JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE SHORT COMMUNICATION J Tissue Eng Regen Med 2010; 4: 242–245. Published online 8 December 2009 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.236
How chondrogenic are human umbilical cord matrix cells? A comparison to adipose-derived stem cells F. Hildner1,3,4 , S. Wolbank1,3,4 , H. Redl1,3,4 , M. van Griensven1,3,4 * and A. Peterbauer2,3,4 1
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Centre, Linz, Vienna, Austria Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria 3 Austrian Cluster for Tissue Regeneration, Vienna, Austria 4 European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Vienna, Austria 2
Abstract The umbilical cord matrix as well as liposuction material have been demonstrated to contain cells capable of differentiating towards the mesodermal lineage. High availability and low donor site morbidity appear promising for the use of human umbilical cord matrix cells (HUCMs) and adiposederived stem cells (ASCs) in cell-based therapies. In the present study we focused on cartilage regeneration and compared HUCMs and ASCs regarding their potential to differentiate towards the chondrogenic lineage. Cells were isolated by explantation culture or enzymatic digestion, phenotypically characterized by flow cytometry and differentiated as 3D micromass pellets for up to 35 days. Under tested conditions, ASCs demonstrated significantly higher glycosaminoglycan synthesis compared to HUCMs. qRT–PCR data gave evidence that chondrogenic genes are expressed by both ASCs and HUCMs. However, higher expression levels of ASCs suggest that this cell type has higher potential for differentiation towards a cartilage-like phenotype than HUCMs. In conclusion, both cell types, HUCMs and ASCs, are easily available, possess typical properties of mesenchymal stem cells and are thus promising for cell-based therapies. However, in terms of cartilage regeneration, ASCs might be more suitable than HUCMs. Copyright 2009 John Wiley & Sons, Ltd. Received 18 February 2009; Revised 18 September 2009; Accepted 15 October 2009
Keywords adipose-derived stem cells; umbilical cord; Wharton’s jelly; mesenchymal stem cells; chondrogenesis; differentiation; cartilage
One of the most challenging tasks in the field of regenerative medicine is to overcome the weak self-repair potential of injured cartilage. Cell-based therapies demonstrated that integrated repair tissue with a successful clinical result in >90% of patients can be generated by autologous chondrocyte transplantation (ACT) (Peterson et al., 2003). However, for ACT, autologous tissue is isolated from healthy cartilage which affects donor site morbidity and increases the risk for osteoarthritis. Moreover, two articular surgical procedures are required for ACT. Thus, evaluation of more easily available cell sources is highly important. Adipose tissue is reported to contain mesenchymal stem cells (Zuk et al., 2001) and can be *Correspondence to: M. van Griensven, Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrasse 13, A-1200 Vienna, Austria. E-mail:
[email protected] Copyright 2009 John Wiley & Sons, Ltd.
harvested in high amounts, which is accompanied with only low donor site morbidity. Wang et al. (2004) demonstrated the existence of mesenchymal stem cells also in the human umbilical cord matrix, called the Wharton’s jelly. Since umbilical cords are usually discarded after birth, this tissue represents an optimal cell source that can be harvested without donor site morbidity. Human umbilical cord matrix cells (HUCMs) express surface markers similar to other mesenchymal cells and can be differentiated towards the chondrogenic, adipogenic, neuronal and osteogenic lineages (Karahuseyinoglu et al., 2007). Other umbilical cord-derived cell types that also possess multilineage differentiation potential include human umbilical cord perivascular cells (HUCPVCs) and umbilical cord blood stem cells (Baksh et al., 2007; Rebelatto et al., 2008). Recently, the immune properties of Wharton’s jelly-derived cells were investigated and the
How chondrogenic are human umbilical cord matrix cells?
results suggest that, similar to adipose-derived stem cells (ASCs), they might be tolerated in allogenic transplantation (Weiss et al., 2008; Wolbank et al., 2007). Studies investigating the chondrogenic differentiation potential of HUCMs demonstrated glycosaminoglycan- and cartilagespecific collagen production in micromass pellet cultures (Karahuseyinoglu et al., 2007) or polyglycolic scaffolds (Bailey et al., 2007). In addition to histological and biochemical analyses, the present study investigated 10 cartilage-associated genes and quantified mRNA expression of HUCMs in comparison to ASCs. Human umbilical cords and liposuction material from three different donors were obtained after approval by the Upper Austrian Ethical Board from a local hospital or a private plastic surgeon and processed within 24 h. Cells were isolated by explantation culture (HUCMs) or enzymatic digestion (ASCs), as previously described (Carlin et al., 2006; Wolbank et al., 2007) and flow cytometric analyses for CD14, CD34, CD44, CD45, CD73, CD90, CD105, HLA-ABC and HLA-DR were performed on a FACS Calibur (BD, Heidelberg, Germany). Antibodies were purchased from Abcam, Austria (CD105) or from BD. HUCM expansion medium was used, as described previously (Carlin et al., 2006). For the expansion of ASCs, EGM-2 (Lonza, Basel, Switzerland) was