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. 2020 Jun 8;15(6):e0234087. doi: 10.1371/journal.pone.0234087

The evaluation of a tissue-engineered cardiac patch seeded with hips derived cardiac progenitor cells in a rat left ventricular model

Yuichi Matsuzaki 1,#, Shinka Miyamoto 1,#, Hideki Miyachi 1, Tadahisa Sugiura 1, James W Reinhardt 1, Chang Yu-Chun 1, Jacob Zbinden 1, Christopher K Breuer 1,2, Toshiharu Shinoka 1,3,*
Editor: Yiru Guo4
PMCID: PMC7279601  PMID: 32511282

Abstract

Background

Ventricular septal perforation and left ventricular aneurysm are examples of potentially fatal complications of myocardial infarction. While various artificial materials are used in the repair of these issues, the possibility of associated infection and calcification is non-negligible. Cell-seeded biodegradable tissue-engineered patches may be a potential solution. This study evaluated the feasibility of a new left ventricular patch rat model to study neotissue formation in biodegradable cardiac patches.

Methods

Human induced pluripotent stem cell-derived cardiac progenitor cells (hiPS-CPCs) were cultured onto biodegradable patches composed of polyglycolic acid and a 50:50 poly (l-lactide-co-ε-caprolactone) copolymer for one week. After culturing, patches were implanted into left ventricular walls of male athymic rats. Unseeded controls were also used (n = 10/group). Heart conditions were followed by echocardiography and patches were subsequently explanted at 1, 2, 6, and 9 months post-implantation for histological evaluation.

Result

Throughout the study, no patches ruptured demonstrating the ability to withstand the high pressure left ventricular system. One month after transplantation, the seeded patch did not stain positive for human nuclei. However, many new blood vessels formed within patches with significantly greater vessels in the seeded group at the 6 month time point. Echocardiography showed no significant difference in left ventricular contraction rate between the two groups. Calcification was found inside patches after 6 months, but there was no significant difference between groups.

Conclusion

We have developed a surgical method to implant a bioabsorbable scaffold into the left ventricular environment of rats with a high survival rate. Seeded hiPS-CPCs did not differentiate into cardiomyocytes, but the greater number of new blood vessels in seeded patches suggests the presence of cell seeding early in the remodeling process might provide a prolonged effect on neotissue formation. This experiment will contribute to the development of a treatment model for left ventricular failure using iPS cells in the future.

Introduction

Due to the complication of myocardial infarction in adults, problems such as left ventricular rupture and ventricular septal perforation may present as fatal complications, mandating surgical repair. [1] The materials currently utilized are not degradable, lack growth potential, and do not promote attachment of endotherial and smooth muscle cells or macrophage migration. [2] Tissue engineering may provide a new way to regenerate organs and deliver tissue-specific cell types to the site of injury, thereby replicating the structure and function of native myocardium. [3] Tissue engineered constructs often consist of a scaffold and seeded cells; as the scaffold degrades, new biocompatible tissue is deposited. [4] Human iPS (hiPS) cells are an infinite source of cardiomyocytes due to their high potential for differentiation and are therefore reported to be one of the most promising cell sources for cardiac regeneration therapy. [5,6,7,8,9] Previously, we reported the development of tissue engineering technology using induced, or artificial, pluripotent stem (iPS) cells and demonstrated that this method is feasible and safe. [10,11] Limitations of these early studies include the use of differentiated cardiomyocytes that may as a result have limited mitotic potential and use of a right ventricular model. [11] We created a new cardiac patch in which hiPS cell-derived cardiac progenitor cells (hiPS-CPC) are seeded on a sheet of bioabsorbable polymer and implanted in a rat left ventricular myocardial defect model.The patch was implanted in the apex wall of the left ventricle so that the luminal surface of the graft received direct blood supply and allowed host cardiomyocyte ingrowth at the anastomosis. The purpose of this study was to verify whether it is possible to obtain improved neoplasia by seeding progenitor cells and to verify whether the material can compensate for left ventricular injury.

Methods

Preparation of the tissue-engineered cardiac patch

A scaffold composed of a woven fabric of polyglycolic acid (PGA) and 50:50 poly (l-lactic-co-ε-caprolactone) copolymer (PLCL) was constructed as previously described. [12] The scaffold with a thickness of 0.6 to 0.7 mm was cut into a 6-mm circular patch using a biopsy punch (Fig 1A). The structure of the sponge was checked by imaging using scanning electron microscopy (SEM, Fig 1B and 1C). hiPS-CPCs (Cellular Dynamics International (CDI), Madison, Wisconsin) were cultured on the biodegradable patch in a 96-well plate at a density of 1.0 × 106 cells per well for one week and cell culture media was changed every two days. Because these cells were myocardial progenitor cells, they did not beat before transplantation. After culturing, patches were implanted in the left ventricular wall of male nude athymic rats, as described below.

Fig 1.

Fig 1

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Example of cardiac patch composed of knit mesh of PGA between 2 layers of PLCL; A: 6-mm diameter biodegradable patch, B: SEM images of the sponge layer 100X magnification. C: SEM images of the sponge layer 500X magnification.This patch consists of a knit mesh of PGA sandwiched between 2 layers of PLCL.

