Xue Xiong; Xiao Yang; Hongwei Dai; Gang Feng; Yuanyuan Zhang; Jianping Zhou; Wenwen Zhou

    Human periodontal ligament stem cells (hPDLSCs) are one of the most promising types of seed cells in periodontal tissue regeneration. Suitable biomaterials are additional essential components that must cooperate with seed cells for in vivo expansion or in vitro implantation. Extracellular matrix (ECM) derived from mesenchymal stem cells (MSCs) was recently reported to be a promising substrate with which to culture MSCs that could be applied in biomaterial scaffolds or bioink. Human urine-derived stem cells (hUSCs) have several advantages; their collection is non-invasive and easy, and hUSCs are low in cost, potentially making them a suitable and efficient source of ECM. The purpose of this study was to characterize the biological properties of ECM derived from hUSCs (UECM) and evaluate the effects of UECM on hPDLSCs. hPDLSCs grown on ECM derived from hPDLSCs (PECM) and fibronectin-coated tissue culture plastic (TCP) served as control groups. Both hUSCs and hPDLSCs were seeded on TCP and stimulated to produce ECM. After 8 days of stimulation, the samples were decellularized, leaving only ECM. Then, hPDLSCs were seeded onto UECM-, PECM-, and fibronectin-coated TCP and untreated TCP. UECM consists of dense bundles of fibers which contain abundant fibronectin. Both UECM and PECM promoted hPDLSC proliferation, attachment, spreading, and differentiation. Between UECM and PECM, UECM enhanced proliferation, osteogenesis, and angiogenesis to a greater extent. Though fibronectin appeared to be the abundant component of UECM, its performance was inferior to that of UECM. Our study provides an original perspective on different cell-specific ECMs and suggests UECM as a suitable biomaterial in which to culture hPDLSCs as UECM enhances their biological functions.

    更新日期:2019-12-19
  • Bone marrow-derived neural crest precursors improve nerve defect repair partially through secreted trophic factors
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-18
    Haiyan Shi; Xiaoli Li; Junling Yang; Yahong Zhao; Chengbin Xue; Yaxian Wang; Qianru He; Mi Shen; Qi Zhang; Yumin Yang; Fei Ding

    Emerging evidence suggests that neural crest-derived cells (NCCs) present important functions in peripheral nerve regeneration to correct the insufficiency of autogenous Schwann cells. Postmigratory NCCs have been successfully isolated from adult rat bone marrow in our previous work. In this study, we aim to provide neural crest-derived Schwann cell precursors (SCPs) for repair of nerve defects in adult rats, and partially reveal the mechanisms involved in neuroregeneration of cell therapy. A clonal cell line of neural crest precursors of rat bone marrow origin (rBM-NCPs) with SCP identity was expanded in adherent monolayer culture to ensure the stable cell viability of NCPs and potentiate the repair of nerve defects after rBM-NCPs implantation based on tissue engineering nerve grafts (TENG). Here the behavioral, morphological, and electrophysiological detection was performed to evaluate the therapy efficacy. We further investigated the treatment with NCP-conditioned medium (NCP-CM) to sensory neurons after exposure to oxygen-glucose-deprivation (OGD) and partially compared the expression of trophic factor genes in rBM-NCPs with that in mesenchymal stem cells of bone marrow origin (rBM-MSCs). It was showed that the constructed TENG with rBM-NCPs loaded into silk fibroin fiber scaffolds/chitosan conduits repaired 10-mm long sciatic nerve defects more efficiently than conduits alone. The axonal regrowth, remyelination promoted the reinnervation of the denervated hind limb muscle and skin and thereby alleviated muscle atrophy and facilitated the rehabilitation of motor and sensory function. Moreover, it was demonstrated that treatment with NCP-CM could restore the cultured primary sensory neurons after OGD through trophic factors including epidermal growth factor (EGF), platelet-derived growth factor alpha (PDGFα), ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor alpha (VEGFα). In summary, our findings indicated that monolayer-cultured rBM-NCPs cell-based therapy might effectively repair peripheral nerve defects partially through secreted trophic factors, which represented the secretome of rBM-NCPs differing from that of rBM-MSCs.

    更新日期:2019-12-19
  • MicroRNA-145 engineered bone marrow-derived mesenchymal stem cells alleviated erectile dysfunction in aged rats
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-18
    Qiwei Liu; Yubin Cui; Haojian Lin; Daoyuan Hu; Tao Qi; Bo Wang; Zhansen Huang; Jun Chen; Ke Li; Hengjun Xiao

    Aging is one of the dominant factors contributing to erectile dysfunction (ED), and effective treatments for age-associated ED are urgently demanded. In this study, the therapeutic efficiency of bone marrow-derived mesenchymal stem cells (BMSCs) overexpressing microRNA-145 (miR-145) was evaluated in ED. Sixty male Sprague-Dawley rats (24 months old) were randomly divided into 4 treatment groups (n = 15/group): PBS (control), BMSCs, BMSCs transfected with a blank vector (vector-BMSCs), and BMSCs transfected with a lentivirus overexpressing miR-145 (OE-miR-145-BMSCs). Fourteen days after transplantation of BMSCs, erectile function was evaluated by measuring intra-cavernous pressure (ICP) and mean arterial pressure (MAP). Subsequently, penile erectile tissues were harvested and subjected to Masson staining, qRT-PCR, immunofluorescence staining, dual luciferase assay, and Western blot analysis. Fourteen days after transplantation, the ICP/MAP was 0.79 ± 0.05 in the OE-miR-145-BMSC group, 0.61 ± 0.06 in the BMSC group, 0.57 ± 0.06 in the vector-BMSC group, and 0.3 ± 0.01 in the PBS group. Treatment with OE-miR-145-BMSCs significantly improved ED (P < 0.05), and the treatment increased the smooth muscle content in the penis tissues of ED rats (P < 0.05). In the OE-miR-145-BMSC group, the expression levels of α-SMA, desmin, and SM-MHC were higher than they were in the other ED groups (P < 0.05). In addition, the levels of collagen 1, MMP2, and p-Smad2 in the BMSC-treated group, especially in the OE-miR-145-BMSC group, were lower than those in the control group (P < 0.05). MicroRNA-145 engineered BMSCs effectively attenuate age-related ED. Transplantation of miR-145-overexpressing BMSCs may provide a promising novel avenue for age-associated ED therapy.

