Modeling Myeloma Dissemination In Vitro with hMSC-interacting Subpopulations of INA-6 Cells and Their Aggregation/Detachment Dynamics.

Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow-derived mesenchymal stromal cells (hMSC) stimulate myeloma cell expansion (e.g., IL6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination. We present an in vitro model using primary hMSCs cocultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNA sequencing, cell viability, and apoptosis. Results were correlated with gene expression data (n = 837) and survival of patients with myeloma (n = 536). On dispersed hMSCs, INA-6 saturate hMSC surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC-INA-6 interactions, which modulated apoptosis time dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6). Isolated nMA-INA6 indicated hMSC autonomy through superior viability after IL6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples. Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow-retentive phenotype with potential clinical relevance. SIGNIFICANCE: Novel methods describe in vitro dissemination of myeloma cells as detachment of daughter cells after cell division. Myeloma adhesion genes were identified that counteract in vitro detachment with potential clinical relevance.

Novel molecular cues for dental defects in hypophosphatasia.

Mineralization disorders with a broad range of etiological factors represent a huge challenge in dental diagnosis and therapy. Hypophosphatasia (HPP) belongs to the rare diseases affecting predominantly mineralized tissues, bones and teeth, and occurs due to mutations in the ALPL gene, which encodes tissue-nonspecific alkaline phosphatase (TNAP). Here we analyzed stem cells from bone marrow (BMSCs), dental pulp (DPSCs) and periodontal ligament (PDLSCs) in the absence and presence of efficient TNAP inhibitors. The differentiation capacity, expression of surface markers, and gene expression patterns of donor-matched dental cells were compared during this in vitro study. Differentiation assays showed efficient osteogenic but low adipogenic differentiation (aD) capacity of PDLSCs and DPSCs. TNAP inhibitor treatment completely abolished the mineralization process during osteogenic differentiation (oD). RNA-seq analysis in PDLSCs, comparing oD with and without TNAP inhibitor levamisole, showed clustered regulation of candidate molecular mechanisms that putatively impaired osteogenesis and mineralization, disequilibrated ECM production and turnover, and propagated inflammation. Combined alteration of cementum formation, mineralization, and elastic attachment of teeth to cementum via elastic fibers may explain dental key problems in HPP. Using this in vitro model of TNAP deficiency in DPSCs and PDLSCs, we provide novel putative target areas for research on molecular cues for specific dental problems in HPP.

Contact of myeloma cells induces a characteristic transcriptome signature in skeletal precursor cells -Implications for myeloma bone disease.

Physical interaction of skeletal precursors with multiple myeloma cells has been shown to suppress their osteogenic potential while favoring their tumor-promoting features. Although several transcriptome analyses of myeloma patient-derived mesenchymal stem cells have displayed differences compared to their healthy counterparts, these analyses insufficiently reflect the signatures mediated by tumor cell contact, vary due to different methodologies, and lack results in lineage-committed precursors. To determine tumor cell contact-mediated changes on skeletal precursors, we performed transcriptome analyses of mesenchymal stem cells and osteogenic precursor cells cultured in contact with the myeloma cell line INA-6. Comparative analyses confirmed dysregulation of genes which code for known disease-relevant factors and additionally revealed upregulation of genes that are associated with plasma cell homing, adhesion, osteoclastogenesis, and angiogenesis. Osteoclast-derived coupling factors, a dysregulated adipogenic potential, and an imbalance in favor of anti-anabolic factors may play a role in the hampered osteoblast differentiation potential of mesenchymal stem cells. Angiopoietin-Like 4 (ANGPTL4) was selected from a list of differentially expressed genes as a myeloma cell contact-dependent target in skeletal precursor cells which warranted further functional analyses. Adhesion assays with full-length ANGPTL4-coated plates revealed a potential role of this protein in INA-6 cell attachment. This study expands knowledge of the myeloma cell contact-induced signature in the stromal compartment of myelomatous bones and thus offers potential targets that may allow detection and treatment of myeloma bone disease at an early stage.

Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line.

Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5Y(TNAPhigh) neuroblastoma cells compared to SH-SY5Y(TNAPlow) cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5Y(TNAPhigh) cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5Y(TNAPhigh) cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5Y(TNAPhigh) cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies.

Unexpected high intrafamilial phenotypic variability observed in hypophosphatasia.

