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The risk of severe disease caused by co-infection with SARS-CoV-2 and influenza virus (IAV) raises an annual concern for global public health. Extracellular vesicles (EV) derived from mesenchymal stem cells (MSC) possess anti-inflammatory properties that can attenuate the inflammatory cytokine levels induced by viral infection. However, the effects of MSC-EV treatment on SARS-CoV-2 and IAV co-infection have not been elucidated. In the present study, we co-induced lung epithelial cells (EpiC) with SARS-CoV-2 Spike protein (S) and H1N1 influenza viral HA protein (HA) and found robust upregulation of inflammatory cytokines in comparison to those induced by either S or HA protein. Consequently, treatment of lung endothelial cells (EC) with conditioned medium from EpiC co-induced by both S and HA proteins resulted in increased apoptosis and impaired angiogenic ability, suggesting the effects of co-induction on epithelial-endothelial crosstalk. In addition, lung EpiC co-induced by both S and HA proteins showed paracrine effects on the recruitment of immune cells, including monocytes, macrophages and neutrophils. Of Note, EV derived from Wharton Jelly's MSC (WJ-EV) transferred miR-146a to recipient lung EpiC, which impaired TRAF6 and IRAK1, resulting in the downregulation of NF-κB pathway and secretion of inflammatory cytokines, rescuing the epithelial-endothelial crosstalk, and reducing the elevation of immune cell recruitment. Moreover, the anti-inflammatory properties of WJ-EV are affected by type 2 Diabetes Mellitus. WJ-EV derived from donors with type 2 Diabetes Mellitus contained less miR-146a and showed impaired ability to downregulate the NF-κB pathway and inflammatory cytokines in recipient cells. Taken together, our findings demonstrate the role of miR-146a in targeting the NF-κB pathway in the anti-inflammatory abilities of WJ-EV, which is a promising strategy to rescue the epithelial-endothelial crosstalk altered by co-infection with SARS-CoV-2 and IAV., (© 2024. The Author(s).)

Introduction: The growing problem of antimicrobial resistance (AMR) poses a significant challenge to public health; This is partly due to the lack of advancements in the development of novel antibiotics and the pressing need for alternative treatment options. Mesenchymal stem cells (MSC) possess secretory components that enhance the immune response and peptides that disrupt the bacteria constitution. The isolation of various human tissues has facilitated the investigation of the diverse potentials of MSC and their components. Further research is needed to fully understand the spectrum and efficacy of these elements and their differences. The primary aim of this study was to perform a thorough review of the current literature related to the antimicrobial properties of MSC and their associated components. The objective was to establish an insight into the results and effects of utilizing MSC in relation to bacterial colonization, and to present an overview of previously documented findings., Methods: This systematic literature review was conducted using the PubMed, Embase, and Web of Science databases. Data on the effect of MSC or their derivatives were measured by calculating the percentage of bacterial counts reduction after treatment with MSC in comparison to the control., Results: A total of 3,911 articles were screened, and 31 eligible publications were selected for inclusion in the analysis. In the current systematic review, the majority of the experimental designs showed positive outcomes in terms of bacterial load reduction when MSC or their derivatives were used, with bone marrow being the most effective tissue. The rest of the findings exhibited heterogeneity in the spectrum of outcomes that could be attributed to the effects of using various tissues derived MSC in both in vivo and in vitro studies., Conclusion: The findings of our study indicate the potential antibacterial characteristics of MSC. The direct antimicrobial activity of these cells was demonstrated by our results, which quantitatively showed a decrease in bacterial growth after treatment with MSC. However, additional research is required to clarify the factors that determine the efficacy of their antimicrobial activity and their various components., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Castro Ramos, Widjaja Lomanto, Vuong, Ohneda and Fukushige.)

Type 2 diabetes mellitus (T2DM) is associated with endothelial dysfunction, which results in delayed wound healing. Mesenchymal stem cells (MSCs) play a vital role in supporting endothelial cells (ECs) and promoting wound healing by paracrine effects through their secretome-containing extracellular vesicles. We previously reported the impaired wound healing ability of adipose tissue-derived MSC from T2DM donors; however, whether extracellular vesicles isolated from T2DM adipose tissue-derived MSCs (dEVs) exhibit altered functions in comparison to those derived from healthy donors (nEVs) is still unclear. In this study, we found that nEVs induced EC survival and angiogenesis, whereas dEVs lost these abilities. In addition, under high glucose conditions, nEV protected ECs from endothelial-mesenchymal transition (EndMT), whereas dEV significantly induced EndMT by activating the transforming growth factor-β/Smad3 signaling pathway, which impaired the tube formation and in vivo wound healing abilities of ECs. Interestingly, the treatment of dEV-internalized ECs with nEVs rescued the induced EndMT effects. Of note, the internalization of nEV into T2DM adipose tissue-derived MSC resulted in the production of an altered n-dEV, which inhibited EndMT and supported the survival of T2DM db/db mice from severe wounds. Taken together, our findings suggest the role of dEV in endothelial dysfunction and delayed wound healing in T2DM by the promotion of EndMT. Moreover, nEV treatment can be considered a promising candidate for cell-free therapy to protect ECs in T2DM.

Introduction: SARS-CoV-2 infection increases the risk of worse outcomes in cancer patients, including those with breast cancer. Our previous study reported that the SARS-CoV-2 membrane protein (M-protein) promotes the malignant transformation of triple-negative breast cancer cells (triple-negative BCC)., Methods: In the present study, the effects of M-protein on the ability of extracellular vesicles (EV) derived from triple-negative BCC to regulate the functions of tissue stem cells facilitating the tumor microenvironment were examined., Results: Our results showed that EV derived from M-protein-induced triple-negative BCC (MpEV) significantly induced the paracrine effects of adipose tissue-derived mesenchymal stem cells (ATMSC) on non-aggressive BCC, promoting the migration, stemness phenotypes, and in vivo metastasis of BCC, which is related to PGE2/IL1 signaling pathways, in comparison to EV derived from normal triple-negative BCC (nEV). In addition to ATMSC, the effects of MpEV on endothelial progenitor cells (EPC), another type of tissue stem cells, were examined. Our data suggested that EPC uptaking MpEV acquired a tumor endothelial cell-like phenotype, with increasing angiogenesis and the ability to support the aggressiveness and metastasis of non-aggressive BCC., Discussion: Taken together, our findings suggest the role of SARS-CoV-2 M-protein in altering the cellular communication between cancer cells and other non-cancer cells inside the tumor microenvironment via EV. Specifically, M-proteins induced the ability of EV derived from triple-negative BCC to promote the functions of non-cancer cells, such as tissue stem cells, in tumorigenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nguyen, Vuong, Fukushige, Usuda, Takagi, Yamashita, Obata-Yasuoka, Hamada, Osaka, Tsukada, Hiramatsu and Ohneda.)