    更新日期:2019-12-19
  • PAMAM-cRGD mediating efficient siRNA delivery to spermatogonial stem cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-18
    Tianjiao Li; Qiwen Chen; Yi Zheng; Pengfei Zhang; Xiaoxu Chen; Junna Lu; Yinghua Lv; Shiguo Sun; Wenxian Zeng

    Spermatogonial stem cells (SSCs) are the cornerstone of sperm production and thus perpetual male fertility. In clinics, transplantation of patient’s own SSCs into testes is a promising technique to restore fertility when male germ cells have been depleted by gonadotoxic therapies. Auto-transplantation of genetically modified SSCs even has the potential to treat male infertility caused by genetic mutations. However, SSCs are refractory to transfection approaches. Poly(amidoamine) (PAMAM) dendrimers have the unique three-dimensional architecture, surface charge, and high density of surface groups that are suitable for ligand attachment, thereby facilitating target delivery. The goal of this study was to elucidate whether PAMAM dendrimers can efficiently deliver short interfering RNAs (siRNAs) to SSCs. We introduced cyclic arginine-glycine-aspartic acid (cRGD) peptides to the fifth generation of PAMAM dendrimers (G5) to generate PAMAM-cRGD dendrimers (G5-cRGD). The characterization of G5-cRGD was detected by Fourier transform infrared spectroscope (FTIR), transmission electron microscope (TEM), and the Cell Counting Kit-8 (CCK-8) assay. Confocal microscopy and flow cytometry were used to evaluate the delivery efficiency of siRNA by G5-cRGD to SSCs. The results showed that G5-cRGD encompassing siRNA could self-assemble into spherical structures with nanoscale size and possess high transfection efficiency, excellent endosomal escape ability, and low cytotoxicity, superior to a commercial transfection reagent Lipofectamine® 2000. Moreover, we demonstrated that G5-cRGD efficiently delivered siRNAs and triggered gene silencing. This study thus provides a promising nanovector for siRNA delivery in SSCs, facilitating the future clinical application of SSC auto-transplantation with genetically modified cells with a hope to cure male infertility that is caused by genetic disorders.

    更新日期:2019-12-19
  • LncRNA Neat1 mediates miR-124-induced activation of Wnt/β-catenin signaling in spinal cord neural progenitor cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-18
    Yi Cui; Yanyun Yin; Zhifeng Xiao; Yannan Zhao; Bing Chen; Bin Yang; Bai Xu; Hongwei Song; Yunlong Zou; Xu Ma; Jianwu Dai

    Emerging evidence suggests that miR-124 performs important biological functions in neural stem cells (NSCs); it regulates NSC behavior and promotes the differentiation of NSCs into neurons, but the exact molecular mechanism remains unknown. And also, the role of miR-124 during spinal cord injury regeneration is unclear. In order to explore the function of miR-124 in neural differentiation, the molecular markers (Tuj1, Map2, and GFAP) correlated with the differentiation of NSCs were detected by immunofluorescence staining both in cultured mouse spinal cord progenitor cells (SC-NPCs) and in spinal cord injury (SCI) animal models. The migration ability and apoptosis of cultured SC-NPCs were also evaluated by Transwell migration assay and TUNEL assay. In addition, the relative expression of lnRNA Neat1- and Wnt/β-catenin signaling-related genes were detected by quantitative real-time PCR. In this study, we revealed that lncRNA Neat1 is involved in regulating Wnt/β-catenin signaling that is activated by miR-124 in SC-NPCs. LncRNA Neat1 was also found to play an important role in regulating neuronal differentiation, apoptosis, and migration of SC-NPCs. Furthermore, we demonstrated that overexpression of miR-124 resulted in elevated Neat1 expression, accompanied with the functional recovery of locomotion in a mouse model of spinal cord injury. Our results confirm the therapeutic effectiveness of miR-124 on the functional recovery of injured spinal cord, supporting the rationale and feasibility of miR-124 for spinal cord injury treatment in future clinical therapy. Furthermore, we concluded that the miR-124-Neat1-Wnt/β-catenin signaling axis is involved in regulating the cell function of SC-NPCs, and this may offer novel therapeutic avenues for future treatment of SCI.

    更新日期:2019-12-19
  • A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-18
    Afnan Munshi; Jelica Mehic; Marybeth Creskey; Jonathan Gobin; Jun Gao; Emma Rigg; Gauri Muradia; Christian C. Luebbert; Carole Westwood; Andrew Stalker; David S. Allan; Michael J. W. Johnston; Terry Cyr; Michael Rosu-Myles; Jessie R. Lavoie

    Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance. Small EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers. Nanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors. The identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

    更新日期:2019-12-19
  • YAP as a key regulator of adipo-osteogenic differentiation in human MSCs
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-18
    Chanchao Lorthongpanich; Kanjana Thumanu; Kantpitchar Tangkiettrakul; Nittaya Jiamvoraphong; Chuti Laowtammathron; Nattaya Damkham; Yaowalak U-pratya; Surapol Issaragrisil

    Mesenchymal stem cells (MSCs) are multipotent stem cells that are able to differentiate into several cell types, including cartilage, fat, and bone. As a common progenitor, MSC differentiation has to be tightly regulated to maintain the balance of their differentiation commitment. It has been reported that the decision process of MSCs into fat and bone cells is competing and reciprocal. Several factors have been suggested as critical factors that affect adipo-osteogenic decision, including melatonin and smad4. Yes-associated protein (YAP) is an important effector protein in the Hippo signaling pathway that acts as a transcriptional regulator by activating the transcription of the genes involved in cell proliferation and anti-apoptosis. The non-canonical role of YAP in regulating bone homeostasis by promoting osteogenesis and suppressing adipogenesis was recently demonstrated in a mouse model. However, it is unclear whether YAP is also crucial for modulating human MSC differentiation to fat and bone. The expression level of YAP during MSC differentiation was modulated using pharmaceutical molecule and genetic experiments through gain- and loss-of-function approaches. We demonstrated for the first time that YAP has a non-canonical role in regulating the balance of adipo-osteogenic differentiation of human MSCs. The result from synchrotron radiation-based Fourier transform infrared (FTIR) microspectroscopy showed unique metabolic fingerprints generated from YAP-targeted differentiated cells that were clearly distinguished from non-manipulated control. These results, thus, identify YAP as an important effector protein that regulates human MSC differentiation to fat and bone and suggests the use of FTIR microspectroscopy as a promising technique in stem cell research.

