PMC

(A) 143B ρ⁰ cells and R6G-treated 143B ρ⁺ cells showed very low intracellular ATP content. Restored cells (TM143B) from both conditions contained much higher ATP content. (B) Oxygen consumption rates show a pattern similar to that of intracellular ATP content. Restored cells from both conditions had much higher oxygen consumption rates.

(A) The cells survived after coculture showed genetic identities with 143B ρ⁰ cell nuclei. The boxed numbers and corresponding peaks represent locations of polymorphisms for each short tandem-repeat marker. (B) Since ρ⁰ cells did not have any detectable mtDNA, the mtDNA of the cells survived after coculture came solely from the MSCs. The hypervariable region sequences of the cells were identical with the MSCs. The arrows indicate sequence variations compared to the Cambridge reference sequence. (C) PCR-RFLP for 10394 Dde I and 10397 Alu I of mtDNA. The recuperated cells in the coculture experiment with R6G treatment to a cybrid harboring mtDNA with +10394 Dde I and +10397 Alu I were revealed to have mtDNAs with both −10394 Dde I and −10397 Alu I, both of which were identical to the MSCs.

(A) MSCs in DMEM had a fibroblast-like appearance. (B) MSCs survived in uridine-free media, (C) but they were sensitive to BrdU. (D) The 143B ρ⁰ cells in permissive media supplemented with uridine had an epithelial cell-like appearance. (E) The 143B ρ⁰ cells could not survive in uridine-free media, (F) but they were resistant to BrdU treatment. (G) At day 4 of Stage II coculture in uridine⁻ BrdU⁺ media, fibroblast-like MSCs could not proliferate and gradually died with a senescent appearance. (H) After 7∼10 days in the uridine⁻ BrdU⁺ media, we observed multiple foci of colonizing cells, which had distinct epithelial-like morphology of 143B cells on microscopic examination; (I) thereafter, they grew exponentially to reach confluence. All are magnified at ×40.

A scanning electron microscopic examination showed a marked difference in the morphology of the MSCs in each conditioned medium (magnified at ×1,000; scale bars represent 10 µm). The MSCs in their own growth medium (A); after 24 h (B) and 48 h (C) in conditioned media, which were prepared by treating 143B ρ⁰ cells with uridine-free media for 48 h. The MSCs showed an increased migration in the conditioned media without uridine supplementation (D). A transmission electron microscopic examination was done after two days of Stage I coculture (magnified at ×9,000; scale bars represent 1 µm). There were intercellular contacts between ρ⁰ and the MSCs by either cytoplasmic process (E) or broad surface contact (F).

Mitochondrial dysfunction was induced by long-term treatment of ethidium bromide (EtBr), which is known to remove mtDNA while sparing nuclear DNA, or by short-term treatment of rhodamine 6G (R6G), which tightly binds to the mitochondrial inner membrane and destroys the mitochondrial respiratory function. Gel image shows that the mtDNA was still present until five days after R6G treatment (inset). We cocultured MSCs with 143B-derived cells with severely compromised mitochondrial function (Δmt) in uridine⁻ BrdU⁻ media for five days (Stage I) and in uridine⁻ BrdU⁺ media thereafter (Stage II). Cybrid cells harboring either the A3243G or 4,977 bp deletion mutation were also cultured in a similar way, with slight modification. For details, refer to the section. The gray-colored box refers to the condition, which was expected to be fatal for the cells.