Glucocorticoids are common anti-inflammatory factors; however, they have been reported to have side effects that delay the wound healing process. In a previous study, we found that mesenchymal stem cells isolated from the adipose tissue of patients with long-term glucocorticoid treatment (sAT-MSC) showed impaired wound healing ability due to the downregulation of SDF-1. In this study, we aimed to clarify the mechanisms by which SDF-1 is regulated in sAT-MSC by focusing on the roles of hypoxia-inducible factors (HIFs). Our data suggested that sAT-MSC showed impairment of HIF-1α and the upregulation of HIF-2α. Notably, HIF-2α impairment resulted in the compensatory overexpression of HIF-1α and its target gene SDF-1, which improved the wound healing ability of sAT-MSC. In addition, using knockdown/knockout heterozygous HIF-2α kd/null mice (kd/null), the functions of HIF-2α in the ischemic wound healing process were clarified. With a 50% reduction in the expression of HIF-2α, kd/null mice showed significantly induced wound healing effects, which are involved in the promotion of the inflammatory phase. Specifically, kd/null mice showed the compensatory overexpression of HIF-1α, which upregulated the expression of SDF-1 and enhanced the recruitment of inflammatory cells, such as neutrophils. Our study highlighted the novel function of HIF-2α in the inflammation phase of the wound healing process through the HIF-1α/SDF-1 axis, suggesting that the physiological state of the impaired expression of HIF-2α is a new concept for wound therapy.

Triple-negative breast cancer (TNBC) is a highly aggressive and invasive type of breast cancer. In addition, type 2 diabetes mellitus (T2DM) is recognized as a risk factor for cancer metastasis, which is associated with mortality in patients with breast cancer. Cancer-associated fibroblasts (CAFs) generated from adipose tissue-derived mesenchymal stem cells (AT-MSCs) play a vital role in the progression of TNBC. However, to date, whether T2DM affects the ability of AT-MSCs to differentiate into CAFs is still unclear. In this study, we found that in coculture with TNBC cells [breast cancer cells (BCCs)] under hypoxic conditions, AT-MSCs derived from T2DM donors (dAT-MSCs) were facilitated to differentiate into CAFs, which showed fibroblastic morphology and the induced expression of fibroblastic markers, such as fibroblast activation protein, fibroblast-specific protein, and vimentin. This was involved in the higher expression of transforming growth factor beta receptor 2 (TGFβR2) and the phosphorylation of Smad2/3. Furthermore, T2DM affected the fate and functions of CAFs derived from dAT-MSCs. While CAFs derived from AT-MSCs of healthy donors (AT-CAFs) exhibited the markers of inflammatory CAFs, those derived from dAT-MSCs (dAT-CAFs) showed the markers of myofibroblastic CAFs. Of note, in comparison with AT-CAFs, dAT-CAFs showed a higher ability to induce the proliferation and in vivo metastasis of BCCs, which was involved in the activation of the transforming growth factor beta (TGFβ)-Smad2/3 signaling pathway. Collectively, our study suggests that T2DM contributes to metastasis of BCCs by inducing the myofibroblastic CAFs differentiation of dAT-MSCs. In addition, targeting the TGFβ-Smad2/3 signaling pathway in dAT-MSCs may be useful in cancer therapy for TNBC patients with T2DM.

Introduction: The therapeutic effects of endothelial progenitor cells (EPC) in neovascularization have been suggested; however, to date, few studies have been conducted on the ability of EPC-derived extracellular vesicles (EV) to rescue the ischemic tissues. In order to examine the functional sources of EV for cell-free therapy of ischemic diseases, we compared the functions of EPC-EV and those of Wharton's Jelly-derived mesenchymal stem cell (WJ-EV) in the flap mouse model. Results and conclusion: Our results demonstrated that in the intravenous injection, EPC-EV, but not WJ-EV, were uptaken by the ischemic tissues. However, EPC-EV showed poor abilities to induce neovascularization and the recovery of ischemic tissues. In addition, compared to EPC-EV, WJ-EV showed a higher ability to rescue the ischemic injury when being locally injected into the mice. In order to induce the secretion of high-functional EPC-EV, EPC were internalized with hypoxic pre-treated WJ-EV, which resulted in a transformed hwEPC. In comparison to EPC, hwEPC showed induced proliferation and upregulation of angiogenic genes and miRNAs and promoted angiogenic ability. Interestingly, hwEPC produced a modified EV (hwEPC-EV) that highly expressed miRNAs related to angiogenesis, such as miR-155, miR-183, and miR-296. Moreover, hwEPC-EV significantly induced the neovascularization of the ischemic tissues which were involved in promoting the proliferation, the expression of VEGF and miR-183, and the angiogenic functions of endothelial cells. Of note, hwEPC-EV were highly uptaken by the ischemic tissues and showed a greater effect with regard to inducing recovery from ischemic injury in the intravenous administration, compared to EPC-EV. Therefore, hwEPC-EV can be considered a functional candidate for cell-free therapy to treat the distal ischemic tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ngo, Chang, Vuong, Yamashita, Obata-Yasuoka, Hamada, Osaka, Hiramatsu and Ohneda.)

Triple negative breast cancer (TNBC) is associated with worse outcomes and results in high mortality; therefore, great efforts are required to find effective treatment. In the present study, we suggested a novel strategy to treat TNBC using mesenchymal stem cell (MSC)-derived extracellular vesicles (EV) to transform the behaviors and cellular communication of TNBC cells (BCC) with other non-cancer cells related to tumorigenesis and metastasis. Our data showed that, BCC after being internalized with EV derived from Wharton's Jelly MSC (WJ-EV) showed the impaired proliferation, stemness properties, tumorigenesis and metastasis under hypoxic conditions. Moreover, these inhibitory effects may be involved in the transfer of miRNA-125b from WJ-EV to BCC, which downregulated the expression of HIF1α and target genes related to proliferation, epithelial-mesenchymal transition, and angiogenesis. Of note, WJ-EV-internalized BCC (wBCC) showed transformed behaviors that attenuated the in vivo development and metastatic ability of TNBC, the angiogenic abilities of endothelial cells and endothelial progenitor cells and the generation of cancer-associated fibroblasts from MSC. Furthermore, wBCC generated a new EV with modified functions that contributed to the inhibitory effects on tumorigenesis and metastasis of TNBC. Taken together, our findings suggested that WJ-EV treatment is a promising therapy that results in the generation of wBCC to interrupt the cellular crosstalk in the tumor environment and inhibit the tumor progression in TNBC., (© 2022. The Author(s).)

