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a Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA;
b Seoul National University School of Medicine, Seoul, Korea;
c Department of Gynecology and Obstetrics, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA
Key Words. Pluripotent stem cells • Embryoid bodies • EG cells • Differentiation
Correspondence: Jennifer Elisseeff, Ph.D., Department of Biomedical Engineering, Johns Hopkins University, 3400N. Charles Street, Clark 106, Baltimore, Maryland 21218, USA. Telephone: 410-516-4915; Fax: 410-516-8152; e-mail: jhe{at}bme.jhu.edu
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ABSTRACT |
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INTRODUCTION |
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When ES and EG cells differentiate in vitro, they are often in the form of complex three-dimensional spheres termed embryoid bodies (EBs) [3]. Early developmental processes occur within the EBs, resulting in collections of precursor and differentiated cells from a wide variety of lineages, including cells of the hematopoietic lineage, neurons, and skeletal muscle. Differentiation of cells in EBs is generally random and uncontrolled, making the resulting cells difficult to apply to regenerative strategies.
One of the most important challenges in stem cell research is to understand and control cell-differentiation processes. EB–derived (EBD) cells are generated from the disaggregation of EBs and by selection of cells that proliferate well in six basic growth conditions [4]. One example, the LVEC culture, was established on type I collagen-coated tissue-culture dishes in the presence of 5% serum, basic fibroblast growth factor (bFGF), insulin-like growth factorI (IGF-I), vascular endothelial cell growth factor (VEGF), and epidermal growth factor (EGF). EBD cells are a mixed cell culture, containing precursors and progenitor cells of various lineages, and a majority of clonal EBD cell lines retain the expression of multiple lineages. Unlike their ES and EG precursors, EBD cells are not immortal and do not form teratomas when injected into mice, making them a potentially useful therapeutic tool [4]. The first studies to evaluate chondrogenic differentiation of ES cells used a mouse line in monolayer culture. Kramer et al. [5, 6] demonstrated expression of cartilage matrix proteins after exposure to bone morphogenetic protein (BMP). More recent, Levenberg et al. [7] seeded human ES cells on polyglycolic acid scaffolds and differentiated the cells toward a cartilage phenotype using transforming growth factor beta (TGFß). The potential of EBD cells to differentiate into musculoskeletal tissues is unknown.
The aim of the current study is to evaluate the differentiation capability of human EBD cells (LVECs) using the micromass pellet culture system. The three-dimensional culture condition resembles more closely the in vitro surroundings during EB differentiation [8]. Increased cell–cell interaction of cell aggregates is designed to approximate the cell environment of condensing mesenchymes in the developing limb in vivo, which is supported by the report that mouse ES cells derived from EBs differentiated into chondrocytes, progressively developing into hypertrophic, calcifying cells [9]. The approach of micromass cultures has been established as a conventional method for chondroinduction of mesynchemal stem cells (MSCs) by disassociation of cells that are pelleted for culture [10]. In addition, the pellet culture system has also been used to create cartilaginous modules from primary cultured chondrocytes and embryonic mesenchymal cells.
Differentiation of LVECs was investigated in a chemically defined chondrogenic differentiation medium with addition of two growth factors that promote chondrogenesis: BMP2 and TGFß3 [11]. Under these conditions, LVECs lost expression of differentiated neural and vascular markers. Protein production of markers for mesenchymal tissues was upregulated. Gene expression of 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, demonstrating the ability of LVEC selection and transdifferentiation to cells of mesenchymal lineages.