Left ventricle wall biodegradable patch implant procedure

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Nationwide Children’s Hospital (Protocol Number: AR12-00079). All surgery was performed under anesthesia and all efforts were made to minimize suffering. Adult male nude athymic rats (Jackson Laboratories, Bar Harbor, ME) weighing 230–300 g were used for the left ventricle wall replacement procedure. General anesthesia was induced with ketamine (50 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.); anesthesia was maintained using isoflurane (1.5%) in oxygen. Animals were intubated with a 16-gauge catheter and respiration was maintained at 60 cycles per minute with a tidal volume of 2.5 ml. The surgical procedure was performed using aseptic techniques with sterile instruments. First, the skin of the chest was sterilized with a povidone-iodine solution and the heart was exposed through a median sternotomy. A purse string suture was placed in the left ventricle apex wall with 7–0 polypropylene sutures (Ethicon, Somerville, NJ, USA). Both ends of the suture were passed through a 22-gauge plastic vascular cannula, which was used as a tourniquet (Fig 2A and 2B). The tourniquet was tightened and two-thirds of the distended part of the left ventricle apex wall inside the purse string suture was cut to create a flap. To indicate that a transluminal defect had been created in the left ventricle, the tourniquet was briefly released to determine whether massive bleeding occurred and a core of a 16-gauge catheter was inserted in left ventricle (Fig 2C–2E). Next, the cardiac patch was sutured along the margin of the purse string suture with 7–0 polypropylene to cover the hole in the left ventricle (Fig 2F) and the ventricular flap was then sutured onto the patch (Fig 2G). After suturing, the tourniquet was released and the purse string suture was removed. Additional sutures were added, as needed, to achieve hemostasis (Fig 2H). After expanding the lungs using positive end-expiratory pressure, the sternum was closed parasternally with four interrupted 5–0 polypropylene sutures (Ethicon). The muscle layer and skin were closed with 4–0 Vicryl absorbable sutures (Ethicon). For the first 3 days after surgery, buprenorphine (0.05 mg/kg) and cefuroxime (100 mg/kg) were administered subcutaneously twice daily. Animals were randomly divided into two groups: 1) hiPS-CPC seeded group and 2) unseeded group (n = 10/group). Two animals in each group were sacrificed at 1 and 2 months post-surgery and 3 animals in each group were sacrificed at 6 and 9 months post-surgery.

Fig 2. Various stages of patch implantation.

Fig 2

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(A): A purse string suture placement in the left ventricle apex wall; (B): tourniquet tightening; (C): cutting the distended part of the left ventricle apex wall; (D) flap creation; (E): needle used to examine hole creation; (F): cardiac patch implantation; (G): closing the flap (H): purse string suture removal.

Echocardiographic analysis

Echocardiographic measurements were obtained preoperatively and at 1, 2, 6, and 9 months postoperatively. Animals were anesthetized with isoflurane (2% isoflurane with 100% oxygen gas inhalation through a nose cone). Echocardiography was performed to assess ventricular function (Vevo Visual Sonics 770; Visual Sonics, Toronto, ON, Canada). Two-dimensional imaging in a parasternal long axis plane was obtained. Left ventricular systolic and diastolic volumes were estimated by the modified Simpsons method to calculate LV ejection fraction (Analysis package from Vevo Visual Sonics).

Histology, immunohistochemistry, and immunofluorescence

After sacrifice, the left ventricle was separated from the heart by axial dissection, and a sagittal transection centered on the apical patch was performed. Explanted cardiac patches were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 5 μm. Sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, von Kossa to examine calcification, and by immunohistochemistry and immunofluorescence (IF). The degree of calcification was determined by area measurements using ImageJ software. Measurements are reported as a percentage of positive area over the entire patch area.

For immunohistochemistry, tissue sections were blocked for endogenous peroxidase activity prior to staining. Platelet endothelial cell adhesion molecule (CD31, 1:1000, Abcam, Cambridge, MA) was used to identify endothelial cells. Antibody binding was detected with appropriate biotinylated secondary antibodies followed by streptavidin-horseradish peroxidase. Positive staining was assessed using 3,3-diaminobenzidine peroxidase substrate (Vector Laboratories SK4105, Burlingame, CA). Nuclei were counterstained with Gill’s hematoxylin. Angiogenesis measurements reported as count of positive CD31 vascular over the entire patch area.

Statistical analysis

Numeric values are listed as mean ± standard deviation (SD). Statistical analyses of ejection fractions were conducted via two-way ANOVA; all other data were analyzed via Student’s t-test. A p value less than 0.05 was selected as the cutoff for statistical significance.

Results

Postoperative course and gross observations

No deaths occurred during the postoperative course in either group and no gross evidence of thrombosis was present in any of the animal explants. At the time of explantation, all rat hearts exhibited minimal thoracic adhesions with no recognizable pattern of adhesive tissue present in either groups. There was no evidence of aneurysm formation at the site of the implanted patch in the left ventricle at any time point (Fig 3).

Fig 3. Macroscopic view of operated rat hearts.

Fig 3

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Over time, some fibrous adhesions on the pericardial surface formed. The patch was sutured in the heart interior. There were no significant differences in macroscopic findings between the two groups.

Echocardiographic analysis

At all-time points, there was no significant difference in ejection fraction between the two groups (Fig 4). Diastolic volumes increased over time in both groups, but there was no significant difference. In the seeded group, there was a trend towards smaller ventricular systolic volumes, but this difference was also not statistically significant.

