Mouse cadaveric heart decellularization and characterization

Figure 1a illustrates the overall schema for this study. We modified a previously published decellularization protocol10 of porcine hearts to decellularize cadaveric mouse hearts. Trypsin and detergents, including SDS and Triton X-100, were used to lyse the cellular content in mouse hearts (Fig. 1b). We only treated mouse hearts with peracetic acid with a low concentration for 5 min, followed with multiple washes. This process minimizes the damages on ECM proteins and properly maintains the 3D architecture of decellularized hearts, as previously described10. A completely decellularized heart was obtained with the yield of a transparent ECM scaffold (Fig. 1b, panel 7). Coronary vessel tree within the DC-ECM was preserved post decellularization and was visualized with the injection of trypan blue solution through the connected cannula (Fig. 1b, panel 8). Histological analysis by hematoxylin and eosin (H&E) staining detected no remaining nuclei in the whole decellularized hearts (Fig. 1c). DNA content within the decellularized hearts decreased to ~3% of that from the cadaveric mouse hearts (Fig. 1d). The acellular DC-ECMs were then subjected to scanning electron microscope (SEM) (Fig. 1e). ECM composition of the decellularized heart was preserved and exhibited a filament-like appearance (Fig. 1e, top right panel). Aortic wall and valve leaflets remained intact in the decellularized hearts (Fig. 1e, bottom middle and right panels). Typical ECM components such as Fibronectin, Laminin and Collagen II remained post decellularization and were detected by immunostaining (Fig. 1f). No DAPI-positive nuclei were observed in the decellularized hearts (Fig. 1f), indicating the complete removal of intact heart cells. Both ECM structure and the amount of residual DNA in the decellularized mouse hearts were similar to those reported for decellularized rat hearts9.

Figure 1: Decellularization of mouse heart. (a) A scheme for the whole study. (b) Photographs showing each step of decellularization process: before decellularization (1); after deionized water perfusion (2); after PBS perfusion (3); after enzymatic perfusion (4); after 1% SDS solution perfusion (5); after 3% Triton X-100 solution perfusion (6); after acidic perfusion (7); perfusion of DC-ECMs with trypan blue solution to visualize the intact coronary vasculature (8). (c) H&E staining of sections from the cadaveric mouse heart (top) and DC-ECM (bottom). Scale bars, 10 μm. (d) DNA content quantification of cadaveric mouse hearts and DC-ECMs. Error bars show s.e.m of three independent experiments. **P<0.005 (n=3, unpaired Student’s t-test). (e) SEM of cadaveric and decellularized hearts. Left ventricular (LV; top panel) and aorta (bottom panel). Myofibers (mf) were present in the cadaveric heart (white stars) but not in the DC-ECMs. Red stars indicate the aortic valve leaflets. (f) Immunostaining of cadaveric mouse hearts and DC-ECMs. Fibronectin (upper), Laminin (middle), and Collagen II (lower). No nuclear staining (DAPI) was observed in DC-ECMs. Scale bars, 50 μm. Full size image

Differentiation of MCPs from human iPSCs

Figure 2a outlines the overall workflow used to repopulate the decellularized mouse hearts with human iPSCs. At stage 1, we adapted our serum-free protocol18 to induce the differentiation of MCPs from human Y1-iPSCs19 (Supplementary Fig. S1a,b). This protocol utilizes the known role of different growth factors in early cardiovascular development to manipulate the differentiation of human ES/iPS cells to MCPs, as well as the specification of MCPs to cardiovascular lineage cells, including CMs, SMCs and ECs (Supplementary Fig. S1c and Supplementary Movie 1). Embryoid bodies (EBs) were generated from Y1 iPSCs with addition of bone morphogenetic protein 4 (BMP4, 1 ng ml−1) for 24 h (day 0–1), followed by treatment with BMP4 (10 ng ml−1), basic fibroblast growth factor (bFGF, 5 ng ml−1) and ActivinA (1.5 ng ml−1) from days 1–4. Vascular endothelial growth factor A (VEGF, 10 ng ml−1) and dickkopf homologue 1 (DKK1, 150 ng ml−1) were then added from day 4 till day 6 (Fig. 2a). At stage 2, the day 6 EBs were dissociated into single cells. Fluorescence-activated cell sorting (FACS) analysis detected an average of 63% KDRlow/C-KITneg cells, which represented the human iPSC-derived MCPs19, from the dissociated EBs (Supplementary Fig. S1d). When further differentiated as monolayers in six-well plates, the dissociated EBs generated >50% CMs, 30% SMCs and 5% ECs with the presence of VEGF (10 ng ml−1) and DKK1 (150 ng ml−1) (Supplementary Movie 2), or >10% CMs, 50% SMCs, 25% ECs in the presence of VEGF (20 ng ml−1) and bFGF (20 ng ml−1) (Supplementary Fig. S1e). These results indicated that cardiovascular lineage specification from iPS-MCPs could be controlled by exogenous growth factors as previously described with human ES cells18. Similar to our previous studies19, smooth muscle actin (SMA) is expressed in SMCs and CMs. Thus, the SMA+ population in Supplementary Fig. S1e represented both iPSC-derived CMs and SMCs. At Stage 3, we perfused the MCP-repopulated mouse decellularized hearts with VEGF (10 ng ml−1) and DKK1 (150 ng ml−1) to increase the reconstruction of heart muscles, or with VEGF (20 ng ml−1) and bFGF (20 ng ml−1) to promote re-endothelialization of the heart constructs.

