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Impaired osteogenesis in Menkes disease-derived induced pluripotent stem cells.

Kim D, Choi J, Han KM, Lee BH, Choi JH, Yoo HW, Han YM - Stem Cell Res Ther (2015)

Bottom Line: Knockdown of ATP7A also impaired osteogenesis in WT-MSCs.Lysyl oxidase activity was also decreased in MD-MSCs during osteoblast differentiation.Our findings indicate that ATP7A dysfunction contributes to retardation in MSC development and impairs osteogenesis in MD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Korea Advanced Institute of Science Technology (KAIST), Daejeon, 305-701, Republic of Korea. kdkd86@kaist.ac.kr.

ABSTRACT

Introduction: Bone abnormalities, one of the primary manifestations of Menkes disease (MD), include a weakened bone matrix and low mineral density. However, the molecular and cellular mechanisms underlying these bone defects are poorly understood.

Methods: We present in vitro modeling for impaired osteogenesis in MD using human induced pluripotent stem cells (iPSCs) with a mutated ATP7A gene. MD-iPSC lines were generated from two patients harboring different mutations.

Results: The MD-iPSCs showed a remarkable retardation in CD105 expression with morphological anomalies during development to mesenchymal stem cells (MSCs) compared with wild-type (WT)-iPSCs. Interestingly, although prolonged culture enhanced CD105 expression, mature MD-MSCs presented with low alkaline phosphatase activity, reduced calcium deposition in the extracellular matrix, and downregulated osteoblast-specific genes during osteoblast differentiation in vitro. Knockdown of ATP7A also impaired osteogenesis in WT-MSCs. Lysyl oxidase activity was also decreased in MD-MSCs during osteoblast differentiation.

Conclusions: Our findings indicate that ATP7A dysfunction contributes to retardation in MSC development and impairs osteogenesis in MD.

No MeSH data available.


Related in: MedlinePlus

Impaired osteogenesis in MD-MSCs. a Representative images of ALP activity in WT- and MD-MSCs during OB differentiation. ALP activity was observed as red granules. D, days after osteogenesis induction. b Representative images of alizarin red S staining in WT- and MD-MSCs during OB differentiation. Alizarin red S staining presented as red granules. c Representative images of Von Kossa staining in WT- and MD-MSCs during OB differentiation. Von Kossa staining was observed as black dots. Scale bars = 500 μm. d Relative expression of osteogenic genes RUNX2, OPN, and OCN in MD-MSCs during osteogenesis. The data are presented as the mean ± SE (n = 3). e, f Effects of ATP7A knock-down on osteogenesis in WT-MSCs. e Representative images of ALP activity, alizarin red S staining, and Von Kossa staining after transfection of siRNAs targeting ATP7A gene. Scramble siRNA (si-SCR) was also transfected in WT-MSCs as a control. f Relative expression of RUNX2, OPN, and OCN after ATP7A knockdown. The data are represented as the mean ± SE (n = 3). *p < 0.05, **p < 0.01. ALP alkaline phosphatase, D day of differentiation, MD1/2 Menkes disease patient 1/2, WT wild type
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Fig4: Impaired osteogenesis in MD-MSCs. a Representative images of ALP activity in WT- and MD-MSCs during OB differentiation. ALP activity was observed as red granules. D, days after osteogenesis induction. b Representative images of alizarin red S staining in WT- and MD-MSCs during OB differentiation. Alizarin red S staining presented as red granules. c Representative images of Von Kossa staining in WT- and MD-MSCs during OB differentiation. Von Kossa staining was observed as black dots. Scale bars = 500 μm. d Relative expression of osteogenic genes RUNX2, OPN, and OCN in MD-MSCs during osteogenesis. The data are presented as the mean ± SE (n = 3). e, f Effects of ATP7A knock-down on osteogenesis in WT-MSCs. e Representative images of ALP activity, alizarin red S staining, and Von Kossa staining after transfection of siRNAs targeting ATP7A gene. Scramble siRNA (si-SCR) was also transfected in WT-MSCs as a control. f Relative expression of RUNX2, OPN, and OCN after ATP7A knockdown. The data are represented as the mean ± SE (n = 3). *p < 0.05, **p < 0.01. ALP alkaline phosphatase, D day of differentiation, MD1/2 Menkes disease patient 1/2, WT wild type

