Nathaniel S. Hwang

In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells

Development of clinically relevant regenerative medicine therapies using human embryonic stem cells (hESCs) requires production of a simple and readily expandable cell population that can be directed to form functional 3D tissue in an in vivo environment. We describe an efficient derivation method and characterization of mesenchymal stem cells (MSCs) from hESCs (hESCd-MSCs) that have multilineage differentiation potential and are capable of producing fat, cartilage, and bone in vitro. Furthermore, we highlight their in vivo survival and commitment to the chondrogenic lineage in a microenvironment comprising chondrocyte-secreted morphogenetic factors and hydrogels. Normal cartilage architecture was established in rat osteochondral defects after treatment with chondrogenically-committed hESCd-MSCs. In view of the limited available cell sources for tissue engineering applications, these embryonic-derived cells show significant potential in musculoskeletal tissue regeneration applications.

Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells.

Embryonic stem (ES) cells have the ability to self‐replicate and differentiate into cells from all three germ layers, holding great promise for tissue regeneration applications. However, controlling the differentiation of ES cells and obtaining homogenous cell populations still remains a challenge. We hypothesize that a supportive three‐dimensional (3D) environment provides ES cell‐derived cells an environment that more closely mimics chondrogenesis in vivo. In the present study, the chondrogenic differentiation capability of ES cell‐derived embryoid bodies (EBs) encapsulated in poly(ethylene glycol)‐based (PEG) hy‐drogels was examined and compared with the chondrogenic potential of EBs in conventional monolayer culture. PEG hydrogel‐encapsulated EBs and EBs in monolayer were cultured in vitro for up to 17 days in chondrogenic differentiation medium in the presence of transforming growth factor (TGF)‐β1 or bone morphogenic protein‐2. Gene expression and protein analyses indicated that EB‐PEG hydrogel culture upregulated cartilage‐relevant markers compared with a monolayer environment and induction of chondrocytic phenotype was stimulated with TGF‐β1. Histology of EBs in PEG hydrogel culture with TGF‐β1 demonstrated basophilic extracellular matrix deposition characteristic of neocartilage. These findings suggest that EB‐PEG hydrogel culture, with an appropriate growth factor, may provide a suitable environment for chondrogenic differentiation of intact ES cell‐derived EBs.

Nanotopography-guided tissue engineering and regenerative medicine ☆

Human tissues are intricate ensembles of multiple cell types embedded in complex and well-defined structures of the extracellular matrix (ECM). The organization of ECM is frequently hierarchical from nano to macro, with many proteins forming large scale structures with feature sizes up to several hundred microns. Inspired from these natural designs of ECM, nanotopography-guided approaches have been increasingly investigated for the last several decades. Results demonstrate that the nanotopography itself can activate tissue-specific function in vitro as well as promote tissue regeneration in vivo upon transplantation. In this review, we provide an extensive analysis of recent efforts to mimic functional nanostructures in vitro for improved tissue engineering and regeneration of injured and damaged tissues. We first characterize the role of various nanostructures in human tissues with respect to each tissue-specific function. Then, we describe various fabrication methods in terms of patterning principles and material characteristics. Finally, we summarize the applications of nanotopography to various tissues, which are classified into four types depending on their functions: protective, mechano-sensitive, electro-active, and shear stress-sensitive tissues. Some limitations and future challenges are briefly discussed at the end.

Differential Response of Adult and Embryonic Mesenchymal Progenitor Cells to Mechanical Compression in Hydrogels

Cells in the musculoskeletal system can respond to mechanical stimuli, supporting tissue homeostasis and remodeling. Recent studies have suggested that mechanical stimulation also influences the differentiation of MSCs, whereas the effect on embryonic cells is still largely unknown. In this study, we evaluated the influence of dynamic mechanical compression on chondrogenesis of bone marrow‐derived MSCs and embryonic stem cell‐derived (human embryoid body‐derived [hEBd]) cells encapsulated in hydrogels and cultured with or without transforming growth factor β‐1 (TGF‐β1). Cells were cultured in hydrogels for up to 3 weeks and exposed daily to compression for 1, 2, 2.5, and 4 hours in a bioreactor. When MSCs were cultured, mechanical stimulation quantitatively increased gene expression of cartilage‐related markers, Sox‐9, type II collagen, and aggrecan independently from the presence of TGF‐β1. Extracellular matrix secretion into the hydrogels was also enhanced. When hEBd cells were cultured without TGF‐β1, mechanical compression inhibited their differentiation as determined by significant downregulation of cartilage‐specific genes. However, after initiation of chondrogenic differentiation by administration of TGF‐β1, the hEBd cells quantitatively increased expression of cartilage‐specific genes when exposed to mechanical compression, similar to the bone marrow‐derived MSCs. Therefore, when appropriately directed into the chondrogenic lineage, mechanical stimulation is beneficial for further differentiation of stem cell tissue engineered constructs.

