Preliminary Exploration of the Conditions and Inflammatory Model Construction for Bactrian Camels Induced Pluripotent Stem Cells into Osteoblasts in vitro
JIANG Di-Di, LI Zong-Shuai, GAO Kun, MA Tian, LIU Wan-Ruo, ZHANG Yong*
College of Veterinary Medicine/Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Gansu Agricultural University, Lanzhou 730070, China
Abstract：Stem cells have the ability of self-replication and multidirectional differentiation, which is of great significance in the field of regenerative medicine. As a unique species adapted to extreme environments, the induced pluripotent stem cells (iPSCs) of the bactrian camel (Camelus bactrianus) contain a special differentiation and control mechanism. This study centered on the optimal culture protocol for the osteogenic induced differentiation of bactrian camel induced pluripotent stem cells (bciPSCs). The optimal culture protocol for osteoblast induced differentiation were screened, and then explored the optimal drugs and concentrations for constructing inflammation models using osteoblast (OB) cells obtained from this protocol. The pluripotency and differentiation ability of bciPSCs kept in the laboratory were firstly determined by qRT-PCR, immunofluorescence and karyotyping; then bciPSCs were induced and differentiated by 2 direct and indirect induction methods and 2 different culture mediums. The OB obtained were tested by qRT-PCR, immunofluorescence, Western blot, etc. to determine the effect of osteogenic differentiation and compare the purity of the OB obtained to determine the optimal induction method; The cells obtained by the optimal method of osteogenic induction were selected to construct the inflammation model, and the OB were treated with lipopolysaccharide (LPS) and Interleukin-1β (IL-1β), and then screened the optimal drug and concentration by CCK-8, qRT-PCR, and Western blot. The results showed that compared with bactrian camel embryonic fibroblasts (CEF), the expression of pluripotency genes in laboratory-preserved bciPSCs was extremely significantly up-regulated (P<0.01). During the passaging process, the cells did not undergo morphological and pluripotency changes, indicating their ability to differentiate into three blastomeres and their suitability for subsequent studies. BciPSCs were directionally induced to differentiate via 2 methods: Direct induction and indirect induction. After induction, the expression of osteogenic marker genes—including osteocalcin bone gamma-carboxyglutamate protein (BGLAP), collagen typeⅠ α2 (COL1A2), runt-related transcription factor 2 (Runx2), and secreted phosphoprotein 1 (SPP1)—was significantly up-regulated (P<0.05). Additionally, the expression levels of osteopontin (OPN) and Runx2 were also significantly increased (P<0.05), and both were positively expressed in immunofluorescence staining and osteogenesis-specific staining. Through intergroup comparisons, the group of cells exhibiting the most pronounced osteogenic characteristics was selected for subsequent studies; The cell viability was significantly decreased after 24 h of LPS and IL-1β treatment (P<0.01), and the expression levels of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL6) and IL-1β were significantly increased (P<0.05), with 70 ng/mL of IL-1β up-regulated inflammatory factors more significantly, which could be used as a condition for constructing an inflammation model. This study provides biological materials and reference data for the study of osteoarthritis disease pathogenesis and drug screening.
蒋娣娣, 李宗帅, 高坤, 马甜, 刘婉若, 张勇. 双峰驼诱导多能干细胞体外分化为成骨细胞条件及炎症模型构建的初探[J]. 农业生物技术学报, 2025, 33(10): 2195-2208.
JIANG Di-Di, LI Zong-Shuai, GAO Kun, MA Tian, LIU Wan-Ruo, ZHANG Yong. Preliminary Exploration of the Conditions and Inflammatory Model Construction for Bactrian Camels Induced Pluripotent Stem Cells into Osteoblasts in vitro. 农业生物技术学报, 2025, 33(10): 2195-2208.
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