The novel and safe small molecule IM176OUT05 (IM) improves the acquisition and maintenance of stem cell pluripotency

The newly synthesized novel small molecule IM (N-1-(2-methyl) phenethyl biguanide hydrochloride), which has a biguanide core, is a hydrophobic cation (Fig. 1a). IM did not show cytotoxicity in cancer cells up to 100 μM in media with a normal glucose concentration (Supplementary Figure S1a), but the cells were susceptible to IM under glucose-deprived conditions in which cells rely on mitochondrial ETC for energy generation (Supplementary Figure S1b). Therefore, IM likely inhibits mitochondrial function, and indeed, IM reduced the OCR as a surrogate of mitochondrial ETC activity with an IC50 of 3.2 μM (Fig. 1b).

Fig. 1: IM176OUT05 (IM) improves the acquisition and maintenance of stem cell pluripotency. a Chemical structure of IM. b Dose-response curve of the inhibition of the ETC by IM in the A549 lung carcinoma cell line. Cells were treated with serially diluted IM for 24 h, and the basal OCR was measured using a Sea Horse XF Analyzer. c MEFs and d HFFs were reprogrammed into iPSCs with OSKM reprogramming factors in the presence of 10 nM IM or rotenone. Representative images of AP+ colonies are shown (top). The total number of AP+ colonies was counted on day 14 (D14, MEFs) or day 28 (D28, HFFs) of reprogramming (bottom). e mESCs cultured under the non-self-renewing conditions (−LIF) or self-renewing conditions (+LIF) were treated with 10 nM IM for 4 days. Representative images of AP+ colonies (top) and the total number of AP+ colonies are shown (bottom). f hESCs were maintained under the self-renewing condition (CM). hESCs cultured under non-self-renewing conditions (UM) were treated with 10 nM IM for 6 days. Representative images of AP+ colonies (top) are shown, and the relative AP expression was quantified by scanning densitometry (bottom). *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test) Full size image

As previously shown, a subtoxic dose of rotenone (an ETC complex I inhibitor) promoted efficient somatic cell reprogramming;23 therefore, we compared the effect of IM on iPSC generation with that of rotenone treatment (Fig. 1c). MEFs (Supplementary Figure S2a) and HFFs (Supplementary Figure S2b) were reprogrammed into iPSCs by introducing OSKM reprogramming factors. IM was applied at concentrations between 1 nM and 100 μM during the iPSC generation (Supplementary Figure S2) because IM did not show cellular toxicity in doses lower than 100 μM in MEFs (Supplementary Figure S1c) and OSKM-transduced MEFs (Supplementary Figure S1d). The reprogramming efficiency, which was determined by AP staining, was slightly increased but not significantly changed in the samples treated with the micromolar concentration range of IM (Supplementary Figure S2a and b). However, the number of AP+ colonies was substantially increased following treatment with IM in the nanomolar concentration range (Supplementary Figure S2a and b). IM (10 nM) and rotenone (10 nM) increased the reprogramming efficiency 9.8-fold and 8.5-fold in the MEFs (Fig. 1c) and 3.0-fold and 2.4-fold in the HFFs (Fig. 1d) over that in each untreated control, respectively. These promoting effects were also observed under conditions using three reprogramming factors without c-Myc (OSK) but were not observed under conditions without reprogramming factors, with single factors, or with OS only transduction (Supplementary Figure S2c). Additionally, the application of IM in the nanomolar concentration range was beneficial to maintain stemness in both mouse and human ESCs (Supplementary Figure S3a and b). IM (10 nM) supported the maintenance of the undifferentiated state of ESCs even under a non-self-renewing condition, namely −LIF for mESCs (Fig. 1e) and UM for hESCs (Fig. 1f). Thus, the optimal low dose of IM can improve the acquisition and maintenance of stem cell pluripotency during the generation process of both mouse and human iPSCs.

Because IM is a derivative of biguanide, we compared the effects of other known biguanides on stem cell pluripotency (Supplementary Figure S4). Metformin slightly increased the reprogramming efficiency at 10 nM as determined by AP staining and Oct4-GFP expression in OG2 (Oct4-GFP transgenic mice)-MEFs; however, metformin did not have an effect comparable to that of 10 nM IM (Supplementary Figure S4a). Phenformin, another biguanide, had no effect on the reprogramming efficiency of MEFs (Supplementary Figure S4b). Additionally, the maintenance of the stemness of mESCs (Supplementary Figure S4c) and hESCs (Supplementary Figure S4d) was slightly favorable following the application of metformin at the nanomolar concentration range, but this effect did not reach the level observed following the application of 10 nM IM.

