Toshiki Kato

Serum-free isolation and culture system to enhance the proliferation and bone regeneration of adipose tissue-derived mesenchymal stem cells

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.

FOXA1 expression affects the proliferation activity of luminal breast cancer stem cell populations

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.

Hypoxia promotes the phenotypic change of aldehyde dehydrogenase activity of breast cancer stem cells

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−/Aldefluorpos) 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−/Aldefluorneg). Moreover, we found Aldefluorpos BCSCs had a greater hypoxic response and subsequent induction of HIF‐1α expression compared to the Aldefluorneg BCSCs. We also found that knocking down HIF‐1α, but not HIF‐2α, in Aldefluorpos 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 Aldefluorneg 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 Aldefluorneg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes.

Comparative Analysis of Cellular and Growth Factor Composition in Bone Marrow Aspirate Concentrate and Platelet-Rich Plasma

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-β1 (TGF-β1), 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-β1, 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.

Glucocorticoid Impaired the Wound Healing Ability of Endothelial Progenitor Cells by Reducing the Expression of CXCR4 in the PGE2 Pathway

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.

Corrigendum to “SDF-1 improves wound healing ability of glucocorticoid-treated adipose tissue-derived mesenchymal stem cells” [Biochem. Biophys. Res. Commun. 493/2 (2017) 1010–1017]

a Laboratory of Regenerative Medicine and Stem Cell Biology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan b Department of Orthopedic Surgery, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan c Division of Regenerative Medicine for Musculoskeletal System, Department of Orthopedic Surgery, Faculty of Medicine, University of Tsukuba, Japan d Program in Human Biology, School of Integrative Global Majors, University of Tsukuba, Japan

Isl1β over-expression with key β cell transcription factors enhances glucose-responsive hepatic insulin production and secretion

Adenoviral gene transfer of key β cell developmental regulators including Pdx1, Neurod1, and Mafa (PDA) has been reported to generate insulin-producing cells in the liver. However, PDA insulin secretion is transient and glucose unresponsive. Here, we report that an additional β cell developmental regulator, insulin gene enhancer binding protein splicing variant (Isl1β), improved insulin production and glucose-responsive secretion in PDA mice. Microarray gene expression analysis suggested that adenoviral PDA transfer required an additional element for mature β cell generation, such as Isl1 and Elf3 in the liver. In vitro promoter analysis indicated that splicing variant Isl1, or Isl1β, is an important factor for transcriptional activity of the insulin gene. In vivo bioluminescence monitoring using insulin promoter-luciferase transgenic mice verified that adenoviral PDA + Isl1β transfer produced highly intense luminescence from the liver, which peaked at day 7 and persisted for more than 10 days. Using insulin promoter-GFP transgenic mice, we further confirmed that Isl1β supplementation to PDA augmented insulin-producing cells in the liver, insulin production and secretion, and β cell‒related genes. Finally, the PDA + Isl1β combination ameliorated hyperglycemia in diabetic mice for 28 days and enhanced glucose tolerance and responsiveness. Thus, our results suggest that Isl1β is a key additional transcriptional factor for advancing the generation of insulin-producing cells in the liver in combination with PDA.

Microvesicles derived from Alde-Low EPCs support the wound healing capacity of AT-MSCs.

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.