Analysis of stem cell marker gene expression in mBM-MASCs. (A) FACS analysis of uninduced mBM-MASCs1 and -2. Expression of Sca-1, c-Kit, CD34, CD45, Ter119, Flk-1, and H-2D^d surface markers was analyzed by staining with phycoerythrin (PE)-conjugated monoclonal antibodies. mBM-MASCs1 and -2 differed in their expressions of Sca-1 and CD34. No expression of CD34 was found in mBM-MASCs1. Negative peaks in the CD34 and Sca-1 graphs represent phycoerythrin-conjugated isotype controls. (B) RT-PCR analysis of the expression of alleged stemness genes in mBM-MASCs1 and -2, embryonic stem (ES) cells, and NIH 3T3 cells. mBM-MASCs1 and -2 showed robust expressions of OCT3/4, Rex-1, b-myb, nanog, and SSEA-1 (stage-specific embryonic antigen) (lanes 1 and 2). Embryonic stem cells (lane 4) were used a positive control, and NIH 3T3 cells (lane 3) were used as a negative control.

Induction of skeletal and cardiac muscle marker genes in mBM-MASCs by different Wnt molecules. (A) RT-PCR analysis of the expression of skeletal muscle myogenic factors. RNA was isolated from mBM-MASCs1 and -2, which were cocultured for 7 days with different amitotic Wnt-expressing cells (lanes 1 to 8), with the parental cell line lacking Wnt expression (lanes 10 and 11), and with skeletal (skel.) muscle cells (lane 9). Different expression patterns of myogenic factors were induced. Treatment with Wnt molecules resulted in the activation of at least one myogenic factor. (B) RT-PCR analysis of the expression of cardiac muscle marker genes using the same experimental setup. Wnt11 was the only Wnt molecule that activated expression of β-MHC and BNP (lanes 4 and 10), while other Wnt molecules stimulated Nkx-2.5, Hand2, and GATA-4 expression. All Wnt molecules failed to induce α-MHC and ANP. MyHC, MHC.

CDO induces the expression of skeletal muscle but not of cardiac muscle marker genes in mBM-MASCs. (A) RT-PCR analysis of the expression of skeletal muscle myogenic factors. RNA was isolated from mBM-MASCs1 and -2 that were cocultured for 7 days with amitotic CDO-expressing cells (lanes 1 and 2), with the parental cell line lacking CDO expression (lanes 3 and 4), and with skeletal muscle cells (lane 5). CDO treatment led to the robust expression of myogenic regulatory factors. (B) RT-PCR analysis of the expression of cardiac muscle marker genes using the same experimental setup. CDO (lane 2) failed to induce cardiac markers such as GATA4 and β-MHC, while Wnt11 alone (lane 1) or in combination with CDO (lane 4) had this ability. (C) Immunofluorescent staining of MASCs for MHC expression after cocultivation with CDO-expressing cells and with the parental cell line lacking CDO expression. Nuclei were counterstained with Hoechst 33258 (c, d, and f) to locate all cells on the plate. (D) Immunofluorescent staining of MASCs for cTnT expression after cocultivation with Wnt11- and CDO-expressing cells and with the parental cell line lacking CDO or Wnt11 expression. Nuclei were counterstained with Hoechst 3444 (c to f) to locate all cells on the plate. (E) RT-PCR analysis of CDO expression in feeder cells and MASCs. (F) RT-PCR analysis of Wnt4 expression in MASCs after induction with CDO. Note the induction of Wnt4 expression after CDO treatment. MyHC, MHC.

Inhibition of PKC abrogates activation of most cardiac markers by Wnt11 in mBM-MASCs. RT-PCR analysis of the expression of cardiac markers after coculturing of Wnt11-expressing cells with mBM-MASCs and treatment with various concentrations of bisindolylmaleimide I and staurosporine (lanes 1 to 8). RNAs isolated from cocultures without the addition of inhibitors and from heart tissue were used as controls. Expression of cardiac marker genes, with the exception of the GATA-4 gene, was efficiently repressed by the inhibitors.