IM facilitates the transition of glycolytic metabolism and induction of the expression of pluripotency-related genes

Subsequently, we examined the cellular changes following IM treatment during the early stage of iPSC generation (Fig. 2a). Compared with the untreated controls, IM treatment not only increased the reprogramming efficiency but also facilitated the reprogramming kinetics (Supplementary Figure S5). Following IM treatment, colonies started to appear earlier on day 7, and the size of the colonies was larger than that in the untreated controls on days 9 and 11 (Supplementary Figure S5a). The time course required for colony selection was also shortened by approximately 10 days after transduction in the IM-treated group but was delayed over 5 days in the untreated controls (Supplementary Figure S5b). Additionally, the numbers and size of the Nanog+ or SSEA1+ fluorescent clusters were increased in the IM-treated group compared with those in the untreated controls at an early time point of reprogramming (from 7 to 9 days after transduction) (Supplementary Figure S5c).

Fig. 2: IM inhibits mitochondrial OXPHOS and increases lactate production by inducing the expression of glycolysis- and pluripotency-related genes. a Schematic diagram of the reprogramming process. b OSKM-transduced MEFs were reseeded in poly-l-lysine-coated 96-well XF plates on D4. On the following day (D5), the medium was replaced with mESC medium and each indicated chemical. After 2 days (D7), the OCR was measured using an XFe96 Flux analyzer. An ATP synthase inhibitor (1.5 μM oligomycin, ETC complex V inhibitor), uncoupler (5 μM FCCP), and complex I inhibitor (0.5 μM rotenone) + complex III inhibitor (0.5 μM antimycin A) were sequentially added at each indicated time point. c Lactate production was measured in each treatment group on day 7 of reprogramming. d The ATP concentration was quantified in each treatment group on days 7 (D7) and 10 (D10) of reprogramming. e The expression of the indicated gene in each treatment group was measured using real-time PCR analysis on days 7 and 10 of reprogramming. β-Actin expression was used as an internal control. f ChIP assays were performed on day 7 of reprogramming with or without IM treatment. MEFs and miPSCs were used as negative/positive controls for each histone mark. Histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3) were precipitated, and the Nanog and Oct4 promoter loci were determined by real-time PCR. Input samples were used as a relative control. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test) Full size image

Furthermore, we investigated the changes in bioenergetics following IM treatment during the early stage of reprogramming. The basal OCRs (before inhibitor treatment) were slightly inhibited by treatment with 10 nM or 100 nM IM compared with those in the DMSO controls on day 7 of reprogramming, and the maximal OCRs were considerably inhibited by IM treatment (Fig. 2b). Rotenone potently reduced both the basal and maximal OCR in a dose-dependent manner (Fig. 2b). The basal ECAR was inversely correlated with the OCR, and IM and rotenone treatment clearly increased the acidification of the medium, revealing increased glycolytic activity (Supplementary Figure S5d). The intracellular production of lactate directly shows that, compared with the untreated controls on day 7, the end-product of glycolysis was increased in the cells treated with 10 nM and 100 nM IM or rotenone (Fig. 2c). The cellular ATP content was reduced by IM treatment on day 7 as a decrease in the energy-generating mitochondrial function accompanying OCR reduction (Fig. 2d). However, the ATP content was recovered on day 10 due to compensation by increased glycolysis, which is an alternative energy-generating pathway that overcomes energy deficits, in the IM-treated cells (Fig. 2d). Therefore, these results show that 10 nM IM slightly interfered with oxygen consumption but was sufficient to accelerate the glycolytic metabolic transition. The changes in gene expression were correspondently observed; compared with each control, the expression levels of NDUS3 (an ETC complex I enzyme) and ATP5B (an ETC complex V enzyme) were downregulated in the IM-treated cells on day 7 but were recovered on day 10 (Fig. 2e). By contrast, the expression levels of HK2 and LDHA (major enzymes of glycolysis) were upregulated in IM-treated cells on day 7, and further induction was found during the progression of reprogramming on day 10. Notably, the expression levels of Nanog and Rex1 (pluripotency-related genes) were potently upregulated in IM-treated cells during the early stages of reprogramming and was saturated in all groups on day 10. More evidently, the occupancies of the active histone mark (H3K4me3) and repressive histone mark (H3K27me3 and H3K9me2) were enriched and decreased, respectively, at the Nanog and Oct4 loci following IM treatment on day 7 (Fig. 2f and Supplementary Figure S5e). These observations suggest that IM can promote glycolytic metabolic reprogramming and pluripotency induction during the early stage of the cellular reprogramming process. Thus, we explored the effect of the application of IM on tissue regeneration, specifically hair follicle regrowth.