    更新日期:2019-12-19
  • Cladophora glomerata methanolic extract promotes chondrogenic gene expression and cartilage phenotype differentiation in equine adipose-derived mesenchymal stromal stem cells affected by metabolic syndrome
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-17
    Lynda Bourebaba; Izabela Michalak; Meriem Baouche; Katarzyna Kucharczyk; Krzysztof Marycz

    Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential. In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome. Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis. The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation. Chondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.

    更新日期:2019-12-18
  • LncRNA KLF3-AS1 in human mesenchymal stem cell-derived exosomes ameliorates pyroptosis of cardiomyocytes and myocardial infarction through miR-138-5p/Sirt1 axis
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-17
    Qing Mao; Xiu-Lin Liang; Chuan-Long Zhang; Yi-Heng Pang; Yong-Xiang Lu

    Myocardial infarction (MI) is a severe disease with increased mortality and disability rates, posing heavy economic burden for society. Exosomes were uncovered to mediate intercellular communication after MI. This study aims to explore the effect and mechanism of lncRNA KLF3-AS1 in exosomes secreted by human mesenchymal stem cells (hMSCs) on pyroptosis of cardiomyocytes and MI. Exosomes from hMSCs were isolated and identified. Exosomes from hMSCs with transfection of KLF3-AS1 for overexpression were injected into MI rat model or incubated with hypoxia cardiomyocytes. Effect of KLF3-AS1 on MI area, cell viability, apoptosis, and pyroptosis was determined. The relationship among miR-138-5p, KLF3-AS1, and Sirt1 was verified by dual-luciferase reporter assay. Normal cardiomyocytes were transfected with miR-138-5p inhibitor or sh-Sirt1 to clarify whether alteration of miR-138-5p or sh-Sirt1 can regulate the effect of KLF3-AS1 on cardiomyocytes. Exosomes from hMSCs were successfully extracted. Transfection of KLF3-AS1 exosome in rats and incubation with KLF3-AS1 exosome in hypoxia cardiomyocytes both verified that overexpression of KLF3-AS1 in exosomes leads to reduced MI area, decreased cell apoptosis and pyroptosis, and attenuated MI progression. KLF3-AS1 can sponge miR-138-5p to regulate Sirt1 expression. miR-138-5p inhibitor transfection and KLF3-AS1 exosome incubation contribute to attenuated pyroptosis and MI both in vivo and in vitro, while transfection of sh-Sirt1 could reverse the protective effect of exosomal KLF3-AS1 on hypoxia cardiomyocytes. LncRNA KLF3-AS1 in exosomes secreted from hMSCs by acting as a ceRNA to sponge miR-138-5p can regulate Sirt1 so as to inhibit cell pyroptosis and attenuate MI progression.

    更新日期:2019-12-18
  • The combinatory effect of sinusoidal electromagnetic field and VEGF promotes osteogenesis and angiogenesis of mesenchymal stem cell-laden PCL/HA implants in a rat subcritical cranial defect
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Jingyuan Chen; Chang Tu; Xiangyu Tang; Hao Li; Jiyuan Yan; Yongzhuang Ma; Hua Wu; Chaoxu Liu

    Restoration of massive bone defects remains a huge challenge for orthopedic surgeons. Insufficient vascularization and slow bone regeneration limited the application of tissue engineering in bone defect. The effect of electromagnetic field (EMF) on bone defect has been reported for many years. However, sinusoidal EMF (SEMF) combined with tissue engineering in bone regeneration remains poorly investigated. In the present study, we investigated the effect of SEMF and vascular endothelial growth factor (VEGF) on osteogenic and vasculogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs). Furthermore, pretreated rBMSC- laden polycaprolactone-hydroxyapatite (PCL/HA) scaffold was constructed and implanted into the subcritical cranial defect of rats. The bone formation and vascularization were evaluated 4 and 12 weeks after implantation. It was shown that SEMF and VEGF could enhance the protein and mRNA expression levels of osteoblast- and endothelial cell-related markers, respectively. The combinatory effect of SEMF and VEGF slightly promoted the angiogenic differentiation of rBMSCs. The proteins of Wnt1, low-density lipoprotein receptor-related protein 6 (LRP-6), and β-catenin increased in all inducted groups, especially in SEMF + VEGF group. The results indicated that Wnt/β-catenin pathway might participate in the osteogenic and angiogenic differentiation of rBMSCs. Histological evaluation and reconstructed 3D graphs revealed that tissue-engineered constructs significantly promoted the new bone formation and angiogenesis compared to other groups. The combinatory effect of SEMF and VEGF raised an efficient approach to enhance the osteogenesis and vascularization of tissue-engineered constructs, which provided a useful guide for regeneration of bone defects.

    更新日期:2019-12-17
  • In vivo conversion of rat astrocytes into neuronal cells through neural stem cells in injured spinal cord with a single zinc-finger transcription factor
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Masoumeh Zarei-Kheirabadi; Mahdi Hesaraki; Sahar Kiani; Hossein Baharvand

    Spinal cord injury (SCI) results in glial scar formation and irreversible neuronal loss, which finally leads to functional impairments and long-term disability. Our previous studies have demonstrated that the ectopic expression of Zfp521 reprograms fibroblasts and astrocytes into induced neural stem cells (iNSCs). However, it remains unclear whether treatment with Zfp521 also affects endogenous astrocytes, thus promoting further functional recovery following SCI. Rat astrocytes were transdifferentiated into neural stem cells in vitro by ZFP521 or Sox2. Then, ZFP521 was applied to the spinal cord injury site of a rat. Transduction, real-time PCR, immunohistofluorescence, and function assessments were performed at 6 weeks post-transduction to evaluate improvement and in vivo lineage reprogramming of astrocytes. Here, we show that Zfp521 is more efficient in reprogramming cultured astrocytes compared with Sox2. In the injured spinal cord of an adult rat, resident astrocytes can be reprogrammed into neurons through a progenitor stage by Zfp521. Importantly, this treatment improves the functional abilities of the rats as evaluated by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and further by calculation of its subscores. There was enhanced locomotor activity in the hind limbs, step length, toe spread, foot length, and paw area. In addition, motor evoked potential recordings demonstrated the functional integrity of the spinal cord. These results have indicated that the generation of iNSCs or neurons from endogenous astrocytes by in situ reprogramming might be a potential strategy for SCI repair.

