Fibrochondrogenesis in Two Embryonic Stem Cell Lines: Effects of Differentiation Timelines
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EMBRYONIC STEM CELLS Fibrochondrogenesis in Two Embryonic Stem Cell Lines: Effects of Differentiation Timelines GWENDOLYN M. HOBEN,a,b EUGENE J. KOAY,a,b KYRIACOS A. ATHANASIOUa aDepartment of Bioengineering, Rice University, Houston, Texas, USA; bBaylor College of Medicine, Houston, Texas, USA Key Words. Fibrocartilage • Human embryonic stem cells • Tissue engineering ABSTRACT Human embryonic stem cells (hESCs) are an exciting cell tiation for 1 week resulted in small constructs with poor source for fibrocartilage engineering. In this study, the structural integrity that could not be mechanically tested. effects of differentiation time and cell line, H9 versus The compressive stiffness of the constructs obtained from BG01V, were examined. Embryoid bodies (EBs) were EBs differentiated for 3 or 6 weeks did not vary signifi- fibrochondrogenically differentiated for 1, 3, or 6 weeks cantly as a function of either differentiation time or cell and then used to engineer tissue constructs that were line. In contrast, the tensile properties were markedly grown for an additional 4 weeks. Construct matrix was greater with the H9 cell line, 1,562–1,940 versus 32–80 fibrocartilaginous, containing glycosaminoglycans (GAGs) and kPa in the BG01V constructs. These results demonstrate collagens I, II, and VI. A differentiation time of 3 or 6 the dramatic effects of hESC line and differentiation time weeks produced homogeneous constructs, with matrix on the biochemical and functional properties of tissue- composition varying greatly with cell line and differenti- engineered constructs and show progress in fibrocartilage ation time: from 2.6 to 17.4 g of GAG per 106 cells and tissue engineering with an exciting new cell source. STEM from 22.3 to 238.4 g of collagen per 106 cells. Differen- CELLS 2008;26:422–430 Disclosure of potential conflicts of interest is found at the end of this article. studies, bone morphogenic protein-2 and TGF-1 have been INTRODUCTION studied for their efficacy in inducing chondrogenic differentia- tion [12–16]. An additional component of this differentiation Injuries to the fibrocartilages of the body, especially the knee has been the microenvironment for differentiation. This micro- meniscus, most commonly result in disabling arthritis [1, 2]. environment can be in terms of differentiating the embryoid Tissue engineering of a replacement tissue offers a possible bodies (EBs) in suspension, on a two-dimensional surface, or a remedy. Most tissue-engineering strategies have used primary three-dimensional scaffold such as a hydrogel or polymer scaf- cells, but over time, the field has begun shifting toward stem fold. This microenvironment can also include the presence of cells. This shift occurred because of a lack of sufficient autol- other cell types for the purpose of differentiation (Table 1). How ogous healthy tissue to provide enough cells for a tissue-engi- long to differentiate the cells prior to using them in a tissue- neered construct. Moreover, the goal of taking only a small engineering strategy is another factor that must be considered. biopsy of native tissue and expanding those cells to reach the Time frames as short as 8 days have been used [12], whereas needed number has been confounded by issues of dedifferenti- Khoo et al. [17] found that hESCs spontaneously differentiated ation, low synthetic capacity, and limited expansion [3–6]. down a cartilaginous lineage after 60 days. Although there is a These issues are even more pronounced in fibrocartilage com- wide array of studies examining each of these components with pared with hyaline cartilage, as fibrochondrocytes in vitro show adult stem cells [18, 19], the field is just beginning with hESCs. inferior matrix production compared with chondrocytes [7, 8]. In this study, we used chondrogenic medium as the primary Toward a goal of tissue engineering fibrocartilages, such as the differentiation treatment for hESC EBs in suspension. The time knee meniscus, temporomandibular joint disc, and intervertebral for differentiation was varied from 1 to 6 weeks, with the disc, both adult and embryonic stem cells may have the capacity hypothesis that with increasing differentiation time, more fibro- to overcome these issues, but they also bring their own chal- cartilaginous matrix would be produced in the EBs, and the cells lenges. from these EBs could be used to create biochemically and One of the biggest challenges is differentiating the cells. A functionally fibrocartilaginous tissue constructs. To address this common treatment in many differentiation studies is the use of hypothesis, the differentiated cells from the EBs were placed in serum-free or low-serum “chondrogenic” medium containing high-density culture as part of a self-assembly process [20], and insulin, ascorbic acid, and dexamethasone [9–11]. The addition the resulting constructs were biochemically and functionally of a transforming growth factor- (TGF-) superfamily