Utilization of transgenic models in the evaluation of osteogenic differentiation of embryonic stem cells

Abstract

Previous studies reported that embryonic stem cells (ESCs) can be induced to differentiate into cells showing a mature osteoblastic phenotype by culturing them under osteo-inductive conditions. It is probable that osteogenic differentiation requires that ESCs undergo differentiation through an intermediary step involving a mesenchymal lineage precursor. Based on our previous studies indicating that adult mesenchymal progenitor cells express αSMA, we have generated ESCs from transgenic mice in which an αSMA promoter directs the expression of red fluorescent protein (RFP) to mesenchymal progenitor cells. To track the transition of ESC-derived MSCs into mature osteoblasts, we have utilized a bone-specific fragment of rat type I collagen promoter driving green fluorescent protein (Col2.3GFP). Following osteogenic induction in ESCs, we have observed expression of alkaline phosphatase and subsequent mineralization as detected by von Kossa staining. After one week of osteogenic induction, ESCs begin to express αSMARFP. This expression was localized to the peripheral area encircling a typical ESC colony. Nevertheless, these αSMARFP positive cells did not show activation of the Col2.3GFP promoter, even after 7 weeks of osteogenic differentiation in vitro. In contrast, Col2.3GFP expression was detected in vivo, in mineralized areas following teratoma formation. Our results indicate that detection of alkaline phosphatase activity and mineralization of ESCs cultured under osteogenic conditions is not sufficient to demonstrate osteogenic maturation. Our study indicates the utility of the promoter-visual transgene approach to assess the commitment and differentiation of ESCs into the osteoblast lineage.

Chromatin dynamics regulate mesenchymal stem cell lineage specification and differentiation to osteogenesis

“…Primary MSCs were isolated from femurs and tibias of 6 – 8 weeks old SMAA-mCherry mice [14], which were kindly provided by Drs I. Kalajzic and D. Rowe. The bone marrow was flushed into ascorbic acid-free MEM-alpha medium (Hyclone, Novato, CA, USA) supplemented with 1x Pen/Strep antibiotics (Invitrogen, Carlsbad, CA, USA).…”

The role of transduced bone marrow cells overexpressing BMP-2 in healing critical-sized defects in a mouse femur

“…A plasmid of a bicistronic LV vector (pLV-RFPch-BMP-2-cmyc) was constructed for gene expression of an RFPch coding sequence 25,26 and a cmyc epitope-tagged BMP-2 gene (BMP-2-cmyc) (see Figure 6). The RFPch and BMP-2-cmyc cDNAs were connected by T2A sequence during overlap extension PCR procedure.…”

Mineral trioxide aggregate improves healing response of periodontal tissue to injury in mice

“…RNA was extracted using Trizol reagent (Life Technologies) . Reverse transcription was performed using the iScript ™ cDNA Synthesis Kit (Bio‐Rad, Hercules, CA, USA).…”

“…RNA was extracted using Trizol reagent (Life Technologies). 24 Reverse transcription was performed using the iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). The expression of osteogenic genes osteocalcin (Oc) (Mm03413826_mH) and bone sialoprotein (Bsp) (Mm00492555_m1) was assessed by RT-qPCR using Taqman primerprobe sets (Life Technologies).…”