    更新日期:2019-12-17
  • Mesenchymal stem cell-derived exosomal microRNA-133b suppresses glioma progression via Wnt/β-catenin signaling pathway by targeting EZH2
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Haiyang Xu; Guifang Zhao; Yu Zhang; Hong Jiang; Weiyao Wang; Donghai Zhao; Jin Hong; Hongquan Yu; Ling Qi

    Mesenchymal stem cells (MSCs) play a significant role in cancer initiation and metastasis, sometimes by releasing exosomes that mediate cell communication by delivering microRNAs (miRNAs). This study aimed to investigate the effects of exosomal miR-133b derived from MSCs on glioma cell behaviors. Microarray-based analysis identified the differentially expressed genes (DEGs) in glioma. The expression patterns of EZH2 and miR-133b along with interaction between them were clarified in glioma. The expression of miR-133b and EZH2 in glioma cells was altered to examine their functions on cell activities. Furthermore, glioma cells were co-cultured with MSC-derived exosomes treated with miR-133b mimic or inhibitor, and EZH2-over-expressing vectors or shRNA against EZH2 to characterize their effect on proliferation, invasion, and migration of glioma cells in vitro. In vivo assays were also performed to validate the in vitro findings. miR-133b was downregulated while EZH2 was upregulated in glioma tissues and cells. miR-133b was found to target and negatively regulate EZH2 expression. Moreover, EZH2 silencing resulted in inhibited glioma cell proliferation, invasion, and migration. Additionally, MSC-derived exosomes containing miR-133b repressed glioma cell proliferation, invasion, and migration by inhibiting EZH2 and the Wnt/β-catenin signaling pathway. Furthermore, in vivo experiments confirmed the tumor-suppressive effects of MSC-derived exosomal miR-133b on glioma development. Collectively, the obtained results suggested that MSC-derived exosomes carrying miR-133b could attenuate glioma development via disrupting the Wnt/β-catenin signaling pathway by inhibiting EZH2, which provides a potential treatment biomarker for glioma.

    更新日期:2019-12-17
  • Relative safety of various spermatogenic stem cell purification methods for application in spermatogenic stem cell transplantation
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Jia Tian; Ke Ma; Cheng-bin Pei; Shao-hua Zhang; Xue Li; Yue Zhou; Bei Yan; Hong-yan Wang; Liang-hong Ma

    Spermatogonial stem cell (SSC) transplantation technology as a promising option for male fertility preservation has received increasing attention, along with efficient SSC purification technology as a necessary technical support; however, the safety of such application in patients with tumors remains controversial. In this study, we used a green fluorescent protein mouse xenograft model of B cell acute lymphocytic leukemia. We isolated and purified SSCs from the testicular tissue of model mice using density gradient centrifugation, immune cell magnetic bead separation, and flow cytometry. The purified SSCs were transplanted into convoluted seminiferous tubules of the nude mice and C57BL/6 male mice subjected to busulfan. The development and proliferation of SSCs in the recipient testis were periodically tested, along with whether B cell acute lymphocytic leukemia was induced following SSC implantation. The genetic characteristics of the offspring obtained from natural mating were also observed. In testicular leukemia model mice, a large number of BALL cells infiltrated into the seminiferous tubule, spermatogenic cells, and sperm cells in the testis tissue decreased. After spermatogonial stem cell transplantation, the transplanted SSCs purified by immunomagnetic beads and flow cytometry methods colonized and proliferated extensively in the basement of the seminiferous tubules of mice; a large number of spermatogenic cells and sperm were found in recipient testicular tissue after 12 weeks of SSC transplantation. In leukemia detection in nude mice after transplantation in the three SSC purification groups, a large number of BALL cells could be detected in the blood of recipient mice 2–3 weeks after transplantation in the density gradient centrifugation group, but not in the blood of the flow cytometry sorting group and the immunomagnetic bead group after 16 weeks of observation. In this study, we confirmed that immunomagnetic beads and flow cytometry methods of purifying SSCs from the testicular tissue of the testicular leukemia mouse model could be safely applied to the SSC transplantation technology without concomitant tumor implantation. The results thus provide a theoretical basis for the application of tumor SSC cryopreservation for fertility preservation in patients with tumors.

    更新日期:2019-12-17
  • Human fetal skin-derived stem cell secretome enhances radiation-induced skin injury therapeutic effects by promoting angiogenesis
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Xiaoli Rong; Jiannan Li; Yanyan Yang; Liyan Shi; Tiechao Jiang

    Radiation dermatitis is a refractory skin injury caused by radiotherapy. Human fetal skin-derived stem cell (hFSSC) is a preferable source for cell therapy and skin tissue regeneration. In the present study, we investigated the repair effect of using hFSSC secretome on a radiation skin injury model in rats. We prepared the hFSSC secretome and studied its effects on the proliferation and tube formation of human umbilical vein endothelial cell (HUVEC) in vitro. Furthermore, we used a Sr-90 radiation-induced skin injury model of rats and evaluated the effects of hFSSC secretome on radiation skin injury in vivo. The results showed that hFSSC secretome significantly promoted the proliferation and tube formation of HUVEC in vitro; in addition, hFSSC secretome-treated rats exhibited higher healing quality and faster healing rate than the other two control groups; the expression level of collagen type III α 1 (Col3A1), transforming growth factor β3 (TGF-β3), angiotensin 1 (Ang-1), angiotensin 2 (Ang-2), vascular endothelial growth factor (VEGF), and placental growth factor (PLGF) was significantly increased, while collagen type I α 2 (Col1A2) and transforming growth factor β1 (TGF-β1) were decreased in hFSSC secretome group. In conclusion, our results provided the first evidence on the effects of hFSSC secretome towards radiation-induced skin injury. We found that hFSSC secretome significantly enhanced radiation dermatitis angiogenesis, and the therapeutic effects could match with the characteristics of fetal skin. It may act as a kind of novel cell-free therapeutic approach for radiation-induced cutaneous wound healing.

