Mesencult osteogenic stimulatory kit

Manufactured by STEMCELL

Sourced in Canada

The MesenCult Osteogenic Stimulatory Kit is a laboratory product designed to support the differentiation of mesenchymal stem cells into osteoblasts, the cells responsible for bone formation.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Mesencult acf chondrogenic differentiation medium, by STEMCELL (1 mentions) Mesencult adipogenic differentiation medium, by STEMCELL (1 mentions) Stempro adipogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions) Stempro chondrogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Mesencult adipogenic differentiation kit, by STEMCELL (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) α mem, by Thermo Fisher Scientific (1 mentions) Mc3t3 e1, by American Type Culture Collection (1 mentions) Alizarin red staining solution, by Merck Group (1 mentions)

Close spelling variants of this product

MesenCult basal medium + mouse MesenCult osteogenic stimulatory kit MesenCult™ Osteogenic Stimulatory Kit (Mouse);

6 protocols using mesencult osteogenic stimulatory kit

Defining Multipotent Mesenchymal Stem Cells

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To confirm the multipotentiality of MSCs used in our research, experiments were performed in accordance with the minimal criteria for defining multipotent MSCs proposed by the International Society for Cellular Therapy (ISCT) (17 (link)).
The cultured plastic-adherent cells expressing the markers CD73 (sic passim; eBioScience, San Diego, CA, USA), CD90 and CD105 but not expressing the markers CD14, CD19, CD34, CD45 and HLA-DR were able to differentiate into adipocytes, osteoblasts and chondrocyte induced by products of Stem Cell Technologies (Vancouver, BC, Canada), which are respectively MesenCult^™ Adipogenic Differentiation Medium (human) and MesenCult^™ Osteogenic Stimulatory kit (human) and MesenCult^™−ACF Chondrogenic Differentiation Medium. Manufacturer's manuals were referred.
To determine whether the expanded MSC cultures maintained multipotency differentiation characteristics, we tested both HD-MSCs and AD-MSC for differentiation into adipogenic, osteogenic and chondrogenic cell lines. MSCs cultured in adipogenic differentiation medium showing lipid droplets were stained by Oil Red O staining. Osteogenic differentiation was demonstrated by calcium deposition, which was stained by Alizarin Red S. Histological sections of chondrogenic pellet were stained with Alcian Blue and Nuclear Fast Red. Undifferentiated AD-MSCs and HD-MSCs were used as controls.

Yang J., Zou S., Liao M, & Qu L. (2017). Transcriptome sequencing revealed candidate genes relevant to mesenchymal stem cells' role in aortic dissection patients. Molecular Medicine Reports, 17(1), 273-283.

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Osteogenic and Adipogenic Differentiation Assays

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Osteogenic differentiation was induced by using the MesenCult osteogenic stimulatory kit (Stem Cell Technologies, #05504). The medium was changed every 2 days. Alkaline phosphatase (Alp) activity was visualized after 14 days of differentiation as described previously³⁰ (link). Briefly, after washing with water, cells were fixed for 30 s with acetone citrate solution (60% acetone, 40% 1:50 diluted citrate solution [Sigma 854C-20 mL]), then washed, and stained by adding 300 μL per well of staining solution (dissolve 1 capsule Fast Violet [Sigma 851-10cap] in 48 mL H₂O + 2 mL Naphthol AS-mix [Sigma 855-20 mL]). After incubation for 30 min in the dark at room temperature, cells were washed with water, and bright-field images were taken. Expression of Alp, Osx, and Runx2 was measured by qPCR after 14 days of differentiation.
Adipogenic differentiation was induced by MesenCult adipogenic stimulatory kit (Stem Cell Technologies, #05507), with medium change every 4 days. Adipogenesis was assessed after 14 days of differentiation by Nile Red stain and quantifying gene expression of adiponectin, leptin, and Pparγ.

Lorenz J., Richter S., Kirstein A.S., Kolbig F., Nebe M., Schulze M., Kiess W., Spitzbarth I., Klöting N., Le Duc D., Baschant U, & Garten A. (2024). Pten knockout in mouse preosteoblasts leads to changes in bone turnover and strength. JBMR Plus, 8(3), ziad016.

