Disease-specific phenotypes in iPSC-derived neural stem cells with POLG mutations (2020)
Figure 6

Figure 6: POLG NSCs exhibited decreased TFAM level and mtDNA depletion, but not mtDNA deletion, consistent with iPSC-derived DA neurons.

A, B: Flow cytometric analysis of TOMM20 (A, n=3, technical replicates per clone for control; n=4, technical replicates per clone for WS5A and CP2A) and TFAM protein expression (B, n=3, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) in NSCs. Expressed as specific TFAM level (total TFAM/TOMM20).

C: Relative mtDNA copy number analyzed by RT-qPCR using primers and fluorogenic probes for regions of ND1 and nuclear gene APP in all NSCs (n=4, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=7, technical replicates per clone for CP2A).

D, E: Flow cytometric analysis of TOMM20 expression level (D, n=4, technical replicates per clone) and TFAM protein expression (E, n=4, technical replicates for control and WS5A; n=5, technical replicates for CP2A) in fibroblasts. Expressed as specific TFAM level (total TFAM/TOMM20).

F: Relative mtDNA copy number analyzed by RT-qPCR for regions of ND1 and nuclear gene APP in fibroblasts (n=3, technical replicates for control; n=4, technical replicates for WS5A and CP2A).

G: Long-PCR for detection of mtDNA deletions in NSCs and their parental fibroblasts from WS5A and CP2A patients.

H: Representative images of confocal microscopy with immunofluorescence labeling of DA neuron-specific marker TH (green) and neuron-specific marker MAP2 (red) for iPSC-derived DA neurons (scale bars, 25 µm). Nuclei are stained with DAPI (blue).

I: Histogram of the positive cell population stained with DA marker TH in iPSC-derived DA neurons using flow cytometric analysis.

J: Flow cytometric measurement of specific TFAM level (total TFAM/TOMM20) in iPSC-derived DA neurons from Detroit 551 control, WS5A and CP2A POLG patients (n=8, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=4, technical replicates per clone for CP2A).

K: Relative mtDNA copy number analyzed by RT-qPCR for regions of ND1 and nuclear gene APP in all iPSC-derived DA neurons (n=3, technical replicates per clone).

Data information: The data points in A-B represent NSCs generated from 3 different control iPSCs including 2 different clones from Detroit 551 control, 1 clone from control AG05836, 3 clones from WS5A patient iPSCs and 2 clones from CP2A patient iPSCs. Data points in C represent NSCs from 5 different controls, including 3 clones from Detroit 551 control, 1 clone from control AG05836, 1 clone from control CCD-1079Sk, 3 clones from WS5A patient iPSCs and 2 clones from CP2A patient iPSCs. The data points in J and K represent DA neurons generated from 3 clones from Detroit 551 control, 3 clones from WS5A patient iPSCs and 2 clones from CP2A patient iPSCs. Data are presented as mean ± SEM for the number of samples. Mann-Whitney U test was used for the data presented in A and J. Two-sided Student's t-test was used for the data presented in B-F and K. Significance is denoted for P values of less than 0.05. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.