    更新日期:2019-12-17
  • Acute myeloid leukemia cells secrete microRNA-4532-containing exosomes to mediate normal hematopoiesis in hematopoietic stem cells by activating the LDOC1-dependent STAT3 signaling pathway
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Chen Zhao; Feng Du; Yang Zhao; Shanshan Wang; Ling Qi

    MicroRNA (miR)-containing exosomes released by acute myeloid leukemia (AML) cells can be delivered into hematopoietic progenitor cells to suppress normal hematopoiesis. Herein, our study was performed to evaluate the effect of exosomal miR-4532 secreted by AML cells on hematopoiesis of hematopoietic stem cells. Firstly, differentially expressed miRs related to AML were identified using microarray analysis. Subsequently, AML cell lines were collected, and CD34+ HSCs were isolated from healthy pregnant women. Then, miR-4532 expression was measured in AML cells and AML cell-derived exosomes and CD34+ HSCs, together with evaluation of the targeting relationship between miR-4532 and LDOC1. Then, AML cells were treated with miR-4532 inhibitor, and exosomes were separated from AML cells and co-cultured with CD34+ HSCs. Gain- and loss-function approaches were employed in CD34+ HSCs. Colony-forming units (CFU) and expression of dickkopf-1 (DKK1), a hematopoietic inhibiting factor associated with pathogenesis of AML, were determined in CD34+ HSCs, as well as the extents of JAK2 and STAT3 phosphorylation and LDOC1 expression. miR-4532 was found to be upregulated in AML cells and AML cell-derived exosomes, while being downregulated in CD34+ HSCs. In addition, exosomes released by AML cells targeted CD34+ HSCs to decrease the expression of CFU and increase the expression of DKK1. miR-4532 was delivered into CD34+ HSCs to target LDOC1 via AML cell-released exosomes. AML cell-derived exosomes containing miR-4532 inhibitor increased CFU but reduced DKK1 in CD34+ HSCs. Inhibition of miR-4532 or JAK2, or ectopic expression of LDOC1 upregulated CFU and downregulated DKK1 expression as well as the extents of JAK2 and STAT3 phosphorylation in CD34+ HSCs. In conclusion, AML cell-derived exosomes carrying miR-4532 repress normal HSC hematopoiesis via activation of the LDOC1-dependent STAT3 signaling pathway.

    更新日期:2019-12-17
  • Current understanding of the administration of mesenchymal stem cells in acute kidney injury to chronic kidney disease transition: a review with a focus on preclinical models
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Lingfei Zhao; Fei Han; Junni Wang; Jianghua Chen

    Incomplete recovery from acute kidney injury (AKI) can result in long-term functional deficits and has been recognized as a major contributor to chronic kidney disease (CKD), which is termed the AKI-CKD transition. Currently, an effective intervention for this disorder is still lacking. Principally, therapeutic strategies targeting the AKI-CKD transition can be divided into those reducing the severity of AKI or promoting the regenerative process towards beneficially adaptive repair pathways. Considering the fact that mesenchymal stem cells (MSCs) have the potential to address both aspects, therapeutic regimens based on MSCs have a promising future. In light of this information, we focus on the currently available evidence associated with MSC therapy involved in the treatment of the AKI-CKD transition and the underlying mechanisms. All of these discussions will contribute to the establishment of a reliable therapeutic strategy for patients with this problem, who can be easily ignored by physicians, and will lead to a better clinical outcome for them.

    更新日期:2019-12-17
  • Genetically modified mesenchymal stem cell therapy for acute respiratory distress syndrome
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Jibin Han; Yuxiang Liu; Hong Liu; Yuanyuan Li

    Acute respiratory distress syndrome (ARDS) is a devastating hypoxemic respiratory failure, characterized by disruption of the alveolar-capillary membrane barrier. Current management for ARDS remains supportive, including lung-protective ventilation and a conservative fluid strategy. Mesenchymal stem cells (MSCs) have emerged as a potentially attractive candidate for the management of ARDS through facilitating lung tissue regeneration and repair by releasing paracrine soluble factors. Over the last decade, a variety of strategies have emerged to optimize MSC-based therapy. Among these, the strategy using genetically modified MSCs has received increased attention recently due to its distinct advantage, in conferring incremental migratory capacity and, enhancing the anti-inflammatory, immunomodulatory, angiogenic, and antifibrotic effects of these cells in numerous preclinical ARDS models, which may in turn provide additional benefits in the management of ARDS. Here, we provide an overview of recent studies testing the efficacy of genetically modified MSCs using preclinical models of ARDS.

    更新日期:2019-12-17
  • Activation of type I interferon antiviral response in human neural stem cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Jhao-Yin Lin; Rei-Lin Kuo; Hsing-I Huang

    Neural stem cells (NSCs) residing in the central nervous system play an important role in neurogenesis. Several viruses can infect these neural progenitors and cause severe neurological diseases. The innate immune responses against the neurotropic viruses in these tissue-specific stem cells remain unclear. Human NSCs were transfected with viral RNA mimics or infected with neurotropic virus for detecting the expression of antiviral interferons (IFNs) and downstream IFN-stimulated antiviral genes. NSCs are able to produce interferon-β (IFN-β) (type I) and λ1 (type III) after transfection with poly(I:C) and that downstream IFN-stimulated antiviral genes, such as ISG56 and MxA, and the viral RNA sensors RIG-I, MDA5, and TLR3, can be expressed in NSCs under poly(I:C) or IFN-β stimulation. In addition, our results show that the pattern recognition receptors RIG-I and MDA5, as well as the endosomal pathogen recognition receptor TLR3, but not TLR7 and TLR8, are involved in the activation of IFN-β transcription in NSCs. Furthermore, NSCs infected with the neurotropic viruses, Zika and Japanese encephalitis viruses, are able to induce RIG-I-mediated IFN-β expression. Human NSCs have the ability to activate IFN signals against neurotropic viral pathogens.