Coronavirus disease 2019 (COVID-19) has spread faster due to the emergence of SARS-CoV-2 variants, which carry an increased risk of infecting patients with comorbidities, such as breast cancer. However, there are still few reports on the effects of SARS-CoV-2 infection on the progression of breast cancer, as well as the factors and mechanisms involved. In the present study, we investigated the impact of SARS-CoV-2 proteins on breast cancer cells (BCC). The results suggested that SARS-CoV-2 M protein induced the mobility, proliferation, stemness and in vivo metastasis of a triple-negative breast cancer (TNBC) cell line, MDA-MB-231, which are involved in the upregulation of NFκB and STAT3 pathways. In addition, compared to MDA-MB-231 cells, the hormone-dependent breast cancer cell line MCF-7 showed a less response to M protein, with the protein showing no effects of promoting proliferation, stemness, and in vivo metastasis. Of note, coculture with M protein-treated MDA-MB-231 cells significantly induced the migration, proliferation, and stemness of MCF-7 cells, which are involved in the upregulation of genes related to EMT and inflammatory cytokines. Therefore, SARS-CoV-2 infection might promote the ability of aggressive BCC to induce the malignant phenotypes of the other non-aggressive BCC. Taken together, these findings suggested an increased risk of poor outcomes in TNBC patients with a history of SARS-CoV-2 infection, which required a long-term follow-up. In addition, the inhibition of NFκB and STAT3 signaling pathways is considered as a promising candidate for the treatment of worsen clinical outcomes in TNBC patients with COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nguyen, Kawahara, Vuong, Fukushige, Yamashita and Ohneda.)

Cytokine storm is recognized as one of the factors contributing to organ failures and mortality in patients with COVID-19. Due to chronic inflammation, COVID-19 patients with diabetes mellitus (DM) or renal disease (RD) have more severe symptoms and higher mortality. However, the factors that contribute to severe outcomes of COVID-19 patients with DM and RD have received little attention. In an effort to investigate potential treatments for COVID-19, recent research has focused on the immunomodulation functions of mesenchymal stem cells (MSCs). In this study, the correlation between DM and RD and the severity of COVID-19 was examined by a combined approach with a meta-analysis and experimental research. The results of a systematic review and meta-analysis suggested that the odd of mortality in patients with both DM and RD was increased in comparison to those with a single comorbidity. In addition, in the experimental research, the data showed that high glucose and uremic toxins contributed to the induction of cytokine storm in human lung adenocarcinoma epithelial cells (Calu-3 cells) in response to SARS-CoV Peptide Pools. Of note, the incorporation of Wharton's jelly MSC-derived extracellular vesicles (WJ-EVs) into SARS-CoV peptide-induced Calu-3 resulted in a significant decrease in nuclear NF-κB p65 and the downregulation of the cytokine storm under high concentrations of glucose and uremic toxins. This clearly suggests the potential for WJ-EVs to reduce cytokine storm reactions in patients with both chronic inflammation diseases and viral infection.

The outbreak of the COVID-19 pandemic has had an unprecedented impact on humanity as well as research activities in life sciences and medicine. Between January and August 2020, the number of coronavirus-related scientific articles was roughly 50 times more than that of articles published in the entire year of 2019 in PubMed. It is necessary to better understand the dynamics of research on COVID-19, an emerging topic, and suggest ways to understand and improve the quality of research. We analyze the dynamics of coronavirus research before and after the outbreaks of SARS, MERS, and COVID-19 by examining all the published articles from the past 25 years in PubMed. We delineate research networks on coronaviruses as we identify experts' background in terms of topics of previous research, affiliations, and international co-authorships. Two distinct dynamics of coronavirus research were found: 1) in the cases of regional pandemics, SARS and MERS, the scope of cross-disciplinary research remained between neighboring research areas; 2) in the case of the global pandemic, COVID-19, research activities have spread beyond neighboring disciplines with little transnational collaboration. Thus, COVID-19 has transformed the structure of research on coronaviruses as an emerging issue. Knowledge on COVID-19 is distributed across the widest range of disciplines, transforming research networks well beyond the field of medicine but within national boundaries. Given the unprecedented scale of COVID-19 and the nationalization of responses, the most likely way forward is to accumulate local knowledge with the awareness of transdisciplinary research dynamics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ohniwa, Kijima, Fukushige and Ohneda.)

Cancer metastasis is the leading cause of mortality among breast cancer patients. Type 2 diabetes mellitus (T2DM) has been suggested as a risk factor of breast cancer; however, whether or not T2DM is associated with breast tumor metastasis remains unclear. In this study, we examined the involvement of T2DM with breast cancer metastasis by a combined approach of a meta-analysis and experimental research. The results of a systematic review and meta-analysis suggested that diabetes significantly increases the risk of lymph node metastasis by 1.10-fold ( P  < 0.01). Consistently, our data from experimental research showed that T2DM induced paracrine effects of mesenchymal stem cells (MSCs), a key contributor to cancer progression, to stimulate metastasis of breast cancer cells (BCCs) by two independent mechanisms. First, T2DM induced the excess secretion of interleukin 6 (IL6) from MSCs, which activated the JAK/STAT3 pathway in BCCs, thus promoting the metastasis of BCCs. Second, beside the EGR-1-/IL6-dependent mechanism, T2DM altered the functions of MSC-derived extracellular vesicles (EVs), which are highly associated with the metastasis of BCCs. Our present study showed that T2DM is a risk factor for breast cancer metastasis, and MSC-derived EVs might be useful for developing a novel anti-breast cancer therapy strategy.

Aging induces numerous cellular disorders, such as the elevation of reactive oxygen species (ROS), in a number type of cells, including mesenchymal stem cells (MSCs). However, the correlation of ROS and impaired healing abilities as well as whether or not the inhibition of elevating ROS results in the rejuvenation of elderly MSCs is unclear. The rejuvenation of aged MSCs has thus recently received attention in the field of regenerative medicine. Specifically, extracellular vesicles (EVs) act as a novel tool for stem cell rejuvenation due to their gene transfer ability with systemic effects and safety. In the present study, we examined the roles of aging-associated ROS in the function and rejuvenation of elderly MSCs by infant EVs. The data clearly showed that elderly MSCs exhibited the downregulation of superoxide dismutase (SOD)1 and SOD3, which resulted in the elevation of ROS and downregulation of the MEK/ERK pathways, which are involved in the impairment of the MSCs' ability to decrease necrotic area in the skin flap model. Furthermore, treatment with the antioxidant Edaravone or co-overexpression of SOD1 and SOD3 rescued elderly MSCs from the elevation of ROS and cellular senescence, thereby improving their functions. Of note, infant MSC-derived EVs rejuvenated elderly MSCs by inhibiting ROS production and the acceleration of cellular senescence and promoting the proliferation and in vivo functions in both type 1 and type 2 diabetic mice.

Embryonic stem (ES) cells have been utilized as an excellent model for the study of neural development. However, the dynamic changes of ES cell-derived neural stem cells (ES-NSCs), under the effects of prolonged cell culture and hypoxic conditions, are still obscured. In the present study, using the combination of serum-free culture of embryoid body-like aggregates (SFEB) culture and cell sorting by Sox-1, the ES-NSCs were easily isolated and showed in vitro temporal neural specification, which resulted in distinct cell fates after neural differentiation. Early passaged ES-NSCs gave rise to neurons, whereas late-passaged ES-NSCs gave rise to glial cells, similar to the in vivo dynamic changes during the neural development. Remarkably, hypoxia treatment induced the neural differentiation of ES-NSCs but did not affect the cell fate. Under hypoxic conditions, early passaged ES-NSCs showed the upregulation of neuronal markers, whereas late-passaged ES-NSCs showed the upregulation of a glial marker. In addition, the knockdown of the hypoxia-inducible factor 1α expression impaired the neuronal differentiation of early passaged ES-NSCs under hypoxic conditions. These data demonstrated the distinct effects of prolonged culture and hypoxic stimuli on the neural differentiation of ES-NSCs; prolonged culture was involved in the cell fate after neural differentiation, while hypoxia treatment efficiently promoted neural differentiation.

