Vet Comp Orthop Traumatol 2025; 38(04): A1-A35
DOI: 10.1055/s-0045-1810316
PODIUM ABSTRACTS

Comprehensive Evaluation of Canine and Human Bone Marrow-Derived Multipotent Stromal Cell Osteogenic Differentiation Using RNA-Sequencing

L. K. Dobson
1   Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States
,
C. N. Berns
1   Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States
,
B. W. Davis
2   Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States
,
C. A. Gregory
3   Department of Cell Biology and Genetics, College of Medicine, Texas A&M University, Bryan, Texas, United States
,
W. B. Saunders
1   Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States
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    Introduction: During in vitro osteogenic differentiation, human MSCs (hMSCs) readily differentiate in response to established methods. In contrast, canine MSCs (cMSCs) respond poorly and require bone morphogenic protein-2 (BMP-2) supplementation. In the present study, RNA sequencing (RNA-Seq) was used to assess the transcriptional activity of cMSCs and hMSCs during in vitro osteogenesis.

    Materials and Methods: Previously isolated and characterized canine and human bone marrow-MSCs were thawed and expanded to confluency. Early-stage osteogenesis was evaluated on day 7 using alkaline phosphatase (ALP) activity and late-stage differentiation was assessed on day 21 via Alizarin Red (ALZ) staining. Total RNA was isolated from cMSCs and hMSCs on days 0, 7, and 21. RNA-Seq libraries were prepared and sequenced targeting 50 million reads per library. Differentially expressed genes (DEGs) were identified between each time point, and between undifferentiated, control (day 0) and early-stage (day 7) or late-stage osteogenic (day 21) cell populations. Select DEGs were validated by qPCR.

    Results: Canine MSCs exhibited weak ALP activity without BMP-2, whereas hMSCs exhibited strong ALP activity in all conditions. RNA-Seq identified that cMSCs produce endogenous canine BMP-2 transcripts. However, cMSCs without BMP-2 supplementation exhibited reduced expression at critical early time points (day 7). Finally, cMSCs exhibited a higher differential expression of BMP antagonists as compared with hMSCs. Classical downstream markers of osteogenesis were identified in both MSCs.

    Discussion/Conclusion: RNA-Seq facilitated the identification of novel canine osteogenic transcriptional activity and gene expression and identified key differences between cMSCs and hMSCs. These results are critically important for canine bone tissue engineering.

    Acknowledgment

    None.


     

    Publication History

    Article published online:
    15 July 2025

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