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.

Morphogenetic signals from chondrocytes promote chondrogenic and osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are potentially useful cells for musculoskeletal tissue engineering. However, controlling MSC differentiation and tissue formation in vivo remains a challenge. There is a significant need for well‐defined and efficient protocols for directing MSC behaviors in vivo. We hypothesize that morphogenetic signals from chondrocytes may regulate MSC differentiation. In micromass culture of MSCs, incubation with chondrocyte‐conditioned medium (CCM) significantly enhanced the production of cartilage specific matrix including type II collagen. In addition, incubation of MSCs with conditioned medium supplemented with osteogenic factors induced more osteogenesis and accumulation of calcium and increased ALP activity. These findings reveal that chondrocyte‐secreted factors promote chondrogenesis as well as osteogenesis of MSCs during in vitro micromass culture. Moreover, when MSCs expanded with chondrocyte‐conditioned medium were encapsulated in hydrogels and subsequently implanted into athymic mice, basophilic extracellular matrix deposition characteristic of neocartilage was evident. These results indicate that articular chondrocytes produce suitable morphogenetic factors that induce the differentiation program of MSCs in vitro and in vivo. J. Cell. Physiol. 212: 281–284, 2007.

Derivation of Chondrogenically-Committed Cells from Human Embryonic Cells for Cartilage Tissue Regeneration

Background Heterogeneous and uncontrolled differentiation of human embryonic stem cells (hESCs) in embryoid bodies (EBs) limits the potential use of hESCs for cell-based therapies. More efficient strategies are needed for the commitment and differentiation of hESCs to produce a homogeneous population of specific cell types for tissue regeneration applications. Methodology/Principal Findings We report here that significant chondrocytic commitment of feeder-free cultured human embryonic stem cells (FF-hESCs), as determined by gene expression and immunostaining analysis, was induced by co-culture with primary chondrocytes. Furthermore, a dynamic expression profile of chondrocyte-specific genes was observed during monolayer expansion of the chondrogenically-committed cells. Chondrogenically-committed cells synergistically responded to transforming growth factor-β1 (TGF-β1) and β1-integrin activating antibody by increasing tissue mass in pellet culture. In addition, when encapsulated in hydrogels, these cells formed cartilage tissue both in vitro and in vivo. In contrast, the absence of chondrocyte co-culture did not result in an expandable cell population from FF-hESCs. Conclusions/Significance The direct chondrocytic commitment of FF-hESCs can be induced by morphogenetic factors from chondrocytes without EB formation and homogenous cartilage tissue can be formed in vitro and in vivo.

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.

RESPONSE OF ZONAL CHONDROCYTES TO EXTRACELLULAR MATRIX-HYDROGELS

We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)‐based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS‐based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen‐based hydrogels demonstrated zonal‐dependent cellular responses.

Musculoskeletal differentiation of cells derived from human embryonic germ cells.

Stem cells have the potential to significantly improve cell and tissue regeneration therapies, but little is understood about how to control their behavior. We investigated the potential differentiation capability of cells derived from human embryonic germ (EG) cells into musculoskeletal lineages by providing a three‐dimensional environment with increased cell–cell contact and growth factors. Cells were clustered into pellets to mimic the mesenchyme condensation process during limb development. LVEC cells, an embryoid body–derived (EBD) cell culture generated from EG cells, were cultured in micromass pellets for 21 days in the presence of bone morphogenetic protein 2 (BMP2) and/or transforming growth factor beta‐3 (TGFβ3). Gene expression for cartilage‐, bone‐, and muscle‐specific matrix proteins—including collagen types I, II, III, IX, X; aggrecan; cartilage proteoglycan link protein; cartilage oligomeric protein; chondroitin sulfate‐4‐S; and myf5—was upregulated in the pellets treated with TGFβ3, while mRNAs for neurofilament heavy (NFH), a neuron marker, and flk‐1, a hematopoietic marker, decreased. Total collagen and proteoglycan production exhibited a time‐dependent increase in the pellets treated with TGFβ3, further confirming the expression of characteristic musculoskeletal markers. Furthermore, our results indicate the ability to select or differentiate stem cells toward a musculoskeletal lineage from a heterogenous EBD cell line.