Fig 4. Echographic analyses over a period of 36 weeks compared to baseline: LV Ejection fraction (EF); LV diastolic volume; LV systolic volume.

Fig 4

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There are no aneurysmal changes in either group.

Histology, immunohistochemistry, and immunofluorescence

Visualization of Masson’s trichrome stain show collagen and muscle fibers. At eight weeks, some patch material still remains and is covered by collagen. Over the course of 24 weeks, the cardiac patch gradually degraded and could not be detected (Fig 5A). We assessed PGA fiber degradation and collagen formation using polarized light microscopy with Picro-Sirius Red (PSR) stained histological sections from 1, 2, 6 and 9 months after implantation. Under polarized light, the PGA fibers are birefringent and can be visualized. The PGA fibers can be seen as highly organized bundles within the scaffold at 1M after implantation, but by 2M the fibers had fragmented and nearly degraded. No fragments of the PGA fibers were visible at 6 months after implantation (Fig 5B). The wall thickness of the LV ventricle apex was very thin. Using immunofluorescence, explanted patches were stained with anti-human nuclear antigen to detect hiPS-CM and troponin to detect cardiomyocytes. While images did reveal cells that stained positive for anti-human nuclear antigen, the pattern of staining was cytoplasmic, and not restricted to the nucleus as would be expected for this antigen. We therefore concluded that these cells represented false positives and remaining true hiPS-CM were not observed. (Fig 5C). At this time point, H&E staining showed cell infiltration. The number of cells present in the patch at each time point was compared, and there was no significant difference between the seeded group and the non-seeded group (Fig 6C-1). At 6 months, multinuclear giant cells and granular tissue can be observed indicating ongoing chronic inflammation (Fig 6A). After 6 months, von Kossa staining revealed no calcification in the patch (Fig 6B), but there was calcification present on the inner surface of the left ventricle. There was no significant difference in degree of calcification between two groups (Fig 6C-2), but there was a significant difference in the number of CD31 positive vessels (p = 0.03, Fig 6C-3).

Fig 5.

Fig 5

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(A) Masson’s trichrome staining of sections indicating collagen (blue) and muscle (red) demonstrating cell infiltration and material degradation. (B) PSR staining of section indicating inmature collagen (yellow) and mature collagen (red), White fiber (PGA fiber) demonstrating PGA degraded at the 2-month time point (red bar 500μm), (C) Evaluation of iPS cells and cardiomyocytes on patches one month after transplantation using immunofluorescence. Red; human nuclear antigen, Green: troponin T Blue: DAPI.

Fig 6.

Fig 6

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(A): Cells can infiltrate and attach to the scaffold four weeks after implantation. Hematoxylin and eosin staining revealed multinuclear giant cells (yellow arrows). (B): Von Kossa staining shows phosphate commonly associated with calcification in the LV at 24 and 36 weeks after implantation. (C):1. Number of cells in bioabsorbable patch at each time point. 2. Evaluation of calcification at 6 months after transplantation (n = 3). 3. Evaluation of the number of CD31 positive vessels 1 month and 6 months after implantation (n = 3/time point).

Discussion

Clinically, non-biodegradable materials are utilized as patches for left ventricular repair associated with sequelae after myocardial infarction; however, there are a host of problems related to durability, thrombosis, biocompatibility, and infection that requires reoperation. [2,12] For example, PTFE patches have been accepted worldwide as one of the most reliable non-biodegradable synthetic materials in terms of durability, low thrombotic properties, and comfortable handling during surgery, but there are many reports of bacterial infections. In general, cardiac surgeons have concerns about implanting foreign materials in the heart as infective endocarditis can be a lethal complication after open heart surgery. Even now, some surgeons inevitably use Dacron patch or PTFE patch to repair for ventricular septal rupture after myocardial infarction. In addition to these risks, left ventricular function after myocardial infarction continues to deteriorate following the use of non-degradable myocardial patches. These clinical demands motivated us to develop new bioabsorbable materials for use in cardiac surgery. Complications associated with non-biodegradable materials and the potential to regenerate functional myocardium associated with tissue engineering have fostered the development of surgical models and evaluation of various scaffold designs. Due to challenges and limitations in the use of exogenous cell-based biomaterials, cell-free strategies are being investigated as an alternative. [13] This approach requires the attachment and growth of host parenchymal cells as well as their production and organization of extracellular matrix. Materials of natural origin, such as collagen and fibrin, have been evaluated as scaffolds for tissue regeneration after myocardial infarction. These are applied surgically as patches, or less invasively as gel forming injectable ECM proteins. Acellular scaffolds are advantageous over cellular scaffolds in that: (1) these are off-the-shelf products that can be immediately implanted (e.g., SynerGraft®), (2) elicit a limited immune response, and (3) cost far less to produce. [14,15] Biodegradable synthetics, like the polymers that comprise our scaffold, are another category or materials widely used in tissue engineering applications due to their mechanical properties, material uniformity, stability, and lower risk of infection compared to natural biomaterials. Biodegradable synthetic polymers can be modified with high precision to meet tissue-specific properties such as appropriate degradation rates, porosity, and mechanical strength. [16–19] Cell seeding can provide additional benefits to neotissue formation that include increasing functional capacity of resulting neotissue, accelerating neo tissue, formation, and modulating the immune response. However, it is well known in cell culture studies that cells are more likely to survive if they are able to attach to a surface as demonstrated in our experiments. [20] Proper selection of both a scaffold and seeded cells will continue to be an important determinant in the success of regenerative medicine. [4]

In the past, heart tissue was considered non-renewable; however, recent studies have suggested that cardiomyocytes, although rare, can replicate. In previous studies, Nakane et al. A designed heart tissue composed of three different cell types differentiated from hiPS cardiomyocytes (CM), endothelial cells, and vascular wall cells. [21] Compared to sham surgery, these structures have led to better preservation of viable myocardium and maintenance of wall thickness. This approach focuses on the outside of the heart, but the same methods applied inside the heart may be worth investigating.