IM promotes hair regrowth in mice

Preliminarily, we tested whether IM could enhance hair regrowth in mice without toxicity or other side effects (Supplementary Figure S6). The hair cycle was synchronized by the depilation of telogen phase hairs from 7-week-old C57BL/6 mice1, and various concentrations of IM were topically applied daily to the dorsal skin of the mice (Supplementary Figure S6). On day 9, dramatic changes were observed in the area treated with 1% IM, and black pigmentation and hair growth were robustly detected. Hair regrowth was not observed in the control areas or areas treated with 0%, 0.1%, or 0.5% IM, although pigmentation developed on day 11. Next, we compared the abilities of IM and minoxidil, which is an approved drug to treat hair loss, to promote hair regrowth in both male and female mice. IM treatment had a strong promoting effect on hair regrowth, especially in female mice (Fig. 3). By day 8, the skin color was clearly distinguished with darkening in IM-treated mice compared with that in either the control or minoxidil-treated mice. By day 10, hair regrowth was distinctly promoted by IM treatment (Fig. 3) and was reproducibly observed in an independent group of female mice (Supplementary Figure S7). These phenomena were similarly observed in the male mice, but the effect of IM was comparable to that of minoxidil in the male mice (Supplementary Figure S8). Rashes and scars were detected in a few mice after depilation (Supplementary Figure S8b, D0); however, adverse effects, such as skin problems induced by IM treatment, were not observed in any of the animals in the in vivo experiments.

Fig. 3: IM promotes hair regrowth in mice. C57BL/6 mice in the telogen phase (7 weeks old) were depilated. Placebo control (−), 1% IM, or 1% minoxidil was topically applied daily to the dorsal skin. Representative photos of mice showing skin color darkness and hair regrowth on days 8, 10, 12, and 16 (left). The level of pigmentation was quantified by the intensity of the darkness of the back skin in the same area (right). *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test) Full size image

IM facilitates the cycle of hair follicle regeneration in mice

The tissues were histomorphometrically analyzed based on the classification by Chase1,30 to show that IM stimulated the progression of the hair follicle cycle (Fig. 4a). On day 20, most hair follicles in the control mice were quantitatively in the anagen III stage, but most hair follicles in the IM-treated mice were at a later stage of anagen, mainly anagen stages V and VI, in longitudinal sections (Fig. 4b). Additionally, the number of hair follicles was obviously increased in the IM-treated mice compared with that in the control mice in transverse sections on day 7 (Fig. 4c), and the number in the IM-treated or minoxidil-treated mice was greater than that in the control mice on day 20 (Fig. 4d). Moreover, keratin 15 (K15), which is a marker of hair follicle stem cells31, was strongly expressed in the hair follicle bulge area of the IM-treated mice compared with that in either the control or minoxidil-treated mice on day 7 (Fig. 5a). The expression of β-catenin, which mediates hair follicle regeneration32, was also increased in the IM-treated mice on day 7. At this time point, Ki67-positive proliferating cells were already apparent in the IM-treated mice, indicating that hair follicle cycling and further expansion of proliferating progenitors were augmented by IM (Fig. 5a). The K15+/β-catenin+ populations were quantified by FACS analysis, and these populations occupied 3.2%, 11.7%, and 6.0% of single cells in the skin of the control and IM-treated, and minoxidil-treated mice, respectively (Fig. 5b). A 3.7-fold increase was observed in the IM-treated mice over the control mice (Fig. 5b). By day 20, K15 and β-catenin were strongly detected in all groups of mice (Fig. 5c), and the K15+/β-catenin+ populations were represented at over 24% in all groups (Fig. 5d). Shh, which is another essential factor for hair follicle development33, was clearly detected in the IM-treated or minoxidil-treated mice (Fig. 5c). The Ki67+/Shh+ populations accounted for <5% in all groups of mice on day 7 (Fig. 5b), but this percentage was significantly increased on day 20 by 13.8%, 36.7%, and 34% in the single cells in the skin of the control, IM-treated, or minoxidil-treated mice, respectively (Fig. 5d). On day 20, the Ki67+/Shh+ populations were increased by 2.7-fold in the IM-treated mice over those in the control mice (Fig. 5d).

Fig. 4: IM facilitates the cycle of hair follicle regeneration in mice. a Representative images of H&E-stained longitudinal sections of each treated mouse skin on days 0 and 20 after depilation. b Progression of the hair follicle cycle on day 20 was quantitatively evaluated. Individual hair follicles were classified based on the classification by Chase. c Representative images of H&E-stained transverse sections of each treated mouse skin on days 0, 7, 14, and 20 after depilation. d Hair follicles were counted on days 7 and 20. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test). Scale bar = 200 μm Full size image

Fig. 5: IM promotes the expression of hair follicle cycling-related markers. a Immunohistochemistry of K15, β-catenin, and Ki67 in each treated mouse skin on day 7. Enlarged images of K15 in the top panels (dotted line) are shown in the second panels. b FACS analysis of K15/β-catenin and Ki67/Shh in single cells of each treated mouse skin on day 7. c Immunohistochemistry of K15, β-catenin, and Shh in each treated mouse skin on day 20. d FACS analysis of K15/β-catenin and Ki67/Shh in single cells of each treated mouse skin on day 20. DAPI staining was used to identify the nuclei (blue). Scale bar = 50 μm Full size image

