In the present study, we have provided mechanistic insights into the impacts of infarcted myocardial ECM stiffness on the endothelial cell lineage commitment of bone marrow-derived CD34+ and CD34 cell subsets. Our results demonstrate that infarcted myocardium-like ECM stiffness regulates the cytoskeletal arrangement, cell survival, cell-ECM adhesion, and cell differentiation in both CD34+ cells and CD34 cells derived from BMMNCs. Moreover, there were significant differences in the specification of the endothelial cell lineage between the two cell subsets induced on the flexible culture substrates, which were distinct from conventional glass rigid substrates. The matrix stiffness of 42 kPa, corresponding to myocardial ECM at days 7–14 after MI, was more suitable for the induction of FITC-UEA-1 and DiI-acLDL double-positive cells, as well as the expression of endothelial cell lineage markers such as CD31, vWF, Flk-1, and VE-cadherin in both CD34+ and CD34 subsets. Meanwhile, in the cell culture system with the flexible substrates, the CD34+ cell subset showed higher endothelial lineage commitment compared with the CD34 subset under various culture conditions. Thus, it is clear that an optimal ECM stiffness promotes the endothelial lineage commitment of bone marrow-derived CD34+ cells in vitro. The combination of an optimal cell subset and a suitable ECM stiffness may provide a potentially useful strategy to enhance cell-based cardiac repair after MI.

For the repair of ischemia or infarcted myocardium, the addition of the proper stem/progenitor cells to the suitable microenvironment seems to be promising via promoting neovascularization [1416]. The definition and classification of stem/progenitor cells mainly depends on the cell surface antigens. CD34 is an important cell surface marker of hematopoietic progenitor cells. Bone marrow-derived CD34+ cells have the potent potential to differentiate into endothelial lineage cells which are deeply involved in neovascularization [17]. Meanwhile, the phenotype CD34 functions to mediate the attachment of cells to the ECM [18]. In the present study, we found significant differences in cell attachment to the flexible substrates between the CD34+ cell subset and the CD34 subset. Furthermore, as compared with the CD34 subset, the CD34+ subset was more easily induced into the endothelial cell lineage, which potentially facilitates angiogenesis as well as cardiac repair. The difference in specification efficiency between the two cell subsets might result from the differences in cell attachment to the flexible substrates. Furthermore, in terms of the CD34+ cell subset, the present study shows a significant difference in the number of cells expressing endothelial phenotypes (not the percentage) on the flexible substrates. However, there exists a consistently high percentage of cells expressing endothelial phenotypes on the 15- to 72-kPa substrates. This suggests that the significant differences in endothelial phenotype expression might result from differences in the cell adherence capacity. In the present study, CD34+ cells seemed to more easily adhere to the flexible substrates than the CD34 cells. Mechanically, the characteristics of the CD34 antigen in improving cell adherence to the ECM might provide an explanation for the preference of CD34+ cell survival and specification on the flexible substrates. In contrast, CD34 cells presented a lower survival and specification ratio. In addition, CD34+ cells showed distinct focal adhesion, cytoskeletal organization, and cellular morphology on the flexible substrates with varied stiffness. Moreover, cytoskeletal architecture and cell-ECM adhesions become increasingly organized with increasing stiffness. Paxillin, as a connection between ECM and cells, regulates cell fate and even cell specification by influencing cell attachment and cytoskeleton formation (mainly referring to the F-actin network) [19]. Cell adhesion and apoptosis were both regulated by cell-ECM interaction. Disruption of the cell-ECM interaction promotes cell apoptosis [20]. In the present study, CD34+ cells had a lower apoptotic rate on the moderately stiff or stiff substrates (42 kPa or 72 kPa) than those on the soft substrates (4 kPa and 15 kPa). Based on the analysis on the cytoskeleton and cell morphology, the differences in cell apoptosis might relate to the higher adhesive strength of CD34+ cells to the relatively rigid substrates or the stronger cell-ECM interaction.

On the other hand, cell-ECM interaction is also widely believed to play an important role in cell survival and differentiation [21]. Stem/progenitor cells are able to sense and respond to the surrounding tissue physical microenvironment, which is known as the ECM stiffness [22]. Accumulating data have shown that tissue ECM stiffness plays an important role in stem cell adhesion, survival, and lineage commitment [23, 24]. Following MI, myocardial ECM stiffness might be an important physical condition impacting the efficacy of cell implantation by influencing these cellular biological behaviors [8, 9, 25]. Thus, the simulation in vitro to the myocardial physical microenvironment post-MI is thought to be essential in detecting implanted cell biology and validating an optimal cell therapy strategy. Indeed, in the present study, infarcted myocardium-like ECM stiffness showed a significant influence on the potential pro-angiogenesis ability of bone marrow-derived CD34+ cells. Furthermore, myocardial ECM at days 7–14 post-MI might offer an optimal physical microenvironment for commitments of engrafted pluripotent cells to endothelial cell lineage, which suggests that the beneficial effect on infarcted myocardium repair might be time- or stiffness-dependent. Moreover, CD34+ cells under induction of ECM stiffness present a similar stiffness-dependent differentiation principle as BMMNCs, and might consequently exert an important role in regulating the efficacy of cell implantation for the damaged myocardium, as reported in our previous study [9]. Furthermore, these findings might partially explain the optimal timing of stem cell implantation after MI.

Notably, almost all of the previously published randomized controlled trials (RCTs) consistently performed cell therapy at days 0 to 7 after MI [26]. Moreover, our previous meta-analysis on these RCTs indicated the more favorable effect of bone marrow-derived stem cell engraftment at 4–7 days after MI on improving left ventricular ejection farction (LVEF) and decreasing left ventricular (LV) end-systolic dimensions than a procedure performed within 24 h following MI [27]. Since days 7–14 post-MI is a “time-domain blank zone” in previous clinical studies on cell-based cardiac repair, it may be important to further investigate the biological behavior of engrafted cells and the efficacy of cell therapy within this “time-domain blank zone” due to the unavoidable therapeutic delay for acute MI. Our previous studies verified the optimal efficacy of cell therapy at 7–14 days after MI, which might relate to ECM stiffness-dependent angiogenesis [9]. Overall, the procedure of cell therapy either too early or too late after acute MI was not productive in terms of promoting cardiac repair due to the absence of the “suitable” stiffness of myocardial ECM. Although the previous clinical trials confirmed the efficacy of bone marrow-derived cell implantation within 24 h or more than 30 days after MI [28, 29], the magnitudes of the beneficial effects were significantly different. Based on our present findings, the time-dependent changes in myocardial ECM stiffness after MI may contribute to the differing efficacy of cell therapy at these different time points.

The limitations of our study deserve comment. Firstly, the adhesion ability of cells on the flexible (4–72 kPa) substrates was relatively low. The less cell-cell contact might influence cell proliferation. Thus, we did not further investigate the impact of the flexible substrates on cell proliferation in this study. Secondly, due to insufficient cell availability and restricted conditions of the special culture system, the impact of substrate stiffness on cell migration was not elucidated. Additionally, the findings in the present study were achieved in a two-dimensional in vitro model, and needs to be verified under three-dimensional conditions in vivo.