Rumi Sawada

Calcium‐incorporated titanium surfaces influence the osteogenic differentiation of human mesenchymal stem cells

In this study, a titanium surface was chemically modified with calcium ions and assessed for its influence on osteogenic differentiation and molecular responses of human mesenchymal stem cells (hMSCs). Titanium disks were treated with NaOH (NaOH treatment), NaOH + CaCl2 (CaCl2 treatment), or NaOH + Ca(OH)2 (Ca(OH)2 treatment). Ca(OH)2 treatment caused significantly greater calcium incorporation onto the titanium surface and apatite formation than CaCl2 treatment. The morphology of hMSCs differed on CaCl2- and Ca(OH)2-treated disks. The osteopontin (OPN) expression in hMSCs cultured on CaCl2-treated titanium was significantly higher than that in cells cultured on NaOH-treated disks; OPN expression was significantly higher in cells cultured on Ca(OH)2-treated disks than on un-, NaOH-, and CaCl2-treated disks. Osteocalcin (OCN) protein expression in hMSCs cultured on Ca(OH)2-treated disks was significantly higher than that on all the other disks. Comparative expression profiling by DNA microarray and pathway analyses revealed that calcium modification of the titanium surface induced integrin β3 after OPN upregulation and promoted Wnt/β-catenin signaling in hMSCs. In addition, Ca(OH)2 treatment upregulated the expression of bone morphogenetic protein 2, cyclooxygenase 2, and parathyroid hormone-like hormone in comparison to CaCl2 treatment. These observations suggest that calcium-modified titanium surfaces affect osteogenic differentiation in hMSCs and that Ca(OH)2 treatment induced osteogenic differentiation in hMSCs, whereas CaCl2 treatment had a limited effect.

The effect of sulfated hyaluronan on the morphological transformation and activity of cultured human astrocytes.

We demonstrated the effect of synthesized sulfated hyaluronan (SHya), which is composed of a sulfated group and hyaluronan, and basic fibroblast growth factor 2 (FGF-2) on normal human astrocytes (NHA) activity and its morphological transformation in vitro study. Astrocyte is a kind of glial cell and stellated astrocyte (activating astrocyte) supports axons network, neurons survival and synaptic plasticity. Treatment of SHya hardly affected NHA proliferation. However combination treatment of SHya and FGF-2 increased NHA proliferation. Treatment of SHya promoted transformation of normal astrocyte into a stella morphology (stellation) and combination treatment of SHya and FGF-2 promoted stellation than that of SHya only. Treatment of SHya increased glial fibrillary acidic protein (GFAP), nestin mRNA and GFAP protein expression in the stellated NHA. The cell-cell adhesion of NHA increased by treatment of SHya. Treatment of SHya increased heparin-binding trophic factors FGF-2, midkine, and some other trophic factors mRNA level in the NHA. These results suggested that the treatment of SHya promoted NHA activity due to enhancing neurotrophins production and the morphological transformation of NHA and the effect of SHya on astrocytes partly involved FGF-2 activity. These findings indicate that SHya may be involved in the astrocyte activity and support neurons survivals.

The Metabolism and Distribution of Docosapentaenoic Acid (n-6) in the Liver and Testis of Growing Rats

To investigate the metabolism and distribution of docosapentaenoic acid (22:5n-6, DPA) in the liver and testis of growing rats, 22:5n-6 was administered to their dams. Newborn rats with a low hepatic arachidonic acid (20:4n-6, AA) level were generated by administrating a diet rich in docosahexaenoic acid (22:6n-3, DHA) but n-6 fatty acid (FA) free to pregnant dams. After parturition, 22:5n-6 or linoleic acid (18:2n-6, LA) was administered with a high level of 22:6n-3 to the dams until weaning. At weaning, the hepatic 20:4n-6 level was significantly highest in the DPA-DHA but not LA-DHA diet-fed animals. The hepatic delta-6 desaturase (D6D) mRNA abundance was significantly lower in both the LA-DHA and DPA-DHA diet-fed animals, connoted with the 20:4n-6 content recovered by 22:5n-6 that did not involve D6D and supporting the occurrence of retroconversion in the liver of the growing rats. The low D6D level in the 3-week-old testis was not in proportion to the elevated 22:5n-6 level, implying that early testicular 22:5n-6 accumulation might require supply from the circulation system.