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Trilineage Differentiation of iMSCs

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For induction of trilineage differentiation, iMSC_OA (passage 4) were harvested and reseeded in appropriate concentrations in plates containing MesenCult Osteogenic Stimulatory kit (Stem Cell Technologies, Vancouver, BC, Canada), StemPro Adipogenesis Differentiation kit (Gibco, BRL), StemPro Chondrogenesis Differentiation kit (Gibco, BRL) for osteogenesis, adipogenesis and chondrogenesis respectively and cultured as a monolayer, according to manufacturer’s instructions. Assessment of the differentiated cells was carried out with staining techniques. Alizarid Red and Alkaline Phosphatase stains assessed osteogenesis, Oil Red for adipogenesis and Hematoxylin and Eosin (H&E) for chondrogenesis.

Konteles V., Papathanasiou I., Tzetis M., Goussetis E., Trachana V., Mourmoura E., Balis C., Malizos K, & Tsezou A. (2023). Integration of Transcriptome and MicroRNA Profile Analysis of iMSCs Defines Their Rejuvenated State and Conveys Them into a Novel Resource for Cell Therapy in Osteoarthritis. Cells, 12(13), 1756.

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Isolation and Characterization of Human Mesenchymal Stem Cells

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huMSCs were kindly provided by the Stem Cell Laboratory of the Center of Reproductive Medicine (Tongji Medical College, Huazhong University of Science and Technology, China). Frozen huMSCs between P3 and P9 were freshly seeded in 10-cm culture dishes (1 × 10⁶ cells per dish) in Iscove’s modified Dulbecco’s medium (IMDM, Genom, China) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, USA), penicillin (100 U/ml), and streptomycin (100 mg/ml; Gibco, USA). Briefly, the phenotypes of huMSCs were specifically identified by FACS, and the osteogenic and adipogenic capacities of the mesenchymal stem cells were assessed with a MesenCult Osteogenic Stimulatory Kit (STEMCELL Technologies Inc., Canada) and a MesenCult Adipogenic Differentiation Kit (STEMCELL Technologies Inc., Canada). P3 to P9 were used for the experiments.

Wang L., Yu C., Chang T., Zhang M., Song S., Xiong C., Su P, & Xiang W. (2020). In situ repair abilities of human umbilical cord–derived mesenchymal stem cells and autocrosslinked hyaluronic acid gel complex in rhesus monkeys with intrauterine adhesion. Science Advances, 6(21), eaba6357.

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In Vitro Osteogenic Differentiation

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We further performed in-house validation experiments for the key findings of this study. The preosteoblastic cell line MC3T3-E1 was purchased from the American type culture collection (ATCC, USA) and was cultured in α-minimal essential medium (α-MEM; Gibco, Thermo Fisher Scientific, United States) with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific, United States) solution. Next, the cells were rinsed with PBS and the medium was replaced with osteogenic differentiation medium (MesenCult™ Osteogenic Stimulatory Kit, Stemcell). Cells were harvested at 0, 2, 3, 7, and 14 days after induction.

Wang S., Gong Y., Wang Z., Meng X., Luo Z., Papasian C.J., Greenbaum J., Li Y., Liang Q., Chen Y., Li X., Xiang Q., Zhang H., Liu Y., Cheng L., Hu Y., Tan L., Shen H., Xiao H, & Deng H. (2023). Regulon active landscape reveals cell development and functional state changes of human primary osteoblasts in vivo. Human Genomics, 17, 11.

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Quantifying Osteogenic Differentiation of Murine Stromal Cells

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Bone marrow stromal cells were flushed from the long bones C57BL6 mice with αMEM supplemented with 10% (v/v) fetal bovine serum, plated in 75 cm² flasks and cultured for 7 days. Medium was renewed every 2-3 days to remove hematopoietic cells in suspension. Primary osteoprogenitor cells were harvested at pre-confluence using trypsin solution and cell viability was determined by the Trypan Blue Exclusion assay. Then, cells were plated at a 5x10⁵ cells/mL, under osteogenic conditions (MesenCult Osteogenic Stimulatory Kit, Stem Cell Technologies, USA). Medium was renewed every 3 days. At day 12 of culture under osteogenic conditions, cells were fixed with 4% (v/v) paraformaldehyde for 10 minutes, at room temperature, and stained with alizarin-red staining solution (Sigma-Aldrich, USA) for 30 minutes, at room temperature with gentle shaking. Then, calcium deposits were eluted with 10% (w/v) cetypyridinium chloride in sodium phosphate (pH 7.0) and quantified at 570 nm.

Gomes A.C., Sousa D.M., Oliveira T.C., Fonseca Ó., Pinto R.J., Silvério D., Fernandes A.I., Moreira A.C., Silva T., Teles M.J., Pereira L., Saraiva M., Lamghari M, & Gomes M.S. (2023). Serum amyloid A proteins reduce bone mass during mycobacterial infections. Frontiers in Immunology, 14, 1168607.

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