Yongsung Hwang

Engineering the cell-material interface for controlling stem cell adhesion, migration, and differentiation.

The effective utilization of stem cells in regenerative medicine critically relies upon our understanding of the intricate interactions between cells and their extracellular environment. While bulk mechanical and chemical properties of the matrix have been shown to influence various cellular functions, the role of matrix interfacial properties on stem cell behavior is unclear. Here, we report the striking effect of matrix interfacial hydrophobicity on stem cell adhesion, motility, cytoskeletal organization, and differentiation. This is achieved through the development of tunable, synthetic matrices with control over their hydrophobicity without altering the chemical and mechanical properties of the matrix. The observed cellular responses are explained in terms of hydrophobicity-driven conformational changes of the pendant side chains at the interface leading to differential binding of proteins. These results demonstrate that the hydrophobicity of the extracellular matrix could play a considerably larger role in dictating cellular behaviors than previously anticipated. Additionally, these tunable matrices, which introduce a new control feature for regulating various cellular functions offer a platform for studying proliferation and differentiation of stem cells in a controlled manner and would have applications in regenerative medicine.

Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling

Significance A mechanistic understanding of how calcium phosphate (CaP) minerals contribute to osteogenic commitment of stem cells and bone tissue formation is a necessary requirement for developing efficient CaP-based synthetic matrices to treat bone defects. This study unravels a previously unknown mechanism, phosphate-ATP-adenosine metabolic signaling, by which the CaP-rich mineral environment in bone tissues promotes osteogenic differentiation of human mesenchymal stem cells. In addition to a mechanical perspective on how biomaterials can influence stem cell differentiation through metabolic pathways, this discovery opens up new avenues for treating critical bone defects and bone metabolic disorders. Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.

Mesenchymal stem cell differentiation and roles in regenerative medicine

Adult stem cells with multi or unipotent differentiation potential are present in almost all tissues of adult organisms. The main function of these stem cells is to support normal repair and rejuvenation of diseased and aging tissues. Mesenchymal stem cells (MSCs) isolated from the bone marrow have the potential to differentiate into multiple connective tissues. Advancements in understanding tissue specific differentiation of MSCs in conjunction with global genomic and proteomic profiling of MSCs have not only provided insights into their biology but also made MSC based clinical trials a reality for treating various debilitating diseases and genetic disorders. The emerging evidence that MSCs are immunosuppressive makes them an even more attractive candidate for regenerative medicine as rejections of transplants by the recipient could be a limiting step for moving the stem cells based therapies from “bedside to bed side.” To a large extent the therapeutic potential of MSCs is attributed to their differentiation ability. The fate and commitment of MSCs are regulated by various instructive signals from their immediate vicinity or microenvironment, which comprises many biological molecules (soluble and insoluble) and biomechanical forces. These biochemical and biophysical factors play a pivotal role in determining the efficacy of MSC differentiation and their contribution to the repair process. In this review, we discuss the characteristics of MSCs, their differentiation potential toward different skeletal tissues (cartilage and bone), and their emerging role in regenerative medicine. Copyright

Poly(ethylene glycol) cryogels as potential cell scaffolds: effect of polymerization conditions on cryogel microstructure and properties

We report the synthesis and characterization of interconnected macroporous network structures of poly(ethylene glycol) (PEG) using cryogelation techniques. Novel monolithic networks containing a gradient of pore size in a layered fashion were created from a single precursor by manipulating their polymerization kinetics. Maintaining conditions that promote the rate of gelation compared to that of the nucleation of ice crystals leads to formation of either conventional hydrogel-like network structures or continuous heterogeneous networks containing layers of hydrogel-like and cryogel-like microstructures. In contrast, conditions promoting a faster rate of the nucleation of ice crystals compared to rate of gelation result in cryogels with a nearly homogeneous interconnected macroporous network. The rates of polymerization and nucleation of ice crystals were altered using a number of different parameters such as concentration of initiator, freezing temperature, and degree of supercooling. Compared to conventional hydrogels, the cryogels exhibit higher stress and strain at break; their mechanical and equilibrium swelling properties show a strong correlation with the network microstructure. Cell viability studies suggest no detrimental effect of these scaffolds on cell attachment and their distribution. Furthermore, a time dependent increase in chondrocyte proliferation was observed in cryogels over a long period of culture.

Templated mineralization of synthetic hydrogels for bone-like composite materials: role of matrix hydrophobicity.

