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Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs).

Saeed H, Iqtedar M - J. Biomed. Sci. (2015)

Bottom Line: However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has not been reported before.To examine these effects, Terc (-/-) BMSCs (bone marrow stromal stem cells) were employed which exhibited reduced proliferation during in vitro osteogenesis along with increased population doubling time and level compared to wild type (WT) BMSCs during the normal culture.More profound changes were observed in genes engaged in extracellular matrix production: Col1a1, Col1a2, Mmp10, Serpinh1 and Col4a1.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Research Laboratory, KMEB, Department of Endocrinology and Metabolism, Odense University Hospital, Odense, Denmark. hamid.pharmacy@pu.edu.pk.

ABSTRACT

Background: Telomerase deficiency has been associated with inadequate differentiation of mesenchymal stem cells. However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has not been reported before.

Results: To examine these effects, Terc (-/-) BMSCs (bone marrow stromal stem cells) were employed which exhibited reduced proliferation during in vitro osteogenesis along with increased population doubling time and level compared to wild type (WT) BMSCs during the normal culture. Osteogenic super array at day 10 of osteoblast differentiation revealed that telomerase deficiency strongly affected the osteoblast commitment by down-regulating Runx2, Twist and Vdr - known transcription regulators of osteogenesis. Similarly, in Terc (-/-) BMSCs a marked reduction in other genes engaged in various phases of osteoblast differentiation were observed, such as Fgfr2 involved in bone mineralization, Phex and Dmp1 engaged in ossification, and Col11a1 and Col2a1 involved in cartilage condensation. A similar trend was observed for genes involved in osteoblast proliferation (Tgfb1, Fgfr2 and Pdgfa) and bone mineral metabolism (Col1a1, Col2a1, Col1a2 and Col11a1). More profound changes were observed in genes engaged in extracellular matrix production: Col1a1, Col1a2, Mmp10, Serpinh1 and Col4a1.

Conclusion: Taken together, these data suggest that telomerase deficiency causes impairment of BMSCs differentiation into osteoblasts affecting commitment, proliferation, matrix mineralization and maturation. Thus, modulating telomerase in BMSCs with advanced aging could improve BMSCs responsiveness towards osteoblast differentiation signals, optimal for osteoblast commitment, proliferation and maturation processes.

No MeSH data available.


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Telomerase deficient BMSCs exhibited impairedin vitroosteoblast differentiation and proliferation with reduced population doubling time and level. A) Short term proliferation of Terc-/ and WT BMSCs, equal number of cells were plated at day 0 and counted at day 1, 3 and 7. B) Long term proliferation of Terc-/- and WT BMSCs. Population doubling time (PDT); Terc-/- BMSCs PDT of 2.1 days compared to PDT of 1.89 days by WT BMSCs at early passage and later increased to 23 and 3.8 days, respectively. C)In vitro osteoblast differentiation of Terc-/- and WT BMSCs - according to the protocol described in material and methods. Alkaline phosphatase (ALP) (upper row) and Alizarin Red (lower row) staining at day 10 of osteoblast differentiation. Data are represented of means ± SD of three independent experiments. *p ≤ 0.05.
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Fig1: Telomerase deficient BMSCs exhibited impairedin vitroosteoblast differentiation and proliferation with reduced population doubling time and level. A) Short term proliferation of Terc-/ and WT BMSCs, equal number of cells were plated at day 0 and counted at day 1, 3 and 7. B) Long term proliferation of Terc-/- and WT BMSCs. Population doubling time (PDT); Terc-/- BMSCs PDT of 2.1 days compared to PDT of 1.89 days by WT BMSCs at early passage and later increased to 23 and 3.8 days, respectively. C)In vitro osteoblast differentiation of Terc-/- and WT BMSCs - according to the protocol described in material and methods. Alkaline phosphatase (ALP) (upper row) and Alizarin Red (lower row) staining at day 10 of osteoblast differentiation. Data are represented of means ± SD of three independent experiments. *p ≤ 0.05.

Mentions: We have previously shown that Terc-/- BMSCs exhibit stunted proliferation as evident by BrdU labelling during normal culture [12], while proliferation was not assessed during in vitro osteoblast differentiation. To examine the proliferation rate during in vitro osteogenesis, we differentiated WT and Terc-/- BMSCs into osteoblasts and counted cells at different time points. Terc-/- BMSCs demonstrated reduced proliferation rate compared to WT controls at day 3 and day 7 (Figure 1A). Moreover, population doubling analysis during normal culture revealed that Terc-/- BMSCs took more time to double with population doubling level (PDL) of 2.44 initially to PDL of 0.214 after 40 days compared to WT BMSC’s PDL of 2.64, which later dropped to PDL of 1.3 after 40 days (Figure 1B). Similarly, population doubling time (PDT) of Terc-/- BMSCs was 2.1 days, and later increased to 23 days at later passages, while PDT of WT BMSCs was around 1.89 days at early passage and 3.8 days around day 40 (Figure 1B). Also cumulative population doubling (CPD) stands at 8.62 for Terc-/- BMSCs at day 40 compared to 15.4 for WT BMSCs at day 40 (Figure 1B).Figure 1


Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs).

