Inhibitors of tyrosine phosphatases and apoptosis reprogram lineage-marked differentiated muscle to myogenic progenitor cells

Abstract

Muscle regeneration declines with aging and myopathies, and reprogramming of differentiated muscle cells to their progenitors can serve as a robust source of therapeutic cells. Here, we used the Cre-Lox method to specifically label postmitotic primary multinucleated myotubes and then utilized small molecule inhibitors of tyrosine phosphatases and apoptosis to dedifferentiate these myotubes into proliferating myogenic cells, without gene overexpression. The reprogrammed, fusion competent, muscle precursor cells contributed to muscle regeneration in vitro and in vivo and were unequivocally distinguished from reactivated reserve cells because of the lineage marking method. The small molecule inhibitors downregulated cell cycle inhibitors and chromatin remodeling factors known to promote and maintain the cell fate of myotubes, facilitating cell fate reversal. Our findings enhance understanding of cell-fate determination and create novel therapeutic approaches for improved muscle repair.

Figure 1. Lineage marking of primary myotubes by Cre-Lox method A Schematic of the system

a. Wild type myoblasts (MB) derived from C57BL/6 mice were infected with Ad-Cre and subsequently co-cultured with Lox-YFP MB obtained from Rosa 26-YFP reporter mice in differentiation medium (DM) to form myotubes. The fusion of these two populations of MB led to the excision of stuffer sequence (green circle) by Cre recombinase activity to give rise to lineage marked YFP expressing myotubes (green). Self fusion among the two populations of MB will give rise to YFP negative myotubes (colorless). These lineage marked myotubes were then used in de-differentiation studies. Fusion-dependent, Cre-Lox mediated labeling of myotubes upon co-culture of Ad-Cre MB with Lox-YFP MB in DM. b. As described in Figure a, wild type MB were co-cultured with Lox-YFP MB (1:2 ratio) in DM to induce formation of myotubes. Endogenous YFP fluorescence in myotubes was observed by 72-96 hours as shown by epifluorescent images. In control infection with control Ad-RFP virus, no YFP fluorescence was observed. No YFP expression was observed upon co-culture of Ad-Cre MB and Lox YFP MB in GM where myoblasts did not undergo physiological fusion to form myotubes. c. Western blotting to determine YFP expression using lysates from Lox-YFP MB, Ad-Cre MB co-cultured with Lox-YFP MB in GM and parallel in DM. YFP protein was observed in the myotubes which arose from fusion of Ad-Cre and Lox-YFP MB in DM. d. qRT-PCR analysis for YFP gene expression. RNA was extracted from Ad-Cre and Lox-YFP MB co-cultured in GM and in DM for 96 hours to detect the levels of YFP and Cre recombinase by qRT-PCR. Data was normalized to internal control GAPDH. Error bars indicate mean and standard deviation, n=3. YFP mRNA levels was only observed in Ad-Cre-Lox-YFP⁺ myotubes while Cre recombinase expressed in both the co-cultures of Ad-Cre and Lox-YFP MB in GM and in DM. The fusion-dependent marking of myotubes was clearly and robustly mediated by this adaptation of the Cre-Lox method, and no mononucleated cells expressed YFP.. See also Figure S1

Figure 2. Immunodetection of YFP and muscle specific markers

a-d. YFP⁺ myotubes obtained after Cre-Lox fusion express muscle differentiation marker and does not incorporate BrdU. Cre-Lox YFP⁺ myotubes cultures were co-immunostained with muscle differentiation markers eMyHC (a) myogenin (b) p21 (c) and DNA synthesis label BrdU (d) along with anti-YFP antibody. Representative images are shown.

Figure 3. BpV with Q-VD de-differentiates the irreversibly-labeled YFP⁺ myotubes to YFP⁺ proliferating mononucleated cells

a. Myotube de-differentiation strategy. MB infected with Ad-Cre were co-cultured with Lox-YFP MB in DM for 4 days to give rise to YFP⁺ myotubes. These were treated with 10uM BpV + 10uM Q-VD in parallel with other experimental conditions for two days in DM. The treated myotubes were then switched to myoblast GM which was replaced fresh every day. YFP⁺ mononucleated cells were observed around day 10. De-differentiation of YFP⁺ myotubes to YFP⁺ proliferative cells. b. YFP⁺ myotubes cultures were treated with the BpV+ Q-VD and photographed every day. The addition of BpV+ Q-VD led to morphological changes and when switched to GM these cells expanded as YFP⁺ mononucleated cells in 72 hours (white arrow shows YFP⁺ mononucleated cells). Representative high magnification images of de-differentiation experiment over the course of 10 days with live Hoechst is shown by epifluorescent microscopy. c. No-treatment (UT): Untreated YFP⁺ myotubes were grown in similar conditions and did not show any de-differentiation events. These data demonstrate that inhibitor mix is necessary and sufficient for de-differentiation of genetically labeled myotubes into expanding mononucleated cells. d. Reprogrammed YFP⁺ mononucleated cells rapidly divide. Cre-Lox-YFP⁺ myotubes reprogrammed as depicted in Figure 3b and c, were pulsed with BrdU for 24 hours and co-stained with anti YFP and BrdU antibodies. Arrows indicate representative BrdU⁺YFP⁺ cells in treated conditions. Untreated cultures of YFP⁺ myotubes do not show any YFP⁺ mononucleated cells though BrdU incorporation is seen in non YFP cycling mononucleated cells. Inset shows magnified images. e. Quantification of percent of BrdU⁺ /YFP⁺ mononucleated cells out of total number of YFP⁺ myotubes (shown are the mean and standard deviations ,n=3 p< 0.05). Note that many reserve myoblasts re-entered cell cycle and incorporated BrdU in GM (both in the presence of BpV+ Q-VD and in control untreated cultures); these cells, however, were reliably distinguished in our experiments by the absence of YFP. See also Figure S2, Figure S3, Figure S4 and Figure S5

