Int J Stem Cells.  2024 Aug;17(3):284-297. 10.15283/ijsc23151.

Impaired Osteogenesis in Human Induced Pluripotent Stem Cells with Acetaldehyde Dehydrogenase 2 Mutations

• Lim J
• Han H
• Jung SI
• Rim YA
• Ju JH

• KMID: 2558606
• DOI: http://doi.org/10.15283/ijsc23151

Abstract

Acetaldehyde dehydrogenase 2 (ALDH2) is the second enzyme involved in the breakdown of acetaldehyde into acetic acid during the process of alcohol metabolism. Roughly 40% of East Asians carry one or two ALDH2*2 alleles, and the presence of ALDH2 genetic mutations in individuals may affect the bone remodeling cycle owing to accumulation of acetaldehyde in the body. In this study, we investigated the effects of ALDH2 mutations on bone remodeling. In this study, we examined the effects of ALDH2 polymorphisms on in vitro osteogensis using human induced pluripotent stem cells (hiPSCs). We differentiated wild-type (ALDH2*1/*1-) and ALDH2*1/*2-genotyped hiPSCs into osteoblasts (OBs) and confirmed their OB characteristics. Acetaldehyde was administered to confirm the impact caused by the mutation during OB differentiation. Calcium deposits formed during osteogenesis were significantly decreased in ALDH2*1/*2 OBs. The expression of osteogenic markers were also decreased in acetaldehyde-treated OBs differentiated from the ALDH2*1/*2 hiPSCs. Furthermore, the impact of ALDH2 polymorphism and acetaldehyde-induced stress on inflammatory factors such as 4-hydroxynonenal and tumor necrosis factor α was confirmed. Our findings suggest that individuals with ALDH2 deficiency may face challenges in acetaldehyde breakdown, rendering them susceptible to disturbances in normal bone remodeling therefore, caution should be exercised regarding alcohol consumption. In this proof-of-concept study, we were able to suggest these findings as a result of a disease-in-a-dish concept using hiPSCs derived from individuals bearing a certain mutation. This study also shows the potential of patient-derived hiPSCs for disease modeling with a specific condition.

Keyword

Induced pluripotent stem cells; Aldehyde dehydrogenase; Acetaldehyde; Osteoblasts; Inflammation

Figure

• 

  Fig. 1 Characterization of ALDH2*1/*1-hiPSCs and ALDH2*1/*2-hiPSCs. (A) Schematic representation of hiPSC generation. (B) DNA sequencing results of ALDH2*1/*1- and ALDH2*1/*2-hiPSCs confirming ALDH2 mutation. (C) Measurement of ALDH2 activity in ALDH2*1/*1-hiPSCs (n=3) and ALDH2*1/*2-hiPSCs (n=3). (D) Bright field microscope images and alkaline phosphatase (AP) staining of ALDH2*1/*1- and ALDH2*1/*2-hiPSCs. Scale bar=200 μm. (E, F) Gene expression of pluripotency markers in ALDH2*1/*1-hiPSCs (n=3) and ALDH2*1/*2-hiPSCs (n=3), determined by RT-PCR and qRT-PCR. GAPDH was used as a loading control. Relative gene expression values were calculated using the delta-delta Ct (2(-ΔΔCt)) method. (G) Immunocytochemical staining for pluripotency markers OCT4, SOX2, KLF4, TRA-1-60, TRA-1-81, and SSEA4. Scale bar=100 μm. Data are presented as the mean±SEM. ##p<0.01 (t-test) indicates statistical significance. PBMC: peripheral blood mononuclear cell, ALDH2: acetaldehyde dehydrogenase 2, hiPSCs: human induced pluripotent stem cells, RT-PCR: real-time polymerase chain reaction, qRT-PCR: quantitative RT-PCR.

• 

  Fig. 2 Differentiation of ALDH2*1/*1 hiPSCs and ALDH2*1/*2 hiPSCs-derived OBs. (A) Schematic representation of the osteogenic differentiation process of ALDH2*1/*1-hiPSCs and ALDH2*1/*2-hiPSCs. (B) Bright-field microscopy images showing the mineralized cell morphology. Scale bar=40 μm. (C) Calcium deposition and hydroxyapatite quantification in ALDH2*1/*1-OBs and ALDH2*1/*2-OBs on day 7, visualized using Alizarin Red S and OsteoImage staining. Scale bar=40 μm. (D) Quantitative assessment of Alizarin Red S staining by absorbance measurement at 405 nm. (E) Verification of OsteoImage staining using the ImageJ software. (F) Quantified expression levels of pluripotent marker OCT4 and osteogenic marker genes, including RUNX2, COL1A1, and OCN, in ALDH2*1/*1-hiPSCs (n=3) and ALDH2*1/*2-hiPSCs (n=3) after 7 days of osteogenic differentiation. Data are presented as the mean±SEM. Statistical significance: #p<0.05, ##p<0.01, ###p<0.001 (t-test), **p<0.01 (two-way ANOVA). E8: Essential 8, ALDH2: acetaldehyde dehydrogenase 2, hiPSCs: human induced pluripotent stem cells, OBs: osteoblasts.

• 

  Fig. 3 Effects of acetaldehyde on OB differentiation. (A) Schematic representation of OB differentiation in ALDH2*1/*1-hiPSCs and ALDH2*1/*2-hiPSCs with or without 4 mM acetaldehyde. (B) Cell viability of the ALDH2*1/*1- and ALDH2*1/*2-hiPSCs derived OBs against acetaldehyde. (C) Measurement of ALDH2 activity in acetaldehyde-treated OBs. (D) Alizarin Red S staining confirming calcium deposition in OBs, measured by absorbance at 405 nm. (E) Quantitative data showing the expression of osteogenic markers, including RUNX2, COL1A1, OCN, and ALP, in OBs treated with or without 4 mM acetaldehyde for 3 days. (F) Protein expression of RUNX2, OCN, and ALDH2 in OBs with or without 4 mM acetaldehyde. GAPDH was used as a loading control. (G) Quantification of western blot results using the Adobe Photoshop. Data are presented as the mean±SEM. Statistical significance is indicated as #p<0.05, ##p<0.01, ###p<0.001 (t-test), *p<0.05, **p<0.01, ***p<0.001 (determined by two-way ANOVA). E8: Essential 8, ALDH2: acetaldehyde dehydrogenase 2, OBs: osteoblasts.

• 

  Fig. 4 Impact of acetaldehyde on ALDH2*1/*2-OBs: oxidative stress and altered TNFα, OPG, and RANKL levels. (A) Quantification of TNFα, OPG, and RANKL expression in OBs treated with or without 4 mM acetaldehyde. (B) Protein expression of 4HNE, TNFα, OPG, and RANKL in OBs with or without 4 mM acetaldehyde. GAPDH was used as a loading control. (C) Quantification of western blot results using the Adobe Photoshop. (D) Calculation of OPG and RANKL ratio based on quantified western blot results. (E) Measurement of TNFα in the cultrued medium of each group. Data are presented as the mean±SEM. #p<0.05, ##p<0.01, ###p<0.001 (t-test), *p<0.05, ***p<0.001 (two-way ANOVA) indicate statistical significance. TNFα: tumor necrosis factor α, OPG: osteoprotegerin, 4HNE: 4-Hydroxynonenal, OBs: osteoblasts.

Reference

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