Bone-mimetic mineral-polymer composite materials have several applications ranging from artificial bone grafts to scaffolds for bone tissue engineering; templated mineralization is an effective approach to fabricate such composites. In this study, we synthesized bone-like composites using synthetic hydrogels having pendant side chains terminating with carboxyl groups as a template for mineralization. The role of matrix hydrophobicity on mineralization was examined using poly(ethylene glycol) hydrogels modified with varying lengths of anionic pendant side chains (CH(2) horizontal lineCHCONH(CH(2))(n)COOH, where n = 1, 3, 5, and 7). The ability of these hydrogels to undergo templated mineralization was found to be strongly dependent upon the length of the pendant side chain as is evident from the extent of calcification and morphology of the minerals. Moreover, mineralized phases formed on the hydrogels were confirmed to resemble apatite-like structures. In addition to demonstrating the importance of material hydrophobicity as a design parameter for the development of bone-like synthetic materials, our study also provides a potential explanation for the in vitro differences between the apatite-nucleating capacity of aspartate-rich osteopontin and glutamate-rich bone sialoprotein.

Smart hydrogels as functional biomimetic systems

Stimuli-responsive (smart) hydrogels have attracted widespread attention as biomimetic systems due to their ability to respond to subtle changes in external and internal stimuli ranging from physical triggers such as temperature and electric field to chemical triggers like glucose and pH. Besides their intriguing behavior, the main interest in such smart hydrogels lies in their potential industrial and biomedical applications. Some of these applications include injectable biomaterials, tunable surfaces for cell sheet engineering, sensors, and actuators. In this review, we discuss the fundamental principles underlying the stimuli-responsive behavior of hydrogels and how these properties have led to major technological innovations. We also review recent advancements in the field of hydrogels, including self-healing and stimuli-responsive degradation in hydrogels. We conclude by providing a perspective on the potential use of smart hydrogels as multifunctional, bioactuating systems for cell and tissue engineering.

Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSC) are a promising cell source with pluripotency and self-renewal properties. Design of simple and robust biomaterials with an innate ability to induce lineage-specificity of hiPSC is desirable to realize their application in regenerative medicine. In this study, the potential of biomaterials containing calcium phosphate minerals to induce osteogenic differentiation of hiPSC was investigated. hiPSC cultured using mineralized gelatin methacrylate-based matrices underwent osteogenic differentiation ex vivo, in both two-dimensional and three-dimensional cultures, in growth medium devoid of any osteogenic-inducing chemical components or growth factors. The findings that osteogenic differentiation of hiPSC can be achieved through biomaterial-based cues alone present new avenues for personalized regenerative medicine. Such biomaterials that could not only act as structural scaffolds, but could also provide tissue-specific functions such as directing stem cell differentiation commitment, have great potential in bone tissue engineering.

Oligo(trimethylene carbonate)-poly(ethylene glycol)-oligo(trimethylene carbonate) triblock-based hydrogels for cartilage tissue engineering.

A triblock co-polymer of oligo(trimethylene carbonate)-block-poly(ethylene glycol) 20000-block-oligo(trimethylene carbonate) diacrylate (TMC20) was used as a photo-polymerizable precursor for the encapsulation of primary articular chondrocytes. The efficacy of TMC20 as a biodegradable scaffold for cartilage tissue engineering was compared with non-degradable poly(ethylene glycol) 20000 diacrylate (PEG20) hydrogel. Chondrocytes encapsulated in PEG hydrogels containing oligo(trimethylene carbonate) (OTMC) moieties underwent spontaneous aggregation during in vitro culture, which was not observed in the PEG hydrogel counterparts. The aggregation of cells was found to be dependent on the initial cell density, as well as the mesh size of the hydrogels. Similarly, cell aggregation was also found in biodegradable PEG hydrogels containing caprolactone moieties. The aggregation of cells in TMC20 hydrogels resulted in enhanced cartilage matrix production compared with their PEG20 counterparts over 3 weeks of culture. Taken together, these results indicate that PEG hydrogels containing degradable OTMC moieties promote the aggregation and biosynthetic activity of encapsulated chondrocytes, indicating their potential as scaffolds for the repair of cartilage tissue.

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.

WNT3A promotes myogenesis of human embryonic stem cells and enhances in vivo engraftment

The ability of human embryonic stem cells (hESCs) to differentiate into skeletal muscle cells is an important criterion in using them as a cell source to ameliorate skeletal muscle impairments. However, differentiation of hESCs into skeletal muscle cells still remains a challenge, often requiring introduction of transgenes. Here, we describe the use of WNT3A protein to promote in vitro myogenic commitment of hESC-derived cells and their subsequent in vivo function. Our findings show that the presence of WNT3A in culture medium significantly promotes myogenic commitment of hESC-derived progenitors expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), as evident from the expression of myogenic markers, including DES, MYOG, MYH1, and MF20. In vivo transplantation of these committed cells into cardiotoxin-injured skeletal muscles of NOD/SCID mice reveals survival and engraftment of the donor cells. The cells contributed to the regeneration of damaged muscle fibers and the satellite cell compartment. In lieu of the limited cell source for treating skeletal muscle defects, the hESC-derived PDGFRA+ cells exhibit significant in vitro expansion while maintaining their myogenic potential. The results described in this study provide a proof-of-principle that myogenic progenitor cells with in vivo engraftment potential can be derived from hESCs without genetic manipulation.