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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

Differentiation of MD-iPSCs into MSCs. a Schematic protocol for differentiation of MD-iPSCs into MSCs. b Formation of embryoid bodies (EBs) from MD-iPSCs. MD1- and MD2-iPSCs formed EBs with typical morphologies. Scale bars = 500 μm. c Representative images of iPSC-derived cells at 0, 1, 2, and 3 weeks after α-MEM induction. Retardation of MSC differentiation was observed both in the MD1- and MD2-iPSC groups compared with the WT-iPSC group. Scale bars = 500 μm. d Expression of MSC markers in MD-MSCs. MD1- and MD2-MSCs showed lower expression of CD105 compared with WT-MSCs. Gating strategy for this analysis is summarized in Additional file 3 (Figure S2). α-MEM α-minimum essential medium, D day of differentiation, DMEM Dulbecco’s modified Eagle’s medium, FBS fetal bovine serum, ITS ITS liquid media supplement, MD1/2 Menkes disease patient 1/2, SB SB431542, SR Knockout Serum Replacement, wk weeks, WT wild type
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Fig2: Differentiation of MD-iPSCs into MSCs. a Schematic protocol for differentiation of MD-iPSCs into MSCs. b Formation of embryoid bodies (EBs) from MD-iPSCs. MD1- and MD2-iPSCs formed EBs with typical morphologies. Scale bars = 500 μm. c Representative images of iPSC-derived cells at 0, 1, 2, and 3 weeks after α-MEM induction. Retardation of MSC differentiation was observed both in the MD1- and MD2-iPSC groups compared with the WT-iPSC group. Scale bars = 500 μm. d Expression of MSC markers in MD-MSCs. MD1- and MD2-MSCs showed lower expression of CD105 compared with WT-MSCs. Gating strategy for this analysis is summarized in Additional file 3 (Figure S2). α-MEM α-minimum essential medium, D day of differentiation, DMEM Dulbecco’s modified Eagle’s medium, FBS fetal bovine serum, ITS ITS liquid media supplement, MD1/2 Menkes disease patient 1/2, SB SB431542, SR Knockout Serum Replacement, wk weeks, WT wild type

Mentions: MSCs were differentiated from MD-iPSCs using an EB-based method (Fig. 2a). In this method, EBs were treated with an inhibitor of transforming growth factor-beta signaling, SB431542 (SB), to enhance differentiation into cardiac mesoderm and neuro-ectoderm lineages. Treatment with SB efficiently blocked SMAD2 phosphorylation in all WT, MD1 and MD2 EBs (Additional file 5: Figure S4A). SB-treated EBs were morphologically normal in the three groups (Fig. 2b), and showed upregulated expression of a cardiac mesodermal gene, cTNT, and a neuro-ectodermal gene, NEUROD1, compared to undifferentiated cells (Additional file 5: Figure S4B). After attachment of SB-treated EBs to fibronectin-coated dishes, development of mesenchymal cells appeared to be retarded in MD-iPSCs (MD1- and MD2-iPSCs) compared with that of WT-iPSCs. Mesenchymal morphology could be observed at 1 week after α-MEM induction in the WT-iPSC group, whereas mesenchymal morphology was observed at 2 weeks in the MD1- and MD2-iPSC groups (Fig. 2c). Differences in mesenchymal development between the WT- and MD-iPSC groups were also apparent after FACS analysis (Fig. 2d). CD105 expression in the MD1- and MD2-iPSC groups was relatively low by 3 weeks during mesenchymal development compared with the WT-iPSC group. The MD1- and MD2-iPSC groups also showed a slight reduction in CD90 expression after 1 week of α-MEM induction, but no difference was detected in the expression of other MSC markers, such as CD44 and CD73, between the WT- and MD-iPSC groups. These results demonstrate that the induction of MD-EBs towards the mesenchyme may be delayed in the early stage. Intriguingly, however, MD1- and MD2-MSCs achieved the normal MSC morphology and cell density of WT-MSCs after a long-term culture of 5 weeks (Fig. 3a). Furthermore, the expression level of CD105 in MD-MSCs was similar to that of WT-MSCs (Fig. 3b), indicating the complete maturation of MSCs. In addition, mature MD-MSCs had normal cellular functions, including cell growth (Fig. 3c), viability (Fig. 3d), apoptosis (Fig. 3e) and cell cycle (Fig. 3f) compared with WT-MSCs. Thus, ATP7A mutations did not influence fundamental cellular functions in MSCs. Genetic mutations of the ATP7A gene were confirmed again in the MD1- and MD2-MSCs (Additional file 6: Figure S5A and S5B, respectively). The ATP7A protein was not detected in either the MD1- or MD2-MSCs (Additional file 6: Figure S5C), and MD-MSCs exhibited higher levels of intracellular copper than WT-MSCs (Additional file 2: Table S4).Fig. 2