Cancer stem cells (CSCs) are a subpopulation of cancer cells responsible for tumor maintenance and relapse due to their ability to resist various anticancer effects. Owing to the resistance of CSCs to the effects of targeted therapy, an alternative strategy that targets post-translational glycosylation may be an improved approach to treat cancer as it disrupts multiple coordinated signaling that maintains the stemness of CSCs. Glucosamine acts as an anticancer agent possibly by inhibiting N-linked glycosylation. The aim of the present study was to investigate the effect of glucosamine on the stemness of breast CSCs, which is regulated by signal transducer and activator of transcription 3 (STAT3) signaling. Human aldehyde dehydrogenase-positive (ALDH + ) breast CSCs and MCF7 cells were treated with various concentrations (0.25, 1 or 4 mM) of glucosamine for 24 h. Subsequently, cell viability was determined by performing a trypan blue exclusion assay, pluripotency gene [ALDH 1 family member A1 ( ALDH1A1 ), octamer-binding transcription factor 4 ( OCT-4 ), and Krüppel-like factor 4 ( KLF4 )] expression was determined using the reverse transcription-quantitative polymerase chain reaction, and STAT3 and phosphorylated STAT3 (pSTAT3) levels were determined by performing western blot analysis. Furthermore, the number of mammosphere-forming units (MFUs) in ALDH + breast CSCs and MCF7 cells was determined. It was determined that glucosamine treatment decreased the viability of ALDH + breast CSCs. Glucosamine treatment also decreased the stemness of ALDH + breast CSCs and MCF7 cells, as indicated by decreased ALDH1A1, OCT-4 and KLF4 expression level, and a decreased number of MFUs. This effect of glucosamine may be associated with a decreased pSTAT3/STAT3 ratio, indicating that glucosamine inhibited STAT3 activation; therefore, the results of the present study indicated that glucosamine treatment may be an improved approach to target the stemness of CSCs.

Background: Endothelial progenitor cells (EPCs) can be used to treat ischemic disease in cell-based therapy owing to their neovascularization potential. Glucocorticoids (GCs) have been widely used as strong anti-inflammatory reagents. However, despite their beneficial effects, side effects, such as impairing wound healing are commonly reported with GC-based therapy, and the effects of GC therapy on the wound healing function of EPCs are unclear. Methods: In this study, we investigated how GC treatment affects the characteristics and wound healing function of EPCs. Results: We found that GC treatment reduced the proliferative ability of EPCs. In addition, the expression of CXCR4 was dramatically impaired, which suppressed the migration of EPCs. A transplantation study in a flap mouse model revealed that GC-treated EPCs showed a poor homing ability to injured sites and a low activity for recruiting inflammatory cells, which led to wound healing dysfunction. Impairment of prostaglandin E2 (PGE2) synthases, cyclooxygenase (COX2) and microsomal PGE2 synthase 1 (mPEGS1) were identified as being involved in the GC-induced impairment of the CXCR4 expression in EPCs. Treatment with PGE2 rescued the expression of CXCR4 and restored the migration ability of GC-treated EPCs. In addition, the PGE2 signal that activated the PI3K/AKT pathway was identified to be involved in the regulation of CXCR4 in EPCs under the effects of GCs. In addition, similar negative effects of GCs were observed in EPCs under hypoxic conditions. Under hypoxic conditions, GCs independently impaired the PGE2 and HIF2α pathways, which downregulated the expression of CXCR4 in EPCs. Our findings highlighted the influences of GCs on the characteristics and functions of EPCs, suggesting that the use of EPCs for autologous cell transplantation in patients who have used GCs for a long time should be considered carefully.

Objective: Earlier studies from our group using hypoxia-inducible factor 3α knockout mice showed impairments in lung remodeling and lung endothelial cells. Another research from our group demonstrated that impaired expression of hypoxia-inducible factor 2α induced compensatory expression of hypoxia-inducible factor 1α in hypoxia-inducible factor 2α knockdown mice. The present study uncovers more insights by extending the investigation, utilizing mice with both hypoxia-inducible factor 3α knockout and hypoxia-inducible factor 2α knockdown., Results: No mice with both hypoxia-inducible factor 3α knockout and hypoxia-inducible factor 2α knockdown died immediately after birth. The mice with both hypoxia-inducible factor 3α knockout and hypoxia-inducible factor 2α knockdown exhibited impaired alveolar sacs and lung alveolar structure and decreased endothelial cell numbers. Analysis of relative mRNA expression revealed depressed expressions of hypoxia-inducible factor 1α, vascular cell adhesion molecule 1, vascular endothelial cadherin, angiopoietin 2, Tie-2, and vascular endothelial growth factor in the lungs of mice with both hypoxia-inducible factor 3α knockout and hypoxia-inducible factor 2α knockdown compared to that in wild-type mice. Further analysis is needed to elucidate the impaired development occurred in the lung endothelial cells.

In the body, different types of adipose tissue perform different functions, with brown and beige adipose tissues playing unique roles in dissipating energy. Throughout life, adipocytes are regenerated from progenitors, and this process is impaired by aging. One of the progenitors of adipocytes are mesenchymal stem cells (MSCs), which have recently become a promising tool for stem cell therapy. However, whether or not aging impairs the brown/beige adipocyte differentiation of adipose tissue-derived MSCs (AT-MSCs) remains unclear. In the present study, we isolated AT-MSCs from two different age groups of donors (infants and elderly subjects) and examined the effects of aging on the AT-MSC brown/beige adipocyte differentiation ability. We found that none of the AT-MSCs expressed Myf5, which indicated the beige (not brown) differentiation ability of cells. Of note, an inverse correlation was noted between the beige adipocyte differentiation ability and age, with AT-MSCs derived from elderly donors showed the most severely reduced function due to induced cellular senescence. The impaired expression of Sirtuin 1 (Sirt1) and Sirt3 proved to be responsible for the induction of senescence in elderly AT-MSCs; however, only Sirt1 was directly involved in the regulation of beige adipocyte differentiation. The overexpression of Sirt1 impaired the p53/p21 pathway, thereby preventing elderly AT-MSCs from entering senescence and restoring the beige differentiation ability. Thus, our study represents the important role of Sirt1 and senescence in the regulation of beige adipocyte differentiation during aging., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The purpose of this study was to quantify the stem cell and growth factor (GF) contents in the bone marrow aspirate concentrate (BMAC) and platelet-rich plasma (PRP) prepared from whole blood using a protocol established in our laboratory. We examined 10 patients with osteonecrosis of the femoral head who were treated by autologous BMAC transplantation at our hospital between January 2015 and June 2015. We quantified CD34+ and CD31-CD45-CD90+CD105+ cells in BMAC and PRP by flow cytometry. Additionally, we measured various GFs, that is, basic fibroblast growth factor (b-FGF), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), transforming growth factor- β 1 (TGF- β 1), and bone morphogenetic protein-2 (BMP-2) in BMAC and PRP using enzyme-linked immunosorbent assays and statistical analyses. CD34+ and CD31-45-90+105+ cells accounted for approximately 1.9% and 0.03% of cells in BMAC and no cells in PRP. The concentration of b-FGF was higher in BMAC than in PRP ( P < 0.001), whereas no significant differences in the levels of PDGF-BB, VEGF, TGF- β 1, and BMP-2 were observed between the two types of sample. BMAC had an average of 1.9% CD34+ and 0.03% CD31-45-90+105+ cells and higher levels of b-FGF than those of PRP.

