Mitochondrial fusion by pharmacological manipulation impedes somatic cell reprogramming to pluripotency: new insight into the role of mitophagy in cell stemness

Abstract

Recent studies have suggested a pivotal role for autophagy in stem cell maintenance and differentiation. Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) has been also suggested to bio-energetically take advantage of mitochondrial autophagy (mitophagy). We have preliminary addressed how mitophagy might play a role in the regulation of induced pluripotency using mdivi-1 (for mitochondrial division inhibitor), a highly efficacious small molecule that selectively inhibits the self-assembly of DRP1, a member of the dynamin family of large GTPases that mediates mitochondrial fission. At mdivi-1 concentrations that rapidly induced the formation of mitochondrial net-like or collapsed perinuclear mitochondrial structures, we observed that the reprogramming efficiency of mouse embryonic fibroblasts transduced with the Yamanaka three-factor cocktail (OCT4, KLF4, and SOX2) is drastically reduced by more than 95%. Treatment of MEFs with mdivi-1 at the early stages of reprogramming before the appearance of iPSC colonies was sufficient to completely inhibit somatic cell reprogramming. Therefore, the observed effects on reprogramming efficiencies were due likely to the inhibition of the process of reprogramming itself and not to an impairment of iPSC colony survival or growth. Moreover, the typical morphology of established iPSC colonies with positive alkaline phosphatase staining was negatively affected by mdivi-1 exposure. In the presence of mdivi-1, the colony morphology of the iPSCs was lost, and they somewhat resembled fibroblasts. The alkaline phosphatase staining was also significantly reduced, a finding that is indicative of differentiation. Our current findings provide new insight into how mitochondrial division is integrated into the reprogramming factors-driven transcriptional network that specifies the unique pluripotency of stem cells.

Figure 1. Top. mdivi-1 blocks the machinery of mitochondrial fission

Mitochondria fission is crucially regulated by the activity of DRP1, which has a sequence homology with the GTPases dynamins that regulate vesicular trafficking and endocytosis. Although the exact molecular mechanism of DRP1 in the process of mitochondrial fission is still subject of debate, one of the well-accepted models is that DRP1 acts as a mechanoenzyme that self-assemble into spirals and onto lipid bilayers forming DRP1 decorated lipid tubes that undergo a large conformational change upon GTP addition resulting in membrane constriction and, therefore, mitochondrial division. DRP1 is a protein that is mainly distributed in the cytoplasm, but there is a fraction that localizes to specific points of the external mitochondrial membrane; these points mark the fission sites in dividing mitochondria. mdivi-1 [3-(2,4-Dichloro-5-methoxy-phenyl)-2-thioxo-1H-quinazolin-4-one] is a cell-permeable quinazolinone compound that inhibits DRP1 and effectively induces mitochondrial fusion into net-like structures (IC50 = 50 μmmol/L in COS cultures) in a reversible manner. mdivi-1 blocks DRP1 GTPase activity and self-assembly by an allosteric modulation-based mechanism. Bottom. mdivi-1 inhibits mitochondrial division in MEFs. MEFs were labeled with DsRed-Mito for the visualization of mitochondrial morphology. Untreated control cells showed a relatively tubular morphology that is maintained because mitochondrial fission and fusion occur in a balanced frequency, in contrast to the extremely long nets of interconnected mitochondria that collapse and aggregate after treatment with the DRP1 inhibitor mdivi-1.

Figure 2. (A) Mouse embryonic fibroblasts (MEFs) fail to reprogram into induced pluripotent stem cells (iPSCs) in the presence of the DRP1 inhibitor mdivi-1

Left. Early passage MEFs infected with retroviruses encoding OCT4, SOX2, and KLF4 (OSK) were cultured in ES medium in the continuous presence or absence of mdivi-1 (10 and 50 μmol/L), as specified. Top. The numbers of AP+ colonies were counted 14 days after the initial infection and were plotted for each condition relative to the controls (x-fold), as specified. The error bars indicate the SEM. Right. Phase-contrast microphotographs of representative MEFs transduced with OSK at different time-points during the reprogramming process in the absence or presence of continuous mdivi-1 (50 μmol/L). The arrows indicate emerging iPSC-like colonies. (B) DRP1 inactivation impedes early stem cell genetic reprogram-ming. The early passage MEFs infected with retroviruses encoding the OSK stemness factors were grown in ES medium in the intermittent presence or absence of mdivi-1 (50 μmol/L), as specified. Left. The numbers of AP+ colonies were counted 14 days after the initial infection and are plotted for each condition relative to the controls (x-fold), as specified. The error bars indicate the SEM. Right. Phase-contrast microphotographs of representative MEFs transduced with OSK at different time-points during the reprogramming process in the absence or presence of intermittent mdivi-1 (50 μmol/L), as specified. Arrows indicate emerging iPSC-like colonies.

Figure 3. DRP1 activity is required for the maintenance of iPSCs

Microphotographs show typical colony morphology of iPSCs with positive AP staining (red). In mdivi-1-treated cultures (5 days), normal undifferentiated phenotype with distinct iPSC colonies was not maintained. Remarkably, the colony morphology of the iPSCs was lost and differentiated cells with negative AP staining and some flattened fibroblast-like cells were formed after treatment with varying concentrations of the DRP1 inhibitor mdivi-1, as specified. Detection of AP activity, which is indicative of the non-differentiated state of iPSCs, was carried out using a commercial AP staining kit according to manufacturer's instructions.