growth characterized (Fig. 1). Two different hESC lines, H9 and factor has also been commonly used for chondrogenic differen- BG01V, were compared in this study as an initial examination tiation. For example, in human embryonic stem cell (hESC) of the level of variation in cartilaginous lineage differentiation Correspondence: Kyriacos A. Athanasiou, Ph.D., P.E., Rice University, Department of Bioengineering, MS-142, P.O. Box 1892, Houston, Texas 77251-1892, USA. Telephone: 713-348-6385; Fax: 713-348-5877; e-mail: [email protected] Received August 6, 2007; accepted for publication November 10, 2007; first published online in STEM CELLS EXPRESS November 21, 2007. ©AlphaMed Press 1066-5099/ 2008/$30.00/0 doi: 10.1634/stemcells.2007-0641 STEM CELLS 2008;26:422–430 www.StemCells.com Hoben, Koay, Athanasiou 423 Table 1. Differentiation strategies for chondrogenesis with hESCs Differentiation treatments (serum, medium, growth Cell line factors etc.) Differentiation time Reference BG01V 1% serum chondrogenic medium, TGF-3 applied for 4 wks Koay et al. 2007 1 wk followed by TGF-1 ϩ IGF-I, or BMP-2 applied to EBs, EBs were then self-assembled into constructs and grown an additional 4 wks H1, H9 Serum-free chondrogenic medium, ϮBMP-2, applied 3 wks Toh et al. 2007 to EBs grown in 2D or micromasses BG02 EBs grown in 20% KSRhESC medium without bFGF 3 wks in hydrogel Hwang et al. 2006 for 10 days, transferred to 2D and grown in 10% serum medium up to passage 5–7, then pelleted ϮTGF-1, BMP-2, or combination H1 Grew EBs for 5 days in 10% serum media then 4 wks Vats et al. 2006 trypsinized and grown in 2D: grew nasal chondrocytes on inserts in 10% serum media, then seeded on PDLLA foams and implanted in nude mice H9 Grew EBs for 89 days in TGF-1 supplemented 8–9 days in suspension Levenberg et al. 2003 chondrogenic medium and then seeded onto then 2 wks on polymer polymer scaffolds for 2 wks H9 Grew EBs in suspension for 5 days then 10 days Schuldiner et al. 2000 differentiated in monolayer for 10 days with TGF-1 ES1-hES3 Grew Ebs in suspension 28 days, 60 days, 104 days Khoo et al. 2005 BMP-2: bone morphogenic protein 2, EBs: embryoid bodies, hESC; human embryonic stem cells; IGF, insulin-like growth factor; KSR: knockout serum replacer, MSC: mesenchymal stem cells, PDLLA: poly - D,-I lactide, TGF-1: transforming growth factor -1. Figure 1. Study design: Two hESC lines, H9 and BG01V (BG), were cultured in monolayer and then collected as embryoid bodies (EBs) off the mouse embryonic fibroblast feeder layers. The EBs were then cultured in chondrogenic medium with 1% serum for 1, 3, or 6 wks (referred to as the differentiation time) and then dissociated to single cells and self-assembled in agarose molds. The resulting constructs were evaluated following 2 and 4 wks in self-assembly culture, referred to as assembly time. Constructs are referred to in the text according to their cell line and their differentiation time in EB form. Abbreviations: h9, H9 human embryonic stem cells; hESC, human embryonic stem cell; wk, week. between hESC lines. Such variation certainly has implications L-glutamine (Invitrogen), 1% nonessential amino acids, 0.4 mM pro- for future clinical work, but it also has important considerations line, 50 g/ml L-ascorbate-2 phosphate, 100 g/ml sodium pyruvate, for in vitro studies, particularly since the BG01V line has less 1% insulin, transferring, and selenium ϩ (ITSϩ) (BD Biosciences, San stringent culture requirements [21, 22]. Jose, CA, http://www.bdbiosciences.com), and 100 nM dexametha- sone. To facilitate EB formation, the resulting suspension was placed on 2% agarose (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich. com)-coated 24-well plates for 4 hours. At that time, the suspension EXPERIMENTAL PROCEDURES was collected into Petri dishes. EBs were grown in Petri dishes with changes of half the medium every 2 days. EB growth was monitored by Cell Culture light microscopy with a stage micrometer on an Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany, http://www.zeiss.com). H9 human embryonic stem cells (H9) (WiCell Research Institute, Madison, WI, http://www.wicell.org) were cultured according to the manufacturer’s instructions on irradiated Carworth Farms-1 mouse Construct Preparation embryonic fibroblasts (MEFs) (Charles River Laboratories, Wilming- EBs were collected after 1, 3, and 6 weeks of differentiation time. ton, MA, http://www.criver.com). Colonies were passaged using 0.1% A portion of the EBs was set aside for analysis, and the remaining type IV collagenase (Invitrogen, Carlsbad, CA, http://www.invitrogen. EBs were digested to form a cell suspension. First, EBs were com) every 4–6 days. BG01V hESCs (BG) (American Type Culture washed in DMEM and digested with 0.05% trypsin-EDTA (Sigma- Collection, Manassas, VA, http://www.atcc.org) were cultured accord- Aldrich) for 1 hour with stirring. Any remaining matrix was then ing to manufacturer’s instructions, and they were similarly grown on digested with 0.2% type II collagenase (Worthington Biochemical, irradiated MEFs and passaged every 4–6 days.