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP + MSCs in various organs. In vivo, EGFP + mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP + cells exhibited a perivascular distribution pattern. EGFP + cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP + specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP + population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glucocorticoids are steroid hormones used as anti-inflammatory treatments. However, this strong immunomodulation causes undesirable side effects that impair bones, such as osteoporosis. Glucocorticoid therapy is a major risk factor for developing steroid-induced osteonecrosis of the femur head (ONFH). Since ONFH is incurable, therapy with mesenchymal stem cells (MSCs) that can differentiate into osteoblasts are a first-line choice. Bone marrow-derived MSCs (BM-MSCs) are often used as a source of stem cell therapy for ONFH, but their proliferative activity is impaired after steroid treatment. Adipose tissue-derived MSCs (AT-MSCs) may be an attractive alternative source; however, it is unknown whether AT-MSCs from steroid-induced ONFH (sAT-MSCs) have the same differentiation ability as BM-MSCs or normal AT-MSCs (nAT-MSCs). In this study, we demonstrate that nAT-MSCs chronically exposed to glucocorticoids show lower alkaline phosphatase activity leading to reduced osteogenic differentiation ability. This impaired osteogenesis is mediated by high expression of Dickkopf1 (Dkk-1) that inhibits wnt/β-catenin signaling. Increased Dkk-1 also causes impaired osteogenesis along with reductions in bone regenerative capacity in sAT-MSCs. Of note, plasma Dkk-1 levels are elevated in steroid-induced ONFH patients. Collectively, our findings suggest that glucocorticoid-induced expression of Dkk-1 could be a key factor in modulating the differentiation ability of MSCs used for ONFH and other stem cell therapies.

Glucocorticoids cause the delayed wound healing by suppressing inflammation that is required for wound healing process. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) play an important role for wound healing by their cytokine productions including stromal derived factor 1 (SDF-1). However, it has not been clear how glucocorticoids affect the wound healing ability of AT-MSCs. In this study, we found that glucocorticoid downregulated SDF-1 expression in AT-MSCs. In addition, glucocorticoid-treated AT-MSCs induced less migration of inflammatory cells and impaired wound healing capacity compared with glucocorticoid-untreated AT-MSCs. Of note, prostaglandin E2 (PGE2) synthesis-related gene expression was downregulated by glucocorticoid and PGE2 treatment rescued not only SDF-1 expression in the presence of glucocorticoid but also their wound healing capacity in vivo. Furthermore, we found SDF-1-overexpressed AT-MSCs restored wound healing capacity even after treatment of glucocorticoid. Consistent with the results obtained from glucocorticoid-treated AT-MSCs, we found that AT-MSCs isolated from steroidal osteonecrosis donors (sAT-MSCs) who received chronic glucocorticoid therapy showed less SDF-1 expression and impaired wound healing capacity compared with traumatic osteonecrosis donor-derived AT-MSCs (nAT-MSCs). Moreover, the SDF-1 level was also reduced in plasma derived from steroidal osteonecrosis donors compared with traumatic osteonecrosis donors. These results provide the evidence that concomitant application of AT-MSCs with glucocorticoid shows impaired biological modulatory effects that induce impaired wound healing., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chronic kidney disease (CKD) results in a delay in wound healing because of its complications such as uremia, anemia, and fluid overload. Mesenchymal stem cells (MSCs) are considered to be a candidate for wound healing because of the ability to recruit many types of cells. However, it is still unclear whether the CKD-adipose tissue-derived MSCs (CKD-AT-MSCs) have the same function in wound healing as healthy donor-derived normal AT-MSCs (nAT-MSCs). In this study, we found that uremic toxins induced elevated reactive oxygen species (ROS) expression in nAT-MSCs, resulting in the reduced expression of hypoxia-inducible factor-1α (HIF-1α) under hypoxic conditions. Consistent with the uremic-treated AT-MSCs, there was a definite imbalance of redox state and high expression of ROS in CKD-AT-MSCs isolated from early-stage CKD patients. In addition, a transplantation study clearly revealed that nAT-MSCs promoted the recruitment of inflammatory cells and recovery from ischemia in the mouse flap model, whereas CKD-AT-MSCs had defective functions and the wound healing process was delayed. Of note, the expression of prolyl hydroxylase domain 2 (PHD2) is selectively increased in CKD-AT-MSCs and its inhibition can restore the expression of HIF-1α and the wound healing function of CKD-AT-MSCs. These results indicate that more studies about the functions of MSCs from CKD patients are required before they can be applied in the clinical setting.

Endothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell subtypes continually grouped under the term "EPC." It would be highly advantageous to agree on standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping, potency assays, and clear separation from hematopoietic angiogenic cells which are not endothelial progenitors. In this review, we seek to discourage the indiscriminate use of "EPCs," and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells and myeloid angiogenic cells are examples of two distinct and well-defined cell types that have been considered EPCs because they both promote vascular repair, albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing "EPC" nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognized for their role in vascular repair in health and disease and, in some cases, progress toward use in cell therapy. Stem Cells Translational Medicine 2017;6:1316-1320., (© 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Stable breast cancer cell (BCC) lines are valuable tools for the identification of breast cancer stem cell (BCSC) phenotypes that develop in response to several stimuli as well as for studying the basic mechanisms associated with the initiation and maintenance of BCSCs. However, the characteristics of individual, BCC-derived BCSCs varies and these cells show distinct phenotypes depending on the different BCSC markers used for their isolation. Aldehyde dehydrogenase (ALDH) activity is just such a recognized biomarker of BCSCs with a CD44 + /CD24 - phenotype. We isolated BCSCs with high ALDH activity (CD44 + /CD24 - /Aldefluor pos ) from a primary culture of human breast cancer tissue and observed that the cells had stem cell properties compared to BCSCs with no ALDH activity (CD44 + /CD24 - /Aldefluor neg ). Moreover, we found Aldefluor pos BCSCs had a greater hypoxic response and subsequent induction of HIF-1α expression compared to the Aldefluor neg BCSCs. We also found that knocking down HIF-1α, but not HIF-2α, in Aldefluor pos BCSCs led to a significant reduction of the stem cell properties through a decrease in the mRNA levels of genes associated with the epithelial-mesenchymal transition. Indeed, HIF-1α overexpression in Aldefluor neg BCSCs led to Slug and Snail mRNA increase and the associated repression of E-cadherin and increase in Vimentin. Of note, prolonged hypoxic stimulation promoted the phenotypic changes of Aldefluor neg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Purpose: Retinal hypoxia plays a crucial role in ocular neovascular diseases, such as diabetic retinopathy, retinopathy of prematurity, and retinal vascular occlusion. Fluorescein angiography is useful for identifying the hypoxia extent by detecting non-perfusion areas or neovascularization, but its ability to detect early stages of hypoxia is limited. Recently, in vivo fluorescent probes for detecting hypoxia have been developed; however, these have not been extensively applied in ophthalmology. We evaluated whether a novel donor-excited photo-induced electron transfer (d-PeT) system based on an activatable hypoxia-selective near-infrared fluorescent (NIRF) probe (GPU-327) responds to both mild and severe hypoxia in various ocular ischemic diseases animal models., Methods: The ocular fundus examination offers unique opportunities for direct observation of the retina through the transparent cornea and lens. After injection of GPU-327 in various ocular hypoxic diseases of mouse and rabbit models, NIRF imaging in the ocular fundus can be performed noninvasively and easily by using commercially available fundus cameras. To investigate the safety of GPU-327, electroretinograms were also recorded after GPU-327 and PBS injection., Result: Fluorescence of GPU-327 increased under mild hypoxic conditions in vitro. GPU-327 also yielded excellent signal-to-noise ratio without washing out in vivo experiments. By using near-infrared region, GPU-327 enables imaging of deeper ischemia, such as choroidal circulation. Additionally, from an electroretinogram, GPU-327 did not cause neurotoxicity., Conclusions: GPU-327 identified hypoxic area both in vivo and in vitro.

Mesenchymal stem cells (MSCs) are defined as multipotent cells that can give rise to various kinds of differentiated mesenchymal cells, and are thus considered to be useful for clinical therapy. However, the big hurdles of MSC therapy are the inability of MSCs to reach the appropriate tissues or sites with high efficiency and engraftment after transplantation. In this study, we investigated how adipose tissue-derived MSCs (AT-MSCs) improve their homing ability after intravenous injection. We previously found that human endothelial progenitor cells with low aldehyde dehydrogenase activity (Alde-Low EPCs) are suitable for the treatment of ischemic tissues. In addition, we demonstrated that microvesicles (MVs) derived from Alde-Low EPCs possessed the ability to improve the homing ability of non-functional Alde-High EPCs, resulting in wound healing. We initially transfected MVs derived from Alde-Low EPCs (EMVs) to human AT-MSCs, which were originally unable to cure ischemic tissues by intravenous transplantation. Remarkably, AT-MSC transfected EMVs dramatically repaired the ischemic skin flap compared with AT-MSC derived-MV (MMVs) transfected AT-MSCs or control AT-MSCs. We then found that the expression of CXCR4, an important chemokine receptor for cell migration, was highly elevated in EMV-transfected AT-MSCs. Moreover, AT-MSCs transfected with EMVs, but not control AT-MSCs, migrated to wound sites after intravenous injection. Consequently, CD45(+) inflammatory cells were successfully recruited at the wound sites after the injection of EMV-transfected AT-MSCs. These results demonstrate that EMVs are a useful source to improve the homing ability and wound healing ability of MSCs at the wound sites., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The prevalence of type 2 diabetes mellitus (T2DM), which leads to diabetic complications, has been increasing worldwide. The possible applications of T2DM-derived stem cells in cell therapy are limited because their characteristics are still not fully understood. In this study, we characterized adipose tissue-derived mesenchymal stem cells (AT-MSCs) from diabetic patients (dAT-MSCs) and found that insulin receptor substrate-1 (IRS-1) was highly phosphorylated at serine 636/639 in dAT-MSCs. Moreover, we found that early growth response factor-1 (EGR-1) and its target genes of PTEN and GGPS1 were highly expressed in dAT-MSCs in comparison to healthy donor-derived AT-MSCs (nAT-MSCs). We observed impaired wound healing after the injection of dAT-MSCs in the ischemic flap mouse model. The expressions of EGR-1 and its target genes were diminished by small hairpin RNA-targeted EGR-1 (shEGR-1) and treatment with a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) inhibitor (PD98059). Importantly, dAT-MSCs with shEGR-1 were able to restore the wound healing ability in the mouse model. Interestingly, under hypoxic conditions, hypoxia-inducible factor-1α (HIF-1α) can bind to the EGR-1 promoter in dAT-MSCs, but not in nAT-MSCs. Together, these results demonstrate that the expression of EGR-1 was upregulated in dAT-MSCs through two pathways: the main regulatory pathway is the MAPK/ERK pathway, the other is mediated by HIF-1α through direct transcriptional activation at the promoter region of the EGR1 gene. Our study suggests that dAT-MSCs may contribute to microvascular damage and delay wound healing through the overexpression of EGR-1. Interrupting the expression of EGR-1 in dAT-MSCs may be a useful treatment for chronic wounds in diabetic patients.

