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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

Generation of MD-iPSCs. a Expression of pluripotent markers in MD-iPSCs. MD1- and MD2-iPSCs had normal morphologies and expressed pluripotent markers. Scale bars = 500 μm. b Transcriptional expression of transgenes such as OCT4, SOX2, cMYC, and KLF4 in MD-Fib, MD-inf, and MD-iPSCs. Transcription of the transgenes was detected only in infected MD1 and MD2 fibroblasts. c Teratoma formation of MD-iPSCs in immunodeficient mice. H&E staining was performed to detect diverse cell types and tissues (neural rosette, ectoderm; adipose tissue, mesoderm; and secretory gland, endoderm). Scale bars = 100 μm. d Epigenetic reprogramming in MD-iPSCs. Promoters of pluripotent genes were highly demethylated in MD1- and MD2-iPSCs compared with fibroblasts. Each circle represents the methylation status of single CpG dinucleotides: empty circle, unmethylated; filled circle, methylated. ALP alkaline phosphatase, iPSC induced pluripotent stem cell, Inf infected fibroblasts, Fib normal fibroblasts, MD1/2 Menkes disease patient 1/2, Tg transgene
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Fig1: Generation of MD-iPSCs. a Expression of pluripotent markers in MD-iPSCs. MD1- and MD2-iPSCs had normal morphologies and expressed pluripotent markers. Scale bars = 500 μm. b Transcriptional expression of transgenes such as OCT4, SOX2, cMYC, and KLF4 in MD-Fib, MD-inf, and MD-iPSCs. Transcription of the transgenes was detected only in infected MD1 and MD2 fibroblasts. c Teratoma formation of MD-iPSCs in immunodeficient mice. H&E staining was performed to detect diverse cell types and tissues (neural rosette, ectoderm; adipose tissue, mesoderm; and secretory gland, endoderm). Scale bars = 100 μm. d Epigenetic reprogramming in MD-iPSCs. Promoters of pluripotent genes were highly demethylated in MD1- and MD2-iPSCs compared with fibroblasts. Each circle represents the methylation status of single CpG dinucleotides: empty circle, unmethylated; filled circle, methylated. ALP alkaline phosphatase, iPSC induced pluripotent stem cell, Inf infected fibroblasts, Fib normal fibroblasts, MD1/2 Menkes disease patient 1/2, Tg transgene

Mentions: MD-iPSCs were generated from dermal fibroblasts of patients MD1 and MD2 by ectopic expression of OCT4, SOX2, cMYC, and KLF4. A MD1-iPSC clone, a MD2-iPSC clone, and WT-iPSCs were used in this study. MD1- and MD2-iPSCs had a typical morphology with tightly packed clusters and sharp boundaries and expressed pluripotency-associated marker genes (Fig. 1a and Additional file 4: Figure S3B). Exogenous genes were silenced after iPSC generation (Fig. 1b). MD1- and MD2-iPSCs differentiated into various cell types of the three germ layers in vitro (Additional file 4: Figure S3C) and formed teratomas after subcutaneous injection into nude mice (Fig. 1c). Furthermore, methylation of CpG dinucleotides in the promoter of OCT4, NANOG, REX1 genes were highly demethylated in MD1- and MD2-iPSCs compared with each patient’s fibroblasts (Fig. 1d), indicating successful epigenetic reprogramming. MD1-iPSCs had a normal karyotype, and MD2-iPSCs showed a polymorphic variant (pericentric inversion of chromosome 9) that was the same as the karyotype of the MD2 patient (Additional file 4: Figure S3D). Mutations of the ATP7A gene were confirmed again at the genomic and transcriptional levels in MD1- and MD2-iPSCs (Additional file 4: Figure S3E and S3F).Fig. 1


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)

Generation of MD-iPSCs. a Expression of pluripotent markers in MD-iPSCs. MD1- and MD2-iPSCs had normal morphologies and expressed pluripotent markers. Scale bars = 500 μm. b Transcriptional expression of transgenes such as OCT4, SOX2, cMYC, and KLF4 in MD-Fib, MD-inf, and MD-iPSCs. Transcription of the transgenes was detected only in infected MD1 and MD2 fibroblasts. c Teratoma formation of MD-iPSCs in immunodeficient mice. H&E staining was performed to detect diverse cell types and tissues (neural rosette, ectoderm; adipose tissue, mesoderm; and secretory gland, endoderm). Scale bars = 100 μm. d Epigenetic reprogramming in MD-iPSCs. Promoters of pluripotent genes were highly demethylated in MD1- and MD2-iPSCs compared with fibroblasts. Each circle represents the methylation status of single CpG dinucleotides: empty circle, unmethylated; filled circle, methylated. ALP alkaline phosphatase, iPSC induced pluripotent stem cell, Inf infected fibroblasts, Fib normal fibroblasts, MD1/2 Menkes disease patient 1/2, Tg transgene
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4562349&req=5

Fig1: Generation of MD-iPSCs. a Expression of pluripotent markers in MD-iPSCs. MD1- and MD2-iPSCs had normal morphologies and expressed pluripotent markers. Scale bars = 500 μm. b Transcriptional expression of transgenes such as OCT4, SOX2, cMYC, and KLF4 in MD-Fib, MD-inf, and MD-iPSCs. Transcription of the transgenes was detected only in infected MD1 and MD2 fibroblasts. c Teratoma formation of MD-iPSCs in immunodeficient mice. H&E staining was performed to detect diverse cell types and tissues (neural rosette, ectoderm; adipose tissue, mesoderm; and secretory gland, endoderm). Scale bars = 100 μm. d Epigenetic reprogramming in MD-iPSCs. Promoters of pluripotent genes were highly demethylated in MD1- and MD2-iPSCs compared with fibroblasts. Each circle represents the methylation status of single CpG dinucleotides: empty circle, unmethylated; filled circle, methylated. ALP alkaline phosphatase, iPSC induced pluripotent stem cell, Inf infected fibroblasts, Fib normal fibroblasts, MD1/2 Menkes disease patient 1/2, Tg transgene
Mentions: MD-iPSCs were generated from dermal fibroblasts of patients MD1 and MD2 by ectopic expression of OCT4, SOX2, cMYC, and KLF4. A MD1-iPSC clone, a MD2-iPSC clone, and WT-iPSCs were used in this study. MD1- and MD2-iPSCs had a typical morphology with tightly packed clusters and sharp boundaries and expressed pluripotency-associated marker genes (Fig. 1a and Additional file 4: Figure S3B). Exogenous genes were silenced after iPSC generation (Fig. 1b). MD1- and MD2-iPSCs differentiated into various cell types of the three germ layers in vitro (Additional file 4: Figure S3C) and formed teratomas after subcutaneous injection into nude mice (Fig. 1c). Furthermore, methylation of CpG dinucleotides in the promoter of OCT4, NANOG, REX1 genes were highly demethylated in MD1- and MD2-iPSCs compared with each patient’s fibroblasts (Fig. 1d), indicating successful epigenetic reprogramming. MD1-iPSCs had a normal karyotype, and MD2-iPSCs showed a polymorphic variant (pericentric inversion of chromosome 9) that was the same as the karyotype of the MD2 patient (Additional file 4: Figure S3D). Mutations of the ATP7A gene were confirmed again at the genomic and transcriptional levels in MD1- and MD2-iPSCs (Additional file 4: Figure S3E and S3F).Fig. 1

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