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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 ?-smooth muscle actin (?SMA), we have generated ESCs from transgenic mice in which an ?SMA 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 (ALP) and subsequent mineralization as detected by von Kossa staining. After 1 week of osteogenic induction, ESCs begin to express ?SMARFP. This expression was localized to the peripheral area encircling a typical ESC colony. Nevertheless, these ?SMARFP 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 ALP 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.

SUBMITTER: Repic D

PROVIDER: S-EPMC3893759 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Repic Dario D Torreggiani Elena E Franceschetti Tiziana T Matthews Brya G BG Ivcevic Sanja S Lichtler Alexander C AC Grcevic Danka D Kalajzic Ivo I

Connective tissue research 20130826 4-5

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 α-smooth muscle actin (αSMA), we have generated ESCs fr ...[more]

PMID: 23782451

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Project description:BackgroundEmbryonic Sertoli cells (eSCs) play an important role in sex determination and in male gonad development which makes them a very useful cell type for therapeutic applications. However, the deriving mechanism of Sertoli cells has been unclear and challenging to create a large number of quality eSCs. Therefore, this study aimed to create the eSCs induced from mouse embryonic stem (mES) cells by regulating defined factors and to explore the relevant regulatory mechanism.MethodsSix inducing factors, Sry, Sox9, SF1, WT1, GATA4, and Dmrt1, were respectively transduced into mES cells by lentiviral infection according to the experimental design. The test groups were identified by development stage-specific markers, AMH, Emx2, SF1, and FasL, using flow cytometry. Induced eSCs were determined by FasL and AMH biomarkers under immunofluorescence, immunocytochemistry, and flow cytometry. Moreover, the pluripotency markers, gonad development-related markers, epithelial markers and mesenchymal markers in test groups were transcriptionally determined by qPCR.ResultsIn this study, the co-overexpression of all the six factors effectively produced a large population of eSCs from mES cells in 35 days of culturing. These eSCs were capable of forming tubular-like and ring-like structures with functional performance. The results of flow cytometry indicated that the upregulation of GATA4 and WT1 contributed to the growth of somatic cells in the coelomic epithelium regarded as the main progenitor cells of eSCs. Whereas, SF1 facilitated the development of eSC precursor cells, and Sry and Sox9 promoted the determination of male development. Moreover, the overexpression of Dmrt1 was essential for the maintenance of eSCs and some of their specific surface biomarkers such as FasL. The cellular morphology, biomarker identification, and transcriptomic analysis aided in exploring the regulatory mechanism of deriving eSCs from mES cells.ConclusionConclusively, we have elucidated a differentiation roadmap of eSCs derived from mES cells with a relevant regulatory mechanism. Through co-overexpression of all these six factors, a large population of eSCs was successfully induced occupying 24% of the whole cell population (1 × 105 cells/cm2). By adopting this approach, a mass of embryonic Sertoli cells can be generated for the purpose of co-culture technique, organ transplantation, gonadal developmental and sex determination researches.

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

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

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