    更新日期:2019-12-17
  • Modeling appendicular skeletal cartilage development with modified high-density micromass cultures of adult human bone marrow-derived mesenchymal progenitor cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Alessandro Pirosa; Karen L. Clark; Jian Tan; Shuting Yu; Yuanheng Yang; Rocky S. Tuan; Peter G. Alexander

    Animal cell-based systems have been critical tools in understanding tissue development and physiology, but they are less successful in more practical tasks, such as predicting human toxicity to pharmacological or environmental factors, in which the congruence between in vitro and clinical outcomes lies on average between 50 and 60%. Emblematic of this problem is the high-density micromass culture of embryonic limb bud mesenchymal cells, derived from chick, mouse, or rat. While estimated predictive value of this model system in toxicological studies is relatively high, important failures prevent its use by international regulatory agencies for toxicity testing and policy development. A likely underlying reason for the poor predictive capacity of animal-based culture models is the small but significant physiological differences between species. This deficiency has inspired investigators to develop more organotypic, 3-dimensional culture system using human cells to model normal tissue development and physiology and assess pharmacological and environmental toxicity. We have developed a modified, miniaturized micromass culture model using adult human bone marrow-derived mesenchymal progenitor cells (hBM-MPCs) that is amenable to moderate throughput and high content analysis to study chondrogenesis. The number of cells per culture was reduced, and a methacrylated gelatin (gelMA) overlay was incorporated to normalize the morphology of the cultures. These modified human cell-based micromass cultures demonstrated robust chondrogenesis, indicated by increased Alcian blue staining and immunodetectable production of collagen type II and aggrecan, and stage-specific chondrogenic gene expression. In addition, in cultures of hBM-MPCs transduced with a lentiviral collagen type II promoter-driven GFP reporter construct, levels of GFP reporter activity correlated well with changes in endogenous collagen type II transcript levels, indicating the feasibility of non-invasive monitoring of chondrogenesis. The modified hBM-MPC micromass culture system described here represents a reproducible and controlled model for analyzing mechanisms of human skeletal development that may later be applied to pharmacological and environmental toxicity studies.

    更新日期:2019-12-17
  • SOX19b regulates the premature neuronal differentiation of neural stem cells through EZH2-mediated histone methylation in neural tube development of zebrafish
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Xian Li; Wenjuan Zhou; Xinyue Li; Ming Gao; Shufang Ji; Wenyu Tian; Guangyu Ji; Jingyi Du; Aijun Hao

    Neural tube defects (NTDs) are the most serious and common birth defects in the clinic. The SRY-related HMG box B1 (SoxB1) gene family has been implicated in different processes of early embryogenesis. Sox19b is a maternally expressed gene in the SoxB1 family that is found in the region of the presumptive central nervous system (CNS), but its role and mechanism in embryonic neural stem cells (NSCs) during neural tube development have not yet been explored. Considering that Sox19b is specific to bony fish, we intended to investigate the role and mechanism of Sox19b in neural tube development in zebrafish embryos. Morpholino (MO) antisense oligonucleotides were used to construct a Sox19b loss-of-function zebrafish model. The phenotype and the expression of related genes were analysed by in situ hybridization and immunolabelling. Epigenetic modifications were detected by western blot and chromatin immunoprecipitation. In this study, we found that zebrafish embryos exhibited a reduced or even deleted forebrain phenotype after the expression of the Sox19b gene was inhibited. Moreover, we found for the first time that knockdown of Sox19b reduced the proliferation of NSCs; increased the transcription levels of Ngn1, Ascl1, HuC, Islet1, and cyclin-dependent kinase (CDK) inhibitors; and led to premature differentiation of NSCs. Finally, we found that knockdown of Sox19b decreased the levels of EZH2/H3K27me3 and decreased the level of H3K27me3 at the promoters of Ngn1 and ascl1a. Together, our data demonstrate that Sox19b plays an essential role in early NSC proliferation and differentiation through EZH2-mediated histone methylation in neural tube development. This study established the role of transcription factor Sox19b and epigenetic factor EZH2 regulatory network on NSC development, which provides new clues and theoretical guidance for the clinical treatment of neural tube defects.

    更新日期:2019-12-17
  • The superiority of conditioned medium derived from rapidly expanded mesenchymal stem cells for neural repair
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Ya-Tzu Chen; May-Jywan Tsai; Nini Hsieh; Ming-Jei Lo; Meng-Jen Lee; Henrich Cheng; Wen-Cheng Huang

    Spinal cord injury (SCI) is a complex and severe neurological condition. Mesenchymal stem cells (MSCs) and their secreted factors show promising potential for regenerative medicine. Many studies have investigated MSC expansion efficacy of all kinds of culture medium formulations, such as growth factor-supplemented or xeno-free medium. However, very few studies have focused on the potential of human MSC (hMSC) culture medium formulations for injured spinal cord repair. In this study, we investigated the effect of hMSC-conditioned medium supplemented with bFGF, EGF, and patient plasma, namely, neural regeneration laboratory medium (NRLM), on SCI in vitro and in vivo. Commercial and patient bone marrow hMSCs were obtained for cultivation in standard medium and NRLM separately. Several characteristics, including CD marker expression, differentiation, and growth curves, were compared between MSCs cultured in standard medium and NRLM. Additionally, we investigated the effect of the conditioned medium (referred to as NRLM-CM) on neural repair, including inflammation inhibition, neurite regeneration, and spinal cord injury (SCI), and used a coculture system to detect the neural repair function of NRLM-MSCs. Compared to standard culture medium, NRLM-CM had superior in inflammation reduction and neurite regeneration effects in vitro and improved functional restoration in SCI rats in vivo. In comparison with standard culture medium MSCs, NRLM-MSCs proliferated faster regardless of the age of the donor. NRLM-MSCs also showed increased adipose differentiative potential and reduced CD90 expression. Both types of hMSC CM effectively enhanced injured neurite outgrowth and protected against H2O2 toxicity in spinal cord neuron cultures. Cytokine arrays performed in hMSC-CM further revealed the presence of at least 120 proteins. Among these proteins, 6 demonstrated significantly increased expression in NRLM-CM: adiponectin (Acrp30), angiogenin (ANG), HGF, NAP-2, uPAR, and IGFBP2. The NRLM culture system provides rapid expansion effects and functional hMSCs. The superiority of the derived conditioned medium on neural repair shows potential for future clinical applications.

