Regenerative medicine approaches to the treatment of cardiovascular disease

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1 Regenerative medicine approaches to the treatment of cardiovascular disease Author: Noah Weisleder, Ph.D. Abstract: Ischemic heart disease related myocardial infarction (MI) remains the leading cause of mortality in western countries and increasingly in the rest of the world. However, there are no therapies currently available to directly treat MI-induced death of cardiomyocytes to improve cardiac function in the infarct zone.cardiac tissue regeneration in the infarct zone using stem cells is a promising approach for directly treating MI. However, several challenges remain in this area including poor cell retention at the injection site, dismal cell viability, and inadequate differentiation into cardiomyocytes (particularly for non-pluripotent stem cells). Although pluripotent stem cells, like embryonic stem (ES) and induced pluripotent stem (ips) cells, possess the best capacity to differentiate into cardiomyocytes and other cardiac cells, they can also form tumors after injection. We have developed novel coaxial electrospray and 3D nonplanar microfluidic technologies to encapsulate mouse ES cells for miniaturized 3D culture in vitro. This technology improves ES cell survival and proliferation, allowing the ES cells to merge and form one cell aggregate in each microcapsule. in these aggregates display higher pluripotency for further direct differentiation into cardiac lineage than the cells cultured under conventional 3D or 2D conditions. After pre-differentiation into the cardiac lineage the aggregates were further encapsulated in an alginate-chitosan micro-matrix (ACM) for intramyocardial implantation to treat MI in a mouse model. The ACM encapsulated cells were found to differentiate into cardiomyocytes in the infarct zone, which increases survival time and cardiac structure/function. Remarkably, while injection of nonencapsulated cell aggregates would result in tumor formation (87.5%), it was not observed at all with ACM encapsulated cells.the use of this technology to assist with the delivery of stem cells may have translational value of the treatment of MI in patients. Keywords:myocardial, heart attack, nanotechnology, encapsulation, stem cell, regenerative medicine Introduction: Ischemic heart disease or myocardial infarction (MI) due to coronary arteriosclerosis remains the single largest cause of mortality in western countries and is increasingly common in other parts of the world, to a level that it is expected to become the leading global cause of death within a decade 1-3. While there are interventions available to reestablish coronary perfusion and treat arrhythmias associated with MI, there is no currently available approach to directly treat cardiomyocyte death Developing novel therapeutic approaches that can compensate for the loss of cardiomyocytes and other cardiac resident cells will have broad translational potential in the treatment of MI to prevent or slow the progression into heart failure One such approach is the use of stem cells to regenerate cardiomyocytes lost due to MI Through multiple attempts to use stem cells to treat MI the field has rapidly evolved to the point where there are current clinical trials for cell-based therapies of MI Much of the continuing pre-clinical work focuses on isolating stem cell populations that can differentiate into cardiac resident cells, and in particular cardiomyocytes, or manipulation of culture or delivery conditions to improve survival and differentiation of stem cells to repopulate the myocardial infarct zone 43,44. Various stem cells have been tested with varying levels of success, including ES cells, mesenchymal stem cells, hematopoietic stem cells, resident cardiac stem and progenitor cells, and ips cells 13,14, Preclinical studies with many of these cell populations indicate that some cell types can contribute to replacement of cardiomyocytes 45,46,57-61, in some cases through endogenous mechanisms and paracrine effects Recent clinical trials show some efficacy for stem cell approaches in human patients by paracrine effects that lead to increased angiogenesis and an attenuated inflammatory response Despite the promise of these regenerative approaches there are several key challenges associated with the development of stem cells as a therapeutic approach for MI. First, only a small number of injected cells eventually