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MATERIALS AND METHODS |
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Micromass Culture
Subconfluent (70%–80%) undifferentiated LVECs (passage 12–15) were resuspended at a density of 2 x 105 cells/ml in growth medium; then 1-ml aliquots of the cell suspension were dispensed into 15-ml sterile, conical polypropylene culture tubes (Sarstedt, Nümbrecht, Germany, http://www.sarstedt.com) and centrifuged at 1,000 rpm for 5 minutes to form spherical pellets [10]. The pellets were cultured at 37°C with 5% CO2 in 1 ml of chondrogenic differentiation medium that contained 1% serum in addition to high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10–7 M dexamethasone, 50 µg/ml ascorbate-2-phosphate, 40 µg/ml proline, 100 µg/ml sodium pyruvate, and 50 mg/ml ITS + Premix (Collaborative Biomedical: 6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenious acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid). After a pellet was formed, the medium in each tube was replaced with 0.5 ml defined medium containing either 20 ng/ml of recombinant human BMP2 or 10 ng/ml of TGFß3 (Research Diagnostics, Inc., Flanders, NJ, http://www.researchd.com), or both. Control cultures were maintained without adding BMP2 or TGFß3. Culture medium was changed three times per week. The pellets were harvested periodically over 3 weeks.
Biochemical Analysis
Protein synthesis was determined by [35S]-methionine incorporation. DNA content, collagen, and proteoglycan deposition were characterized using 33258 Hoechst dye, hydroxyproline content, and dimethylmethylene blue (DMMB) spectrophotometric assay, respectively [12]. To investigate whether the increase of pellet size and weight might be a possible effect of TGFß3 on cell proliferation, tetrazolium-based cell growth analysis was performed in LVECs cultured in monolayer with or without BMP2 or TGFß3 for 3 weeks.
Histology and Immunostaining Preparation
Pellets were fixed overnight in 4% paraformaldehyde in phosphate-buffered solution (PBS; pH 7.5) at 40°C and transferred to 70% ethanol until they were embedded in paraffin, according to standard histological technique. Pellets were processed, embedded in paraffin, and cut to 5 µm in thickness. Serial sections were stained with hematoxylin and eosin (H&E) for morphology, Toluidine blue for pericellular proteoglycan, and Masson’s trichrome for collagen detection. For immunostaining, sectioned pellets were deparaffinized and incubated for 30 minutes in goat serum followed by incubation with rabbit anti-type I and anti-type X collagen (Research Diagnostics). After washing in PBS, the sections were incubated for 1 hour with fluorescein isothiocyanate–conjugated or Texas Red dye–conjugated anti-rabbit second-aryantibodies(Jackson Immuno Research Laboratories, West Grove, Pennsylvania; http://www.jacksonimmuno.com).
Specimens were washed in PBS, and coverslips were mounted in glycerol-vinyl alcohol–mount (Zymed Laboratories, San Francisco, http://www.zymed.com).
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from 5 million of undifferentiated LVECs at day 0 or from 50 pellets at given time points with TRIzol (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), following the manufacturer’s instructions. RNA was treated with DNase I using the DNA-free Kit (Invitrogen). Two micrograms of total RNA per 20 µ1 of reaction volume was reverse transcribed into cDNA using the Super Script First-Strand Synthesis System (Invitrogen). The PCR primers are provided in online supplementary Table 1
. The conditions for all PCRs in this study were 35 cycles of 95°C for 30 seconds, 57°C to 60°C for 45 seconds, and 72°C for 1 minute, with one exception of 18s rRNA (12 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute). PCR products were separated by electrophoresis at 100 V on a 1.4% agarose gel in Tris-acetate-EDTA buffer, then visualized with ethidium bromide staining.
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RESULTS |
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), LVECs in pellets significantly reduced the expression of these neural and vascular endothelial markers. NFH, flk-1, and ALP expressions decreased in control cultures of pellets, whereas nestin expression showed little difference, indicating that LVECs might have lost, in part, their original characteristics in monolayer culture when cultured in micromass conditions.
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). The skeletal muscle cell-specific marker myf5, which was hardly detected in monolayer or in control pellets, was clearly expressed in BMP2 and slightly expressed in TGFß3 cultures to suggest differentiation toward this musculoskeletal tissue (Fig. 1A). Also, the basic helix-loop-helix transcription factors, Twist-a and Twist-b, which are required for somatic myogenesis [13] and bone formation [14], were upregulated by all pellet cultures compared with undifferentiated cells (online supplementary, Table 1; not shown). While ALP is a marker for undifferentiated stem cells, it is also a characteristic marker found in bone. ALP expression, quantified by real time PCR, markedly increased in pellets exposed to growth factors (Fig. 1A, 1D), and this is consistent with the activity measured in the pellets biochemically (Fig. 1D).
