Abdallah Basem M, Jensen Charlotte H, Gutierrez Gloria, Leslie Robert G Q, Jensen Thomas G, Kassem Moustapha
Department of Endocrinology, Univerity Hospital of Odense, Odense, Denmark.
J Bone Miner Res. 2004 May;19(5):841-52. doi: 10.1359/JBMR.040118. Epub 2004 Jan 19.
Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells.
Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs.
As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation.
Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPARgamma2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor -1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected.
During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.
Dlk-1/Pref-1被鉴定为人类骨骼干细胞分化的新型调节因子。Dlk1/Pref-1在骨组织和培养的成骨细胞中表达，其组成性过表达导致人骨髓基质细胞的成骨细胞和脂肪细胞分化受到抑制。
人类间充质干细胞（hMSC）向成骨细胞和脂肪细胞分化的分子调控机制尚不清楚。在本研究中，我们检测了δ样1/前脂肪细胞因子-1（Dlk1/Pref-1）在调节hMSC分化中的作用。
作为hMSC的模型，我们用全长人Dlk1/Pref-1 cDNA稳定转导端粒酶永生化hMSC（hMSC-TERT），并通过细胞化学染色、流式细胞术分析和实时PCR评估其对hMSC生长以及向成骨细胞或脂肪细胞分化的影响。采用体外颅骨器官培养试验来证实Dlk/Pref-1对骨形成的体外作用。
发现Dlk1/Pref-1在胎儿和成人骨组织、hMSC以及一些成骨细胞系中表达。使用含有人类Dlk1/Pref-1 cDNA的逆转录病毒载体构建了一个表达高水平Dlk1/Pref-1蛋白的细胞系（hMSC-dlk1）。Dlk1/Pref-1的过表达不影响hMSC的增殖率，但降低了其在体外形成成熟脂肪细胞、矿化基质以及在新生小鼠颅骨器官培养中形成新骨的能力。这些作用与脂肪细胞（脂肪细胞脂肪酸结合蛋白[aP2]、过氧化物酶体增殖物激活受体γ2[PPARγ2]和脂联素[APM1]）和成骨细胞分化（碱性磷酸酶[ALP]、I型胶原[Col1]和骨钙素[OC]）后期基因表达标志物的抑制相关。脂肪细胞（脂肪细胞决定和分化因子-1[ADD1]）和成骨细胞（核心结合因子/ runt相关结合因子2[Cbfa1/Runx2]）的谱系决定标志物未受影响。
在hMSC分化过程中，Dlk1/Pref-1通过抑制成熟成骨细胞和脂肪细胞的形成来维持双能祖细胞池的大小。
