Enhanced chondrogenic differentiation of embryonic stem cells by coculture with hepatic cells.

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.

Collagen mimetic peptide-conjugated photopolymerizable PEG hydrogel.

Collagen mimetic peptide (CMP) with a specific amino acid sequence, -(Pro-Hyp-Gly)(x)-, forms a triple helix conformation that resembles the native protein structure of natural collagens. CMP previously has been shown to associate with type I collagen molecules and fibers via a strand invasion process. We hypothesized that when poly(ethylene glycol) (PEG) hydrogel, a non-adhesive tissue engineering scaffold, is conjugated with CMP, it may retain cell-secreted collagens and also form physical crosslinks that can be manipulated by cells. A photopolymerizable CMP derivative was synthesized and copolymerized with poly(ethylene oxide) diacrylate to create a novel PEG hydrogel. In a model retention experiment, diffusional loss of type I collagen that was added to the hydrogel was limited. Chondrocytes were encapsulated in the hydrogel to examine its use as a tissue engineering scaffold. After 2 weeks, the biochemical analysis of the CMP-conjugated PEG gel revealed an 87% increase in glycosaminoglycan content and a 103% increase in collagen content compared to that of control PEG hydrogels. The histology and immunohistochemistry analyses also showed increased staining of extracellular matrix. These results indicate that the CMP enhances the tissue production of cells encapsulated in the PEG hydrogel by providing cell-manipulated crosslinks and collagen binding sites that simulate natural extracellular matrix.

Nanofiber matrices promote the neuronal differentiation of human embryonic stem cell-derived neural precursors in vitro.

The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs.

Enhanced chondrogenesis of mesenchymal stem cells in collagen mimetic peptide-mediated microenvironment.

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.