One important feature of MSCs, chondrocytes, and fibrochondrocytes is their high expression level of genes for specific types of collagen, which forms the major structural components of bone, cartilage, and other connective tissues. Therefore, we investigated the expression of genes involved in the chondrogenic differentiation of LVEC pellets. Type I collagen (Col I), which is found in bone, tendon, ligament, and meniscal cartilage, was expressed by undifferentiated cells, but was significantly upregulated by BMP2 and/or TGFß3 treatments (Fig. 1B, 1D). Both immature (Col 
1(I)) and mature collagen type I are expressed in developing cartilage. In the study reported here, it was revealed that expressions of Col 1(I) and Col I increased with pellet formation and TGFß3 treatment. Type III collagen (Col III) resides at the insertion sites of the major ligaments and tendons and within the perichondrium and periosteum and is also involved in early chondrogenesis. Its expression was readily detected in undifferentiated monolayer LVECs and was upregulated by the pellet culture of the cells (Fig. 1B). The expression, however, was not elevated with further treatment of BMP2 or TGFß3. Detectable levels of type II collagen mRNA (Col II), the primary collagen specific to hyaline cartilage, was observed upon differentiation and strongly increased in TGFß3-treated pellets (Fig. 1B). Furthermore, the juvenile splice variant of type II collagen (Col IIA), was not observed in cells in monolayer but was expressed after treatment with BMP2 and was more clearly upregulated by TGFß3 (Fig. 1B). The adult cartilage splice variant of collagen (IIB), which is activated in mature chondrocytes, was not found in any of the cultures.
Cartilage-Specific Gene Expression in the Presence of TGFß3
Specific markers for cartilage, a tissue that cannot repair and is a frequent target for regenerative therapies, were upregulated in the micromass culture system. Col IX is cartilage specific, and Col X is expressed in hypertrophic chondrocytes and bone [15]. These markers were not expressed in undifferentiated cells but underwent a rapid increase in the pellet cultures (Fig. 1B, 1C). The expression of cartilage oligomeric protein (COMP) that is characteristic for developing chondrocytes was dramatically induced by pellet culture of the cells, whereas LVECs in monolayer culture showed no detectable expression of COMP (Fig. 1B). These markers increased the most in TGFß3-treated pellets. In contrast, cartilage proteoglycan link protein (LP) and one of the chondroitin sulfate proteoglycans, aggrecan (AGN) were not markedly increased by TGFß3 treatment in pellets compared with control pellets (Fig. 1B). LP was readily observed, while a trace of AGN was seen in undifferentiated LVECs. The RT-PCR results are summarized in Table 1.
For further validation of mesenchymal tissue lineage progression, the mRNA level of genes involved in cartilage development was analyzed over time. Col IX and AGN expressions were temporally increased by TGFß3 treatment in comparison with control in 14 days of culture. Chondromodulin (ChM), a protein abundant in cartilage tissue, was minimally expressed in undifferentiated LVEC culture but substantially increased expression in all pellet cultures (Fig. 1C).
These results suggest that LVEC pellet exposure to BMP2 or TGFß3 promoted the loss of propensity to neural and hematopoietic lineages and significantly enhanced the expression of musculoskeletal tissue markers. Since LVEC is a mixed cell culture, this could mean that the precursor cells to other lineages died off or that the entire population moved toward the musculoskeletal lineages. The experimental design in the pellet culture system could not distinguish between these two possibilities. However, the homogenous immunostaining of types I and X collagen in the pellets suggests that most of the cells are expressing musculoskeletal differentiation markers. This observation, in addition to the homogenous cell morphology, leads us to hypothesize that the majority of the cells are differentiating in a similar manner.
