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Microenvironment modulates osteogenic cell lineage commitment in differentiated embryonic stem cells.

Yamashita A, Nishikawa S, Rancourt DE - PLoS ONE (2010)

Bottom Line: Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best.In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification.In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT

Background: Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.

Methodology/principal findings: ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

Conclusions/significance: We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.

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Related in: MedlinePlus

Adipogenesis during ESC osteoblast differentiation in static culture.(A) Using Oil Red O staining, lipids with dark red appearance were observed in re-attached cells on day 10 of differentiation in static: KSR-basic (a), KSR-Dex (b), KSR-VD3 (c). Oil Red O co-staining with Alizarin Red S shows both lipid and calcification within re-attached cells in KSR-Dex (d) and KSR-VD3 (e); scale bars represent 20 µm. (B) Lipid accumulation per micro-mass spot was determined on day 10 of differentiation (a). Lipid accumulation was also normalized against DNA content to determine lipid synthesis per cell (b); data is expressed as mean ± SD (n = 3) per well. * represents a significant difference between undifferentiated and differentiated ESCs; P<0.05 with Student's t-test. (C) The expression of adipocyte-related mRNAs (PPARγ, aP2) was analyzed using real-time RT-PCR at 0 (ESCs), 7 and 14 days; data is expressed as means ± SD (n = 3) per point.
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pone-0009663-g002: Adipogenesis during ESC osteoblast differentiation in static culture.(A) Using Oil Red O staining, lipids with dark red appearance were observed in re-attached cells on day 10 of differentiation in static: KSR-basic (a), KSR-Dex (b), KSR-VD3 (c). Oil Red O co-staining with Alizarin Red S shows both lipid and calcification within re-attached cells in KSR-Dex (d) and KSR-VD3 (e); scale bars represent 20 µm. (B) Lipid accumulation per micro-mass spot was determined on day 10 of differentiation (a). Lipid accumulation was also normalized against DNA content to determine lipid synthesis per cell (b); data is expressed as mean ± SD (n = 3) per well. * represents a significant difference between undifferentiated and differentiated ESCs; P<0.05 with Student's t-test. (C) The expression of adipocyte-related mRNAs (PPARγ, aP2) was analyzed using real-time RT-PCR at 0 (ESCs), 7 and 14 days; data is expressed as means ± SD (n = 3) per point.

Mentions: As osteoblasts and adipocytes share a common progenitor, we investigated whether adipogenesis might be a disturbing factor that prevented efficient osteoblast differentiation. Adipocytes accumulates lipid within their cells during development. Using Oil Red O staining, we observed significant lipid accumulation in cells by day 10 of osteogenic differentiation KSR-Dex, KSR-VD3, compared to KSR-basic medium (Fig. 2A). Interestingly, when co-stained with Oil Red O and Alizarin Red S, many of the lipid containing cells also showed signs of mineralization. This lipid accumulation was confirmed biochemically using AdipoRed, where we observed a considerable increase in lipid accumulation in osteogenic conditions compared to undifferentiated controls (Fig. 2B). When we examined the expression of adipocyte-related genes using real-time RT-PCR, we observed that both adipocyte markers, PPARγ and aP2, were significantly up-regulated in osteoblast differentiations compared to undifferentiated controls (Fig. 2C).


Microenvironment modulates osteogenic cell lineage commitment in differentiated embryonic stem cells.

Yamashita A, Nishikawa S, Rancourt DE - PLoS ONE (2010)

Adipogenesis during ESC osteoblast differentiation in static culture.(A) Using Oil Red O staining, lipids with dark red appearance were observed in re-attached cells on day 10 of differentiation in static: KSR-basic (a), KSR-Dex (b), KSR-VD3 (c). Oil Red O co-staining with Alizarin Red S shows both lipid and calcification within re-attached cells in KSR-Dex (d) and KSR-VD3 (e); scale bars represent 20 µm. (B) Lipid accumulation per micro-mass spot was determined on day 10 of differentiation (a). Lipid accumulation was also normalized against DNA content to determine lipid synthesis per cell (b); data is expressed as mean ± SD (n = 3) per well. * represents a significant difference between undifferentiated and differentiated ESCs; P<0.05 with Student's t-test. (C) The expression of adipocyte-related mRNAs (PPARγ, aP2) was analyzed using real-time RT-PCR at 0 (ESCs), 7 and 14 days; data is expressed as means ± SD (n = 3) per point.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837348&req=5

pone-0009663-g002: Adipogenesis during ESC osteoblast differentiation in static culture.(A) Using Oil Red O staining, lipids with dark red appearance were observed in re-attached cells on day 10 of differentiation in static: KSR-basic (a), KSR-Dex (b), KSR-VD3 (c). Oil Red O co-staining with Alizarin Red S shows both lipid and calcification within re-attached cells in KSR-Dex (d) and KSR-VD3 (e); scale bars represent 20 µm. (B) Lipid accumulation per micro-mass spot was determined on day 10 of differentiation (a). Lipid accumulation was also normalized against DNA content to determine lipid synthesis per cell (b); data is expressed as mean ± SD (n = 3) per well. * represents a significant difference between undifferentiated and differentiated ESCs; P<0.05 with Student's t-test. (C) The expression of adipocyte-related mRNAs (PPARγ, aP2) was analyzed using real-time RT-PCR at 0 (ESCs), 7 and 14 days; data is expressed as means ± SD (n = 3) per point.
Mentions: As osteoblasts and adipocytes share a common progenitor, we investigated whether adipogenesis might be a disturbing factor that prevented efficient osteoblast differentiation. Adipocytes accumulates lipid within their cells during development. Using Oil Red O staining, we observed significant lipid accumulation in cells by day 10 of osteogenic differentiation KSR-Dex, KSR-VD3, compared to KSR-basic medium (Fig. 2A). Interestingly, when co-stained with Oil Red O and Alizarin Red S, many of the lipid containing cells also showed signs of mineralization. This lipid accumulation was confirmed biochemically using AdipoRed, where we observed a considerable increase in lipid accumulation in osteogenic conditions compared to undifferentiated controls (Fig. 2B). When we examined the expression of adipocyte-related genes using real-time RT-PCR, we observed that both adipocyte markers, PPARγ and aP2, were significantly up-regulated in osteoblast differentiations compared to undifferentiated controls (Fig. 2C).

Bottom Line: Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best.In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification.In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT

Background: Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.

Methodology/principal findings: ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

Conclusions/significance: We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.

Show MeSH
Related in: MedlinePlus