Microvesicles (MVs) derived from mesenchymal stem cells showed the ability to alter the cell phenotype and function. We previously demonstrated that type 2 diabetic adipose tissue-derived mesenchymal stem cells (dAT-MSCs) increase in cell aggregation and adhesion in vitro and impair wound healing in vivo. However, the characterization and function of MVs derived from human non-diabetic AT-MSCs (nAT-MSCs) remain unknown. In this study, we characterized nAT-MSC-derived MVs and their function after the transfection of dAT-MSCs with MVs using the scratch assay and a flap mouse model. We found that human nAT-MSC-derived MVs expressed MSC-surface markers and improved dAT-MSC functions by altering the expression of genes associated with cell migration, survival, inflammation, and angiogenesis as well as miR29c and miR150. Remarkably, the transfection of dAT-MSCs with nAT-MSC-derived MVs improved their migration ability in vitro and wound healing ability in a flap mouse model. These results demonstrate a promising opportunity to modify the function of dAT-MSCs for therapeutic stem cell application in diabetic patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The expression of estrogen receptor is the key in most breast cancers (BC) and binding of estrogen receptor to the genome correlates to Forkhead protein (FOXA1) expression. We herein assessed the correlation between the cancer stem cell (CSC) population and FOXA1 expression in luminal BC. We established luminal BC cells derived from metastatic pleural effusion and analyzed the potency of CSC and related factors with established luminal BC cell lines. We also confirmed that mammosphere cultures have an increased aldehyde dehydrogenase-positive population, which is one of the CSC markers, compared with adherent culture cells. Using a quantitative PCR analysis, we found that mammosphere forming cells showed a higher expression of FOXA1 and stemness-related genes compared with adherent culture cells. Furthermore, the growth activity and colony-forming activity of 4-hydroxytamoxifen-treated BC cells were inhibited in a mammosphere assay. Interestingly, 4-hydroxytamoxifen-resistant cells had significantly increased FOXA1 gene expression levels. Finally, we established short hairpin RNA of FOXA1 (shFOXA1) MCF-7 cells and investigated the relationship between self-renewal potential and FOXA1 expression. As a result, we found no significant difference in the number of mammospheres but decreased colony formation in shFOXA1 MCF-7 cells compared with control. These results suggest that the expression of FOXA1 appears to be involved in the proliferation of immature BC cells rather than the induction of stemness-related genes and self-renewal potency of CSCs., (© 2015 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Endothelial progenitor cells (EPCs) have the ability to form new blood vessels and protect ischemic tissues from damage. We previously reported that EPCs with low activity of aldehyde dehydrogenase (Alde-Low EPCs) possess the greater ability to treat ischemic tissues compared with Alde-High EPCs. The expression level of the hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, was found to be greater in Alde-Low EPCs than in Alde-High EPCs. However, the precise role of the HIF factors in the regulation of EPC activity remains obscure. In this study, we demonstrate a critical role of HIF-2α and its target gene CXCR4 for controlling the migratory activity of EPC to ischemic tissue. We found that coculture of Alde-High EPCs with microvesicles derived from Alde-Low EPCs improved their ability to repair an ischemic skin flap, and the expression of CXCR4 and its ligand SDF1 was significantly increased following the coculture. In Alde-Low EPCs, the expression of CXCR4 was suppressed by short hairpin RNA (shRNA)-mediated HIF-2α, but not HIF-1α downregulation. Chromatin immunoprecipitation assays showed that HIF-2α, but not HIF-1α, binds to the promoter region of CXCR4 gene. The CXCR4 shRNA treatment in Alde-Low EPCs almost completely abrogated their migratory activity to ischemic tissues, whereas the reduction of vascular endothelial growth factor (VEGF) showed much less effect. The CXCR4 overexpression in Alde-High EPCs resulted in a partial, but significant improvement in their repairing ability in an ischemic skin flap. Collectively, these findings indicate that the CXCR4/SDF-1 axis, which is specifically regulated by HIF-2α, plays a crucial role in the regulation of EPC migration to ischemic tissues.

Erythropoiesis is strongly influenced by the interactions between stromal cells and erythroid progenitors, as well as by a key regulatory factor, erythropoietin (EPO). We previously generated mice with a knockdown mutation of Hif-2α (referred to as kd/kd) and found that these kd/kd mice exhibited normocytic anemia, even though the EPO expression was not severely affected. However, the VCAM-1 expression in spleen endothelial cells (EC), which is regulated by HIF-2α, was impaired, resulting in defective erythroid maturation. A deficiency of HIF-2α clearly led to pancytopenia. However, the critical level of HIF-2α required for erythropoiesis has not yet been elucidated. In this study, we generated HIF-2α knockdown/knockout heterozygous mice (kd/null). Strikingly, anemia was observed in the kd/null mice, but the red blood cell indices were significantly improved compared to those of kd/kd mice. In the spleens of kd/null mice, higher HIF-1α activity and expansion of the red pulp area were observed compared to those of kd/kd mice. Importantly, EC isolated from kd/null spleens showed high expression of VEGF receptors, FLK-1 and FLT-1, which are regulated by HIF-1α instead of HIF-2α under hypoxic conditions. We also found higher expression of phosphorylated ERK and higher proliferative activity in the EC isolated from kd/null mice compared to those from kd/kd mice. While the HIF-2α expression was diminished, HIF-1α bound to the HRE region in the promoters of genes that are normally regulated by HIF-2α. These results suggest that there is a compensatory pathway involving HIF-1α that regulates the expression of some HIF-2α target genes., (© 2015 Wiley Periodicals, Inc.)

GATA2 plays a crucial role for the mast cell fate decision. We herein demonstrate that GATA2 is also required for the maintenance of the cellular identity in committed mast cells derived from mouse bone marrow (BMMCs). The deletion of the GATA2 DNA binding domain (GATA2ΔCF) in BMMCs resulted in a loss of the mast cell phenotype and an increase in the number of CD11b- and/or Ly6G/C-positive cells. These cells showed the ability to differentiate into macrophage- and neutrophil-like cells but not into eosinophils. Although the mRNA levels of basophil-specific genes were elevated, CD49b, a representative basophil marker, never appeared on these cells. GATA2 ablation led to a significant upregulation of C/EBPα, and forced expression of C/EBPα in wild-type BMMCs phenocopied the GATA2ΔCF cells. Interestingly, simultaneous deletion of the Gata2 and Cebpa genes in BMMCs restored the aberrant increases of CD11b and Ly6G/C while retaining the reduced c-Kit expression. Chromatin immunoprecipitation assays indicated that GATA2 directly binds to the +37-kb region of the Cebpa gene and thereby inhibits the RUNX1 and PU.1 binding to the neighboring region. Upregulation of C/EBPα following the loss of GATA2 was not observed in cultured mast cells derived from peritoneal fluid, whereas the repression of c-Kit and other mast cell-specific genes were observed in these cells. Collectively, these results indicate that GATA2 maintains cellular identity by preventing Cebpa gene activation in a subpopulation of mast cells, whereas it plays a fundamental role as a positive regulator of mast cell-specific genes throughout development of this cell lineage., (© 2015 by The American Society of Hematology.)

Cell therapy using human mesenchymal stem cells (MSCs) is an attractive approach for many refractory diseases. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are considered as a favorable tool due to its abundance in the body, easy proliferation, and high cytokine production potency. In order to avoid the risks associated with the use of fetal bovine serum (FBS) in culture that includes batch variations and contamination with pathogens, development of serum-free culture system has been initiated. We have formulated a completely serum-free culture medium (SFM) that could be used not only for the expansion of AT-MSCs but also for initial isolation. We demonstrate that the AT-MSCs isolated and cultured in serum-free medium (AT-MSCs/SFM) possess high proliferation capacity and differentiation potency to osteoblast, adipocyte, and chondrocyte lineages in vitro. In in vivo bone fraction model analysis, AT-MSCs/SFM showed higher bone repair potency and quality of the regenerated bone than the cells cultured in serum-containing medium (AT-MSCs/SCM). This was attributed to the (i) presence of translated cells in the bone, as evidenced by in vivo imaging of the illuminated translated cells and (ii) high level of expression and induction capacity of AT-MSCs/SFM for cytokine BMP2, CCL2, and CCL5. Taken together, we report a new serum-free culture system for AT-MSCs that is suitable for cell therapy.