Matrix Synthesis during the Pellet Culture of LVECs
To further examine the potential musculoskeletal differentiation of LVEC pellets, protein analysis and matrix production were evaluated. The general metabolic state of LVECs in pellets was assessed by the measurement of total protein synthesis after pellets were treated with or without BMP2 and/or TGFß3. Pellets were pulse-chased with [35S]-methioninefor3 hours on day 21 of culture and digested with papain, and then the counts per minute (cpm) of each pellet was determined. As presented in Figure 2A, cpm of the pellets treated with TGFß3 alone or TGFß3 in combination with BMP2 showed a significant increase in new protein synthesis (15,232.2 ± 4,250.7 and 15,204.0 ± 3,836.9, respectively) compared with the control (6,346.6 ± 1,965) or BMP2-treated pellet (9,405.8 ± 1,263.6). A time-dependent increase of the [35S]-methionine incorporation was seen in TGFß3-treated pellets supplementary online Table 1; not shown).
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). PG content in pellets treated with growth factors was much higher than in cells from monolayer culture (Fig. 2C). Consistent with the results of total collagen synthesis, the most dramatic increase of PG content was shown in TGFß3-treated pellets with approximately a fivefold increase over that of undifferentiated cells (7.367 ± 0.905). A time-dependent increase of the PG release into the culture medium and in the pellets was observed (Fig. 2E, 2F). Control pellets that were not treated with any growth factor also showed a slight increase of PG. Both total collagen and PG content were significantly higher in control pellets (0.181 ± 0.020 of total collagen and 3.973 ± 0.337 of PG) than in undifferentiated cells (1.456 ± 0.201), suggesting that the pellet culture system itself could provide important environmental cues for cell differentiation, which could be further enhanced with the addition of BMP2 or TGFß3.
Pellet morphology, as monitored by phase contrast microscopy, changed over the culture period and depended on growth factor exposure. Immediately after LVECs were centrifuged to form pellets, the cells were aggregated and appeared flattened at the bottom of the tube. After 24 hours, all pellets thickened and developed into round, dense aggregates. Between days 2 and 7, the pellets became spherical without any increase in size (Fig. 3A, day 3 and 7). During further cultivation, they changed from white and opaque aggregates to a compact structure matrix border, suggesting production of adense ECM[16]. The size of pellets incubated with TGFß3 for 21 days showed a considerable increase in size. Consistent with pellet size, the wet weight per pellet was greatest for TGFß3 pellets (about threefold) over 3 weeks-culture (Fig. 3D). LVECs in monolayer, treated with BMP2, showed no significant difference in proliferation rate compared with the control without treatment. The DNA quantity of pellets on day 21 was about 40%–60% of the initial DNA contents on day 0 (supplementary online Table 1; not shown), similar to results from MSC pellet culture [16]. The largest decrease in DNA quantity (40%) occurred with TGFß3 treatment compared with control pellets, whereas no significance was observed in BMP2 treatment alone or BMP2 in combination with TGFß3. Therefore, the increase in size and weight might be at least in part due to ECM protein synthesis such as collagen and PG in pellets. Furthermore, because initial cell loss is frequently observed in pellet cultures, the decrease in LVEC number may also suggest the loss of precursor cells from non-musculoskeletal lineages.
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) compared with the control pellets, which became smaller over time in the absence of growth factor treatment. Histological examination of the ECM was assessed in paraffin-embedded, serially sectioned pellets. Masson’s trichrome staining was selected to stain for collagen (blue or green) because it is useful to distinguish collagen from muscle (red) or other elements (black). As shown in Fig. 3B, a gradual increase in blue color was observed over time in TGFß3-treated pellets after 14 days of culture. A significant increase in matrix staining was observed on day 21, indicating a time-dependent accumulation of collagen matrix in TGFß3-treated pellets. The metachromatic staining by Toluidine blue was used for morphological assessment of ECM proteoglycan synthesis. The light- to dark-red/purple staining, positive for proteoglycans, was noticeable within the cell pellets treated with TGFß3 for 21 days (Fig. 3C). However, homogeneous staining over the whole area of pellet was rarely seen. In the absence of added growth factors, pellets had little evidence of matrix accumulation. Evaluation of the nuclear staining with H&E in pellet sections after 21 days revealed that separation between cells increased with time in TGFß3 pellets as more ECM was produced (Fig. 3B, 3C). |
DISCUSSION |
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In the study reported here, we used a micromass pellet culture system to investigate the differentiation capacity of EBD cells. This system provides a unique three-dimensional culture condition for undifferentiated cells that resembles more closely in vitro surroundings for EB formation and in vivo development [8]. First, to elucidate whether the expression of markers involved in distinct developmental lineages of human EBD cells could be differentially regulated by micromass culture of the cells, RT-PCR analysis was performed in RNA extracts from undifferentiated cells cultured both as a monolayer and as pellets. Decrease of neuronal (NFH) and hematopoietic (flk-1) marker expressions, in addition to downregulation of ALP by pellet culture of undifferentiated cells, suggests that the human EBD cells have lost their neural and vascular progenitor cell characteristics in pellet culture. We hypothesize that both cell selection and differentiation processes are occurring. The culture conditions may be selecting for the growth of a subpopulation of EBD stem cells with capability for mesenchymal differentiation, as evidenced by the decreasing cell number and increase in characteristic mesenchymal markers such as AGN that are present in the undifferentiated cells. While neural and vascular expression significantly decreased, there is still low expression level, suggesting that cells with the capability to differentiate to different lineages still exist. Differentiation processes are also occurring, as demonstrated by the "activation" of mesenchymal tissue-related markers not originally present in the EBD cells, including COMP, Col IX, and Col X.
