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Techniques that can create three-dimensional (3D) structures to provide architectural support for cells have a significant impact in generating complex and hierarchically organized tissues/organs. In recent times, a number of technologies, including photopatterning, have been developed to create such intricate 3D structures. In this study, we describe an easy-to-implement photopatterning approach, involving a conventional fluorescent microscope and a simple photomask, to encapsulate cells within spatially defined 3D structures. We have demonstrated the ease and the versatility of this approach by creating simple to complex as well as multilayered structures. We have extended this photopatterning approach to incorporate and spatially organize multiple cell types, thereby establishing coculture systems. Such cost-effective and easy-to-use approaches can greatly advance tissue engineering strategies.

Spontaneous differentiation of human embryonic stem cells (hESCs) is generally inefficient and leads to a heterogeneous population of differentiated and undifferentiated cells, limiting the potential use of hESCs for cell-based therapy and studies of specific differentiation programs. Here, we demonstrate biomaterial-dependent commitment of a mesenchymal cell population derived from hESCs toward the osteogenic lineage in vivo. In skeletal development, bone formation from condensing mesenchymal cells involves two distinct pathways: endochondral and intramembraneous bone formation. In this study, we demonstrate that the hESC-derived mesenchymal cells differentiate and regenerate in vivo bone tissues through two different pathways depending upon the local cues present in a scaffold microenvironment. Hydroxyapatite (HA) was incorporated into biodegradable poly(lactic-co-glycolic acid)/poly(l-lactic acid) (PLGA/PLLA) scaffolds to enhance bone formation. The HA microenvironment stabilized the β-catenin and upregulated Runx2, resulting in faster bone formation through intramembraneous ossification. hESC-derived mesenchymal cells seeded on the PLGA/PLLA scaffold without HA, however, showed minimal levels Runx2, and differentiated via endochondral ossification, as evidenced by formation of cartilaginous tissue, followed by calcification and increased blood vessel invasion. These results indicate that the ossification mechanisms of the hESC-derived mesenchymal stem cells can be regulated by the scaffold-mediated microenvironments, and bone tissue can be formed.

There is an extensive clinical need for soft tissue filler materials, such as adipose tissue, for plastic and reconstructive surgery. Due to limitations with autologous adipose transplantation, engineered adipose tissue provides a potential alternative therapy. Embryonic germ cells form embryoid bodies and subsequent embryoid body-derived (EBD) cells have the ability to differentiate toward multiple tissue types. The objective of this study was to demonstrate that EBD cells were capable of adipogenic differentiation in vitro and in vivo using a poly(ethylene glycol)-based hydrogel scaffold. EBD cells underwent adipogenic differentiation in vitro and in vivo. Results were directly compared to adipogenic differentiation of adult bone marrow-derived mesenchymal stem cells (MSCs). Differentiated EBD cells in both monolayer and three-dimensional in vitro culture demonstrated fat granules by light microscopy, stained positive for lipids with oil red-O, and expressed adipocyte-specific genes (lipoprotein lipase [LPL], peroxisome proliferator activated receptor gamma2, and adipocyte-specific fatty acid binding protein [alphaP2]). In vivo constructs demonstrated adipogenic differentiation by alphaP2 and LPL gene expression and oil red-O staining of lipid granules. In conclusion, EBD cells are capable of differentiating toward an adipogenic lineage in vitro and in vivo. EBD cells' adipogenic differentiation is comparable to that of MSCs and demonstrate therapeutic potential for soft tissue augmentation and reconstruction.

A new type of synthetic hydrogel scaffold that mimics certain aspects of structure and function of natural extracellular matrix (ECM) has been developed. We previously reported the conjugation of collagen mimetic peptide (CMP) to poly(ethylene oxide) diacrylate (PEODA) to create a polymer-peptide hybrid scaffold for a suitable cell microenvironment. In this study, we showed that the CMP-mediated microenvironment enhances the chondrogenic differentiation of mesenchymal stem cells (MSCs). MSCs were harvested and photo-encapsulated in CMP-conjugated PEODA (CMP/PEODA). After 3 weeks, the histological and biochemical analysis of the CMP/PEODA gel revealed twice as much glycosaminoglycan and collagen contents as in control PEODA hydrogels. Moreover, MSCs cultured in CMP/PEODA hydrogel exhibited a lower level of hypertrophic markers, core binding factor alpha 1, and type X collagen than MSCs in PEODA hydrogel as revealed by gene expression and immunohistochemisty. These results indicate that CMP/PEODA hydrogel provides a favorable microenvironment for encapsulated MSCs and regulates their downstream chondrogenic differentiation.

