Saos calcification

Series:	IN_VITRO DIFFERENTIATION SERIES
Species:	Human (Homo sapiens)
Genomic View:	Zenbu
Expression table:	FILE
Link to TET:	TET
Sample providers :	Kim Summers
Germ layer:	mesoderm
Primary cells or cell line:	cell line
Time span:	28 days
Number of time points:	18

Overview

The formation of bone is a multi-stage process. It begins with mesenchymal cells committed to becoming cartilage. These cells condense into nodules and differentiate into chondrocytes which proliferate rapidly to form the template for the developing bone. They secrete a cartilege-specific extracellular matrix. blood vessels sinvade the cartilage structure. Mesenchymal precursors surrounding the cartilage cells then differentiate into osteoblasts which begin to form an extracelular matrix specific for bone. At the same time the chondrocytes within the osteoblast shell begin to die from apoptosis leaving a space that becomes the bone marrow. osteoblasts themselves become embedded into the bone matrix and differentiate further into osteocytes. Bone is constantly being remodelled by the action of osteoclasts, cells of the monocyte lineage that degrade the bone and osteoblasts on the surface which replace it with new bone. Thus the formation and maintenance of bone is a dynamic process.During the formation of bone, osteoblasts lay down a matrix of hydroxyapatite, a mineral containing calciumand phosphorous which makes up to 50% of the weight of bone. this process of mineralisation can be simulated in vitro in cell cultures treated with chemicals to induce calcification. Many cell lines, primarily derived from osteosarcomas, can be induced to mineralise in this way, as can primary vascular smooth muscle cells. It is important to understand the process of mineralisation because formation of the bony skeleton is critical to the proper functioning of the vertebrate organism and because ectopic mineralisation can occur in genetic and environmental disease. For example, calcification is a key finding in forms of arterial disease including atherosclerosis.

Sample description

In this study we used the human osteosarcoma cell line Saos-2. This line was first established from an osteosarcoma isolated from an 11 year old female patient in 1973 [1][2]. The line has been commonly used to study extracellular matrix and mechanical mechanisms in both expression and mineralisation studies [3] although the utility of Saos-2 as a human model system for normal osteoblast formation and function has been debated. Varying cellular morphology and proliferation has been observed [4], but Saos-2 demonstrates physiological levels of multiple osteoblastic markers including, osteocalcin (OC), bone sialoprotein (BSP) and decorin (DCN) [3]. In addition, active levels of alkaline phosphatase (ALPL) and the capacity to mineralize render Saos-2 a useful model system in the study of osteoblast function and ECM formation [5].This experiment was designed to determine promoter specificity and gene expression patterns of key regulators early and late in the mineralization process using this human model system. To control for effects of serum cell proliferation and hyperconfluence we also submitted two control samples: mock-treated Saos-2 cells that had medium changes at the same time points but were not treated with BGP/ascorbic acid and MG63 osteosarcoma cells that were treated but failed ot mineralise. MG63 cells lack high levels of alkaline phosphatase and are not able to mineralise under these conditions, but they may respond to the signal by upregulating some required genes. [3][6].The experiment was designed across 18 time points: 0, 15, 30, 45, 60, 80,100,120,150 and 180 minutes, 4, 8, and 24 hour as well as 4, 7, 14, 21, and 28 days following calcification induction. Cells were plated into 6 well plates (NUNC) or flasks (T25 or T75) at approximately 100,000 cells / well and mineralization was induced two days later with 50µg/ml ascorbic acid (Sigma) and 2.5mM ß-glycerophosphate (BGP) (Sigma) in medium with 10% serum. Medium was replaced with fresh medium including 10% serum and BGP/ascorbic acid every two to three days. All time point samples were processed for RNA at the same time relative to medium-change. RNA from the 18 time point samples were isolated through the following methods. The cells were lifted using 1x Trypsin-EDTA solution (Sigma) and RNA was isolated using the RNA Bee protocol (Ambisco). The sample RNA was quantified using a Nanodrop spectrophotometer (Nanodrop, USA). Three biological replicates were performed. Reference: [1] Rodan S.B. et al., Characterizatin of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res, 1987. 46(18): p. 4961-6. [2] Fogh, J., J.M. Fogh, and T. Orfeo, One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst, 1977. 59(1): p. 221-6. [3] Pautke, C., et al., Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts. Anticancer Res, 2004. 24(6): p. 3743-8. [4] Boskey, A.L. and R. Roy, Cell culture systems for studies of bone and tooth mineralization. Chem Rev, 2008. 108(11): p. 4716-33.[5] McQuillan, D.J., et al., Matrix Deposition by a Calcifying Human Osteogenic Sarcoma Cell Line (SAOS-2).Bone, April 1995. 16(4):p.415-26 [6] Billiau, A., et al., Human interferon: mass production in a newly established cell line, MG-63. Antimicrob Agents Chemother, 1977. 12(1): p. 11-5.

