Ovine cord blood accommodates multipotent mesenchymal progenitor cells.
BACKGROUND
Stem cells derived from umbilical cord blood display mesenchymal multipotency and can differentiate into osteoblasts, chondroblasts and adipoblasts in vitro under defined stimuli. Although sheep have been used as experimental models for investigations on xenoreactivity after transplantation of stem cells isolated from human umbilical cord blood, the potential of ovine cord blood stem cells to differentiate has been examined to date.


METHODS
Mononuclear cells from the placentoms of 3 lambs were isolated via density gradient centrifugation and cultivated. After expansion up to 3 passages, the cells were stimulated to differentiate towards osteogenic (dexamethasone, ascorbic-acid-2-phosphate, beta-glycerolphosphate), chondrogenic (TGF-beta3, insulin, transferrin, selenium, dexamethasone, ascorbic-acid-2-phosphate) and adipogenic (indomethacine, insulin, 3-isobutyl-1-methylxanthine, dexamethasone) lines for 20 days. The cells were characterized morphologically by transmission and phase contrast light microscopy during lineage-specific stimulation. Immunocytochemistry and conventional stains were used to detect lineage-typical markers: fat vacuoles and peroxisome proliferation-acitivated receptor gamma2 (PPAR) served to detect adipoblasts, whereas osteopontin (OP) was used to characterize osteoblasts. A positive antibody reaction to collagen II and chondrogenic oligomeric protein (COMP) revealed the presence of chondroblasts.


RESULTS
The osteogenic line formed bone nodules, adipogenic cells developed lipid droplets and the cells of the chondrogenic line showed typical chondroblast-like morphology.


CONCLUSIONS
It was demonstrated that ovine mesenchymal stem cells, derived from umbilical cord blood (sheep unrestricted somatic stem cells, S-USSCs), can be isolated via gradient density centrifugation and expanded in vitro. Under lineage-specific stimulation, S-USSCs differentiated into osteo-, chondro- and adipoblasts with typical morphological characteristics. Significant quantitative differences between the stimulated and control groups in lineage-typical immunocytochemical markers verified these findings.
Jäger
Marcus
M
Department of Orthopaedics, Heinrich-Heine University Hospital Duesseldorf D-40225 Duesseldorf Germany. Jaeger@med.uni-duesseldorf.de
Bachmann
Radu
R
Scharfstädt
Axel
A
Krauspe
Rüdiger
R
D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects; D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects; D003239:Connective Tissue Cells / Q000166:cytology* / Q000187:drug effects; D005312:Fetal Blood / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects; D023421:Models, Animal; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects; D012756:Sheep / Q000097:blood*