This study focused on examining the feasibility of a biodegradable, prosthetic patch seeded with hiPS cells in improving left ventricular function following cardiac insult.

This study investigated the possibility of using a biodegradable PGA-PLCL scaffold for surgical left ventricular repair. Our previous work has shown proven that PGA-PLCL, an FDA-approved material with growth potential, seeded with cultured bone marrow cells used in the venous system is clinically feasible. [22,23] This patch consists of a knit mesh of PGA sandwiched between 2 layers of PLCL. The PGA provides a strong backbone to withstand the blood flow within the body, while PLCL provides a porous surface for cell attachment and infiltration. Given that sufficient nutrient and oxygen delivery through functional vasculature or other means of perfusion is essential for successful cardiac stem cell therapy, we implanted the hiPS-CPC seeded heart patch via left ventricular reconstruction allowing the blood supply to be integrated directly into the luminal surface of the patch. Another consideration of the study was the thickness of graft material. A previous study by J. Riegler et al. demonstrated that the survival rate of seeded cells was lower when implanted in heart tissue grafts thicker than 400μm in mouse model. [24] Considering the difference in animal models, we chose a graft thickness of 600μm.

When we try to create infarction model with the additional incision of left ventricular apex for placing the patch in rats, animal survival rates were low following the procedure. Therefore, we simplified the model to utilize left ventricular apex incision to implant our patches in the blood stream. Following implantation, the patch was able to withstand the high-pressure system without rupturing and there was no evidence of infection in both groups. There is no report of survival experiments in rats which simulating the left ventricle, that is, simulating VSP or left ventricular rupture, and we consider the importance of this research on the method of surviving the rat for one year with no complication. However, long-term postoperative follow-up revealed calcification. These results suggest that cell seeding may not influence formation of calcification. Furthermore, as our previous works with PGA-PLCL tissue engineered constructs have exhibited no calcification in the venous and right ventricular environments, the results suggest that the cause may be due to shear stress of the left ventricle, extensive tissue necrosis, and the inflammatory response that can lead to to calcium chemo-attaction. [25] Immunofluorescence showed that seeded hiPS-CPCs do not remain on the biodegradable patch after 1 month. Prior in vitro studies on co-cultures of adult rat muscle and cardiac stem cells show that stem cells survive longer than adult cardiomyocytes alone due to increased levels of insulin-like growth factor 1 and vascular endothelial growth factors, both of which were associated with decreased apoptosis and increased myocyte survival. [26] The seeded cells may also contribute to differences in cardiac function. Keller et al. showed that scaffold-based 3D human dermal fibroblast cultures can be used as cardiac patches to stimulate revascularization when transplanted into infarcted LV of severe combined immunodeficiency (SCID) mice. [27] In our results, the cell seeding group also showed more revascularization at 6-month timepoints, however there was no statistical difference in left ventricular ejection fraction between the two groups during the 9-month follow-up.

Several limitations exist with this current work. Small sample size is one of the limitations. This study is still at a preliminary stage, and only examines how seeding iPS cells with bioabsorbable material contributes to myocardial regeneration. In the future study, we would like to perform power analysis and increase the number of samples. Our approach utilized cells purchased from CDI for which the genetic information and differentiation induction method are not clear; nor were the cells. [28] Therefore, we may need to generate a line of myocardial progenitor cells with our desired functions. Secondly, additional time-points are required to elucidate the temporal loss of seeded cells. It is possible that the in vivo structural and chemical environment results in the death of hiPS-CPCs.

Although more effort is required to further develop these cardiac patches, we have demonstrated the potential of tissue engineering to improve cardiac function following myocardial infarction.

Conclusion

In summary, we have demonstrated the safety of a biodegradable cardiac patch of hiPS-CPC seeded in left ventricular myocardial contraction in rats. Seeded hiPS-CPCs disappeared from the patch at an early stage and did not contribute to left ventricular contractility, but may affect angiogenesis. These findings need to be further investigated for improved surgical outcomes.

Data Availability

All relevant data are within the paper.

Funding Statement

This research is supported by US National Institutes of Health (NIH) grants: R01HL098228 and GUNZE co ltd. Specifically GUNZE co ltd provided material support through the creation of the biodegradable patch. YM was supported by Department of Defence (DoD) and Funding award from Uehara Memorial Foundation (Tokyo Japan) in 2019. JWR was supported in part by the American Heart Association under Award Number 18POST33990231. The specific roles of these authors are articulated in the 'author contributions' section.

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Decision Letter 0

YIRU GUO

3 Jan 2020

PONE-D-19-33452

The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model

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[This work was supported by in part by a grant from the National Institutes of Health

(R01- HL098228 to Dr. Breuer). Dr. Shinoka receives grant support from Gunze Ltd.