Hypophosphatasia (HPP) is a clinically heterogeneous rare, inherited disorder of bone and mineral metabolism with extensive allelic heterogeneity in the ALPL gene. In this report, we present a family with heterozygous parents (maternal p.(Glu191Lys), paternal p.(Gly334Asp) mutations in the ALPL gene) and four children (one genotypically normal, one heterozygous carrier and two compound heterozygous) showing an unexpected high phenotypic variability. One of the compound heterozygous showed clinical symptoms of the mild childhood form mainly affecting the teeth. The other one was more seriously affected with severe failure to thrive, delayed motor development, need for oxygen supply and profound mineralization deficit compatible with an infantile form of HPP. Functional in vitro studies identified p.(Glu191Lys) as mild (68%, no dominant-negative effect) and p.(Gly334Asp) as severely affected allele (1.2%, dominant-negative effect). In vitro simulation of the children's genetic status showed a residual AP activity of 29%, while the biochemical AP activity in the serum was comparably reduced in both children (22 and 36 U/l). This family report indicates that mapping ALPL mutations within the gene does not necessarily help to predict the clinical severity of the phenotype. Therefore, results of prenatal diagnostics have to be interpreted with caution and prenatal genetic diagnosis and counseling for HPP should be provided within an experienced multidisciplinary team. Research about other confounding factors is urgently needed.

What works? Process evaluation of a school-based fruit and vegetable distribution program in Mississippi.

BACKGROUND: During the 2004-2005 school year, the Mississippi Department of Education, Office of Child Nutrition, initiated a pilot program to distribute free fruit and vegetable snacks to students during the school day. This article describes the first-year implementation of the Mississippi Fruit and Vegetable Pilot Program. METHODS: The process evaluation addressed where, when, and how produce was distributed; what was distributed; challenges and successes; and recommended modifications. Five of the 25 program schools were selected to participate in the evaluation; selection was based on grade levels served and demographic characteristics. Data were collected from program staff (N = 11) and administrators (N = 6) via interviews and logs; student (N = 42) and parent (N = 19) focus groups; student questionnaires (N = 660); and school staff questionnaires (N = 207). RESULTS: Distributing fresh fruit and vegetable snacks at school was well received by staff and students. Most schools distributed the fresh fruit and vegetable snacks at morning break in classrooms or a central courtyard. Twenty-two types of fresh fruit, 4 types of dried fruit, and 7 types of vegetables were served to students during the program year. Commonly distributed fruit included apples, oranges, pears, bananas, and tangerines. Carrots were the staple vegetable, followed by celery. Key challenges included getting students to try new foods and receiving the produce in a timely manner without spoiling. Main successes included seeing students try new fruit and vegetable snacks, having the program run smoothly, and teacher support. CONCLUSIONS: The program fit well within the school structure and could be an effective component of a multifaceted approach to enhancing child nutrition.

Distributing free fresh fruit and vegetables at school: results of a pilot outcome evaluation.

OBJECTIVES: Consumption of fruit and vegetables among children is generally below recommended levels. This evaluation addressed two questions: (1) To what extent did children's attitudes toward, familiarity with, and preferences for fruit and vegetables change during the school year? and (2) To what extent did children's consumption of fruit and vegetables change during the school year? METHODS: During the 2004-2005 school year, the Mississippi Department of Education, Child Nutrition Programs initiated a pilot program to distribute free fruit and vegetables to students (kindergarten through 12th grade) during the school day. Data were collected in 2004-2005 within a one-group pretest/posttest design using a self-report questionnaire (n=725) and 24-hour dietary recalls (n=207) with a sample of students from five schools in Mississippi. Data were analyzed in 2006-2007. RESULTS: Results showed greater familiarity with fruit and vegetables at all grade levels (p<0.05) and increased preferences for fruit among eighth- and 10th-grade students (p<0.01). Eighth-grade students also reported more positive attitudes toward eating fruit and vegetables (p<0.01), increased perceived self-efficacy to eat more fruit (p<0.01), and increased willingness to try new fruit. Finally, results showed increased consumption of fruit, but not vegetables, among eighth- and 10th-grade students (p<0.001). CONCLUSIONS: Distributing free fruit and vegetables at school may be a viable component of a more comprehensive approach for improving students' nutrition attitudes and behaviors. More program emphasis is needed on ways to promote vegetable consumption.

Pulse treatment with zoledronic acid causes sustained commitment of bone marrow derived mesenchymal stem cells for osteogenic differentiation.

The aminobisphosphonate zoledronic acid (ZA) is a bone seeking specific inhibitor of protein farnesylation and geranylgeranylation, which causes inhibition of osteoclast function and apoptosis. It is widely used as an osteoclast targeted antiresorptive treatment of metastatic bone disease, Paget's disease and osteoporosis. Mesenchymal stem cells (MSC) and osteoblast precursors can also be targets of bisphosphonates, but the clinical relevance of these effects is under debate. We show here that ZA in vitro causes inhibition of proliferation and induction of apoptosis in hMSC, when applied in concentrations of 20 and 50 microM for more than 24 h which can be rescued by treatment with 10 microM geranylgeranyl pyrophosphate (GGPP). However, pulse stimulation for 3 and 6 h with these concentrations and subsequent culture for up to 2 weeks under osteogenic conditions exerts sustained regulation of osteogenic marker genes in hMSC. The effect on gene regulation translates into marked enhancement of mineralization, as shown by alizarin red and alkaline phosphatase staining after 4 weeks of osteogenic culture. ZA, when applied as a pulse stimulus, might therefore also stimulate osteogenic differentiation in vivo, since muM plasma concentrations can be achieved by intravenous application of 5 mg in patients. These data set the stage for the future dissection of the effects of ZA and other aminobisphosphonates on cells beyond osteoclasts, with respect to cell differentiation in benign metabolic and to antitumor efficacy in metastatic bone diseases, as well as adverse events due to putative substance accumulation in bone during long-term treatment.

