YING MEI, Ph.D.
Assistant Professor of Bioengineering
Clemson University MUSC Campus
Cardiovascular disease, the leading cause of death and disability worldwide, claims more lives than all kinds of cancers combined. Ischaemic heart disease (IHD) and myocardial infarction (MI) are major contributors to cardiovascular morbidity and mortality; they usually are associated with irreversible death of cardiomyocyte and lead to permanent loss of heart function. Human embryonic stem cell (hESC) and human induced pluripotent stem cell (hIPSC) technologies hold remarkable promise for myocardial regeneration because of their unquestionable capacity to produce de novo cardiomyocytes.
Two key limitations of existing hESC and hIPSC technology are:
Synthetic short peptides derived from extracellular matrix (ECM) proteins and growth factors have been extensively used to enhance cell adhesion and proliferation on synthetic materials. However, their low affinity for cell-surface receptors limits their ability to direct stem-cell differentiation. Using my expertise in peptide synthesis, polymer science, combinatorial approach, and hESC and hIPSC technology, I propose to develop completely synthetic, biologically functional substrates to direct hESCs and hIPSCs to differentiate into clinical-grade cardiomyocytes in a xeno-free, chemically defined condition.
The central hypothesis of this project is that the substrates with a variety of high density peptides derived from multiple cardiogenic ECM proteins and growth factors that are essential to cardiogenesis can effectively induce cardiac differentiation of hESCs and hIPSCs through high affinity, synergic engagements between peptides and cell-surface receptors.
Accordingly, our specific aims are:
Aim 1: Develop completely synthetic, biologically functional culture substrates using combinatorial peptide-polymer arrays for directed cardiac differentiation; and
Aim 2: Develop a rapid, efficient method to differentiate hESCs and hIPSCs into homogeneous populations of functional cardiomyocytes in xeno-free, chemically defined conditions.
We have made significant progress in the project VII. We have laid down a solid foundation for the fabrication of peptide-polymer microarrays. To this end, we have established a robust synthetic route to prepare peptide-acrylate monomers for peptide-polymer microarray fabrication. Further, the peptide-acrylate monomers have been utilized to prepare peptide-functionalized polymeric substrates with elasticity (~10KPa) similar to that of neonatal cardiac tissues. To seek extramural support, I have submitted a R21 proposal based on the Project VII. The proposal was scored in top 30%, which indicated a good possibility to secure a NIH R01 grant in the future. In addition, an invited review article “microarrayed materials for stem cells” was published in Materials Today last October. The COBRE support has also encouraged us to participate in the NSF-Bioprinting project. To this end, we have developed a 3D printing based approach to fabricate vascularized cardiac tissues using neonatal rat cardiomyocytes and human vascular cells. This project has led to a R03 proposal and two manuscripts in preparation. It is noteworthy that 3D printing facilitated cardiac tissue fabrication can be naturally synergized with the COBRE project, which focuses on the derivation of human cardiomyocytes from hESCs and hIPSCs. I expect the synergy between the two projects will lead to multiple NIH/NSF proposals and peer-reviewed publications.
Cardiovascular disease is the leading cause of death worldwide. Presently, the only long-term solution is cardiac transplantation, a procedure severely limited by a shortage of donor hearts1-4. As an alternative, stem-cell mediated myocardial regeneration has been the focus a significant amount of research. Although adult stem cells have been shown to have little potential to generate de novo cardiomyocytes, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) have unquestionable capacity to be differentiated into cardiac lineage5, 6. Stem-cell differentiation is controlled by both soluble and insoluble factors. In particular, significant research has been conducted to engineer soluble factors, such as growth factors and small molecules, to direct cardiac differentiation of hESCs and hIPSCs 7-11. However, little work has been done to optimize insoluble factors, such as the substrates on which cells grow12, 13. The currently used substrates, Matrigel and Synthemax, were originally developed to support self-renewal of hESCs and hIPSCs14, not to promote cardiac differentiation. In addition to the lack of substrates to facilitate cardiac differentiation of hESCs and hIPSCs, another big challenge is that immature cardiomyocytes derived from hESCs and hIPSCs are functionally similar to embryonic/neonatal-stage human cardiomyocytes, not adult cardiomyocytes, and thus have limited clinical application15-18.
The current inability to derive fully mature cardiomyocytes from hESCs and hIPSCs remains a key challenge for stem-cell mediated cardiac regeneration. My long-term career goal is to develop bioengineering approaches for the derivation of a sufficient number of fully mature cardiomyocytes from hESCs and hIPSCs for cardiac tissue regeneration. To achieve this, I propose a two-stage differentiation protocol (Fig. 1). In Stage 1, I propose to induce cardiac differentiation of hESCs and derive immature cardiomyocytes. In Stage 2, the immature cardiomyocytes derived from the first stage will be terminally differentiated into a fully mature phenotype. Here, I propose two convergent specific aims to assess how the environmental factors (e.g., substrates and electrical stimulation) can affect hESC cardiac differentiation and maturation process. The goal of the proposal is seek the mechanism of the effects of environmental factors with the intent to use this information in the future to develop a bioengineering approach for cardiovascular regeneration.
Figure 1: Two-stage cardiac differentiation process to derive fully mature cardiomyocytes from hESCs.
Aim 1: High-throughput assessment of polymeric substrates for enhanced cardiac differentiation of hESCs. We hypothesize that with high-throughput screening of a library of polymeric substrates known to promote hESC clonal growth, substrates capable to enhance cardiac differentiation of hESCs can be identified.
Aim 2: Promote terminal differentiation of hESCs derived immature cardiomyocytes by mimicking key aspects of biochemical and biophysical stimuli in developing hearts. We hypothesize that we can promote maturation (e.g., sarcomerogenesis) of hESC-derived immature cardiomyocytes by mimicking biochemical and biophysical stimuli in developing hearts (e.g., co-culture with pediatric human cardiac fibroblasts, 3D cell spheroid culture, and electrical stimulation).
This approach is innovative: For the first time, we will utilize an emerging polymer microarray technology with high throughput to develop defined substrates to facilitate cardiac differentiation of hESCs. Further, we will recapitulate key aspects of biochemical and biophysical stimuli of developing hearts to derive fully mature cardiomyocytes. The central hypothesis of this proposal is that a library of materials known to promote hESC and hIPSC clonal growth can allow for the identification of substrates capable to facilitate cardiac differentiation of hESCs. Further, I hypothesize we can promote maturation of hESC-derived immature cardiomyocytes by mimicking key aspects of biochemical and biophysical stimuli in developing hearts.
By exploiting human pluripotent stem-cell technology, high throughput materials development, and principles of cardiovascular developmental biology, this research would allow for efficient derivation of fully mature cardiomyocytes from hESCs. It can have major impacts in drug development and cardiac tissue engineering.