(Kyoto, Japan). The is study was performed by the authors, who also had full control

of the study design, methods used, outcome measurements, data analysis, and

production of the written report.].

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 [Dr. Matsuzaki was the recipient of funding award from Uehara Memorial Foundation (Tokyo Japan) in 2019

This work was supported by in part by a grant from the National Institutes of Health (R01- HL098228 to Dr. Breuer). Dr. Shinoka receives grant support from Gunze Ltd. (Kyoto, Japan). The is study was performed by the authors, who also had full control of the study design, methods used, outcome measurements, data analysis, and production of the written report.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Matsuzaki and colleagues submit their manuscript entitled, “The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model” for consideration of publication in PLOS ONE. The authors use a rodent model to test whether a cell-seeded biodegradable tissue-engineered patch provides beneficial effects in LV function. The experiments are well designed with appropriate controls. Unfortunately, but not surprising cell-seeding of iPS cells did not lead to an increase in cardiomyocytes. The authors do demonstrate that a biodegradable patch can be used as a viable option for ventricular rupture.

Comments

1. The potential benefit of the biodegradable patch was the ability to cell-seed the patch. However, since the cell-seeding did not lead to increased myocardial cells, authors should clarify the benefit of their patch over non-absorbable patches.

2. Inclusion of a non-biodegradable patch as a comparison to the biodegradable patch could have been included. Do the authors have any comparison data? Especially in light that at 36 weeks, there was no difference between cell-seeded and no cell-seeding. So is a biodegradable patch add benefit?

3. Was vascularization assessed? Also, was staining performed for fibroblasts?

Reviewer #2: The work described in this manuscript addresses an important issue of designing a cell-seeded cardiac patch to improve cardiac function. Successful development of such a system could improve cardiac function in adults after ventricular rupture. The limited results in this study show that biodegradable patches implanted at the left ventricular apex with or without cardiomyocytes did not improve cardiac function. The reason for this lack of effect could not be clearly identified due to limitations of the study design.

The authors provide no assessment of cardiomyocyte function or organization prior to implantation, so it unclear whether the implanted cardiomyocytes had any contractile function at the time of implantation.

The small sample size in each group (1 and 2 month time points had 2 animals in each group and the 6 and 9 each had 3 animals in each group) precluded detection of differences between the two groups except at 8 weeks for ejection fraction. The sample size is much smaller than the authors used in their prior study, reference 11.

The histological analysis of the implants did not examine for the presence of cardiomyocytes or vascularization in the patches. Thus, the reason for the difference in ejection fraction due to cell seeding could not be evaluated.

Specific Comments

1. Figure 4. Horizontal axis label is needed for each panel.

2. Figure 4. Were the changes in LV systolic and diastolic volumes over time significant? The statistical methods used indicate that t-tests were used, but the results in Figure 4 indicate that a two-factor ANOVA (time and treatment) should be used to evaluate trends with time.

Reviewer #3: This study tested the feasibility of applying stem-cell patches with a biodegradable substrate to damaged ventricular tissue to enhance long-term remodeling. Engineered tissues for cardiac repair are evolving and in-vivo studies such as are valuable results that may help the field advance toward human use.

Although the goals of the study are modest, and methods used to quantify histological changes are limited, the results do provide useful information and show that this type of tissue engineered patch with human iPS cells and some type of biodegradable substrate can integrate with the native myocardium, albeit with limited functional outcomes at the present time.

A major limitation of the study is that the application of these patches, as stated in the introduction, can help with tissue remodeling in situations such as ventricular aneurysm, but the studies were done in a much different ventricular trauma injury model. The acute injury response to the procedure here is much different than the human diseases mentioned in the introduction and as rationale for the study. This difference is not discussed in the paper.

The methods should describe how the microscopic imaging were analyzed and what exactly was quantified, including sample sizes. Also, information on orientation of the sections, since cardiac and scar tissue, the patch and the geometry of the wall are all have direction-dependence structure. Finally, better quantification of the cell numbers and degree of calcification should be done in addition to the qualitative descriptions in the results.

Part of the study is the examine the biodegradability of the patch substrate, but this was not quantified, and only vaguely described in the paper. How do we know the that “some patch material still remains and is covered by collagen” or that it actually degrades? Some kind measure to back up these statements would be useful.

The quantitative results are likely under-powered, with small sample sizes (n=2 or 3). Have the authors done any power analysis or other statistical approaches to determine if the numerical results (echo studies) are actually statistically valid? Changes in LV function is one of the stated goals of the study, and it is hard to interpret the functional results with these sample sizes.

The conclusions paragraph can be improved. The first and third sentences are not conclusions. The second sentence is the only actual statement of a conclusion, and certainly does not summarize the main findings of the paper.

Minor:

Please include page numbers for easier referencing. Also, please use indentation or spacing for new paragraphs.

Abstract, last sentence: “differentiate into cardiomyocytes survive”, correct wording.

Intro, line 4: “cell attachment or migration” migration of what cell type?

Intro, line 14: “Litations”, Limitations??

The discussion starts with references to post-infarction remodeling, but the current study does not use that model. It would be better for the rationale for the current experiments to be better related to the same type of remodeling that would occur with the current animal model. Why is “infection” mentioned, when this is not part of the current study (but durability and biocompatibility are)?

