Congress Abstracts - Society for Developmental Biology

To address this issue, we have taken advantage of a lineage reprogramming approach in which fetal fibroblasts can be converted into
murine chondrocytes by retroviral-mediated expression of transcription factors Sox9, Klf4 and c-Myc and examined whether osteochondroprogenitors
could be formed during 14 days of the reprogramming using Sox9-EGFP knock-in mice as a reporter. We
obtained significant number of Sox9-EGFP/Runx2 positive nodules from day 10 to 14 upon culturing the transduced cells in mTeSR
medium. Consistent with this, real-time PCR analysis also revealed these transduced nodules express osteo-chondroprogenitorsassociated
genes such as Sox9, Runx2, Col2a1, Sox5 and Col1a1. In addition, the Sox9-EGFP/Runx2+ve cells can be differentiated
into chondrogenic and osteogenic lineages both in vivo and in vitro. Taken together, these findings suggest that Sox9, Klf4 and c-Myc
are sufficient to convert mouse fibroblasts into cells that closely resemble osteo-chondroprogenitors.
Program/Abstract # 525
(-)-epicatechin-induced differentiation of human bone marrow mesenchymal stem cells to osteoblasts
Diaz, Hector; Parra, Alberto; Mera, Elvia; Salas, Jose L; Acevedo, Leonardo F; Benitez, Gamaliel; Caceres, Julio R; Najera,
Nallely; Rubio, Angel I O; Palma, Icela; Ceballos, Guillermo M; Gutierrez, Gisela (Escuela Superior de Medicina, Mexico)
Introduction: Osteoporosis is a skeletal disease where bone mass decreases inducing bone fragility and susceptibility to fractures. In
Mexico (2006), the prevalence of osteoporosis in old women, without social security, was of 4.16% costs about 97 million US dollars.
The FDA has approved several drugs for its treatment, but almost all have important side effects. Some clinical studies have showed
that the intake of tea improve bone mineral density in older women, probably due to antioxidants, called flavonoids. It has been
proposed the use of Mesenchymal Stem Cell for elucidate the mechanisms of flavonoides effects, demonstrating that (-)-catechin,
epigallocatechin and (-)-epigallocatechin-3-gallate induced cell differentiation. In this work we study the effect of (-)-epicatechin in
cell differentiation of human Bone Marrow Mesenchymal Stem Cells (hBM-MSCs). Results: Flow cytometry; Positive markers of
hBM-MSCs were: CD105: 10.81%, CD73: 77.08% and CD90: 61.53% and negative markers were: CD34: 0.67%, CD45: 0.15%,
HLA-DR: 0.16%. RT-PCR: Expression of bmp2 was negative in hBM-MSCs without stimuli. All concentrations of OSTEO alone
induced the expression of bmp2. OSTEO concentrate with EPI at 1uM, 10uM and 100uM did not induce the expression of bmp2. The
combinations of OSTEO 50%/100uM EPI, OSTEO 50%/10uM EPI, OSTEO 50%/1uM EPI, OSTEO 30%/100uM EPI, OSTEO
30%/10uM EPI and OSTEO 30%/1uM EPI were positive to bmp2 in different ratios. All samples expressed ostenonectin and runx2.
Discussion: For study the osteogenic effects of EPI we chose hBM-MSCs as a model, because they are able to differentiate into bone,
heart or adipose tissue and other tissues. On the other hand, it was shown that bmp2 has the highest osteogenic activity among the bmp
family members, for this reason we decided to evaluate the effect of EPI on expression of bmp2. Our results demonstrated what BM-
MSCh treated with OSTEO in different concentrations induced the expression of bmp2, the same expression was observed when were
added EPI (1uM, 10uM and 100uM) in presence of OSTEO at 50% or 30%, coinciding with previous reports demonstrating the
osteogenic activity of some polifenols. We were unable of defect the expression of bmp2 in control negative, neither in the samples
treated with OSTEO concentrate in presence of EPI. We think that EPI has an antagonist effect with any compound associated to
differentiation effect of OSTEO. osteonectin was observed in all samples, but its expression was highest in hBM-MSCs treated with
concentrate OSTEO and less in OSTEO concentrations, in agreement with reports related about osteogenic differentiation in hBM-
MSCs, however they do not explain the basal expression of osteonectin in negative control (NC). We suggest that osteonectin is
involved in different pathways because osteonectin also plays important roles in development, wound healing, adipogenesis,
angiogenesis and others, not only in osteogenic induction of hBM-MSCs. We also evaluated the expression of runX2 on osteogenic
differentiation and we observed its expression in all samples. runX2 has been associated to cell cycle exit, this can explain the
expression of runX2 in our NC. Due to osteoblastogenesis is regulated by multiple signaling pathways, we think that canonical and
non-canonical Wnt signaling is the way through which (-)-epicatechin exerts its effect, this because we did not observed marked
difference in the expression of osteonectin and RunX2 but if in bmp2. Conclusions: We propose hBM-MSCs as a suitable model of
osteogenic differentiation. (-)-epicatechin alone at 1uM, 10uM and 100uM is able to induce the expression of bmp2. Although must be
investigate the pathways of osteogenic differentiation induced by EPI of hBM-MSCs and its inhibitory effect in presence of OSTEO.
These results support that (-)-epicatechin can be used as adjuvant in the treatment on osteogenic disease not only in Osteoporosis.
Program/Abstract # 526
PLZF: a master regulator of mesenchymal stem cell lineage commitment
Djouad, Farida; Tejedor, Gautier; Toupet, Karine; Maumus, Marie; Chuchana, Paul; Jorgensen, Christian; Noël, Danièle (INSERM,
France)
Objectives: Mesenchymal stem cells (MSC) are an attractive cell source for cartilage and bone tissue engineering given their ability to
differentiate into chondrocytes and osteoblasts. However, the common origin of these two specialized cell types raised the question
about the identification of an essential regulatory factor determining whether MSC will differentiate into chondrocyte or osteoblast.
Methods: Using affymetrix gene chips we performed a transcriptomic analysis of MSC differentiated into chondrocytes, osteoblasts
and adipocytes to identify a regulatory factor that signs MSC commitment: PLZF. We examined, in vitro, the multi-differentiation
potential of MSC overexpressing PLZF and, in vivo, in a mouse osteochondral defect model we investigated their reparative
potential. Results: We found out that PLZF induced expression in MSC is the mark of their commitment toward the 3 main lineages.
PLZF acts as an upstream regulator of both Sox9 and Runx2 and its overexpression in MSC improved chondrogenesis and
osteogenesis while inhibiting adipogenesis. In vivo, C3-PLZF implantation in mice full thickness osteochondral defects resulted in the
formation of a reparative tissue cartilage- and bone-like. Conclusions: Our findings demonstrate that PLZF is a key factor for MSC
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