2 implant into the infarct zone. It is common for therapeutic efforts to involve systemic or local injection of millions of stem cells into an animal to generate a very small number of viable cells that arrive and/or implant into the injured myocardium following MI. There are several different aspects that contribute to the loss of injected cells, varying from physical shear forces during the delivery and transit of stem cells through the circulatory system to the immunological effects that can result in the death of cells even before they arrive at the target tissues Increasing the efficiency of delivery would reduce the number of stem cells that would have to be generated for each preparation, thus increasing the ability to translate experimental stem cell approaches into viable therapies. Second, exposure to systemic and local stressors results in the loss of a massive percentage of injected stem cells after direct implantation into the local tissue environment. These losses can result from the inflammatory effects that are greatly exacerbated in the infarct zone in the heart following MI. Loss of stem cells can decrease the efficacy of any attempted therapeutic approach, as there will be fewer cells that can actually contribute to the regeneration of the heart. Third, cells that do implant have to differentiate into cell types that have some restorative effect on heart structure and function. There is significant disagreement on the extent that many of these various stem cell types can differentiate into functional cardiomyocytes. Pluripotent stem cells are believed to have the best capability of differentiating into cardiac cells within an appropriate developmental niche Fourth, the use of (particularly pluripotent) stem cells can raise concerns about potential tumorigenesis following injection of the cells. While the degree of risk for tumor formation can vary by type of stem cells and the specific approach used, the issue of tumor formation can complicate the use of any stem cells in regenerative medicine. In an attempt to address these challenges we have made use of novel microencapsulation technologies that can improve the regenerative capacity of mouse ES cells following experimental MI in mice. We developed novel coaxial electrospray and non-planar microfluidic technologies to create cell-laden microcapsules where ES cells (or any other cells) can be suspended in an aqueous liquid core surrounded by a shell made of alginate hydrogel. Mouse ES cells can survive well in the miniaturized core space and proliferate within 7 days to form a single ES cell aggregate in each microcapsule with much higher pluripotency than the ES cells under conventional 3D bulk hydrogel or 2D culture 21,22. These pluripotent ES cell aggregates can be effectively directed into cardiac lineage using BMP-4 and bfgf treatment for 3 days 21,83. These pre-differentiated cell aggregates can then be released out of the microcapsules to allow further differentiation. Released aggregates can then be encapsulated once again in a 3D alginate-chitosan micro-matrix (ACM) to complete the preparation for use in regenerative medicine. While we have initially had in vitro success with using this microencapsulation approaches coupled with pretreatment to induce differentiation of ES cells into cardiomyocytes, we have recently tested this approach in vivo in a mouse model of MI 21,35. Our novel microencapsulation approaches address a number of challenges facing the use of stem cells in regenerative medicine with several innovative aspects. First, the use of biomimetic (to pre-hatching embryos) core-shell microcapsules to encapsulate pluripotent stem cells for culture allows generation of cell aggregates with high pluripotency and uniform size. The latter is also important because it allows generation of a product with consistent characteristics that would comply with FDA regulations for use in clinical trials. Second, the ACM composition combines excellent biocompatibility and the capacity to alter the permeability and degradation kinetics of the materials 27-34, allowing for optimization of the characteristics of the micro-matrix. Third, the capacity for the ACM to create a microenvironment that can both increase differentiation of stem cells and decrease tumorigenesis would be an innovation that could increase the efficacy and safety of this pluripotent stem cell-based approach. Fourth, we find ACM encapsulation allows us to use fewer stem cells compared to other approaches and still have a therapeutic effect, an innovation that would also help to increase the translation of these findings to treatments for MI. Since fewer cells would have to be generated for each treatment, it would make it more likely that this approach could be widely applied in a clinical setting at a lower cost. Results: For our initial proof of concept studies with ACM encapsulated cell aggregates we used a permanent ligation mouse model of MI and conducted intramyocardial injection of with cells/animal into the periphery of the infarct zone immediately following permanent occlusion of the LAD. Figure 1A shows typical pictures of the gross