Discussion, first para: “sustain regular rhythm”, why mention this is such a short “intro” in the discussion, when it is not part of the current study?

Discussion: “In the past, heart tissue was considered non-renewable and the heart was considered the final organ”. Wording is awkward.

Discussion: “….to create a pulsation model.” Not clear what this type of model refers to.

Discussion: “and host species leading to a non-sychronized heartbeat” not-clear that this is the case or that this would happen here. Also typo in “synchronized”

**********

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Reviewer #1: No

Reviewer #2: Yes: George A. Truskey

Reviewer #3: No

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Attachment

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PLoS One. 2020 Jun 8;15(6):e0234087. doi: 10.1371/journal.pone.0234087.r002

Author response to Decision Letter 0

7 Apr 2020

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Matsuzaki and colleagues submit their manuscript entitled, “The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model” for consideration of publication in PLOS ONE. The authors use a rodent model to test whether a cell-seeded biodegradable tissue-engineered patch provides beneficial effects in LV function. The experiments are well designed with appropriate controls. Unfortunately, but not surprising cell-seeding of iPS cells did not lead to an increase in cardiomyocytes. The authors do demonstrate that a biodegradable patch can be used as a viable option for ventricular rupture.

Comments

1. The potential benefit of the biodegradable patch was the ability to cell-seed the patch. However, since the cell-seeding did not lead to increased myocardial cells, authors should clarify the benefit of their patch over non-absorbable patches.

Thank you for pointing out. The discussion section has been modified. (Page 14 line 227-Page 15 line 244).Current non-biodegradable material which is used for LV repair has the risk of thrombosis and the need for frequent reoperation due to infection. Bioabsorbable materials are more useful than others as they serve as scaffolds for ingrowth of functional host myocardium and once degraded there is no longer a foreign material that can act as a nidus for infection.

2. Inclusion of a non-biodegradable patch as a comparison to the biodegradable patch could have been included. Do the authors have any comparison data? Especially in light that at 36 weeks, there was no difference between cell-seeded and no cell-seeding. So is a biodegradable patch add benefit?

Thanks for asking a question. As stated in question 1, we thought that bioabsorbable materials are useful for models of left ventricular formation after myocardial infarction. The design of this experiment focuses most on demonstrating the efficacy of iPS cell seeding using the bioabsorbable material. Therefore, the control group is only the non-seeded bioabsorbable material group. (page14 line 227-page15 line 244)

3. Was vascularization assessed? Also, was staining performed for fibroblasts?

Thank you for pointing out. We decided to evaluate angiogenesis in one-month and 6 month specimens by immunohistochemisty of CD31. It was newly confirmed by CD31 that the number of new blood vessels was higher in the seeded group. We did not stain for fibroblasts. The reason was difficulty in using immunohistochemical markers specific to markers and not other cell types capable of ECM production. (Page13 line 213-221 )

Reviewer #2: The work described in this manuscript addresses an important issue of designing a cell-seeded cardiac patch to improve cardiac function. Successful development of such a system could improve cardiac function in adults after ventricular rupture. The limited results in this study show that biodegradable patches implanted at the left ventricular apex with or without cardiomyocytes did not improve cardiac function. The reason for this lack of effect could not be clearly identified due to limitations of the study design.

The authors provide no assessment of cardiomyocyte function or organization prior to implantation, so it unclear whether the implanted cardiomyocytes had any contractile function at the time of implantation.

Cardiac progenitor cells purchased from CDI, thawed, and directly sprayed on bioabsorbable material. This time, it was Cardiac progenitor cells, not a differentiated cardiomyocyte, so it did not have a contractile function.

The small sample size in each group (1 and 2 month time points had 2 animals in each group and the 6 and 9 each had 3 animals in each group) precluded detection of differences between the two groups except at 8 weeks for ejection fraction. The sample size is much smaller than the authors used in their prior study, reference 11.

Thank you for pointing out. Small sample size is one of the limitations. This study is still at a preliminary stage, and only examines how seeding iPS cells with bioabsorbable material contributes to myocardial regeneration. In the future study, we would like to perform power analysis and increase the number of samples. (Page 18 line 298-302)

The histological analysis of the implants did not examine for the presence of cardiomyocytes or vascularization in the patches. Thus, the reason for the difference in ejection fraction due to cell seeding could not be evaluated.

Thank you for pointing out. When statistical processing was actually performed using the AVOVA, there was no significant difference in the ejection fraction. We now include in the manuscript quantification of vascularization using IHC against CD31 and found that there was a greater number of new blood vessels in the seeded group. (Figure 6) Cardiomyocytes on the patch were also described in Figure 5C. (Page 13 Line 207-221)

Specific Comments

1. Figure 4. Horizontal axis label is needed for each panel. Horizontal axis labels are now included for all panels.

2. Figure 4. Were the changes in LV systolic and diastolic volumes over time significant? The statistical methods used indicate that t-tests were used, but the results in Figure 4 indicate that a two-factor ANOVA (time and treatment) should be used to evaluate trends with time.

We performed statistical processing with ANOVA and there was no longer a significant difference between groups at the 2M time point. The manuscript has been updated to reflect this change. (Page 12 line 190-194)

Reviewer #3: This study tested the feasibility of applying stem-cell patches with a biodegradable substrate to damaged ventricular tissue to enhance long-term remodeling. Engineered tissues for cardiac repair are evolving and in-vivo studies such as are valuable results that may help the field advance toward human use.