Saeed H, Iqtedar M - J. Biomed. Sci. (2015)

Telomerase deficient BMSCs exhibited impairedin vitroosteoblast differentiation and proliferation with reduced population doubling time and level. A) Short term proliferation of Terc-/ and WT BMSCs, equal number of cells were plated at day 0 and counted at day 1, 3 and 7. B) Long term proliferation of Terc-/- and WT BMSCs. Population doubling time (PDT); Terc-/- BMSCs PDT of 2.1 days compared to PDT of 1.89 days by WT BMSCs at early passage and later increased to 23 and 3.8 days, respectively. C)In vitro osteoblast differentiation of Terc-/- and WT BMSCs - according to the protocol described in material and methods. Alkaline phosphatase (ALP) (upper row) and Alizarin Red (lower row) staining at day 10 of osteoblast differentiation. Data are represented of means ± SD of three independent experiments. *p ≤ 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4318164&req=5

Fig1: Telomerase deficient BMSCs exhibited impairedin vitroosteoblast differentiation and proliferation with reduced population doubling time and level. A) Short term proliferation of Terc-/ and WT BMSCs, equal number of cells were plated at day 0 and counted at day 1, 3 and 7. B) Long term proliferation of Terc-/- and WT BMSCs. Population doubling time (PDT); Terc-/- BMSCs PDT of 2.1 days compared to PDT of 1.89 days by WT BMSCs at early passage and later increased to 23 and 3.8 days, respectively. C)In vitro osteoblast differentiation of Terc-/- and WT BMSCs - according to the protocol described in material and methods. Alkaline phosphatase (ALP) (upper row) and Alizarin Red (lower row) staining at day 10 of osteoblast differentiation. Data are represented of means ± SD of three independent experiments. *p ≤ 0.05.
Mentions: We have previously shown that Terc-/- BMSCs exhibit stunted proliferation as evident by BrdU labelling during normal culture [12], while proliferation was not assessed during in vitro osteoblast differentiation. To examine the proliferation rate during in vitro osteogenesis, we differentiated WT and Terc-/- BMSCs into osteoblasts and counted cells at different time points. Terc-/- BMSCs demonstrated reduced proliferation rate compared to WT controls at day 3 and day 7 (Figure 1A). Moreover, population doubling analysis during normal culture revealed that Terc-/- BMSCs took more time to double with population doubling level (PDL) of 2.44 initially to PDL of 0.214 after 40 days compared to WT BMSC’s PDL of 2.64, which later dropped to PDL of 1.3 after 40 days (Figure 1B). Similarly, population doubling time (PDT) of Terc-/- BMSCs was 2.1 days, and later increased to 23 days at later passages, while PDT of WT BMSCs was around 1.89 days at early passage and 3.8 days around day 40 (Figure 1B). Also cumulative population doubling (CPD) stands at 8.62 for Terc-/- BMSCs at day 40 compared to 15.4 for WT BMSCs at day 40 (Figure 1B).Figure 1

Bottom Line: However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has not been reported before.To examine these effects, Terc (-/-) BMSCs (bone marrow stromal stem cells) were employed which exhibited reduced proliferation during in vitro osteogenesis along with increased population doubling time and level compared to wild type (WT) BMSCs during the normal culture.More profound changes were observed in genes engaged in extracellular matrix production: Col1a1, Col1a2, Mmp10, Serpinh1 and Col4a1.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Research Laboratory, KMEB, Department of Endocrinology and Metabolism, Odense University Hospital, Odense, Denmark. hamid.pharmacy@pu.edu.pk.

ABSTRACT

Background: Telomerase deficiency has been associated with inadequate differentiation of mesenchymal stem cells. However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has not been reported before.

Results: To examine these effects, Terc (-/-) BMSCs (bone marrow stromal stem cells) were employed which exhibited reduced proliferation during in vitro osteogenesis along with increased population doubling time and level compared to wild type (WT) BMSCs during the normal culture. Osteogenic super array at day 10 of osteoblast differentiation revealed that telomerase deficiency strongly affected the osteoblast commitment by down-regulating Runx2, Twist and Vdr - known transcription regulators of osteogenesis. Similarly, in Terc (-/-) BMSCs a marked reduction in other genes engaged in various phases of osteoblast differentiation were observed, such as Fgfr2 involved in bone mineralization, Phex and Dmp1 engaged in ossification, and Col11a1 and Col2a1 involved in cartilage condensation. A similar trend was observed for genes involved in osteoblast proliferation (Tgfb1, Fgfr2 and Pdgfa) and bone mineral metabolism (Col1a1, Col2a1, Col1a2 and Col11a1). More profound changes were observed in genes engaged in extracellular matrix production: Col1a1, Col1a2, Mmp10, Serpinh1 and Col4a1.

Conclusion: Taken together, these data suggest that telomerase deficiency causes impairment of BMSCs differentiation into osteoblasts affecting commitment, proliferation, matrix mineralization and maturation. Thus, modulating telomerase in BMSCs with advanced aging could improve BMSCs responsiveness towards osteoblast differentiation signals, optimal for osteoblast commitment, proliferation and maturation processes.

No MeSH data available.


Related in: MedlinePlus