Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells

Nihal AlMuraikhi; Dalia Ali; Aliah Alshanwani; Radhakrishnan Vishnubalaji; Muthurangan Manikandan; Muhammad Atteya; Abdulaziz Siyal; Musaad Alfayez; Abdullah Aldahmash; Moustapha Kassem; Nehad M. Alajez

doi:10.1186/s13287-018-1068-x

Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells

Nihal AlMuraikhi, Dalia Ali, Aliah Alshanwani, Radhakrishnan Vishnubalaji, Muthurangan Manikandan, Muhammad Atteya, Abdulaziz Siyal, Musaad Alfayez, Abdullah Aldahmash, Moustapha Kassem, Nehad M. Alajez

Morphogenesis and Differentiation Program

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Abstract

BACKGROUND: Better understanding of the signaling pathways that regulate human bone marrow stromal stem cell (hBMSC) differentiation into bone-forming osteoblasts is crucial for their clinical use in regenerative medicine. Chemical biology approaches using small molecules targeting specific signaling pathways are increasingly employed to manipulate stem cell differentiation fate. METHODS: We employed alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin R staining to assess mineralized matrix formation of cultured hBMSCs. Changes in gene expression were assessed using an Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. For in vivo ectopic bone formation experiments, hMSCs were mixed with hydroxyapatite-tricalcium phosphate granules and implanted subcutaneously into the dorsal surface of 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius Red staining were used to detect bone formation in vivo. RESULTS: We identified several compounds which inhibited osteoblastic differentiation of hMSCs. In particular, we identified ruxolitinib (INCB018424) (3 μM), an inhibitor of JAK-STAT signaling that inhibited osteoblastic differentiation and matrix mineralization of hMSCs in vitro and reduced ectopic bone formation in vivo. Global gene expression profiling of ruxolitinib-treated cells identified 847 upregulated and 822 downregulated mRNA transcripts, compared to vehicle-treated control cells. Bioinformatic analysis revealed differential regulation of multiple genetic pathways, including TGFβ and insulin signaling, endochondral ossification, and focal adhesion. CONCLUSIONS: We identified ruxolitinib as an important regulator of osteoblast differentiation of hMSCs. It is plausible that inhibition of osteoblast differentiation by ruxolitinib may represent a novel therapeutic strategy for the treatment of pathological conditions caused by accelerated osteoblast differentiation and mineralization.

Original language	English
Article number	319
Journal	Stem Cell Research & Therapy
Volume	9
Number of pages	10
ISSN	1757-6512
DOIs	https://doi.org/10.1186/s13287-018-1068-x
Publication status	Published - 2018

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Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cellsFinal published version, 5.27 MBLicence: CC BY

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AlMuraikhi, N., Ali, D., Alshanwani, A., Vishnubalaji, R., Manikandan, M., Atteya, M., Siyal, A., Alfayez, M., Aldahmash, A., Kassem, M., & Alajez, N. M. (2018). Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells. Stem Cell Research & Therapy, 9, Article 319. https://doi.org/10.1186/s13287-018-1068-x

@article{ddc73c47eaea4a56bc01d092bc9eecaa,

title = "Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells",

abstract = "BACKGROUND: Better understanding of the signaling pathways that regulate human bone marrow stromal stem cell (hBMSC) differentiation into bone-forming osteoblasts is crucial for their clinical use in regenerative medicine. Chemical biology approaches using small molecules targeting specific signaling pathways are increasingly employed to manipulate stem cell differentiation fate. METHODS: We employed alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin R staining to assess mineralized matrix formation of cultured hBMSCs. Changes in gene expression were assessed using an Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. For in vivo ectopic bone formation experiments, hMSCs were mixed with hydroxyapatite-tricalcium phosphate granules and implanted subcutaneously into the dorsal surface of 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius Red staining were used to detect bone formation in vivo. RESULTS: We identified several compounds which inhibited osteoblastic differentiation of hMSCs. In particular, we identified ruxolitinib (INCB018424) (3 μM), an inhibitor of JAK-STAT signaling that inhibited osteoblastic differentiation and matrix mineralization of hMSCs in vitro and reduced ectopic bone formation in vivo. Global gene expression profiling of ruxolitinib-treated cells identified 847 upregulated and 822 downregulated mRNA transcripts, compared to vehicle-treated control cells. Bioinformatic analysis revealed differential regulation of multiple genetic pathways, including TGFβ and insulin signaling, endochondral ossification, and focal adhesion. CONCLUSIONS: We identified ruxolitinib as an important regulator of osteoblast differentiation of hMSCs. It is plausible that inhibition of osteoblast differentiation by ruxolitinib may represent a novel therapeutic strategy for the treatment of pathological conditions caused by accelerated osteoblast differentiation and mineralization.",

