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, 52 (12), 689-694

Mitochondrial Genome Mutations in Mesenchymal Stem Cells Derived From Human Dental Induced Pluripotent Stem Cells

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Mitochondrial Genome Mutations in Mesenchymal Stem Cells Derived From Human Dental Induced Pluripotent Stem Cells

Jumi Park et al. BMB Rep.

Abstract

Ethical and safety issues have rendered mesenchymal stem cells (MSCs) popular candidates in regenerative medicine, but their therapeutic capacity is lower than that of induced pluripotent stem cells (iPSCs). This study compared original, dental tissue-derived MSCs with re-differentiated MSCs from iPSCs (iPS-MSCs). CD marker expression in iPS-MSCs was similar to original MSCs. iPS-MSCs expressed higher in pluripotent genes, but lower levels in mesodermal genes than MSCs. In addition, iPS-MSCs did not form teratomas. All iPSCs carried mtDNA mutations; some shared with original MSCs and others not previously detected therein. Shared mutations were synonymous, while novel mutations were non-synonymous or located on RNA-encoding genes. iPS-MSCs also harbored mtDNA mutations transmitted from iPSCs. Selected iPS-MSCs displayed lower mitochondrial respiration than original MSCs. In conclusion, screening for mtDNA mutations in iPSC lines for iPS-MSCs can identify mutation-free cell lines for therapeutic applications. [BMB Reports 2019; 52(12): 689-694].

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures

Fig. 1
Fig. 1
Characterization of iPS-MSCs and original MSCs. (A) Experimental design of the study. (B) Morphology of all iPSC lines similar to normal PSC morphology. (C) Characterizations of randomly selected iPSCs. OCT4 and SSEA4 were expressed in iPSC1, 2 and 6. (D) The teratoma formed in the mouse injected with iPSC1. Black arrows indicate three germ layers contained in teratoma. Scale bars = 500 μm. (E) Change in cell morphology to a spindle-like shape during differentiation of iPSC1 to MSCs. Scale bars = 500 μm. (F) Expression of CD markers in MSCs, iPSC1, and iPS1-MSCs. Both of MSCs and iPS1-MSCs were 100% positive in CD44. iPSC1 showed reduced expression of MSC positive markers. Negative MSCs markers, including CD34 and CD45, were expressed at less than 2% in all cell types. (G) Expression of pluripotency and mesodermal related genes in MSCs, iPSC1, and iPS1-MSCs. The level of the pluripotent gene OCT4 was higher in iPS1-MSCs than MSCs, while expression levels of the mesodermal genes NCAM, MSX2, and PDGF-β were lower in iPSC1 than MSCs. (H) Differentiation of MSCs and iPS1-MSCs into adipogenic, osteogenic, and chondrogenic lineages. (I) Teratoma formation by iPSC1 and iPS1-MSCs. No teratomas were observed in the mice injected with iPS1-MSCs. Black arrows indicate teratoma injection sites. The letters a, b, and c indicate significant (P < 0.05) differences among MSCs, iPSCs, and iPS-MSCs. Mean ± SEM. MSCs, mesenchymal stem cells; iPSCs, induced pluripotent stem cells; iPS-MSCs, iPSC-derived MSCs; NCAM, neural cell adhesion molecule; MSX2, Msh homeobox 2; PDGFβ, platelet-derived growth factor subunit B.
Fig. 2
Fig. 2
Mitochondrial genome integrity. (A) All iPSC clones harbored meaningful heteroplasmic (> 15%) mutations. (B) Venn diagram showing two mutations of MSCs shared by iPSC clones. (C) Mutations shared by MSCs and iPSC lines. The mt4988C>T in MSCs was present in five iPSC lines with various heteroplasmy, and the mt12528G>A in MSCs were present in two iPSC lines with 50% heteroplasmy and homoplasmy. (D) Stable mtDNA mutation during differentiation into MSCs. One homoplasmic mutation, detected in iPSC1 line, was transmitted to iPS1-MSCs. (E) mtDNA copy number in iPSCs was similar to hESCs. mtDNA copy number of original MSCs was similar in UCM-MSCs and higher than that in BM-MSCs. mtDNA copy number was similar between iPSCs and hESCs. Asterisks indicate statistically significant differences (*P < 0.05). Mean ± SEM. mtDNA, mitochondrial DNA; UCM-MSCs, umbilical cord matrix MSCs; BM-MSCs, bone marrow MSCs; hESCs, human embryonic stem cells.
Fig. 3
Fig. 3
Mitochondrial respiration was lower in iPS-MSCs than in MSCs. (A) Cell morphology and expression of CD44 in iPS-MSC lines. iPS-MSC lines were similar to original MSCs morphologically and displayed positive CD44 expression. The results on expression of CD44 in MSCs and iPS1-MSCs was duplicated in Fig. 1F. Scale bars = 250 μm. (B) Lower OCR levels in iPS-MSC lines than in MSCs. Mean ± SM. (C) The correlation of lower OCR to higher heteroplasmy in non-synonymous or RNA gene mutations. Heteroplasmy percentages refer to the sum of heteroplasmy of non-synonymous and RNA gene mutations in each cell line. OCR, oxygen consumption rates.

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