Hisako Katano

Microgravity induces prostaglandin E2 and interleukin-6 production in normal rat osteoblasts: role in bone demineralization

It has been suggested that microgravity alters bone metabolism. Evidence for this phenomenon includes the negative calcium balance and decreased bone density in astronauts, as well as, inhibition of bone formation in rats flown for 2 to 3 weeks. However, the specific mechanisms that modulate these changes in microgravity are unknown. The purpose of this study was to clarify the mechanism of microgravity-induced bone demineralization using normal rat osteoblasts obtained from femur marrow cultures. The osteoblasts were cultured for 5 days during a Shuttle-Spacelab flight (STS-65). After collection of the culture medium, the cellular DNA and RNA were fixed on board. Enzyme-immunoassay of the culture medium for prostaglandin E2 (PGE2) indicated that microgravity induced a 4.5- to 136-fold increase in flight samples as compared to the ground control cultures. This increase of PGE2 production was consistent with a 3.3- to 9.5-fold elevation of inducible prostaglandin G/H synthase-2 (PGHS-2) mRNA, quantitated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA induction for the constitutive isozyme PGHS-1 was less than that for PGHS-2. The interleukin-6 (IL-6) mRNA was also increased (6.4- to 9.3-fold) in microgravity as compared to the ground controls. Since PGE2 and IL-6 are both known to play a role in osteoclast formation and bone resorption, these data provide molecular mechanisms that contribute to our understanding of microgravity-induced alterations in the bone resorption process.

Immunochemical and biochemical characteristics of enamel proteins in hypocalcified amelogenesis imperfecta

Amelogenesis imperfecta is a hereditary disease of the enamel that is unassociated with generalized defects. Cases of the condition are clinically classified into three groups: hypoplastic, hypomaturation, and hypocalcified. In this study, soluble protein fractions of the enamel from three patients with hypocalcified amelogenesis imperfecta were examined through the use of immunochemical and biochemical techniques. In immunochemical analyses done with a polyclonal anti-amelogenin antibody, all samples from enamel in which there was amelogenesis imperfecta were found to contain considerable amounts of amelogenin peptides. When an enamel sample from one patient was examined by Western-blot transfer and immunobinding analysis, the amelogenin fraction was found to consist of a 26-kDa molecule thought to be normally present in the outer layer of secretory-stage enamel. This enamel was also found to contain albumin as one of the major constituents of the protein fraction. These results suggest that hypocalcified amelogenesis imperfecta may in part be caused by a disturbance in matrix protein degradation during the maturation phase.

An improved quantitative RT-PCR fluorescent method for analysis of gene transcripts in the STS-65 space shuttle experiment

We describe a reverse transcription polymerase chain reaction (RT-PCR) technique using fluorescent dUTP to examine changes in mRNA level in samples. In this procedure, the amplified product is identified by the fluorescent detection system in an automated DNA sequencer, and if the MW of the DNA/RNA or the fluorescent dye is different, several samples can be analyzed in a single lane. The basis for this technique is similar to that of radiolabeled methods, and we applied this technique for the comparison of the expression level of the rat c-myc gene in osteoblasts exposed to microgravity and unit gravity conditions. Using the fluorescent- and radiolabeled methods, the level of rat c-myc mRNA were compared quantitatively and the results demonstrated that the c-myc expression level was not altered by microgravity. Therefore, this fluorescent RT-PCR technique is useful for gene expression analysis particularly when the samples are limited, such as in space flight experiments. The method also allows for rapid assessment of mRNA changes in many samples simultaneously.

Expression of PDGF-β receptor, EGF receptor, and receptor adaptor protein Shc in rat osteoblasts during spaceflight

A number of studies have indicated that microgravity induces osteopenia and modulates functions of mammalian cells. However, the molecular mechanisms underlying these effects of microgravity are still unknown. Rat osteoblasts were cultured for 4 and 5 days during Shuttle-Spacelab flight, and fixed by guanidine isothiocyanate solution on board after treatment with 1α, 25 (OH)2 vitamin D3. The mRNA levels for platelet-derived growth factor (PDGF)-β receptor, epidermal growth factor (EGF) receptor, the growth factor receptor adaptor protein Shc, and c-fos were determined using the method of quantitative reverse transcription-polymerase chain reaction. The mRNA levels for EGF receptor were not altered by microgravity. However, the mRNA levels for PDGF-β receptor, Shc, and c-fos were decreased to 62, 55 and 25% on the 4th day of flight, and 47, 40, and 43% on the 5th day, respectively, as compared to the corresponding ground controls. Expression of the growth factor receptor and the receptor adaptor protein was modulated in rat osteoblasts during spaceflight. Data suggest that signal transduction via growth factor receptors in rat osteoblasts is impaired by microgravity. Dysfunction of osteoblasts might be involved in spaceflight-induced osteopenia.