Figure 2: Recellularization of decellularized mouse heart. (a) A scheme showing the working strategy to recellularize the decellularized mouse hearts with human iPSC-derived MCPs. Human iPSC-derived EBs were differentiated until day 6 and then dissociated into single cells (Stage 1). Dissociated cells (10 million) were seeded into a decellularized mouse heart and perfused (Stage 2). The seeded MCPs in situ differentiated into cardiac lineage cells and reconstructed DC-ECMs. (b) Detection of vessel-like structures from human Y1 iPS cell recellularized DC-ECMs. Red arrows indicate the reconstructed vessel-like structures. Scale bars, 500 μm. (c) EKG recording of human Y1 iPS cell recellularized DC-ECMs when electrically paced at 4 HZ. (d) Detection of the intracellular (CaiT) synchronization driven by local propagation of electrical activity (sites 1, 2 and 3) from Y1 iPSC RC-DC-ECMs. (e) A RC-DC-ECM was embedded in paraffin and cross-sectioned, followed with H&E staining. The up left panel shows the recellularized VW. High magnification panel and the other three panels show recellularized vessel cavities and distribution of MCP-derived cells along the endocardial surface (red arrows). Full size image

Recellularization of decellularized mouse hearts

Approximately 1.0 × 107 of day 6 EB-dissociated single cells were seeded into one decellularized mouse heart (Supplementary Fig. S2a) through the cannula that was connected to the aorta. In order to induce CM formation from the seeded MCPs, heart constructs were periodically (once per 8 h) perfused with VEGF (10 ng ml−1) and DKK1 (150 ng ml−1) through the same cannula. To calculate the retention ratio of repopulated cells, we collected the perfusate and counted the total cell loss of each perfusion. After 7 days, only a few cells were observed in the medium post perfusion, suggesting a stable interaction of repopulated cells with the heart ECM had occurred. Thus, we estimated ~10–15% repopulated cells were preserved in the DC-ECMs after 7 days perfusion. In addition to Y1 iPSC-derived MCPs, we also repopulated the decellularized mouse hearts with human RUES2 ES cell-derived MCPs (Supplementary Fig. S2b,c). We observed reconstructed vessel-like structures within both human iPS cell and ES cell recellularized heart constructs (Fig. 2b, Supplementary Fig. S3a). Both ES- and iPS cell-repopulated heart constructs exhibited spontaneous contractions after 20 days of perfusion (Supplementary Movies 3 and 4), with a similar beating rate of 40–50 beats per min. This observation indicated an identical cardiovascular commitment process from the repopulated MCPs of human ES cells and iPS cells.