During limb development, cartilage serves as anlagen for bone, in addition to forming fibrocartilage, hyaline cartilage, tendon, and ligaments. There are ECM markers characteristic for each of these tissues. The ECM of cartilage contains large amounts of AGN, Col II, and other key matrix components, including COMP. In this study, the marked induction of Col II, Col IX, AGN, COMP, and ChM in the presence of TGFß3 was revealed in LVEC pellets (Fig. 1), supporting the conclusion that the cells were differentiating into musculoskeletal lineages. Prior to induction of chondrogenesis in undifferentiated cells at day 0, transcripts for Col II, Col IIA, Col IX, Col X, COMP, and ChM were hardly detected, whereas those for Col I, Col III, LP, AGN, CS-4S, and CS-6S were readily or weakly detectable.
The genes that showed strong expression in our results—Col 1 (I), Col I, and CS (Fig. 1)—are involved in osteochondrogenesis of MSCs. ChM is not only a major regulator in cartilage development but also a bone-remodeling factor [18]. Our results of expression of hypertrophic chondrocyte markers, as assessed by the increase of ALP activity and upregulation of Col I, Col II, and X expression upon exposure to BMP2 or TGFß3 (Fig. 1), also indicate that LVECs were capable of differentiating into osteogenic lineages, even though there was no detectable mineral deposition assessed by von Kossa’s stain in the pellet section (data not shown). Protein expression of types I and X collagen was confirmed by immunostaining (Fig. 3C). Col III and myf5 expressions further suggest the differentiation of LVECs into ligament/tendon and myogenic lineages, respectively. Although this speculation is based on the results of PCR assay without definitive proof of human EBD cell differentiation into specific lineages, all these results support that LVECs in micromass have a potential to differentiate from EG cells into musculoskeletal lineages.
The maintenance of nestin expression is compatible with MSCs. Nestin is found in neural precursors, in the developing mouse limb buds in vivo, and in differentiating muscle and myocardial cells [19, 20]. In addition, nestin is expressed in immature MSC precursors [21], indicating that its expression is not restricted to developing neural cells but can be a marker found in MSCs. In the current study, nestin was strongly expressed in undifferentiated LVECs as reported [4], but little change was detected in pelleted LVECs with or without treatments of BMP2 or TGFß3 (Fig. 1A). These results suggest that undifferentiated LVECs might have cells producing a complex pattern of neural and mesenchymal cell differentiation, or the LVECs might maintain a capacity similar to that of MSCs to differentiate into mesenchymal cell lineages after responding to mesenchymal environments, including osteochondrogenic stimuli. This notion is supported by the report that stem cells, mesenchymal cells, and neural progenitors were present together during mouse ES cell differentiation after exposure of the cells to chordin, a BMP inhibitor [22].