Intracerebral microinjection of stannous 2-ethylhexanoate affects dopamine turnover in cerebral cortex and locomotor activity in rats

Stannous 2-ethylhexanoate [Sn(Oct)(2)] is used as a catalyst for production of poly-L-lactic acid and copolymers that are implanted in cranial surgery, but reports on its effects on the central nervous system are few. We examined the effects of Sn(Oct)(2) on cell viability in vitro and on neurotransmission and behavior in the rat. Treatment of normal human astrocytes with 10 mg/mL Sn(Oct)(2) reduced mitochondrial activity to 16% of the control. Injection of Sn(Oct)(2) at 6.28 mg/kg BW (2 mg/kg BW Sn) into right lateral ventricle of the rat brain tended to increase the ambulation distance after 30 days when compared with the control group. The turnover of dopamine neurotransmission was increased in the cerebral cortex. These results suggest that Sn(Oct)(2) is cytotoxic to astrocytes in vitro. Injection of Sn(Oct)(2) into the brain had no or very weak immediate neurotoxicity, but long-term exposure to Sn(Oct)(2) increased dopamine neurotransmission turnover.

Enhancing action by sulfated hyaluronan on connexin-26, -32, and -43 gene expressions during the culture of normal human astrocytes

Astrocyte proliferation is strictly controlled during development and in the adult nervous system. In this study, we examined the role of sulfated hyaluronan (SHya) in the proliferation and differentiation of normal human astrocytes (NHAs). Cells were cultured with different concentrations of SHya for 7 days, and the number of viable cells and the presence of neural cell-specific genes were determined to assess their proliferation and development, respectively. With SHya, cell proliferation increased nonsignificantly. Furthermore, remarkable enhancing action by SHya on connexin-26, -32, and -43 gene expressions were observed during the culture of NHAs. It has been suggested that a fraction of NHAs have neural precursor activity that gives rise to astrocytes themselves, oligodendrocytes, and neurons. Our results clearly demonstrated that the expression of specific genes for neural precursor cells, astrocytes, neurons, and oligodendrocytes was significantly increased to 50 mug/mL in SHya-treated cultures when compared with that of the control culture. These findings suggest that SHya plays an important role in the proliferation and differentiation of NHAs and in the production of a novel material for tissue engineering.

Cyclin D2 Promotes the Proliferation of Human Mesenchymal Stem Cells

Background: Human mesenchymal stem cells (hMSCs) hold promise for use in cell-based therapies and tissue engineering. Although hMSCs are thought to be stable ex vivo, it is possible that they undergo an undesirable transformation to a phenotype of unlimited proliferation during ex vivo. In this study, we searched for the factor required for unlimited proliferation of hMSCs. Methods: Changes in gene expression were evaluated between hMSCs and Ewing’s sarcoma cell lines, which may be derived from hMSCs, using GeneChip Human Genome U133 plus 2.0 Array. A gene up-regulated by at least 10-fold in Ewing’s sarcoma cell lines, Cyclin D2, was overexpressed in hMSCs by a lentiviral vector. Results: Overexpression of Cyclin D2 in hMSCs altered cell morphology and promoted cell proliferation. Expression of transforming growth factor-b2 (TGF-b2), which induces senescence in hMSCs, was down-regulated in Cyclin D2- overexpressing hMSCs. Furthermore, Gene Ontology analysis revealed that Cyclin D2 overexpression activated expression of genes associated with proliferation and interphase. Conclusions: Cyclin D2 promotes hMSC proliferation and is a candidate biomaker for hMSC transformation.

Corrigendum to "Characterization of the cell growth analysis for detection of immortal cellular impurities in human mesenchymal stem cells" [Biologicals 43 (2) (March 2015) 146-149].