Although the goals of the study are modest, and methods used to quantify histological changes are limited, the results do provide useful information and show that this type of tissue engineered patch with human iPS cells and some type of biodegradable substrate can integrate with the native myocardium, albeit with limited functional outcomes at the present time.

A major limitation of the study is that the application of these patches, as stated in the introduction, can help with tissue remodeling in situations such as ventricular aneurysm, but the studies were done in a much different ventricular trauma injury model. The acute injury response to the procedure here is much different than the human diseases mentioned in the introduction and as rationale for the study. This difference is not discussed in the paper.

Thank you for pointing out. Certainly, the indication described in the introduction is the creation of patches for use after human myocardial infarction. This study is still at a preliminary stage, and only examines how seeding iPS cells with bioabsorbable material contributes to myocardial regeneration. (Page 18 line 298-302) We believe that this model of rat is useful for evaluating remodeling for about one year. Also many paper exist to attach these iPS patch on the above of LV. But there is no report of survival experiments in rats which simulating the left ventricle, that is, simulating VSP or left ventricular rupture, and we consider the importance of this research on the method of surviving the rat for one year. (Page17 line276-279)

1.The methods should describe how the microscopic imaging were analyzed and what exactly was quantified, including sample sizes.

Also, 2. information on orientation of the sections, since cardiac and scar tissue, the patch and the geometry of the wall are all have direction-dependence structure.

Finally,3. better quantification of the cell numbers and degree of calcification should be done in addition to the qualitative descriptions in the results.

Thank you for pointing out.

1. We have added details on the analysis method of the microscopy. (Page 10 line161-Page11 line172)

2. Details have been added to the section. After the sacrifice, the left ventricle was separated from the heart by axial dissection, and a sagittal transection centered on the apical patch was performed. (Page10 line156-157)

3. Regarding the area of calcification, there was no statistically significant difference between Seeded (N = 6) 16.0 ± 5.2% vs Unseeded 8.14 ± 3.3% (N = 6) (P = 0.09).(Figure 6C-2) We stained CD31 to check for angiogenesis. There is statistically significant differences at 6M timepoint (N=3). It is defined as above the patch and near the anastomosis. (Page 13 line 207- 221) (Figure 6C-3)

Part of the study is the examine the biodegradability of the patch substrate, but this was not quantified, and only vaguely described in the paper. How do we know the that “some patch material still remains and is covered by collagen” or that it actually degrades? Some kind measure to back up these statements would be useful.

Thank you for pointing out. We characterized scaffold degradation and neotissue formation using polarized light images of Picro-Sirius Red (PSR) stained sections from Rat Patch obtained 1 M, 2M, 6M,9M after implantation, which revealed both the degradation of PGA fibers and the deposition and maturation of collagen fibers. The PGA fibers remained highly organized within the scaffold at 1M after implantation, but had thinned and begun to show evidence of early fragmentation at 2M. Only rare thin individual fragments of the PGA fibers were visible by 6M after implantation. (Page 12 line 200-204, Figure 5B)

The quantitative results are likely under-powered, with small sample sizes (n=2 or 3). Have the authors done any power analysis or other statistical approaches to determine if the numerical results (echo studies) are actually statistically valid? Changes in LV function is one of the stated goals of the study, and it is hard to interpret the functional results with these sample sizes.

The small sample size is a limitation of this preliminary study. Power size was not verified. (page 18 line 298-302) A new statistical analysis was performed using ANOVA. (page11 line 175-178)

The conclusions paragraph can be improved. The first and third sentences are not conclusions. The second sentence is the only actual statement of a conclusion, and certainly does not summarize the main findings of the paper.

Thank you for point out. We have modified the conclusions. (Page19 line313 -317)

Minor:

Please include page numbers for easier referencing. Also, please use indentation or spacing for new paragraphs.

Thank you for pointing out. The manuscript now includes page numbers.

Abstract, last sentence: “differentiate into cardiomyocytes survive”, correct wording.

Thank you for pointing out.

This wording has been changed to “Seeded hiPS-CPCs did not differentiate into cardiomyocytes, but the greater number of new blood vessels in seeded patches suggests the presence of cell seeding early in the remodeling process might provide a prolonged effect on neotissue formation.” (page 4, Line 62-67)

Intro, line 4: “cell attachment or migration” migration of what cell type?

This line has been modified to specify macrophage migration. (Page 5 Line 72-74)

Intro, line 14: “Litations”, Limitations??

The spelling of this word has been corrected to “limitations”(page 5 Line 83)

The discussion starts with references to post-infarction remodeling, but the current study does not use that model. It would be better for the rationale for the current experiments to be better related to the same type of remodeling that would occur with the current animal model. Why is “infection” mentioned, when this is not part of the current study (but durability and biocompatibility are)?

Thank you for your comments and great questions to our experiments. When we try to create infarction model with the additional incision of left ventricular apex for placing the patch in rats, animal survival rates were low following the procedure. Therefore, we simplified the model to utilize left ventricular apex incision to implant our patches in the blood stream. (Page17 line 271-274)

In general, cardiac surgeons have concerns about implanting foreign materials in the heart as infective endocarditis can be a lethal complication after open heart surgery. Even now, some surgeons inevitably use Dacron patch or PTFE patch to repair for ventricular septal rupture after myocardial infarction. These clinical demands motivated us to develop new bioabsorbable materials for use in cardiac surgery. This experiment is initial attempt to develop new cell-based materials. (page 14 line227- 237)

Discussion, first para: “sustain regular rhythm”, why mention this is such a short “intro” in the discussion, when it is not part of the current study?