Electrophysiological analysis of engineered heart constructs

Of all the constructs engineered in this study, ~90% exhibited spontaneous contractions and 10% failed to beat because of the low cell retention or contamination during perfusion. In order to examine the electrophysiological characteristics of engineered heart tissues, we first conducted EKG (electrocardiogram) recording (Fig. 2c). In humans, EKG is used to measure the electrical activity of whole heart and diagnose abnormal rhythms of the heart. The engineered heart constructs showed EKG-like electrical signals with irregular wave morphology, indicating the lack of electrical activities controlled by a conduction system. Next, we investigated whether CMs within the engineered heart tissues were electrically coupled by loading the RC-DC-ECMs with an intracellular Ca2+ indicator dye, Rhod-2/AM and mapping CaiT at the high spatial and temporal resolution20. Figure 2d and Supplementary Fig. S3 show the optical recording of CaiT in the heart constructs engineered with human Y1 iPS and RUES2 ES cells. Mapping CaiT in a paced preparation is an outstanding surrogate for mapping action potential propagation and to measure local cell–cell coupling, as previously demonstrated19,20. CaiT mapping detected many regions of uniform wave propagation indicating a possible electrical coupling of CMs and cell–cell communication in heart constructs recellularized with pluripotent stem cells (Fig. 2d, Supplementary Fig. S3b and Supplementary Movie 5). However, CaiT waves also encountered zones of anatomical block indicating regions of uncoupled tissues. The loss of coupling was most likely due to gaps between the recellularized CMs or a low density of CMs, as those uncoupled areas could be re-synchronized by electrical stimulation (Supplementary Fig. S3b, Supplementary Movie 6). Optical mapping results indicated that our engineered heart constructs largely functioned as a syncytium, often with an endogenous pacemaker and otherwise could be electrically paced.

Histological analysis of engineered heart constructs

After 20 days of perfusion, expression of pluripotency marker genes, including NANOG, SOX2 and OCT4, was significantly decreased in heart constructs compared with that in undifferentiated iPS cells, indicating no remaining of pluripotent iPS cells (Supplementary Fig. S2d). Histological analysis of engineered heart constructs revealed MCP-derived cells (detected by H&E) that formed muscle-like or vessel cavities-like structures, as well as a single layer on the endocardial surface (Fig. 2e). To identify the types of cells locating on the different sites of DC-ECMs, sections were immunostained using antibodies recognizing human CMs, SMCs and ECs (Fig. 3a–c). Myofibers were detected in the recellularized ventricular wall (VW) (Fig. 3a), as illustrated with low-magnification images to visualize as large a field-of-view as possible. Two CM markers, sarcomeric α-Actinin and Cardiac Troponin T (CTNT)18 were found throughout the DC-ECMs post recellularization (Fig. 3a). SMA immunostaining illustrated the recellularized VW, apex (AP) and ventricular septum (VS) (Supplementary Fig. S4a). Both SMCs and CMs repopulated the VS and exhibited the branching structures. Immunostaining of smooth muscle myosin heavy chain (SMMHC), a marker of SMC, detected the differentiation and localization of SMCs on the border of CM fibres (Fig. 3b), as well as in some small vessel cavities (data not shown). ECs distribution was observed along the endocardial surface with the formation of a single layer of ECs (Fig. 3c). Both PECAM (CD31) and VE-Cadherin18 expressions were detected on ECs located on the inner surface of the small coronary vessels throughout the engineered heart tissues (Supplementary Fig. S4b). The MCP-derived CMs refilled the decellularized mouse hearts (Fig. 3d) and expressed Connexin-43 (CX43), which is the predominant cardiac gap-junction protein (Fig. 3e). The CX43 expression pattern in our heart tissue is not as homogenous as that in native myocardium and comparable to that in previous bioartificial rat hearts9. Lastly, we quantified the ratios of CMs and ECs in the heart constructs after perfusion with media containing DKK1 (150 ng ml−1) and VEGF (10 ng ml−1) or VEGF (20 ng ml−1) and bFGF (20 ng ml−1). We found the presence of bFGF and DKK1 could substantially promote the specification of ECs or CMs from the reseeded human MCPs, respectively (Fig. 3f). These data indicated that the local heart ECM niches, as well as the extracellular growth factors, could affect the migration and/or lineage commitment of early-stage heart progenitor cells.