PGs are a major component of cartilage ECM. The evaluation of PG release into the culture medium is as important as evaluation of PG content in the cartilage matrix. In our studies, the enhancement of TGFß3 in osteochondrogenic differentiation of LVEC pellets was confirmed by measuring both PG content of the pellets and PG release into the culture medium. PG accumulation in pellets and in the medium was increased by TGFß3 during all incubation periods, and the release was maintained higher in TGFß3 pellets than in the control, suggesting differentiation (Fig. 2E, 2F). However, control pellets or pellets treated with BMP2 alone or co-treated with BMP2 and TGFß3 released approximately the same amount of PG into the medium on day 21. Interestingly, a detectable level of PG was observed in undifferentiated LVECs (Figs. 2, 5). That could be explained by the observation of PGs (AGN, LP, CS-4S, and CS-6S) in monolayer cultured LVECs by RT-PCR analysis (Fig. 1). In addition, it is possible that a pericellular matrix, TRA-1-60, was responsible for DMMB-reactive PG detectable in undifferentiated cells because it is one of the important markers of human pluripotent EG cells [23].
TGFßs are potent antiproliferative agents of many different cell types [24]. Activities of TGFßs include induction of cell-cycle arrest, induction of apoptosis, and induction of expression of cell adhesion molecules and components of the ECM, including collagen, fibronectin, and integrins. When MSCs differentiate into chondrocytes under TGFß3 and BMP6 treatment, there is a continued loss of cells through apoptosis [16]. More interestingly, MSCs exposed to TGFß1 show a significant decline in cell number in three-dimensional culture disks (REF-worster), indicating that TGFßs can suppress cell growth in a three-dimensional system. The TGFßRII expression remained constant in all experimental pellet groups.
The expression of cartilage and other musculoskeletal-specific genes, in addition to the results of biochemical analysis in this study, strongly suggests that LVEC pellets differentiated toward chondrocytic and MSC lineages. Small quantities of serum (1%) instead of the strictly serum-free medium were used in this study, based on reports that mesodermal differentiation of ES cells can be inhibited under serum-free conditions [25]. We could not identify morphologically hypertrophic or mature chondrocytes in pellets treated with TGFß3 (Fig. 3C). There was a positive staining with Toluidine blue in regions of the pellets that were incubated in TGFß3, but not throughout the pellet. Thus, it seems that LVECs could not differentiate further down a specific MSC lineage such as cartilage or bone. One explanation may be that prolonged treatment of TGFß3 in this study might have prevented or retarded phenotype development of chondrocyte maturation at a later stage of cartilage formation, as reported for cartilage nodules generated in the absence of TGFß3 at a later phase of chondrogenic culture of ES cells from an E14 embryo [26]. However, the detection of Col X transcript by PCR (Fig. 1) and immunostaining (Fig. 3C) suggests that hypertrophic differentiation might occur in the presence of TGFß3. We also hypothesize that the cells may need a more complex array of soluble and matrix-related signals to further differentiate. More complex signals, including the enhanced cell–cell contacts provided in the pellet culture system, may be incorporated into three-dimensional tissue engineering biomaterial scaffold systems with a cocktail of growth factors to promote specific differentiation.
In summary, human LVECs showed features of chondrogenic and mesenchymal differentiation when they were grown in micromass culture, as evidenced by tissue-specific gene expression and protein synthesis. Metachromatic staining with Toluidine blue of pellet sections confirmed that differentiated LVECs accumulated ECM. A significant increase in PG and collagen protein synthesis was observed in the presence of TGFß3, indicating that the osteochondro-inductive agent TGFß3 further enhanced LVEC-derived chondrogenic differentiation. This study also shows the flexibility of partially differentiated stem cells to shift their differentiation profile to other developmental lineages, furthering our understanding of stem cell capabilities and their potential use in musculoskeletal regeneration therapies.
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ACKNOWLEDGMENTS |
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Disclosure
Under a licensing agreement between Geron Corporation and JHU, Drs. Gearhart and Shamblott are entitled to a share of royalty received by the university on sales of products described in this presentation. Drs. Gearhart and Shamblott and the University own Geron stock, which is subject to certain restrictions under university policy. The terms of this arrangement are being managed by JHU in accordance with its conflict of interest policies.
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