Thank you for pointed out. As you mentioned this is not part of the current study so we eliminated this sentence.

Discussion: “In the past, heart tissue was considered non-renewable and the heart was considered the final organ”. Wording is awkward.

This section has been modified.　(page15 line246)

Discussion: “….to create a pulsation model.” Not clear what this type of model refers to.

This section has been modified (page16 line269)

Discussion: “and host species leading to a non-sychronized heartbeat” not-clear that this is the case or that this would happen here. Also typo in “synchronized”

This section has been modified.　(page18 line298-page19 line310)

Attachment

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Decision Letter 1

YIRU GUO

24 Apr 2020

PONE-D-19-33452R1

The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model

PLOS ONE

Dear Dr. Shinoka,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jun 08 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

YIRU GUO, M.D., F.A.H.A.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Matsuzaki and colleagues submit their revised manuscript for evaluation for publication in PlosOne. The initial criticism which dampened this reviewer’s enthusiasm for the manuscript was the negative results that cell-seeding of iPS cells did not lead to an increase in cardiomyocytes nor longer term improvement of cardiac function. Additionally, the lack of an adequate control for the biodegradable patch was excluded. The authors have only made minimal changes to the manuscript in the discussion highlighting the negative results, but more importantly not providing adequate discussion of the potential positive effects of the biodegradable patch without cell seeding. There is a an abundance of literature of the use of bioactive scaffolds without the use of cell transplantation for myocardial repair/regeneration.

Reviewer #2: While preliminary, the results show that merely adding the iPS-derived cardiac progenitor cells is insufficient to ensure the survival of these cells in a cardiac patch. This information is worth transmitting to the research community. Study limitations are duly noted.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2020 Jun 8;15(6):e0234087. doi: 10.1371/journal.pone.0234087.r004

Author response to Decision Letter 1

28 Apr 2020

Reviewer #1: Matsuzaki and colleagues submit their revised manuscript for evaluation for publication in PlosOne. The initial criticism which dampened this reviewer’s enthusiasm for the manuscript was the negative results that cell-seeding of iPS cells did not lead to an increase in cardiomyocytes nor longer term improvement of cardiac function. Additionally, the lack of an adequate control for the biodegradable patch was excluded. The authors have only made minimal changes to the manuscript in the discussion highlighting the negative results, but more importantly not providing adequate discussion of the potential positive effects of the biodegradable patch without cell seeding. There is an abundance of literature of the use of bioactive scaffolds without the use of cell transplantation for myocardial repair/regeneration.

Thank you for your comments. As you pointed out, I have made the following changes to the discussion section, incorporating literature on the benefits of cell-free scaffolds.　Thank you for your understanding. (P14, line237- P16, line 261)

Complications associated with non-biodegradable materials and the potential to regenerate functional myocardium associated with tissue engineering have fostered the development of surgical models and evaluation of various scaffold designs. Due to challenges and limitations in the use of exogenous cell-based biomaterials, cell-free strategies are being investigated as an alternative. This approach requires the attachment and growth of host parenchymal cells as well as their production and organization of extracellular matrix. Materials of natural origin, such as collagen and fibrin, have been evaluated as scaffolds for tissue regeneration after myocardial infarction. These are applied surgically as patches, or less invasively as gel forming injectable ECM proteins. Acellular scaffolds are advantageous over cellular scaffolds in that: (1) these are off-the-shelf products that can be immediately implanted (e.g., SynerGraft®), (2) elicit a limited immune response, and (3) cost far less to produce. 14,15 Biodegradable synthetics, like the polymers that comprise our scaffold, are another category or materials widely used in tissue engineering applications due to their mechanical properties, material uniformity, stability, and lower risk of infection compared to natural biomaterials. Biodegradable synthetic polymers can be modified with high precision to meet tissue-specific properties such as appropriate degradation rates, porosity, and mechanical strength.16-19

Reviewer #2: While preliminary, the results show that merely adding the iPS-derived cardiac progenitor cells is insufficient to ensure the survival of these cells in a cardiac patch. This information is worth transmitting to the research community. Study limitations are duly noted.

Thank you for your comments.

Reviewer #3: (No Response)

Attachment

Submitted filename: Review Comments to the Author newest.docx

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Decision Letter 2

YIRU GUO

19 May 2020

The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model

PONE-D-19-33452R2

Dear Dr. Shinoka,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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YIRU GUO, M.D., F.A.H.A.

Academic Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Authors addressed concerns. No further comments or revisions needed. Although a negative study, the manuscript further provides evidence that iPS cells alone are not beneficial for myocardial repair and that a polymer alone may impact LV remodeling after an injury.

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Reviewer #1: No

Acceptance letter

YIRU GUO

28 May 2020

PONE-D-19-33452R2

The Evaluation of a Tissue-Engineered Cardiac Patch Seeded with hiPS Derived Cardiac Progenitor Cells in a Rat Left Ventricular Model

Dear Dr. Shinoka:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

Dr. YIRU GUO

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