Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1.
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.


BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.
Abdallah
Basem M
BM
Department of Endocrinology, Univerity Hospital of Odense, Odense, Denmark.
Jensen
Charlotte H
CH
Gutierrez
Gloria
G
Leslie
Robert G Q
RG
Jensen
Thomas G
TG
Kassem
Moustapha
M
D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D000328:Adult; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001846:Bone Development; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D015536:Down-Regulation; D005260:Female; D005333:Fetus / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012886:Skull / Q000378:metabolism; D014161:Transduction, Genetic
D015415:Biomarkers; D002135:Calcium-Binding Proteins; C512941:DLK1 protein, human; C512925:Dlk1 protein, mouse; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D012097:Repressor Proteins