Osteoblast Responsiveness to 1α,25‐Dihydroxyvitamin D3 during Spaceflight

Abstract: Rat osteoblasts were cultured aboard a space shuttle for 4 and 5 days. Cells were treated with 1 nM 1α,25‐dihydroxyvitamin D3 (VD) for the last 1 day. The conditioned media were harvested. Cells were solubilized with guanidine solution on board. We examined microgravity effects on the production/expression of osteocalcin, bone sialoprotein (BSP), and VD receptor (VD‐R) in osteoblasts. Under VD treatment, the osteocalcin protein level was 243 ± 117 and 1,718 ± 534 pg/μg cellular DNA in flight cultures and ground controls, respectively. Without VD treatment, the osteocalcin protein level was not different between flight cultures and ground controls. The osteocalcin mRNA level in the VD‐treated flight cultures was as low as 16% of that in ground controls. The VD‐R mRNA level in the VD‐treated flight cultures was also decreased to 16% of that in ground controls. Microgravity would suppress the VD‐inducible production of osteocalcin but not the basal productivity. The BSP mRNA level was increased by microgravity. VD/VD‐R binds to the vitamin D‐responsive element (VDRE) on the target genes. The rat osteocalcin gene is positively regulated via “enhancer” VDRE, whereas the rat BSP gene is negatively regulated via “repressor” VDRE. Microgravity might modulate osteoblast responsiveness to VD through the suppression of VD‐R.

Platelet-derived growth factor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells

IntroductionFor expansion of human mesenchymal stem cells (MSCs), autologous human serum is safer than fetal bovine serum in clinical situations. One of the problems with the use of autologous human serum is that its proliferative effect on MSCs varies widely between donors. The threefold goals of this study were: (1) to demonstrate an improved method for preparing human serum; (2) to identify growth factors predictive of proliferative potential; and (3) to identify a cytokine to promote MSC proliferation in human serum.MethodsFresh blood was collected using a closed bag system containing glass beads. The bag was shaken at 20 °C for 30 minutes for rapid preparation, or kept stationary at 4 °C for 24 hours for slow preparation. Passage 0 synovial MSCs derived from four donors were cultured with 10 % conventional rapid preparation serum or modified slow preparation serum from four different donors. To perform the colony-forming unit assay, synovial MSCs were cultured in these serums. The protein expression profile in serum was analyzed using cytokine array. The candidate proteins were speculated from the correlation between the colony-forming ability and protein expression. As an evaluation of the candidate proteins, proliferation ability, surface marker phenotype and differentiation capability of synovial MSCs were examined.ResultsCompared with rapid preparation serum, slow preparation serum resulted in a significantly higher total colony number and twofold higher expression levels of nine proteins (angiopoietin-1, BDNF, EGF, ENA-78, IGFBP-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, RANTES and TfR). Colony number was positively correlated with PDGF-AA/AB concentrations. Exogenous PDGF-AA significantly promoted proliferation of synovial MSCs, whereas PDGF receptor (PDGFR) inhibitor decreased it. Addition of PDGFs or PDGFR inhibitor did not affect surface epitopes of synovial MSCs. Pretreatment with PDGFs or PDGFR inhibitor did not affect chondrogenic, adipogenic, or calcification potentials of synovial MSCs.ConclusionSlow preparation serum contained higher concentrations of PDGF-AA/AB and increased the colony formation number of synovial MSCs. PDGF-AA/AB were indicators of the proliferative potential of human serum. Exogenous PDGF-AA increased proliferation of synovial MSCs without alteration of surface epitopes and differentiation potentials.

Platelet-Activating Factor Receptor Signals in Rat Osteoblasts during Spaceflight

Abstract: The platelet‐activating factor (PAF) is a lipid mediator. The G‐protein‐coupled receptor of PAF (PAF‐R) is activated by inflammatory and stressful conditions in numerous cell types. PAF/PAF‐R is involved in apoptotic and antiapoptotic processes. We examined microgravity effects on the expression of PAF‐R and second messengers in rat osteoblasts. The PAF‐R signals are transmitted via arachidonic acid, phospholipase C (PLC), protein kinase C (PKC), and mitogen‐activated protein kinase. Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized on board. PAF‐R gene expression in flight cultures increased to 2–6‐fold higher than in ground controls. Gene expression of the G‐protein α subunit Gαq in flight cultures increased to 3‐fold and higher than in ground controls. It is known that Gaq stimulates the effecter PLCβ, activating PKC. The mRNA levels of PKCδ and PKCϑ in flight cultures were increased to 2–5‐fold higher than in ground controls. The PKCα mRNA level in flight cultures was increased to 3‐fold higher than in ground controls on the 4th day. Gene expression of catalytic and regulatory subunits of protein kinase A was suppressed in flight cultures. PKCδ and PKCϑ are novel PKCs that can be target substrates of caspases. The PAF‐R gene may act as a mechano‐sensitive gene that is involved in the apoptotic and antiapoptotic processes of osteoblasts under microgravity.