Corrigendum to “Characterization of the cell growth analysis for detection of immortal cellular impurities in human mesenchymal stem cells” [Biologicals 43 (2) (March 2015) 146e149] Ken Kono , Nozomi Takada b, , Satoshi Yasuda b, , Rumi Sawada , Shingo Niimi , Akifumi Matsuyama , Yoji Sato b, d, e, f, g, * a Division of Medical Devices, National Institute of Health Sciences, 1-18-1 Kami-yoga, Setagaya, Tokyo, 158-8501, Japan b Division of Cellular & Gene Therapy Products, National Institute of Health Sciences, 1-18-1 Kami-yoga, Setagaya, Tokyo, 158-8501, Japan c Research on Disease Bioresources, Platform of Therapeutics for Rare Disease and Health Policy, National Institute of Biomedical Innovation, Kobe International Business Center Rm#602, 5-5-2 Minatojima-Minami-Machi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan d Foundation for Biomedical Research and Innovation, Hyogo, Japan e Department of Quality Assurance Science for Pharmaceuticals, Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan f Department of Cellular & Gene Therapy Products, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan g Department of Translational Pharmaceutical Sciences, Graduated School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

Application of cell growth analysis to the quality assessment of human cell-processed therapeutic products as a testing method for immortalized cellular impurities

In human cell-processed therapeutic products (hCTPs) for clinical application, tumorigenic cellular impurities in the manufacturing process are a major concern. Because cellular immortalization is one of the prerequisite steps in tumorigenesis, we tested whether cell growth analysis can be employed to check for immortalized (and potentially tumorigenic) cellular impurities in hCTPs. We monitored the growth of human bone marrow-derived mesenchymal stem cells (BMSCs) mixed with HeLa cells at a ratio of 1/106 or more and compared their growth rates with that of BMSCs alone. The cell growth analysis detected a significant increase in the growth rate of the BMSCs spiked with 0.0001% HeLa within 30 days at a probability of 47%. When human adipose-derived stem cells (ADSCs) were spiked with ASC52telo cells, a human telomerase reverse transcriptase (hTERT)-immortalized adipose-derived mesenchymal stem cell line, at a ratio of 0.001% or more, their growth rates were significantly increased within 15 passages, compared with that of ADSCs alone. These results indicate that cell growth analysis for the detection of immortalized cellular impurities in human somatic stem cells is simple and can be useful for the quality assessment of hCTPs in the manufacturing process.

Effects of Tin Compounds on Human Chondrogenic Activity In Vitro

Organotin compounds, particularly tributyltin chloride (TBT) and dibutyltin dichloride (DBT), are widely distributed toxicants. They inhibit cell proliferation and are known to cause neurotoxicity and genotoxicity in animals and humans. DBT is used as a catalyst in biodegradable polymers. We evaluated the effects of TBT and DBT on chondrogenesis of human articular chondrocytes (HAC) under a micromass culture system. In 4 weeks of culture, the lowest dose of TBT caused an increase in cell proliferation and differentiation. When the dose was increased, its effect on cultured chondrocytes was inhibitory compared with the control cultures. DBT produced little change in cell proliferation but it significantly inhibited cell differentiation compared with the control cultures. The expression of cartilage-specific genes, namely collagen type II and aggrecan was inhibited in TBT- and DBT-treated cultured chondrocytes. TBT was significantly toxic to HAC at 0.16 ppb and DBT at 0.75 ppb.

In Vitro Cytotoxic Effects of Tin Compounds on Normal Human Astrocytes

Astrocyte proliferation is strictly controlled both during development and in the adult nervous system. Stannous chloride (SnCl2) an inorganic tin, stimulated the nervous system when injected into laboratory animals. It also induced extensive DNA damage. In the present study, we investigated the effects of SnCl2 and stannous 2-ethylhexanoate [Sn(Oct)2] at different concentrations on the proliferation and development of normal human astrocytes (NHA). Cells were cultured with SnCl2 or Sn(Oct)2, and the number of viable cells and the presence of neural cell-specific genes were determined to assess their proliferation, gap-junctional intercellular communica-tion (GJIC) function, and development, respectively. Cell proliferation, GJIC function, and the expression of gap junctional proteins were suppressed when NAH were cultured with SnCl2 and Sn(Oct)2. It was reported that the proliferating cells initially express nestin, a gene specific for neural precursor cell which subsequently give rise to neurons, oligodendrocytes, and astrocytes. Here, we examined the expression of neural cell-specific genes using real-time polymerase chain reaction (PCR). Expression of genes specific for neural precursor cells and astrocytes was decreased, while expression of genes specific for neurons and oligodendrocytes was increased with Sn(Oct)2, but all were decreased with SnCl2 compared with the control culture. Our findings suggest that these tin compounds are neurotoxic to astrocytes, resulting in the suppression of the proliferation and development of NHA.