Mentions: To test the effect of ATP7A mutations on osteogenesis during bone formation, MD-MSCs were differentiated into OBs. To monitor OB differentiation, an ALP assay, alizarin red S staining, and Von Kossa staining were performed. In WT-MSCs, ALP activity was clearly observed at 7 days and reached the highest level 14 days after OB induction (Fig. 4a, upper panel). Intriguingly, ALP activity was barely detected at 7 days and was very weak even at 21 days in the MD1-MSCs during OB differentiation (Fig. 4a, middle panel). MD2-MSCs also showed low ALP activity in the process of OB differentiation (Fig. 4a, bottom panel). Thus, we found that MD-MSCs had aberrant ALP activity during OB differentiation. ALP activity is very important for calcium crystallization or mineralization during bone formation [27]. Therefore, we speculated that low ALP activity might lead to abnormal calcium deposition in MD-MSCs. As expected, MD1- and MD2-MSCs showed lower levels of calcium deposition during OB differentiation compared with WT-MSCs (Fig. 4b and c). These results imply that reduced ALP activity accounts for insufficient calcium crystallization or mineralization. Relative expression of the matrix-related genes OPN and OCN was significantly downregulated, whereas the expression of the osteogenic transcription factor RUNX2 was similar in MD-OBs compared with WT-OBs (Fig. 4d). To test whether ATP7A is associated with osteogenesis, a knockdown experiment using siRNA targeting ATP7A was carried out in WT-MSCs. In a preliminary experiment, siRNA use efficiently downregulated ATP7A transcript (Additional file 7: Figure S6). Knockdown of ATP7A in WT-MSCs also showed impaired osteogenesis with downregulation of osteogenic genes (Fig. 4e and f). Thus, knockdown of ATP7A in WT-MSCs recapitulated osteogenic impairments of MD-MSCs. Our results indicate that ATP7A plays an important role in bone formation.Fig. 4


Impaired osteogenesis in Menkes disease-derived induced pluripotent stem cells.

Kim D, Choi J, Han KM, Lee BH, Choi JH, Yoo HW, Han YM - Stem Cell Res Ther (2015)