Enhancing the specific differentiation of pluripotent embryonic stem (ES) cells has been a challenge in the field of tissue engineering. Previously, hepatic cells have been shown to secrete various soluble morphogenic factors to direct mesodermal differentiation of ES cells. In this study, we hypothesized that factors secreted by hepatic cells possess chondrogenic-differentiating effects, and, therefore, the co-culture of hepatic cells would enhance chondrogenesis of ES cells. ES-derived cells (ESDCs) were co-cultured with hepatic cells (HEPA-1C1c7) in three-dimensional bilayered hydrogels. After 3 weeks culture, the histological and biochemical analysis of the HEPA-co-cultured ESDCs revealed a four-fold increase in glycosaminoglycan (GAG) compared to ESDCs cultured alone. This result was supported by real-time PCR analysis, which demonstrated an 80-fold increase in aggrecan expression in co-cultured ESDCs. Additionally, type IIB collagen expression was observed only with co-cultured ESDCs, and immunohistochemical analysis resulted in significantly more positive type II collagen staining with co-cultured ESDCs. Moreover, at day 21, gene expression of other lineages in HEPA-co-cultured ESDCs was either comparable to or lower than those of ESDCs cultured alone. These results indicated that co-culture of ESDCs with hepatic cells significantly enhanced specific chondrogenic differentiation of ESDCs.

Human embryonic stem cells (hESCs) have the potential to self-renew and generate multiple cell types, producing critical building blocks for tissue engineering and regenerative medicine applications. Here, we describe the efficient derivation and chondrogenic differentiation of mesenchymal-like cells from hESCs. These cells exhibit mesenchymal stem cell (MSC) surface markers, including CD29, CD44, CD105, and platelet-derived growth factor receptor-alpha. Under appropriate growth conditions, the hESC-derived cells proliferated without phenotypic changes and maintained MSC surface markers. The chondrogenic capacity of the cells was studied in pellet culture and after encapsulation in poly(ethylene glycol)-diacrylate (PEGDA) hydrogels with exogenous extracellular proteins or arginineglycine- aspartate (RGD)-modified PEGDA hydrogels. The hESC-derived cells exhibited growth factor- dependent matrix production in pellet culture but did not produce tissue characteristic of cartilage morphology. In PEGDA hydrogels containing exogenous hyaluronic acid or type I collagen, no significant cell growth or matrix production was observed. In contrast, when these cells were encapsulated in RGDmodified poly(ethylene glycol)hydrogels, neocartilage with basophilic extracellular matrix deposition was observed within 3 weeks of culture, producing cartilage-specific gene up-regulation and extracellular matrix production. Our results indicate that precursor cells characteristic of a MSC population can be cultured from differentiating hESCs through embryoid bodies, thus holding great promise for a potentially unlimited source of cells for cartilage tissue engineering.

The capabilities of stem cells continue to be revealed, leading to excitement regarding potential new therapies. Regenerative medicine is an area in which stem cells hold great promise for overcoming the challenge of limited cell sources for tissue repair. Biomaterials play an important role in directing tissue growth and may provide another tool to manipulate and control stem cell behavior. Biomaterials are made from natural or synthetic polymers and can be processed into three-dimensional scaffolds designed to promote cell proliferation and/or differentiation that ultimately produces new tissue. Stem cells will have a significant impact on the fields of regenerative medicine and tissue engineering as a powerful cell source that will work, in conjunction with biomaterials, to treat tissue and organ loss. Herein, we survey our latest research on applying embryonic stem (ES) cells to hydrogel biomaterials for engineering musculoskeletal tissues, emphasizing the unique biomaterial requirements of ES cells for differentiation and tissue development.