3 Fibrotic area, % Fibrotic area % morphology of the heart (top row) together with H&E (middle row) and Masson s trichrome (bottom row) stained tissue sections for the MI with different treatments. Being consistent with the literature, injection of non-encapsulated cells (even with pre-differentiation to early cardiac lineage) resulted in obvious tumor formation in injected animals. Interestingly, microscopic examination of the tumor architecture (Fig. 1B and C) revealed that it is a diffuse tissue with loose extracellular matrix rather than typical teratoma with dense tissue architecture, which might be a result ofpre-differentiation of the ES cell aggregates into a (A) No PLA ACM- ACM (B) Myocardium (C) Myocardium 10 m 10 m (D) (E) 10 m 10 m N o P L No PLA S ali ne AC M- Cell s ACM& Treatments Cell s AC M ACM Figure 1.Intramyocardial injection of ACM encapsulated, pre-differentiated ES cell aggregates minimizes fibrosis and depresses tumor formation in mouse myocardial infarction (MI) model produced by permanent ligation (PLA). (A), low magnification images of the heart (top row) and H&E (middle row) and Masson s trichrome (bottom row) stained tissue sections showing tumor formation (7/8 or 87.5%) in the group treated with non-encapsulated cell aggregates and extensive fibrosis in the saline and materials (i.e., ACM without cells) treated groups; high magnification micrographs of H&E (B) and Masson s trichrome (C) stained tissue sections showing the microstructure of tumor tissue adjacent to normal myocardium; (D), typical Masson's trichrome stained MI tissue treated with ACM encapsulated cell aggregates showing minimal fibrosis and possible cardiomyocyte-like cells (arrows) differentiated from the injected cells; (E), typical high magnification micrograph of Masson's trichrome staining showing extensive fibrosis in saline treated group; and (F), quantitative data of fibrosis showing significantly reduced fibrosis in the two groups treated with the pre-differentiated ES cell aggregates, compared to the saline and ACM treated groups. : p< 0.05 mesoderm lineage before injection. More importantly, tumor formation was not observed in any of the 8 animals treated with the ACM encapsulated cell aggregates. In other words, ACM encapsulation of the pre-differentiated cell aggregates could effectively depress tumor formation, potentially addressing one of the major concerns with using pluripotentstem cells for regenerative medicine. Moreover, cardiomyocyte-like cells (arrows, Fig. 1D) possibly differentiated from the transplanted cells were observable in the MI tissue treated with the ACM encapsulated cell aggregates. Besides the absence of tumor formation, the MI tissue treated with encapsulated cell aggregates also had reduced total area of fibrosis (Fig. 1A and D) while it was extensive in both the saline and materials (ACM without cells) treated groups (Fig. 1A and E). These differences between the groups treated with and without the predifferentiated cell aggregates are further quantified to be statistically significant (Fig.1F).

4 Survival, % Survival time, day Additional studies tested cardiac function that reflects the contractility of the left ventricular wall by measuring the left ventricle inner diameter during both systole (LVIDs) and diastole (LVIDd) using echocardiography. The treatment with encapsulated cell aggregates significantly reduced both LIVDs (Fig. 2A) and LVIDd (Fig. 2B) compared to the treatments with saline and ACM in the mouse MI model although they were (C) (D) still larger than that of normal heart. Figure 2.ACM encapsulated pre-differentiated ES cell aggregates improves We also found that treatment with cardiac 75 function and animal survival in mouse MI model created by permanent non-encapsulated ES cell aggregates ligation (PLA). No (A-B), PLA quantitative data showing the treatment with ACM encapsulated cell ACM- aggregates significantly reduced both LVIDs (A) and LVIDd (B) significantly reduced both LIVDs and compared to the saline ACM and materials (ACM) treatments; (C), percentage survival of LVIDd compared to the treatments mice with 25 PLA and four different treatments together with mice with no PLA showing that the treatment with ACM encapsulated cell aggregates improved with saline and ACM in the mouse MI 5 survival while 0 the treatment with non-encapsulated cell aggregates No resulted model. However, the high rate (7/8 or Treatments ACM- in the ACM worst survival; and (D), mean survival time of animals PLA died of PLA for the four 87.5%) of forming tumors (see Fig. different treatments Time showing Days elapsed, Elapsed significantly day extended survival time Treatments for mice treated with ACM encapsulated cells compared to non-encapsulated cells. : p< A) leads to the worst survival percentage (Fig. 2C) and survival time (Fig. 2D) among the four treatment groups with PLA. The mice with MI treated