Impaired osteogenesis in MD-MSCs. a Representative images of ALP activity in WT- and MD-MSCs during OB differentiation. ALP activity was observed as red granules. D, days after osteogenesis induction. b Representative images of alizarin red S staining in WT- and MD-MSCs during OB differentiation. Alizarin red S staining presented as red granules. c Representative images of Von Kossa staining in WT- and MD-MSCs during OB differentiation. Von Kossa staining was observed as black dots. Scale bars = 500 μm. d Relative expression of osteogenic genes RUNX2, OPN, and OCN in MD-MSCs during osteogenesis. The data are presented as the mean ± SE (n = 3). e, f Effects of ATP7A knock-down on osteogenesis in WT-MSCs. e Representative images of ALP activity, alizarin red S staining, and Von Kossa staining after transfection of siRNAs targeting ATP7A gene. Scramble siRNA (si-SCR) was also transfected in WT-MSCs as a control. f Relative expression of RUNX2, OPN, and OCN after ATP7A knockdown. The data are represented as the mean ± SE (n = 3). *p < 0.05, **p < 0.01. ALP alkaline phosphatase, D day of differentiation, MD1/2 Menkes disease patient 1/2, WT wild type
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Fig4: Impaired osteogenesis in MD-MSCs. a Representative images of ALP activity in WT- and MD-MSCs during OB differentiation. ALP activity was observed as red granules. D, days after osteogenesis induction. b Representative images of alizarin red S staining in WT- and MD-MSCs during OB differentiation. Alizarin red S staining presented as red granules. c Representative images of Von Kossa staining in WT- and MD-MSCs during OB differentiation. Von Kossa staining was observed as black dots. Scale bars = 500 μm. d Relative expression of osteogenic genes RUNX2, OPN, and OCN in MD-MSCs during osteogenesis. The data are presented as the mean ± SE (n = 3). e, f Effects of ATP7A knock-down on osteogenesis in WT-MSCs. e Representative images of ALP activity, alizarin red S staining, and Von Kossa staining after transfection of siRNAs targeting ATP7A gene. Scramble siRNA (si-SCR) was also transfected in WT-MSCs as a control. f Relative expression of RUNX2, OPN, and OCN after ATP7A knockdown. The data are represented as the mean ± SE (n = 3). *p < 0.05, **p < 0.01. ALP alkaline phosphatase, D day of differentiation, MD1/2 Menkes disease patient 1/2, WT wild type
Mentions: To test the effect of ATP7A mutations on osteogenesis during bone formation, MD-MSCs were differentiated into OBs. To monitor OB differentiation, an ALP assay, alizarin red S staining, and Von Kossa staining were performed. In WT-MSCs, ALP activity was clearly observed at 7 days and reached the highest level 14 days after OB induction (Fig. 4a, upper panel). Intriguingly, ALP activity was barely detected at 7 days and was very weak even at 21 days in the MD1-MSCs during OB differentiation (Fig. 4a, middle panel). MD2-MSCs also showed low ALP activity in the process of OB differentiation (Fig. 4a, bottom panel). Thus, we found that MD-MSCs had aberrant ALP activity during OB differentiation. ALP activity is very important for calcium crystallization or mineralization during bone formation [27]. Therefore, we speculated that low ALP activity might lead to abnormal calcium deposition in MD-MSCs. As expected, MD1- and MD2-MSCs showed lower levels of calcium deposition during OB differentiation compared with WT-MSCs (Fig. 4b and c). These results imply that reduced ALP activity accounts for insufficient calcium crystallization or mineralization. Relative expression of the matrix-related genes OPN and OCN was significantly downregulated, whereas the expression of the osteogenic transcription factor RUNX2 was similar in MD-OBs compared with WT-OBs (Fig. 4d). To test whether ATP7A is associated with osteogenesis, a knockdown experiment using siRNA targeting ATP7A was carried out in WT-MSCs. In a preliminary experiment, siRNA use efficiently downregulated ATP7A transcript (Additional file 7: Figure S6). Knockdown of ATP7A in WT-MSCs also showed impaired osteogenesis with downregulation of osteogenic genes (Fig. 4e and f). Thus, knockdown of ATP7A in WT-MSCs recapitulated osteogenic impairments of MD-MSCs. Our results indicate that ATP7A plays an important role in bone formation.Fig. 4

Bottom Line: Knockdown of ATP7A also impaired osteogenesis in WT-MSCs.Lysyl oxidase activity was also decreased in MD-MSCs during osteoblast differentiation.Our findings indicate that ATP7A dysfunction contributes to retardation in MSC development and impairs osteogenesis in MD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Korea Advanced Institute of Science Technology (KAIST), Daejeon, 305-701, Republic of Korea. kdkd86@kaist.ac.kr.

ABSTRACT

Introduction: Bone abnormalities, one of the primary manifestations of Menkes disease (MD), include a weakened bone matrix and low mineral density. However, the molecular and cellular mechanisms underlying these bone defects are poorly understood.

Methods: We present in vitro modeling for impaired osteogenesis in MD using human induced pluripotent stem cells (iPSCs) with a mutated ATP7A gene. MD-iPSC lines were generated from two patients harboring different mutations.

Results: The MD-iPSCs showed a remarkable retardation in CD105 expression with morphological anomalies during development to mesenchymal stem cells (MSCs) compared with wild-type (WT)-iPSCs. Interestingly, although prolonged culture enhanced CD105 expression, mature MD-MSCs presented with low alkaline phosphatase activity, reduced calcium deposition in the extracellular matrix, and downregulated osteoblast-specific genes during osteoblast differentiation in vitro. Knockdown of ATP7A also impaired osteogenesis in WT-MSCs. Lysyl oxidase activity was also decreased in MD-MSCs during osteoblast differentiation.

Conclusions: Our findings indicate that ATP7A dysfunction contributes to retardation in MSC development and impairs osteogenesis in MD.

No MeSH data available.


Related in: MedlinePlus