Figure 4. Genetically-labeled progeny of de-differentiated myotubes have functional and genetic attributes of muscle progenitor cells

a. Co-immunostaining of FACS-sorted, proliferating YFP⁺ mononucleated cells for (i) Pax7 and (ii) MyoD along with anti-YFP antibody was performed and representative images are shown. b. Histogram quantifies Pax7 and MyoD expressing YFP⁺ mononucleated cells which represents mean and standard deviation of three independent experiments. c. De-differentiated, FACS sorted, YFP⁺ cells were expanded in GM and cultured in DM for 96 hours where myoblasts typically form myotubes; cultures were co-immunostained with antibodies specific to YFP and to myotube specific marker (i) eMyHC ii) myogenin, as well as (ii) the CDK inhibitor p21. d. Western blotting with antibodies specific for Pax7, MyoD, eMyHC, p21, myogenin and YFP was performed using protein extracts from Cre-Lox-YFP myotubes, de-differentiated YFP⁺ mononucleated cells and re-differentiated YFP⁺ cells as indicated. Actin served as loading control. e. Gene expression analysis of muscle differentiation markers. qRT-PCR data in log scale for Pax7, MyoD, myogenin, p21 and eMyHC depicts the relative gene expression of re-differentiated myotubes to de-differentiated YFP⁺ cells. The data represents the mean and standard error for three independent experiments. See also Figure S6

Figure 5. Reprogrammed YFP+ proliferative cells contribute to in vivo muscle regeneration

FACS sorted YFP+ proliferating mononucleated cells were expanded in GM and injected in cardiotoxin injured Tibialis Anterior (TA) immuno-compromised NOD-SCID mice. 2-3 weeks later, TA muscles were dissected out, sectioned at 10um and co stained with YFP and laminin to visualize YFP+ myofibres. Control buffer and Lox YFP myoblast injected TA muscle did not show any YFP+ myofibres.

Figure 6. Molecular analysis of reprogramming in genetically labeled myotubes

Inhibitor mix treatment down regulates muscle differentiation marker in Cre Lox-YFP myotubes. a and b. 4 day old Ad-Cre-Lox-YFP myotubes were untreated/ treated with inhibitor mix for 48 hours, followed by immuno-detection of myogenin (a), p21 (b) and YFP (green), using antibodies specific for these proteins. Myogenin and p21 were down-regulated in a subset of YFP⁺ myotubes (shown by white arrows). Control myotubes did not change expression of muscle differentiation markers. c and d. The histogram quantifies the percent of YFP⁺/myogenin ⁺, YFP⁺ myogenin⁻ cells and YFP⁺/p21⁺ and YFP⁺/p21⁻ in the experiment shown in Figure 6a and b (n=3 ± S.D.; p***<0.001, p**<0.05). e and f. Ad-Cre-Lox-YFP myotubes untreated/ treated with BpV+ Q-VD for 48 hours were analysed for protein and mRNA levels. Protein lysates were subjected to western blotting for antibodies against p21, myogenin and eMyHC. Actin served as a loading control. q-RT-PCR was performed on RNA lysates for gene expression of p21, p15, p16, myogenin and eMyHC. Data was normalized to GAPDH and represents mean and standard deviation of three independent experiments each done in triplicates (n=3 ± S.D.; p***<0.001, p**<0.05). Untreated sample was taken as 1.

Figure 7. Inhibitor mix treatment modulates chromatin remodeling factors and enzymes

a and b. Clustergram analysis of chromatin remodeling factors and enzymes for Ad-Cre-Lox YFP myotubes treated and untreated with BpV+Q-VD (inhibitor mix) for 48 hours using SA Biosciences/Qiagen PCR arrays. 0.5ug RNA isolated from three independent set of experiments of Ad-Cre-Lox YFP myotubes were reverse transcribed and gene expression profile monitored. c and d. Histogram representation for few set of genes normalized by Hprt gene levels. Control untreated was taken as 1. (n=3 ± S.D.; p***<0.001, p**<0.05). See also Supplementary Table S1 and Table S2