with non-encapsulated started to die on day 2 while it did not happen to the ACM and saline treated mice until day 5 and the ACM encapsulated cell aggregates treated mice until day 8. On day 15, the percentage survival of the MI mice treated with ACM encapsulated cell aggregates was more than twice of that for the other three treatments. Moreover, the survival time of animals following PLA is significantly longer for the mice treated with ACM encapsulated cell aggregates compared to nonencapsulated cell aggregates. Discussion: For these studies, we used mouse ES cells because these cells are thought to have the best potential to differentiate into mouse cardiomyocytes and other cardiac resident cells, making them useful for transplantation into the infarct zone. However, the main disadvantage of using ES cells is that they frequently produce teratomas. We subjected C57Bl6/J mice to permanent surgical occlusion of the LAD and the ACM encapsulated aggregates containing 2 x 10 5 cells were injected into the infarct zone at five minutes after the occlusion of the LAD. We found that these encapsulated cells increase the survival time of the mice following permanent LAD occlusion as well as improving cardiac structure and function. Labeling the encapsulated cells showed the appearance of new cells in the infarct zone that express cardiomyocyte markers such as cardiac troponin T (ctnt), -actinin, and connexin-43, suggesting that new cardiomyocytes are formed from the implanted cells in the infarct zone. Interestingly, we found that while aggregated cells without ACM encapsulation would produce tumor formation in nearly all of the mice (87.5%), no overt tumor formation was observed in any mice injected with the ACM encapsulated aggregates (Fig. 1). These preliminary data led us to conclude that direct injection of pre-differentiated ES cell aggregates encapsulated in biodegradable, biocompatible ACM into an infarct zone allows for enhanced cell retention and viability, improved pathology, and reduced tumorigenesis following experimental induction of MI in mice. No PLA ACM- ACM

5 There are multiple advantages to the use of microscale biomaterials for culture and delivery of stem cells as a treatment for MI. It can produce cell aggregates of uniform size, which is an important consideration for the translation of these findings as clinical development of such an approach would require that characteristics of the delivery system could be standardized. Moreover, creating microencapsulated (< ~ 150 m) aggregates would reduce resistance to the transport of oxygen and nutrients to all the aggregated cells (compared to bulk or macroencapsulation) and enhance transfer of therapeutic products produced by the cells out of the aggregates Moreover, microencapsulated aggregates should have better mechanical stability, which should lead to better cell retention and biocompatibility. At the same time, microencapsulation may also allow for an appropriate niche to form within the microcapsule or micro-matrix that will increase differentiation of stem cells into the cardiac lineage during predifferentiation and cardiac resident cell types after injection 89,90. Biography: Noah Weisleder received his B.S. in Biotechnology and Molecular Biology from Worcester Polytechnic Institute, a Ph.D. in Cell Biology from Baylor College of Medicine and conducted his postdoctoral studies at Robert Wood Johnson Medical School. Currently, Dr. Weisleder is an Associate Professor of Physiology and Cell Biology at The Ohio State University and an Investigator in the Davis Heart and Lung Research Institute. Since that time, Dr. Weisleder has published numerous peer-reviewed publications or book chapters in the fields of muscle physiology, cardiovascular disease, cytoskeletal dynamics, membrane repair and cellular calcium homeostasis in normal physiology and disease states. He has chaired sessions at national and international meetings on muscle physiology and metabolism, and been invited to present his research at several international conferences. Additionally, he is an inventor on five US patents, related international patents and twelve published US patent applications. These inventions became the basis for formation of TRIM-edicine, a biotechnology company developing protein therapeutics targeting regenerative medicine applications, where Dr. Weisleder is a Co-Founder and Chief Scientific Officer. He has received a Fellowship from the American Heart Association, a Pathway to Independence Award from the National Institutes of Health and the Kauffman Foundation Outstanding Postdoctoral Entrepreneur Award. Contact Information: Noah Weisleder, Ph.D. Davis Heart and Lung Research Institute - Room 611A The Ohio State University Wexner Medical Center 473 W. 12th Ave. Columbus, OH Phone (614) Fax (614) noah.weisleder@osumc.edu