Figure 3: Histology analyses of engineered heart tissues. (a) A representative coronal section of recellularized mouse heart immunostained by CTNT and merged with the bright field image. High magnification shows the sarcomeric organization of myofibers. (b) A representative VW section was immunostained with CTNT and SMMHC, a SMC marker. (c) Sections of a recellularized heart were stained by CD31, a surface mark of ECs. White arrows indicate human MCP-derived ECs along endocardial surface. (d) A representative coronal section of the recellularized mouse heart immunostained by anti-CTNT and anti- Laminin antibodies. (e) Co-immunostaining of CM marker α-Actinin and Connexin-43 (CX43), showing expression of gap-junction-associated protein in RC-DC-ECMs. Ventricular wall (VW), left ventricle (LV), right ventricle (RV), ventricle septum (VS), apex (AP). (f) Ratios of CTNT+ and CD31+ cells in the Y1 iPSC RC-DC-ECMs after perfusion with medium containing DKK1 (150 ng ml−1) and VEGF (10 ng ml−1) or VEGF (20 ng ml−1) and bFGF (20 ng ml−1). The ratio of CD31+ or CTNT+ cells in all DAPI+ cells was obtained from at least five views per section. At least 15 sections per construct were calculated. Error bars show s.e.m of three independent experiments. **P<0.01 (unpaired Student’s t-test). Full size image

Impact of heart ECM on CM commitment from human MCPs

Previous studies demonstrated that ECM influences vertebrate heart formation21,22. It would be interesting to know the role of heart ECM in regulating early-stage human cardiovascular differentiation, and our engineered heart constructs provide an ideal model to address these questions. First, we conducted q-PCR to compare the relative expression levels of several cardiac-specific genes in undifferentiated Y1-iPSCs, day 26 Y1 EBs, day 26 heart ECM constructs, human fetal heart and human heart atrium (Fig. 4a). Twenty million day 6 EB cells were generated from Y1 iPS cells, of which 10 million EB cells were used to make a heart construct and the other half were cultured in six-well plates. Both EBs and heart constructs were cultured with the same medium till day 26. This allowed us to compare the CM differentiation from MCPs under two 3D environments. One 3D environment is the decellularized mouse heart ECM, whereas the other represents 3D EBs in the absence of mouse heart ECM. Expression levels of α-MHC, CTNT, CX43 and NK2 homeobox 5 (NKX2.5) in heart ECMs were identical to those from the two human heart tissues and were significantly higher than those from the EBs (Fig. 4a). The expression level of β-MHC in heart ECMs was five-fold higher than that in the day 26 EBs. Next, we compared the expression levels of atrial CM markers, Myosin regulatory light chain 2, atrial isoform (MLC2A), Sarcolipin (SLN)23 and ventricular CM marker, Iroquois homeobox 4 (IRX4)24 in all samples. As expected, the highest expression level of atrial markers and the lowest level of IRX4 were detected in human atrium. Interestingly, we found an increased level of IRX4 in heart ECMs than that in EBs. The relative expression ratio of IRX4 versus SLN in the heart ECMs was comparable to that in fetal heart, but significantly higher than those in EBs and human atrium (Fig. 4b). Additionally, a higher ratio of MLC2A+CMs was observed in the recellularized atria than in the ventricles (38 versus 17%, Fig. 4c). However, the atria of engineered heart constructs were not recellularized as well as the ventricles, which was confirmed by both histological and q-PCR analyses (Supplementary Fig. S5). Therefore, all these results implied a potential role of ventricular heart ECM during the commitment of ventricular CMs from MCPs.

Figure 4: Impact of heart ECM on repopulated MCPs. (a) Transcriptional expression of cardiac-associated genes, including cardiac-specific genes (α-MHC, β-MHC, CTNT, MLC2A, SLN and IRX4), cardiac-related transcription factor (NKX2.5), and gap-junction protein (CX43) (n=3). (b) Relative expression ratio of IRX4 versus SLN was compared (n=3). (c) Quantification of CTNT+ and MLC2A+ cells in the recellularized atria and ventricles. Both atria and ventricles were separated from the heart constructs and subjected to MLC2A (red) and CTNT (green) immunostaining. At least 10 sections of each tissue were calculated. White arrows indicate the MLC2A+ cells in ventricles. (d) Cell apoptosis assessment. EBs and RC-DC-ECMs were stained with anti-CTNT antibody and labelled with TUNEL assay. Quantitative apoptotic nuclei in total DAPI+ or in CMs were calculated and were shown in histogram (right) (n=3). (e) Cell proliferation assessment. EBs and RC-DC-ECMs were stained with anti-CTNT and anti-phospho-Histone H3 antibodies. Quantitative proliferating nuclei in CMs and ratio of CTNT in total DAPI+ cells were calculated and shown in histogram (right) (n=3). Red arrows indicate the proliferating nuclei and white arrows indicate the phospho-Histone H3-negative nuclei. (f) Ultrastructural analysis of CMs in EBs and RC-DC-ECMs by TEM. Width of myofibrillar bundles was measured (right). F, myofibrillar bundle. Z, Z-line. All error bars show s.e.m of three independent experiments. *P>0.05, **P<0.05, ***P<0.01 (unpaired Student’s t-test). Full size image