    更新日期:2019-12-17
  • Genetic tool for fate mapping of Oct4 (Pou5f1)-expressing cells and their progeny past the pluripotency stage
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-16
    Andrey A. Kuzmin; Veronika V. Ermakova; Sergey A. Sinenko; Sergey V. Ponomartsev; Tatiana Y. Starkova; Elena V. Skvortsova; Olga Cherepanova; Alexey N. Tomilin

    Methods based on site-specific recombinases are widely used in studying gene activities in vivo and in vitro. In these studies, constitutively active or inducible variants of these recombinases are expressed under the control of either lineage-specific or ubiquitous promoters. However, there is a need for more advanced schemes that combine these features with possibilities to choose a time point from which lineage tracing starts in an autonomous fashion. For example, the key mammalian germline gatekeeper gene Oct4 (Pou5f1) is expressed in the peri-implantation epiblast which gives rise to all cells within embryos. Thus the above techniques are hardly applicable to Oct4 tracing past the epiblast stage, and the establishment of genetic tools addressing such a limitation is a highly relevant pursuit. The CRISPR/Cas9 tool was used to manipulate the genome of mouse embryonic stem cells (ESCs), and various cell culture technics—to maintain and differentiate ESCs to neural cell, lentivirus-based reprogramming technique—to generate induced pluripotent stem cells (iPSCs). In this paper, we have developed a two-component genetic system (referred to as O4S) that allows tracing Oct4 gene activity past the epiblast stage of development. The first component represents a knock-in of an ubiquitous promoter-driven inducible Cre, serving as a stop signal for downstream tdTomato. Upon activation of Cre activity with 4-hydroxytamoxifen (4-OHT) at any given time point, the recombinase excises a stop signal and poses the second component of the system—the FlpO recombinase, knocked into 3’UTR of Oct4, to be expressed upon activation of the latter gene. Oct4-driven expression of FlpO, in turn, triggers the tdTomato expression and thus, permanently marks Oct4+ cells and their progeny. We have validated the O4S system in cultured ESCs and shown that it is capable, for example, to timely capture an activation of Oct4 gene during the reprogramming of somatic cells into iPSCs. The developed O4S system can be used to detect Oct4 activation event, both permanent and transient, in somatic cell types outside the germline. The approach can be equally adjusted to other genes, provided the first component of the system is placed under transcriptional control of these genes, thus, making it a valuable tool for cell fate mapping in mice.

    更新日期:2019-12-17
  • Magnesium-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells by activating ERK/BMP2/Smads signaling
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-10
    Yuanyuan Kong; Xiaoli Hu; Yingqun Zhong; Ke Xu; Buling Wu; Jianmao Zheng

    Magnesium (Mg2+)-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells (DPSCs), but the regulatory mechanisms remain undefined. The aim of this work was to assess magnesium’s function in the above process and to explore the associated signaling pathway. DPSCs underwent culture in odontogenic medium with the addition of 0, 1, 5, or 10 mM MgCl2. Intracellular Mg2+ levels in DPSCs were evaluated flow cytometrically using Mag-Fluo-4-AM. Mg2+-entry was inhibited by TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB). RNA-Sequencing was carried out for assessing transcriptome alterations in DPSCs during odontogenic differentiation associated with high extracellular Mg2+. KEGG pathway analysis was performed to determine pathways related to the retrieved differentially expressed genes (DEGs). Immunoblot was performed for assessing magnesium’s role and exploring ERK/BMP2/Smads signaling. Mg2+-enriched microenvironment promoted odontogenic differentiation in DPSCs via intracellular Mg2+ increase. Consistently, the positive effect of high extracellular Mg2+ on odontogenic differentiation in DPSCs was blocked by 2-APB, which reduced Mg2+ entry. RNA-sequencing identified 734 DEGs related to odontogenic differentiation in DPSCs in the presence of high extracellular Mg2+. These DEGs participated in many cascades such as MAPK and TGF-β pathways. Consistently, ERK and BMP2/Smads pathways were activated in DPSCs treated with high extracellular Mg2+. In agreement, ERK signaling inhibition by U0126 blunted the effect of high extracellular Mg2+ on mineralization and odontogenic differentiation in DPSCs. Interestingly, BMP2, BMPR1, and phosphorylated Smad1/5/9 were significantly decreased by U0126, indicating that BMP2/Smads acted as downstream of ERK. Mg2+-enriched microenvironment promotes odontogenic differentiation in DPSCs by activating ERK/BMP2/Smads signaling via intracellular Mg2+ increase. This study revealed that Mg2+-enriched microenvironment could be used as a new strategy for dental pulp regeneration.

    更新日期:2019-12-11
  • The shift in the balance between osteoblastogenesis and adipogenesis of mesenchymal stem cells mediated by glucocorticoid receptor
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-05
    Lizhi Han; Bo Wang; Ruoyu Wang; Song Gong; Guo Chen; Weihua Xu

    Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into several tissues, such as bone, cartilage, and fat. Glucocorticoids affect a variety of biological processes such as proliferation, differentiation, and apoptosis of various cell types, including osteoblasts, adipocytes, or chondrocytes. Glucocorticoids exert their function by binding to the glucocorticoid receptor (GR). Physiological concentrations of glucocorticoids stimulate osteoblast proliferation and promote osteogenic differentiation of MSCs. However, pharmacological concentrations of glucocorticoids can not only induce apoptosis of osteoblasts and osteocytes but can also reduce proliferation and inhibit the differentiation of osteoprogenitor cells. Several signaling pathways, including the Wnt, TGFβ/BMP superfamily and Notch signaling pathways, transcription factors, post-transcriptional regulators, and other regulators, regulate osteoblastogenesis and adipogenesis of MSCs mediated by GR. These signaling pathways target key transcription factors, such as Runx2 and TAZ for osteogenesis and PPARγ and C/EBPs for adipogenesis. Glucocorticoid-induced osteonecrosis and osteoporosis are caused by various factors including dysfunction of bone marrow MSCs. Transplantation of MSCs is valuable in regenerative medicine for the treatment of osteonecrosis of the femoral head, osteoporosis, osteogenesis imperfecta, and other skeletal disorders. However, the mechanism of inducing MSCs to differentiate toward the osteogenic lineage is the key to an efficient treatment. Thus, a better understanding of the molecular mechanisms behind the imbalance between GR-mediated osteoblastogenesis and adipogenesis of MSCs would not only help us to identify the pathogenic causes of glucocorticoid-induced osteonecrosis and osteoporosis but also promote future clinical applications for stem cell-based tissue engineering and regenerative medicine. Here, we primarily review the signaling mechanisms involved in adipogenesis and osteogenesis mediated by GR and discuss the factors that control the adipo-osteogenic balance.

    更新日期:2019-12-06
  • Immunomodulatory effects of interferon-γ on human fetal cardiac mesenchymal stromal cells
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-04
    Karl-Henrik Grinnemo; Marie Löfling; Lubov Nathanson; Roland Baumgartner; Daniel F. J. Ketelhuth; Vladimir Beljanski; Lindsay C. Davies; Cecilia Österholm

    Mesenchymal stromal cells (MSCs), due to their regenerative and immunomodulatory properties, are therapeutically used for diseases, including heart failure. As early gestational-phase embryonic tissues exhibit extraordinary regenerative potential, fetal MSCs exposed to inflammation offer a unique opportunity to evaluate molecular mechanisms underlying preferential healing, and investigate their inherent abilities to communicate with the immune system during development. The principal aim of this study was to evaluate the effects of interferon-γ (IFNγ) on the immunomodulatory effects of first-trimester human fetal cardiac (hfc)-MSCs. hfcMSCs (gestational week 8) were exposed to IFNγ, with subsequent analysis of the whole transcriptome, based on RNA sequencing. Exploration of surface-expressed immunoregulatory mediators and modulation of T cell responses were performed by flow cytometry. Presence and activity of soluble mediators were assessed by ELISA or high-performance liquid chromatography. Stimulation of hfcMSCs with IFNγ revealed significant transcriptional changes, particularly in respect to the expression of genes belonging to antigen presentation pathways, cell cycle control, and interferon signaling. Expression of immunomodulatory genes and associated functional changes, including indoleamine 2,3-dioxygenase activity, and regulation of T cell activation and proliferation via programmed cell death protein (PD)-1 and its ligands PD-L1 and PD-L2, were significantly upregulated. These immunoregulatory molecules diminished rapidly upon withdrawal of inflammatory stimulus, indicating a high degree of plasticity by hfcMSCs. To our knowledge, this is the first study performing a systematic evaluation of inflammatory responses and immunoregulatory properties of first-trimester cardiac tissue. In summary, our study demonstrates the dynamic responsiveness of hfcMSCs to inflammatory stimuli. Further understanding as to the immunoregulatory properties of hfcMSCs may be of benefit in the development of novel stromal cell therapeutics for cardiovascular disease.

    更新日期:2019-12-05
  • Mesenchymal stem cells induce dendritic cell immune tolerance via paracrine hepatocyte growth factor to alleviate acute lung injury
    Stem Cell Res. Ther. (IF 4.627) Pub Date : 2019-12-04
    Zhonghua Lu; Wei Chang; Shanshan Meng; Xiuping Xu; Jianfeng Xie; Fengmei Guo; Yi Yang; Haibo Qiu; Ling Liu

    Mesenchymal stem cells (MSCs) have been shown to alleviate acute lung injury (ALI) via paracrine hepatocyte growth factor (HGF) and to induce the differentiation of dendritic cells (DCs) into tolerogenic dendritic cells (DCregs) and participate in the immune response. However, whether MSCs induce the production of DCregs by secreting HGF to alleviate early ALI remains unclear. We observed that the protective effect of mouse bone marrow-derived MSCs against lipopolysaccharide (LPS)-induced ALI was achieved by inducing mature DCs (mDCs) to differentiate into DCregs, and its mechanism is related to the activation of the HGF/Akt pathway. MSCs or MSCs with overexpression or knockdown of HGF were cocultured with DCs derived from mouse bone marrow using a Transwell system for 3 days. Moreover, we used MSCs or MSCs with overexpression or knockdown of HGF to treat LPS-induced ALI mice for 24 h. Flow cytometry was performed to measure the phagocytosis, accumulation, and maturation of DCs, as well as proliferation of T cells. Lung injury was estimated by lung wet weight to body weight ratio (LWW/BW) and histopathological analysis. Furthermore, we used the Akt inhibitor MK-2206 in a coculture system to elucidate the role of the HGF/Akt pathway in regulating the differentiation of DCs into regulatory DCs and relieving lung injury in early ALI mice. Immature DCs (imDCs) were induced to mature after 24 h of LPS (50 ng/ml) stimulation. MSCs or HGF induced the differentiation of mDCs into regulatory DCs characterized by low expression of MHCII, CD86, and CD40 molecules, strong phagocytic function, and the ability to inhibit T cell proliferation. The effect of MSCs on DCregs was enhanced with the increase in HGF secretion and was weakened with the decrease in HGF secretion. DCregs induced by recombinant HGF were attenuated by the Akt inhibitor MK-2206. Lung DC aggregation and mDC ratio increased in LPS-induced ALI mice, while treatment with MSCs decreased lung DC aggregation and maturation and alleviated lung pathological injury. High expression of the HGF gene enhanced the above effect of MSCs, while decreased expression of HGF weakened the above effect of MSCs. MSCs alleviate early ALI via paracrine HGF by inducing mDCs to differentiate into regulatory DCs. Furthermore, the mechanism of HGF-induced differentiation of mDCs into DCregs is related to the activation of the Akt pathway.

    更新日期:2019-12-05
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