Craniometaphyseal Dysplasia Mutations in ANKH Negatively Affect Human Induced Pluripotent Stem Cell Differentiation into Osteoclasts

I-Ping Chen, Raj Luxmi, Jitendra Kanaujiya, Zhifang Hao, Ernst J Reichenberger, I-Ping Chen, Raj Luxmi, Jitendra Kanaujiya, Zhifang Hao, Ernst J Reichenberger

Abstract

We identified osteoclast defects in craniometaphyseal dysplasia (CMD) using an easy-to-use protocol for differentiating osteoclasts from human induced pluripotent stem cells (hiPSCs). CMD is a rare genetic bone disorder, characterized by life-long progressive thickening of craniofacial bones and abnormal shape of long bones. hiPSCs from CMD patients with an in-frame deletion of Phe377 or Ser375 in ANKH are more refractory to in vitro osteoclast differentiation than control hiPSCs. To exclude differentiation effects due to genetic variability, we generated isogenic hiPSCs, which have identical genetic background except for the ANKH mutation. Isogenic hiPSCs with ANKH mutations formed fewer osteoclasts, resorbed less bone, expressed lower levels of osteoclast marker genes, and showed decreased protein levels of ANKH and vacuolar proton pump v-ATP6v0d2. This proof-of-concept study demonstrates that efficient and reproducible differentiation of isogenic hiPSCs into osteoclasts is possible and a promising tool for investigating mechanisms of CMD or other osteoclast-related disorders.

Keywords: craniometaphyseal dysplasia; hiPSC differentiated osteoclasts; osteoclastogenesis; rare genetic bone disorder.

Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1
Figure 1
Differentiating Healthy Control hiPSCs into Osteoclasts (A) Schematic protocol of differentiating hiPSCs into osteoclasts. (B) Embryoid body (EB) formation and mesoderm gene expression. EBs cultured for 4 days (left panel). Scale bar, 200 μm. Expression of mesoderm marker genes in EBs cultured for 1, 2, 3, and 4 days by qPCR. ∗p < 0.05 by one-way ANOVA. Data presented are means ± SD. (C) Myelomonocytic cell expansion. Single cells released from EBs into suspension (top panel). Scale bar, 100 μm. Percentage of cells positive for hematopoietic cell surface markers CD14, CD43, and CD45 in cells released from 10, 13, 17, and 21 day adherent EBs by flow cytometry. Data presented are means ± SD. (D) TRAP+ osteoclasts differentiated from hiPSCs (left panel), resorption pits on bone chips (middle panel), and expression of OC marker genes, TRAP and CTSK by RT-PCR (right panel). HPRT served as internal control. Ctl1, control1; Ctl2, control2; 1w, 2w, 1 or 2 weeks in stage 3. Scale bar, 100 μm (left panel) and 200 μm (middle panel). Three independent experiments (three technical replicates per experiment) for each hiPSC line. Data were pooled from four wild-type hiPSC lines (hiPSCs from two healthy subjects, two hiPSC clones of each individual donor).
Figure 2
Figure 2
Osteoclasts Differentiated from Healthy Control and CMD hiPSCs (A) Comparison of control and CMD osteoclasts derived from hiPSCs. Numbers of TRAP+ multinucleated cells (nuclei ≥3) are shown (mean ± SD) in the left corners of the images. Different letters (a, b) indicate statistically significant difference (p TRAP, CATHEPSIN K, CALCITONIN RECEPTOR mRNA message in osteoclasts differentiated from CMD hiPSCs shown by qPCR. ∗p < 0.05 by two-way ANOVA. Data presented are means ± SD. Three independent experiments (technical replicates) were performed for each hiPSC line.
Figure 3
Figure 3
Osteoclasts Differentiated from Isogenic hiPSC with or without CMD ANKH Mutations (A) Electropherograms showing partial sequences of ANKH exon 9 with insertion of Phe377del into wild-type hiPSCs and correction of Ser375del ANKH in CMD hiPSCs. Blue arrows indicate silent mutations introduced to minimize recutting of a repaired site. (B) Decreased numbers of TRAP+ multinucleated osteoclasts formed from isogenic CMD mutant hiPSCs compared with wild-type controls. Numbers of TRAP+ multinucleated cells (nuclei ≥3) are shown (mean ± SD) in the left corners of the images. ∗ indicates statistical significance compared with the parental isogenic hiPSCs by Student’s t test. Scale bar, 100 μm. (C) Decreased resorbed area on bone chips by osteoclasts derived from isogenic CMD mutant hiPSCs compared with wild-type controls. ∗ on bone images (left panel) indicate resorption pits. Bar figures show the percentage of resorbed area (mean ± SD). Scale bar, 200 μm. ∗ indicates statistical significance compared with the parental isogenic hiPSCs by Student’s t test. (D) Decreased nuclei numbers in CMD hiPSC-derived osteoclasts (n = 70–156 cells per group). Yellow bar indicates the mean value of each group. Different letters (a, b, c) indicate statistically significant difference (p TRAP, CATHEPSIN K, CALCITONIN RECEPTOR, NFATc1 in osteoclasts differentiated from isogenic hiPSCs with a S375del mutation in ANKH by qPCR. ∗p < 0.05 by Student's t test. Data presented are means ± SD. (F) A representative Atp6v0d2 (40 kDa) immunoblot of hiPSCs (left panel) and hiPSC-differentiated osteoclasts (right panel) with or without S375del or F377del ANKH mutations. GAPDH served as loading control. Samples with wild-type and a S375del mutation in ANKH are isogenic hiPSCs. The F377del cell lysate is from patient CMD1 hiPSCs and hiPSC-derived osteoclasts. Numbers below indicate ratio of Atp6v0d2 to GAPDH. (G) A representative ANKH (52 kDa) immunoblot of undifferentiated hiPSCs (left panel) and hiPSC-derived osteoclasts (right panel) with or without S375del or F377del ANKH mutations. GAPDH served as loading control. Samples with wild-type and a S375del mutation are isogenic hiPSCs. The F377del cell lysate is from patient CMD1 hiPSCs and hiPSC-derived osteoclasts. Numbers below indicate the ratio of ANKH to GAPDH. Three independent experiments (technical replicates) were performed for each isogenic hiPSC line.

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