author = "Nihal AlMuraikhi and Dalia Ali and Aliah Alshanwani and Radhakrishnan Vishnubalaji and Muthurangan Manikandan and Muhammad Atteya and Abdulaziz Siyal and Musaad Alfayez and Abdullah Aldahmash and Moustapha Kassem and Alajez, {Nehad M.}",

year = "2018",

doi = "10.1186/s13287-018-1068-x",

language = "English",

volume = "9",

journal = "Stem Cell Research & Therapy",

issn = "1757-6512",

publisher = "BioMed Central",

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TY - JOUR

T1 - Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells

AU - AlMuraikhi, Nihal

AU - Ali, Dalia

AU - Alshanwani, Aliah

AU - Vishnubalaji, Radhakrishnan

AU - Manikandan, Muthurangan

AU - Atteya, Muhammad

AU - Siyal, Abdulaziz

AU - Alfayez, Musaad

AU - Aldahmash, Abdullah

AU - Kassem, Moustapha

AU - Alajez, Nehad M.

PY - 2018

Y1 - 2018

N2 - BACKGROUND: Better understanding of the signaling pathways that regulate human bone marrow stromal stem cell (hBMSC) differentiation into bone-forming osteoblasts is crucial for their clinical use in regenerative medicine. Chemical biology approaches using small molecules targeting specific signaling pathways are increasingly employed to manipulate stem cell differentiation fate. METHODS: We employed alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin R staining to assess mineralized matrix formation of cultured hBMSCs. Changes in gene expression were assessed using an Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. For in vivo ectopic bone formation experiments, hMSCs were mixed with hydroxyapatite-tricalcium phosphate granules and implanted subcutaneously into the dorsal surface of 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius Red staining were used to detect bone formation in vivo. RESULTS: We identified several compounds which inhibited osteoblastic differentiation of hMSCs. In particular, we identified ruxolitinib (INCB018424) (3 μM), an inhibitor of JAK-STAT signaling that inhibited osteoblastic differentiation and matrix mineralization of hMSCs in vitro and reduced ectopic bone formation in vivo. Global gene expression profiling of ruxolitinib-treated cells identified 847 upregulated and 822 downregulated mRNA transcripts, compared to vehicle-treated control cells. Bioinformatic analysis revealed differential regulation of multiple genetic pathways, including TGFβ and insulin signaling, endochondral ossification, and focal adhesion. CONCLUSIONS: We identified ruxolitinib as an important regulator of osteoblast differentiation of hMSCs. It is plausible that inhibition of osteoblast differentiation by ruxolitinib may represent a novel therapeutic strategy for the treatment of pathological conditions caused by accelerated osteoblast differentiation and mineralization.

AB - BACKGROUND: Better understanding of the signaling pathways that regulate human bone marrow stromal stem cell (hBMSC) differentiation into bone-forming osteoblasts is crucial for their clinical use in regenerative medicine. Chemical biology approaches using small molecules targeting specific signaling pathways are increasingly employed to manipulate stem cell differentiation fate. METHODS: We employed alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin R staining to assess mineralized matrix formation of cultured hBMSCs. Changes in gene expression were assessed using an Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. For in vivo ectopic bone formation experiments, hMSCs were mixed with hydroxyapatite-tricalcium phosphate granules and implanted subcutaneously into the dorsal surface of 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius Red staining were used to detect bone formation in vivo. RESULTS: We identified several compounds which inhibited osteoblastic differentiation of hMSCs. In particular, we identified ruxolitinib (INCB018424) (3 μM), an inhibitor of JAK-STAT signaling that inhibited osteoblastic differentiation and matrix mineralization of hMSCs in vitro and reduced ectopic bone formation in vivo. Global gene expression profiling of ruxolitinib-treated cells identified 847 upregulated and 822 downregulated mRNA transcripts, compared to vehicle-treated control cells. Bioinformatic analysis revealed differential regulation of multiple genetic pathways, including TGFβ and insulin signaling, endochondral ossification, and focal adhesion. CONCLUSIONS: We identified ruxolitinib as an important regulator of osteoblast differentiation of hMSCs. It is plausible that inhibition of osteoblast differentiation by ruxolitinib may represent a novel therapeutic strategy for the treatment of pathological conditions caused by accelerated osteoblast differentiation and mineralization.

U2 - 10.1186/s13287-018-1068-x

DO - 10.1186/s13287-018-1068-x

M3 - Journal article

C2 - 30463599

AN - SCOPUS:85056933018

SN - 1757-6512

VL - 9

JO - Stem Cell Research & Therapy

JF - Stem Cell Research & Therapy

M1 - 319

ER -

Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells

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