In order to study the impact of heart ECM on MCP-derived CMs, the ratios of CMs in day 26 EBs versus day 26 heart constructs were compared. Approximately 40% CMs were identified in EBs and ~70% CMs were found in heart constructs (Fig. 4e, right). This suggested that heart ECM may have an impact on the viability of CMs. Next, we assessed the levels of cell apoptosis and proliferation. Approximately 6 and 10% apoptotic cells were detected from all DAPI+ cells within EBs and RC-DC-ECMs, respectively. Nearly 2.5% apoptotic CMs were identified in both EBs and RC-DC-ECMs (Fig. 4d, Supplementary Fig. S6). Given the higher cell density and the lower delivery of nutrition supply in the heart constructs, more apoptotic cells were expected in RC-DC-ECMs than in EBs. Interestingly, we found ~80% of phospho-Histone H3-positive CMs in RC-DC-ECMs and only 40% of phospho-Histone H3 positive CMs in EBs (Supplementary Fig. S7, Fig. 4e). Taken together, our results suggested that heart ECM could stimulate the proliferation of early differentiated CMs or maintain the proliferation of CMs for a longer period of time than in a 3D environment devoid of ECM. The ultrastructures of CMs in EBs and RC-DC-ECMs were investigated by TEM. We found wider myofilament bands within RC-DC-ECMs than those from the EBs (Fig. 4f), indicating that heart ECM may promote the formation of myofibrillar bundles in CMs.

Drug response of engineered heart constructs

As our engineered heart constructs exhibited spontaneous contractions, we next sought to test whether these constructs could respond to pharmaceutical agents, which are known to affect CM physiology. In clinical and experimental settings, the increase of heart beating rate in response to β-adrenergic stimulation has been well documented25. The chronotropic response of our heart constructs to β-adrenergic stimulation was examined by perfusing isoproterenol, a selective β1-adrenergic agonist, with concentrations ranging from 1 nM to 5 μM. As shown in Fig. 5a, isoproterenol administration increased the frequency of spontaneous contractions. Perfusion with 1 μM isoproterenol doubled the contraction rate from ~45 beats per min to 90 beats per min, and induced small premature calcium waves that gradually developing to full Ca2+ transients (Fig. 5b). Next, we tested the response of RC-DC-ECMs to E4031, a selective blocker of the rapid component of the delayed rectifying K+ current, I Kr , known to elicit long QT type 2 and Torsade de Pointes, a form of polymorphic ventricular arrhythmias26. Inhibition of I Kr with E4031 gradually developed Ca2+ instabilities (Fig. 5c), resulting in an arrhythmogenic phenotype with irregular amplitude of Ca2+ transient (Fig. 5d) compared with the baseline conditions. This observation is consistent with the report that Ca2+ instability or lability often precedes ventricular arrhythmias in drug-induced long QT type 2 (ref. 27). Furthermore, we measured the baseline mechanical force, as well as the active force of heart constructs in response to external [Ca2+] out , as previously described28. Supplementary Fig. S8 shows a representative image of a heart construct instrumented to measure mechanical force. Engineered heart tissues generated contractile force by spontaneous contractions and showed increased contractile force in response to increased [Ca2+] out (Fig. 5e,f, Supplementary Movie 7). Active force–frequency relationship showed that active contractile force was increased with electrical pacing between 1 to 1.5 Hz and then decreased at 2 and 2.5 Hz (Fig. 5g, Supplementary Movie 8), which is consistent with the stair-case effect observed in the working myocardium29. The average contraction force of engineered heart constructs under 1-Hz pacing was ~0.18 mN, which was higher than the force generated by spontaneous contractions and comparable to another report on engineered human heart tissues30. Put together, these results indicated that the heart tissues engineered with our strategy displayed electrophysiological and mechanical properties.