Effects of high glucose on mesenchymal stem cell proliferation and differentiation.

High glucose (HG) concentrations impair cellular functions and induce apoptosis. Exposition of mesenchymal stem cells (MSC) to HG was reported to reduce colony forming activity and induce premature senescence. We characterized the effects of HG on human MSC in vitro using telomerase-immortalized MSC (hMSC-TERT) and primary MSC (hMSC). HG (25mM) enhanced hMSC-TERT proliferation in long-term studies in contrast to hMSC where proliferation was unchanged. Thioredoxin-interacting protein, which is involved in apoptosis regulation, was stimulated by glucose in hMSC-TERT. However, apoptosis was not influenced by HG in both cell types. MSC treatment with HG favored osteogenic differentiation. MSC are resistant to HG toxicity, depending on the stemness of MSC. Proliferation and osteogenic differentiation are stimulated by HG. Effects of HG on the transient amplifying compartment of MSC may differ from those in mature cells. Further research is needed to unravel the molecular mechanisms of HG resistance of MSC.

Selenium supplementation restores the antioxidative capacity and prevents cell damage in bone marrow stromal cells in vitro.

Bone marrow stromal cells (BMSCs) and other cell populations derived from mesenchymal precursors are developed for cell-based therapeutic strategies and undergo cellular stress during ex vivo procedures. Reactive oxygen species (ROS) of cellular and environmental origin are involved in redox signaling, cumulative cell damage, senescence, and tumor development. Selenium-dependent (glutathione peroxidases [GPxs] and thioredoxin reductases [TrxRs]) and selenium-independent (superoxide dismutases [SODs] and catalase [CAT]) enzyme systems regulate cellular ROS steady state levels. SODs process superoxide anion to hydrogen peroxide, which is subsequently neutralized by GPx and CAT; TrxR neutralizes other ROS, such as peroxinitrite. Primary BMSCs and telomerase-immortalized human mesenchymal stem cells (hMSC-TERT) express GPx1-3, TrxR1, TrxR2, SOD1, SOD2, and CAT. We show here that in standard cell cultures (5%-10% fetal calf serum, 5-10 nM selenite), the activity of antioxidative selenoenzymes is impaired in hMSC-TERT and BMSCs. Under these conditions, the superoxide anion processing enzyme SOD1 is not sufficiently stimulated by an ROS load. Resulting oxidative stress favors generation of micronuclei in BMSCs. Supplementation of selenite (100 nM) restores basal GPx and TrxR activity, rescues basal and ROS-stimulated SOD1 mRNA expression and activity, and reduces ROS accumulation in hMSC-TERT and micronuclei generation in BMSCs. In conclusion, BMSCs in routine cell culture have low antioxidative capacity and are subjected to oxidative stress, as indicated by the generation of micronuclei. Selenite supplementation of BMSC cultures appears to be an important countermeasure to restore their antioxidative capacity and to reduce cell damage in the context of tissue engineering and transplantation procedures.

Down-regulation by nuclear factor kappaB of human 25-hydroxyvitamin D3 1alpha-hydroxylase promoter.

1,25-(OH)(2) vitamin D(3) is important for calcium homeostasis and cell differentiation. The key enzyme for the activation of liver-derived 25(OH) vitamin D(3) is 25-hydroxyvitamin D(3) 1alpha-hydroxylase. It is expressed mainly in the kidney but also in peripheral tissues. A 1413-bp fragment of the 1alpha-hydroxylase promoter was cloned into luciferase vectors pGL2basic and pGL3basic. Sequence analyses revealed four base exchanges and three base deletions compared with the published sequence which were identically found in five control persons. In silico promoter analyses revealed 17 putative nuclear factor (NF)kappaB sites, 10 of which were found to bind NFkappaB in EMSA experiments. Cotransfection of NFkappaB p50 and p65 subunits resulted in dramatic reduction of the promoter activity of the full-length construct as well as a series of 5'-deletion constructs. Deletion of the farmost 3'-situated NFkappaB-responsive element almost abolished NFkappaB responsiveness. Treatment of human embryonic kidney 293 cells with sulfasalazine, a NFkappaB inhibitor, resulted in enhanced 1alpha-hydroxylase mRNA production. Down-regulation of 1alpha-hydroxylase promoter through NFkappaB signaling may contribute to the pathogenesis of inflammation-associated osteopenia/osteoporosis.