IM activates stem cell metabolism and further expansion of proliferating progenitors during the early phases of hair follicle regeneration

A recent report provided data showing that K15-positive hair follicle stem cells highly express pyruvate dehydrogenase kinase (PDK), which is an enzyme that promotes glycolytic conversion34, whereas Ki67-positive proliferating cells strongly express pyruvate dehydrogenase (PDH), which is an enzyme responsive to mitochondrial OXPHOS12. PDK expression was mainly detected in the K15-positive stem cells in the IM-treated mice but was not detected in either the control or minoxidil-treated mice on day 7 (Fig. 6a). The K15+/PDK+ populations were hardly detected in the control or minoxidil-treated mice but occupied 5.9% of the single cells in the IM-treated mice skin by FACS analysis on day 7 (Fig. 6c). These results showed that the IM treatment increased the glycolytic stem cell population (K15+/PDK+) by 6.6-fold over that in the control mice. PDH expression was obvious in the Ki67-positive proliferating cells in the IM-treated mice on day 7 (Fig. 6a) and was detected in the highly proliferative cells in all groups on day 20 (Fig. 6b). The Ki67+/PDH+ populations were also increased in the IM-treated mice by 13.7%, whereas these populations represented 5.0% and 8.3% of cells, respectively, in the control and minoxidil-treated mice on day 7 (Fig. 6c). Subsequently, we examined the reciprocal expression patterns of each marker, and the discrete expression of K15/PDH and Ki67/PDK was obviously detected in the IM-treated mice on day 20 (Supplementary Figure S9a), clearly distinguishing the PDK-expressing glycolytic K15-positive hair follicle stem cells from PDH-expressing OXPHOS-dependent Ki67-positive proliferating cells. Moreover, FACS analysis apparently revealed that the populations of PDK+ and Ki67+ cells were well separated and that each population was increased by the IM or minoxidil treatment on day 20 (Supplementary Figure S9b). Separated populations of PDH+ and K15+ cells were also detected and increased by IM or minoxidil treatment on day 20 (Supplementary Figure S9b).

Fig. 6: IM stimulates the expression of stem cell metabolism-related and hair follicle regeneration-related genes during the early phases of hair regrowth. Immunohistochemistry of K15, PDK, Ki67, and PDH in each treated mouse skin on a day 7 and b day 20. DAPI staining was used to identify the nuclei (blue). Scale bar = 50 μm. c FACS analysis of K15/PDK and Ki67/PDH in single cells of each treated mouse skin on day 7. d Expression of the indicated gene in each treated mouse was quantified by real-time PCR analysis on days 7 and 20 after depilation. β-Actin expression was used as an internal control. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test) Full size image

Finally, the expression of genes related to metabolism and hair regeneration was analyzed in skin tissues from each treated mouse (Fig. 6d). The expression of glycolysis-related enzymes (HK2 and LDHA) was prominently induced in the IM-treated mice by day 7 and was significantly accelerated in both the IM-treated and minoxidil-treated mice compared with that in the control mice on day 20. At this stage, the expression of OXPHOS-related enzymes (NDUS3 and ATP5B) was elevated in both the IM- and minoxidil-treated mice compared with that in the control mice, implying that IM facilitates glycolytic metabolic transition during the early phases of hair follicle regeneration, resulting in the further promotion of OXPHOS during the later phase and contributing to the active energy metabolism required for hair regrowth. Importantly, the expression of hair follicle development-related genes (Wnt1a, Lef-1, Gli-1, and Versican) was potently upregulated in the IM-treated mouse skin by day 7, and minoxidil also slightly increased the expression of Wnt1a, Lef-1, and Gli-1 compared with that in the control. By day 20, compared with control mice, both the IM-treated and minoxidil-treated mice showed pronounced induction of stemness-related genes (Fig. 6d). Overall, IM promotes an increase in the glycolytic hair follicle stem cell population during the early phases of tissue regeneration, possibly conferring advantages in the progression to the next stages of the regeneration process.

We also compared the effect of a biguanide, metformin, with that of IM on hair regrowth (Supplementary Figure S10). The intensity of the darkness of the back skin was slightly increased in the metformin-treated mice compared with that in the control mice by day 8, but the effects were not significantly, consistent with the in vitro data (Supplementary Figure S4). Additionally, the effect of IM on tissue regeneration was observed in pinnal tissue repair (Supplementary Figure S11). IM dose dependently decreased the wound area in an ear hole punch assay. Thus, IM could possibly activate tissue regeneration other than hair regrowth.