Different Wnt signaling molecules induce expression of MHC in mBM-MASCs. mBM-MASCs1 and -2 were cocultured with amitotic cells that expressed either Wnt1 (A to D), Wnt3 (E to H), Wnt7a (I to L), Wnt7b (M to P), Wnt4 (Q to T), or Wnt11 (U to X) or with the parental cell line (Y to Ö). After 7 to 8 days, cultures were stained with the monoclonal MF-20 antibody against sarcomeric myosin heavy chain and a secondary antibody coupled to Alexa 594 anti-mouse. MHC expression was indiscriminately activated by all Wnt molecules tested. Nuclei were counterstained with Hoechst 33258 to locate all cells on the plate (B, F, J, N, R, V, and Z [second column] and D, H, L, P T, X, and Ö [fourth column]). MyHC, MHC.

Comparison of the numbers of myogenin-expressing mBM-MASCs after induction with different Wnts, CDO, and CA-LEF. (A) FACS-assisted cell counts of GFP-positive mBM-MASCs that had been stably infected with a myogenin promoter-eGFP retroviral reporter virus before treatment with various effectors. (B) Semiquantitative RT-PCR of the expression of different Wnt molecules in inducing feeder cells. (C) Number of myogenin-positive cells after staining with an anti-myogenin antibody. Numbers in panels A and C represent the percentages of positive cells within the whole cell population. Error bars indicate the standard deviations. (D) mBM-MASCs were induced with different Wnt-expressing cells and stained with an anti-myogenin antibody to reveal the presence of the nuclear antigen. Representative examples are shown.

CA-LEF1 induces the expression of skeletal muscle but not of cardiac muscle marker genes in mBM-MASCs. RT-PCR analysis of the expression of skeletal muscle myogenic factor (A) and cardiac marker genes (B) after infection of mBM-MASCs1 and -2 with a retrovirus encoding CA-LEF. CA-LEF1 led to the activation of several skeletal muscle markers but not of cardiac marker genes, with the exception of the Nkx-2.5 gene. (C) mBM-MASCs1 and -2, each containing a myogenin promoter-eGFP reporter construct, were infected with a retrovirus encoding CA-LEF. Activation of the myogenin-eGFP reporter construct is shown under fluorescent light (a and c) and merged with corresponding phase-contrast images (b and d), indicating the activation of skeletal muscle genes.

Epigenetic reprogramming leads to enhanced plasticity and spontaneous differentiation of MASCs. (A) mBM-MASCs2 were stained with the monoclonal MF-20 antibody against sarcomeric myosin heavy chain and a secondary antibody coupled to Alexa 488 after treatment with AZA and/or TSA (a to f) or without treatment (g and h). Nuclei were stained with Hoechst 33258 to locate all cells within a culture (b, d, f, and h). The addition of either drug dramatically increased the number of MHC-positive cells. (B) RT-PCR analysis of the expression of skeletal muscle myogenic factors in mBM-MASCs2 after the addition of AZA, TSA, and AZA/TSA (lanes 1 to 8). RNAs isolated from untreated mBM-MASCs2 (lane 9) and from skeletal muscle tissue (lane 10) were used as negative and positive controls, respectively. Note the lack of Myf5 expression after AZA and AZA/TSA treatment. (C) After treatment with AZA, mBM-MASCs2 were stained with a monoclonal antibody against cTnI and a secondary antibody coupled to Alexa 594 (a and c) in the absence (a) and presence (c) of Hoechst 33258. In panels b and d, treatment with AZA was done with mBM-MASCs2 that contained an α-MHC-eGFP reporter construct. Activation of the α-MHC-eGFP reporter construct is shown under fluorescent light (b) and merged with a corresponding phase-contrast image (d). (D) RT-PCR analysis of the expression of cardiac muscle marker genes in mBM-MASCs1 after the addition of AZA, TSA, and AZA/TSA (lanes 1 to 3). RNAs isolated from untreated mBM-MASCs1 (lane 4) and from heart tissue (lane 5) were used as negative and positive controls, respectively.