The variants of the hypoxia-inducible factor-3α gene HIF-3α and NEPAS are known to repress the transcriptional activities driven by HIF-1α and HIF-2α. Although NEPAS has been shown to play an important role in vascular remodeling during lung development, little is known about the roles of HIF-3α in adult lung function. Here, we examined pulmonary endothelial cells (ECs) isolated from wild-type (WT) and HIF-3α functional knockout (KO) mice. The expression levels of angiogenic factors (Flk1, Ang2 and Tie2) were significantly greater in the HIF-3α KO ECs than those in the WT ECs irrespective of oxygen tension. However, the HIF-3α KO ECs showed impaired proliferative and angiogenic activities. The impaired EC function was likely due to the excess vascular endothelial (VE)-cadherin, an inhibitor of Flk1/PI3 kinase/Akt signaling, as treatment of the cells to a neutralizing antibody partly restored the phenotype of the HIF-3α KO ECs. Importantly, we found that the mRNA levels of HIF-2α and Ets-1 were significantly increased by HIF-3α ablation. Given that both factors are known to activate the VE-cadherin gene, the transcriptional repression of these factors by HIF-3α might be important for silencing the irrelevant expression of the VE-cadherin gene. Collectively, these data show novel and unique roles of HIF-3α for angiogenic gene regulation in pulmonary ECs., (© 2015 The Authors Genes to Cells © 2015 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)

Embryonic stem (ES) cells are useful for elucidating the molecular mechanisms of cell fate decision in the early development of mammals. It has been shown that aggregate culture of ES cells efficiently induces neuroectoderm differentiation. However, the molecular mechanism that leads to selective neural differentiation in aggregate culture is not fully understood. Here, we demonstrate that the oxygen-sensitive hypoxia-inducible transcription factor, Hif-1α, is an essential regulator for neural commitment of ES cells. We found that a hypoxic environment is spontaneously established in differentiating ES cell aggregates within 3 days, and that this time window coincides with Hif-1α activation. In ES cells in adherent culture under hypoxic conditions, Hif-1α activation was correlated with significantly greater expression of neural progenitor-specific gene Sox1 compared with ES cells in adherent culture under normoxic conditions. In contrast, Hif-1α-depleted ES cell aggregates showed severe reduction in Sox1 expression and maintained high expression of undifferentiated ES cell marker genes and epiblast marker gene Fgf5 on day 4. Notably, chromatin immune precipitation assay and luciferase assay showed that Hif-1α might directly activate Sox1 expression. Of additional importance is our finding that attenuation of Hif-1α resulted in an increase of BMP4, a potent inhibitor of neural differentiation, and led to a high level of phosphorylated Smad1. Thus, our results indicate that Hif-1α acts as a positive regulator of neural commitment by promoting the transition of ES cell differentiation from the epiblast into the neuroectoderm state via direct activation of Sox1 expression and suppressing endogenous BMP signaling.

Mesenchymal stem cells (MSC) are multipotent and possess high proliferative activity, and thus are thought to be a reliable cell source for cell therapies. Here, we isolated MSC from adult tissues--bone marrow (BM-MSC), dental tissue (DT-MSC), and adipose tissue (AT-MSC)--to compare how autotransplantation of these MSC effectively supports the repair of bone fracture and ischemic tissue. An analysis by in vitro differentiation assays showed no significant difference among these MSC. The degree of calcification at the joint region of bone fracture was higher in mice transplanted with AT-MSC than in mice transplanted with BM-MSC or DT-MSC. To compare the abilities of MSC, characterize how those MSC affect the repair of ischemic tissue, vascular occlusion was performed by ligation of the femoral artery and vein. Of note, the blood flow in the ischemic region rapidly increased in mice injected with AT-MSC, as contrasted with mice injected with BM- or DT-MSC. The number of CD45- and F4/80-positive cells at the femoral region was higher in AT-MSC recipients than in recipients of BM-MSC or DT-MSC. We evaluated the mRNA expression of angiogenic and migration factors in MSC and found the expression of CCL5 mRNA was higher in AT-MSC than in BM-MSC or DT-MSC. Transplantation of AT-MSC with impaired expression of CCL5 clearly showed a significant delay in the recovery of blood flow compared with the control. These findings have fundamental implications for the modulation of AT-MSC in the repair of vasculature and bone fracture.

Purpose: To investigate the role of reactive oxygen species (ROS) in retinal development during the early postnatal stage of rd1 mice., Methods: Development of the three retinal vascular layers of C57BL/6 J (WT) and C3H/HeN (rd1) mice was evaluated from 9th postnatal day (P9) to P21. Retinal ROS production was semi-quantitatively measured using dihydroethidium fluorescence. Mice were treated with intraperitoneal injections of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) at a dose of 275 mg/kg body weight, and PBS as the control from P3 to P8., Results: Rd1 mice showed retardation of retinal vascular development in the deep layer at P9. No significant difference was observed in the outer nuclear layer thickness of rd1 and WT mice. ROS production in the outer nuclear layer of rd1 mice was significantly higher than that in the outer nuclear layer of WT mice at P9, P13, and P17 (P < .05). TEMPOL facilitated the development of the deep vascular layer when compared with injection of PBS., Conclusions: Retardation of retinal vascular development is observed in rd1 mice; ROS is partially responsible for this finding. When using rd1 mice, we should be aware of this difference in comparison to other retinal degeneration animal models and human pathophysiological changes.

Objective: Involvement of reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase has been documented in the development of hypoxia-induced model of pulmonary arterial hypertension (PAH). Because the PAH-like phenotype was demonstrated in mice deficient in Nox1 gene (Nox1(-/Y)) raised under normoxia, the aim of this study was to clarify how the lack of NOX1/NADPH oxidase could lead to pulmonary pathology., Approach and Results: Spontaneous enlargement and hypertrophy of the right ventricle, accompanied by hypertrophy of pulmonary vessels, were demonstrated in Nox1(-/Y) 9 to 18 weeks old. Because an increased number of α-smooth muscle actin-positive vessels were observed in Nox1(-/Y), pulmonary arterial smooth muscle cells (PASMCs) were isolated and characterized by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In Nox1(-/Y) PASMCs, the number of apoptotic cells was significantly reduced without any change in the expression of endothelin-1, and hypoxia-inducible factors HIF-1α and HIF-2α, factors implicated in the pathogenesis of PAH. A significant decrease in a voltage-dependent K(+) channel, Kv1.5 protein, and an increase in intracellular potassium levels were demonstrated in Nox1(-/Y) PASMCs. When a rescue study was performed in Nox1(-/Y) crossed with transgenic mice overexpressing rat Nox1 gene, impaired apoptosis and the level of Kv1.5 protein in PASMCs were almost completely recovered in Nox1(-/Y) harboring the Nox1 transgene., Conclusions: These findings suggest a critical role for NOX1 in cellular apoptosis by regulating Kv1.5 and intracellular potassium levels. Because dysfunction of Kv1.5 is among the features demonstrated in PAH, inactivation of NOX1/NADPH oxidase may be a causative factor for pulmonary vascular remodeling associated with PAH.

Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the 'GATA factor switching'. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1-knockdown embryos that concomitantly express Gata2-GFP reporter, high-level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1-binding sites widely overlap with the sites pre-occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient-phase genes., (© 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)