Specific markers and properties of synovial mesenchymal stem cells in the surface, stromal, and perivascular regions

BackgroundSynovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. Synovial tissue can be histologically classified into three regions; surface, stromal and perivascular region, but the localization of synovial MSCs has not been fully investigated. We identified markers specific for each region, and compared properties of MSCs derived from each region in the synovium.MethodsThe intensity of immunostaining with 19 antibodies was examined for surface, stromal, and perivascular regions of human synovium from six osteoarthritis patients. Specific markers were identified and synovial cells derived from each region were sorted. Proliferation, surface marker expression, chondrogenesis, calcification and adipogenesis potentials were compared in synovial MSCs derived from the three regions.ResultsWe selected CD55+ CD271− for synovial cells in the surface region, CD55− CD271− in the stromal region, and CD55− CD271+ in the perivascular region. The ratio of the sorted cells to non-hematopoietic lineage cells was 5% in the surface region, 70% in the stromal region and 15% in the perivascular region. Synovial cells in the perivascular fraction had the greatest proliferation potential. After expansion, surface marker expression profiles and adipogenesis potentials were similar but chondrogenic and calcification potentials were higher in synovial MSCs derived from the perivascular region than in those derived from the surface and stromal regions.ConclusionsWe identified specific markers to isolate synovial cells from the surface, stromal, and perivascular regions of the synovium. Synovial MSCs in the perivascular region had the highest proliferative and chondrogenic potentials among the three regions.

High-sensitivity virus and mycoplasma screening test reveals high prevalence of parvovirus B19 infection in human synovial tissues and bone marrow

BackgroundLatent microorganism infection is a safety concern for the clinical application of mesenchymal stem cells (MSCs). The aim of this study is to investigate the frequencies and sensitivities of the latent virus and mycoplasma infections in synovium, bone marrow, peripheral blood cells, and blood plasma and cultured synovial MSCs.MethodsTotal DNA and RNA of the synovium (n = 124), bone marrow (n = 123), peripheral blood cells (n = 121), plasma (n = 121), and 14-day cultured synovial MSCs (n = 63) were collected from patients who underwent total knee arthroplasty or anterior ligament reconstruction after written informed consents were obtained. The multiplex polymerase chain reaction (PCR) primers were designed to quantitatively measure the representative genomes of 13 DNA viruses, 6 RNA viruses, and 9 mycoplasmas. Multi-spliced mRNA detection and virus spike test were also performed to demonstrate the sensitivity of synovial MSCs to the candidate pathogens.ResultsIn synovium and bone marrow, the positive rates of parvovirus B19 genome were significantly higher than in peripheral blood cells (18.7% and 22% vs. 0.8%, respectively). Multi-alignment analysis of amplified and sequenced viral target genes showed the proximity of the parvovirus B19 gene from different tissue in the same patients. Synovial MSCs cultured for 14 days were positive for virus infection only in two patients (2/62 = 3%). Parvovirus B19 multi-spliced mRNAs were not detected in these two samples. Virus spike test demonstrated the sensitivity of synovial MSCs to herpes simplex virus (HSV)1 and cytomegalovirus (CMV), but not to parvovirus B19.ConclusionThis study revealed a relatively high incidence of latent parvovirus B19 in synovium and bone marrow tissue.

Canine mesenchymal stem cells from synovium have a higher chondrogenic potential than those from infrapatellar fat pad, adipose tissue, and bone marrow

Osteoarthritis (OA), a common chronic joint disorder in both humans and canines, is characterized by a progressive loss of articular cartilage. Canines can serve as an animal model of OA for human medicine, and this research can simultaneously establish effective veterinary treatments for canine OA. One attractive treatment that can lead to cartilage regeneration is the use of mesenchymal stem cells (MSCs). However, for canine OA, little information is available regarding the best source of MSCs. The purpose of this study was to identify a promising MSC source for canine cartilage regeneration. We collected synovial, infrapatellar fat pad, inguinal adipose, and bone marrow tissues from six canines and then conducted a donor-matched comparison of the properties of MSCs derived from these four tissues. We examined the surface epitope expression, proliferation capacity, and trilineage differentiation potential of all four populations. Adherent cells derived from all four tissue sources exhibited positivity for CD90 and CD44 and negativity for CD45 and CD11b. The positive rate for CD90 was higher for synovium-derived than for adipose-derived and bone marrow-derived MSCs. Synovium-derived and infrapatellar fat pad-derived MSCs displayed substantial proliferation ability, and all four populations underwent trilineage differentiation. During chondrogenesis, the wet weight was heavier for cartilage pellets derived from synovium MSCs than from the other three sources. The synovium is therefore a promising source for MSCs for canine cartilage regeneration. Our findings provide useful information about canine MSCs that may be applicable to regenerative medicine for treatment of OA.