Quality control

Mineralization was verified using alizarin red staining of calcium at time point 0, 7, 14, 21 and 28 days. The cells were fixed with 4% paraformaldehyde (Sigma) for 10 minutes at room temperature, and rinsed with 1% PBS solution. The cells were stained using 1ml of 2% Alizarin Red (pH 4.2) for 5 minutes at room temperature. The wells were washed 3 times with dH20. The wells were extracted with 10% cetypyridium chloride and the extract used for quantification. The optical density was recorded using Multiskan ascent (Thermo) plate reader at a wavelength of 570nm. The image shows Saos-2 cells at 0, 1, 2, 3 and 4 weeks after initiation of calcification. Red staining shows calcium deposition. The graph shows quantification of calcium deposition in Saos-2 and MG63 cells treated with BGP and ascorbic acid and in untreated Saos-2 cells. Under treatment, Saos-2 cells deposit calcium in the matrix which MG63 do not and resemble the untreated Saos-2 cells. Pictures show the Saos-2 culture at 0 and 3 weeks where dark patches are caused by the deposition of calcium. Marker gene expression Key upregulated marker genes that indicate success of the mineralization: COL1A2 [7], PHOSPHO1 [8], IFITM5 [9],SOST [10], DMP1 [10]All these are markers of mineralisation in osteoblasts or the later stage osteocytes. All markers increased from Day1 or Day 4 and in general remained high for the remainder of the time course, indicating that calcification of the Saos-2 cultures involved induction of genes associated with miineralisation in vivo. Reference: [7] Rodan, G.A., et al., Diversity of osteoblastic phenotype. Ciba Found Symp, 1988. 136: p. 78-91. [8] Houston, B., et al., PHOSPHO1 - A novel phosphatase specifically expressed at sites of mineralisation in bone and cartilage. Bone, 2004. 34(4): p. 629-37. [9] Moffatt P., et al., Bril. A novel bone-specific modulator of mineralization, J Bone Miner Res, 2008. 23:1497-508 [10] Bonewald, L.F., The amazing osteocyte. J Bone Miner Res, 2011. 26(2): p. 229-38.

Profiled time course samples

Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples

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12662-134I7	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr00min	biol_rep1 (A1 T0)
12663-134I8	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr15min	biol_rep1 (A1 T1)
12664-134I9	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr30min	biol_rep1 (A1 T2)
12665-135A1	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr45min	biol_rep1 (A1 T3)
12666-135A2	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	01hr00min	biol_rep1 (A1 T4)
12667-135A3	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	01hr20min	biol_rep1 (A1 T5)
12668-135A4	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	01hr40min	biol_rep1 (A1 T6)
12669-135A5	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	02hr00min	biol_rep1 (A1 T7)
12670-135A6	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	02hr30min	biol_rep1 (A1 T8)
12671-135A7	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	03hr	biol_rep1 (A1 T9)
12672-135A8	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	04hr	biol_rep1 (A1 T10)
12673-135A9	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	08hr	biol_rep1 (A1 T11)
12674-135B1	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	24hr	biol_rep1 (A1 T12)
12675-135B2	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day04	biol_rep1 (A1 T13)
12676-135B3	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day07	biol_rep1 (A1 T14)
12677-135B4	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day14	biol_rep1 (A1 T15)
12678-135B5	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day21	biol_rep1 (A1 T16)
12679-135B6	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day28	biol_rep1 (A1 T17)
12760-136B6	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr00min	biol_rep2 (A2 T0)
12761-136B7	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr15min	biol_rep2 (A2 T1)
12762-136B8	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr30min	biol_rep2 (A2 T2)
12763-136B9	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr45min	biol_rep2 (A2 T3)
12764-136C1	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	01hr00min	biol_rep2 (A2 T4)
12765-136C2	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	01hr20min	biol_rep2 (A2 T5)
12766-136C3	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	01hr40min	biol_rep2 (A2 T6)
12767-136C4	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	02hr00min	biol_rep2 (A2 T7)
12768-136C5	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	02hr30min	biol_rep2 (A2 T8)
12769-136C6	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	03hr	biol_rep2 (A2 T9)
12770-136C7	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	04hr	biol_rep2 (A2 T10)
12771-136C8	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	08hr	biol_rep2 (A2 T11)
12772-136C9	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	24hr	biol_rep2 (A2 T12)
12773-136D1	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day04	biol_rep2 (A2 T13)
12774-136D2	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day07	biol_rep2 (A2 T14)
12775-136D3	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day14	biol_rep2 (A2 T15)
12776-136D4	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day21	biol_rep2 (A2 T16)
12777-136D5	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day28	biol_rep2 (A2 T17)
12858-137D5	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr00min	biol_rep3 (A3 T0)
12859-137D6	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr15min	biol_rep3 (A3 T1)
12860-137D7	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr30min	biol_rep3 (A3 T2)
12861-137D8	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	00hr45min	biol_rep3 (A3 T3)
12862-137D9	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	01hr00min	biol_rep3 (A3 T4)
12863-137E1	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	01hr20min	biol_rep3 (A3 T5)
12864-137E2	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	01hr40min	biol_rep3 (A3 T6)
12865-137E3	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	02hr00min	biol_rep3 (A3 T7)
12866-137E4	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	02hr30min	biol_rep3 (A3 T8)
12867-137E5	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	03hr	biol_rep3 (A3 T9)
12868-137E6	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	04hr	biol_rep3 (A3 T10)
12869-137E7	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	08hr	biol_rep3 (A3 T11)
12870-137E8	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	24hr	biol_rep3 (A3 T12)
12871-137E9	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day04	biol_rep3 (A3 T13)
12872-137F1	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day07	biol_rep3 (A3 T14)
12873-137F2	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day14	biol_rep3 (A3 T15)
12874-137F3	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day21	biol_rep3 (A3 T16)
12875-137F4	Saos-2 osteosarcoma treated with ascorbic acid and BGP to induce calcification	day28	biol_rep3 (A3 T17)