Myocardial interstitial fluid inhibits proliferation and cardiomyocyte differentiation in pluripotent embryonic stem cells

Abstract

Several recent studies have demonstrated that the transplantation of pluripotent murine embryonic stem cells (mESCs) can improve or restore the function of infarcted myocardium. Although the extent of remuscularization and its contribution to the restoration of function are unclear, these outcomes are likely strongly influenced by factors in the infarcted and/or ischemic environment. As an initial step toward understanding how the ischemic environment of host myocardium affects transplanted pluripotent cells, we have taken a reductionist approach wherein mESCs are cultured in medium containing ischemic myocardial interstitial fluid (iMIF). iMIF is generated in canine myocardium during eight hourly episodes of transient ischemia and collected on a daily basis, over a 24-day collection period. iMIF strongly reduced the numbers of pluripotent mESCs after 11 days in culture. This inhibitory effect, which was most pronounced for iMIF pools from early time points of the 24-day collection period, resulted from an inhibition of cell proliferation. iMIF also inhibited the differentiation of pluripotent mESCs into cardiomyocytes. By contrast, the expression of vascular smooth muscle and endothelial cell markers was relatively unaffected, consistent with previous findings that iMIF promotes angiogenesis. Taken together, these results suggest that whereas the ischemic/infarcted environment is favorable to stem cell-mediated angiogenesis, it is hostile to cardiac myogenesis. These findings also imply that observations of mESC-mediated improvement of cardiac function after transplantation of pluripotent cells do not reflect remuscularization.

Fig. 1.

Serum inhibits cardiomyogenesis in cultured embryonic stem cells (ESCs). Murine ESCs were induced to differentiate in monolayer culture by growing for 11 days in either 1) RPMI/B27 supplemented with 100 ng/ml activin-A for 1 day followed by RPMI/B27 containing 10 ng/ml bone morphogenic protein-4 (BMP-4) for 4 days, then RPMI/B27 only for 6 days; 2) RPMI/B27 alone for 11 days; or 3) RPMI/B27 supplemented with 10% FBS for either the first 5 days or 4) for the full 11 days. All media were exchanged for fresh medium daily. Bars depict the extent of immunohistochemically detected expression of sarcomeric myosin heavy chain (MHC), detected using monoclonal antibody MF20 followed by ImageJ quantitation. The extent of MHC expression denoted by each bar is shown relative to cells grown in RPMI/B27 only, which was set at 1.0. Error bars represent means ± SE; numbers in parentheses indicate the number of cultures that were evaluated. Statistical significance was assessed by Student's t-test. *P < 0.05, presence of 10% FBS for the 1st 5 days and full 11 days of the culture period compared with cells grown in RPMI/B27 only.

Fig. 2.

Ischemic myocardial interstitial fluid (iMIF) reduces ESC culture density. Murine ESCs were cultured for 11 days as described in materials and methods, using defined medium supplemented with 10% (vol/vol) iMIF or sham-operated MIF (sMIF) that was harvested from canine myocardium and pooled from the indicated collection days. Medium, including MIFs, were changed daily. A: phase-contrast microscopy performed on the 11th day of the culture period. B: quantitatively depiction of the effect of MIFs on cell density in the cultures, as determined by 4′,6-diamidino-2-phenylindole (DAPI) staining. Randomly selected areas of each culture were photographed under ×40 magnification, and areas occupied by DAPI staining were quantitated via ImageJ analysis. *P < 0.01 and **P < 0.02 compared with RPMI/B27 control group; +P < 0.05 compared with sMIF collected from same days. C: hemocytometer counts of trypsinized ESCs cultured for 11 days in the presence of pooled iMIF and sMIF using an alternative early stage collection pool (days 4–6). *P < 0.05 compared with RPMI/B27 control group; +P < 0.05 compared with sMIF. In B and C, the error bars depict means ± SE, and numbers in parentheses indicate the number of cultures evaluated. Statistical significance was assessed by Student's t-test.

Fig. 3.

Reduced ESC density reflects inhibition of proliferation by iMIF. A, top: ESCs were cultured for 11 days with the indicated sMIF and iMIF pools, followed by immunohistochemical assessment of mitotic cells exhibiting H3P. For the bar graph, randomly selected fields were photographed under ×100 magnification and H3P-positive nuclei were enumerated. *P < 0.05, significant inhibition caused by iMIF compared with ESCs grown in RPMI/B27. A, bottom: representative images of H3P-stained nuclei (MIF collection pool 13–15). B, top: results of H3P and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays performed during the second day of the culture period. Note that these experiments were performed using an alternatively early pool of iMIF and sMIF, collected at days 4–6, to avoid exhausting MIF supplies. The inhibition of ESC proliferation by iMIF within 48 h of plating corroborates the inhibitory effects in A. *P < 0.001, significant inhibition caused by iMIF compared with ESCs grown in RPMI/B27. NS, not significant. By contrast, iMIF did not induce cell death. TUNEL signals were verified to reside in the nucleus by colocalization with the DAPI signal, as depicted by the representative images shown in B, bottom. In all experiments, all media were changed daily. Error bars depict means ± SE. Numbers in parentheses indicate the number of cultures evaluated. Statistical significance was via Student's t-test.

Fig. 4.

iMIF inhibits cardiomyocyte differentiation. A: cells were cultured for 11 days in the presence of sMIF and iMIF pools as indicated, followed by immunostaining to detect cells expressing sarcomeric MHC protein as described in materials and methods. Randomly selected areas of each culture well were photographed under ×100 magnification, and fluorescence from MHC-positive cells was quantitated by ImageJ. All media were exchanged daily. Error bars depict means ± SE. Numbers in parentheses indicate the number of cultures evaluated. Statistical significance was assessed by Student's t-test. *P < 0.01 compared with RPMI/B27. B: representative immunostained images that were used to obtain the data shown in A. C: results from RT-PCR evaluation of cardiomyogenic markers α-MHC and Nkx-2.5. Each subpanel shows radioactive PCR products from triplicate wells. This result was verified by 2 additional repetitions that yielded similar results.

Fig. 5.

iMIF does not affect expression of vascular cell markers. Cells cultured for 11 days with sMIF and iMIF were subjected to RT-PCR to assess expression of the indicated vascular cell markers. VE-cadherin (VE-Cad) and Tie-2 are endothelial cell markers. Calponin-1 (CNN-1) is a smooth muscle marker. Each subpanel shows radioactive PCR products from triplicate wells. This result was verified by repetition of this experiment.