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)

Differentiation of MD-iPSCs into MSCs. a Schematic protocol for differentiation of MD-iPSCs into MSCs. b Formation of embryoid bodies (EBs) from MD-iPSCs. MD1- and MD2-iPSCs formed EBs with typical morphologies. Scale bars = 500 μm. c Representative images of iPSC-derived cells at 0, 1, 2, and 3 weeks after α-MEM induction. Retardation of MSC differentiation was observed both in the MD1- and MD2-iPSC groups compared with the WT-iPSC group. Scale bars = 500 μm. d Expression of MSC markers in MD-MSCs. MD1- and MD2-MSCs showed lower expression of CD105 compared with WT-MSCs. Gating strategy for this analysis is summarized in Additional file 3 (Figure S2). α-MEM α-minimum essential medium, D day of differentiation, DMEM Dulbecco’s modified Eagle’s medium, FBS fetal bovine serum, ITS ITS liquid media supplement, MD1/2 Menkes disease patient 1/2, SB SB431542, SR Knockout Serum Replacement, wk weeks, WT wild type
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
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Fig2: Differentiation of MD-iPSCs into MSCs. a Schematic protocol for differentiation of MD-iPSCs into MSCs. b Formation of embryoid bodies (EBs) from MD-iPSCs. MD1- and MD2-iPSCs formed EBs with typical morphologies. Scale bars = 500 μm. c Representative images of iPSC-derived cells at 0, 1, 2, and 3 weeks after α-MEM induction. Retardation of MSC differentiation was observed both in the MD1- and MD2-iPSC groups compared with the WT-iPSC group. Scale bars = 500 μm. d Expression of MSC markers in MD-MSCs. MD1- and MD2-MSCs showed lower expression of CD105 compared with WT-MSCs. Gating strategy for this analysis is summarized in Additional file 3 (Figure S2). α-MEM α-minimum essential medium, D day of differentiation, DMEM Dulbecco’s modified Eagle’s medium, FBS fetal bovine serum, ITS ITS liquid media supplement, MD1/2 Menkes disease patient 1/2, SB SB431542, SR Knockout Serum Replacement, wk weeks, WT wild type
Mentions: MSCs were differentiated from MD-iPSCs using an EB-based method (Fig. 2a). In this method, EBs were treated with an inhibitor of transforming growth factor-beta signaling, SB431542 (SB), to enhance differentiation into cardiac mesoderm and neuro-ectoderm lineages. Treatment with SB efficiently blocked SMAD2 phosphorylation in all WT, MD1 and MD2 EBs (Additional file 5: Figure S4A). SB-treated EBs were morphologically normal in the three groups (Fig. 2b), and showed upregulated expression of a cardiac mesodermal gene, cTNT, and a neuro-ectodermal gene, NEUROD1, compared to undifferentiated cells (Additional file 5: Figure S4B). After attachment of SB-treated EBs to fibronectin-coated dishes, development of mesenchymal cells appeared to be retarded in MD-iPSCs (MD1- and MD2-iPSCs) compared with that of WT-iPSCs. Mesenchymal morphology could be observed at 1 week after α-MEM induction in the WT-iPSC group, whereas mesenchymal morphology was observed at 2 weeks in the MD1- and MD2-iPSC groups (Fig. 2c). Differences in mesenchymal development between the WT- and MD-iPSC groups were also apparent after FACS analysis (Fig. 2d). CD105 expression in the MD1- and MD2-iPSC groups was relatively low by 3 weeks during mesenchymal development compared with the WT-iPSC group. The MD1- and MD2-iPSC groups also showed a slight reduction in CD90 expression after 1 week of α-MEM induction, but no difference was detected in the expression of other MSC markers, such as CD44 and CD73, between the WT- and MD-iPSC groups. These results demonstrate that the induction of MD-EBs towards the mesenchyme may be delayed in the early stage. Intriguingly, however, MD1- and MD2-MSCs achieved the normal MSC morphology and cell density of WT-MSCs after a long-term culture of 5 weeks (Fig. 3a). Furthermore, the expression level of CD105 in MD-MSCs was similar to that of WT-MSCs (Fig. 3b), indicating the complete maturation of MSCs. In addition, mature MD-MSCs had normal cellular functions, including cell growth (Fig. 3c), viability (Fig. 3d), apoptosis (Fig. 3e) and cell cycle (Fig. 3f) compared with WT-MSCs. Thus, ATP7A mutations did not influence fundamental cellular functions in MSCs. Genetic mutations of the ATP7A gene were confirmed again in the MD1- and MD2-MSCs (Additional file 6: Figure S5A and S5B, respectively). The ATP7A protein was not detected in either the MD1- or MD2-MSCs (Additional file 6: Figure S5C), and MD-MSCs exhibited higher levels of intracellular copper than WT-MSCs (Additional file 2: Table S4).Fig. 2

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