used. Population doubling times were calculated according to the following formula: Td = (t2 − t1 ) × log(2)/ log(q2 /q1 ). Micromass pellets of 3 × 105 ASCs or HUCMs in passages 3–4 were prepared by centrifugation (5 min, 350 × g). Constructs were cultured for up to 35 days in chondrogenic differentiation medium (Lonza) supplemented with 10 ng/ml TGFβ3 (Lonza) and 10 ng/ml BMP-6 (Biomedica, Vienna, Austria). Control pellets were cultured in expansion medium. On days 0 and 14 RNA was isolated according to the TRIReagent protocol (Sigma-Aldrich, Steinheim, Germany) and transcribed to cDNA, as stated in the High Capacity cDNA Archive Kit protocol (Applied Biosystems, Brunn am Gebirge, Austria). Quantification of specific cDNAs was conducted using a LightCycler 480 (Roche, Mannheim, Germany) and Taqman gene expression assays (Applied Biosystems) for the following genes: SOX9 [SRY (sex-determining region Y)-box 9, Hs00165814 m1]; COL2A1 (collagen type II, Hs01064869 m1); COL9A2 (collagen type IX, Hs00899019 m1); AGC1 (aggrecan, Hs01048724 m1); MIA (melanoma inhibitory activity, Hs01064456 g1); COMP (cartilage oligomeric matrix protein, Hs01561085 g1); CRTL1 (cartilage link protein 1, Hs00157103 m1); CSPG2 (chondroitin sulphate proteoglycan II, Hs01007932 m1); COL1A1 (collagen type I, Hs00164004 m1); COL10A1 (collagen type X, Hs00950955 g1). Analyses were performed in triplicate and expression values were normalized to hypoxanthine–guanine phosphoribosyl transferase. The efficiencycorrected quantification was performed automatically, using LightCycler 480 Relative Quantification Software (Roche). For quantitative glycosaminoglycan (GAG) analysis, papain-digested samples were subjected to the Blyscan Sulphated Glycosaminoglycan Assay (Biocolor, Carrickfergus, UK) and DNA was quantified using the Copyright 2009 John Wiley & Sons, Ltd.
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PicoGreen dsDNA Quantitation Kit (Invitrogen, Lofer, Austria). For qualitative GAG analyses, deparaffinized sections were stained with Alcian blue/fast red (Morphisto, Frankfurt am Main, Germany). GAG analysis and histology were performed at the end of the culture. Statistical analyses were performed with GraphPad Prism 3.02, using ANOVA and Tukey’s post hoc test. Values are presented as mean ± standard deviation (SD; n = 3 independent samples/donor). p < 0.05 was considered to be statistically significant. In line with previous studies, flow cytometric analyses revealed similar surface marker expression for ASCs and HUCMs demonstrating positive staining for CD44, CD73, CD90, CD105, HLA-ABC and lack of CD14, CD34, CD45 and HLA-DR (data not shown) (Karahuseyinoglu et al., 2007; Lund et al., 2007; Weiss et al., 2006). Monolayer cultures of HUCMs demonstrated significantly higher proliferation (population doubling time = 2.3 ± 0.6 days) compared to ASCs (population doubling time = 7.1 ± 0.9 days) (Figure 1A). Baksh et al. (2007) compared the proliferative activity of HUCPVCs and bone marrow-derived mesenchymal stem cells (BMSCs) and their findings also demonstrated higher proliferation for the umbilical cord-derived cells. However, Alcian blue staining and GAG quantification indicate significantly higher GAG accumulation during 3D micromass culture for ASCs than for HUCMs (Figure 1B, C). This might be due to the presence of BMP-6, which is known to strongly enhance the chondrogenic differentiation capacity of ASCs (Estes et al., 2006), while it obviously acts less inductively for HUCMs. Various studies used toluidine blue to stain GAGs in HUCM constructs (Hoynowski et al., 2007; Karahuseyinoglu et al., 2007). However, Bailey et al. (2007) more specifically detected chondroitin 4-sulphate and chondroitin 6-sulphate (types IV and VI) by immunohistochemistry. In contrast to our experimental set-up, which used micromass pellet cultures, Bailey et al. seeded HUCMs onto PEG scaffolds and differentiated them in absence of BMP-6. Since chondroitin sulphate constitutes the major GAG component of aggrecan, they suggested that aggrecan was present in large amounts. However, chondroitin sulphate can be found in both aggrecan and versican, whereas the latter is mainly expressed by cells of fibrous tissues. Weak staining for aggrecan was found by Wang et al. (2004); however, no information about the expression of versican was provided. For that reason we studied gene expression of both proteoglycans (Figure 2). Only minute amounts of aggrecan mRNA were detected, while CSPG2 (versican) showed strong expression. Therefore we suppose that versican represents the main GAG component in our HUCM constructs, indicating a more fibrous than hyaline cartilage phenotype. In contrast, ASCs demonstrated strong AGC1 expression on day 14, while CSPG2 was expressed in the same range by both cell types, resulting in a more hyaline matrix composition in ASC compared to HUCM constructs. All investigated genes coding for collagens were upregulated during 3D micromass culture in both ASCs J Tissue Eng Regen Med 2010; 4: 242–245. DOI: 10.1002/term
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Figure 1. (A) Growth curves and population doubling times (PDTs) of HUCMs and ASCs. (B) GAG quantification of micromass pellets cultured in control medium or chondrogenic medium supplemented with 10 ng/ml TGFβ3 and 10 ng/ml BMP-6; groups not sharing a letter are significantly different. (C) Alcian blue staining of micromass pellets. Magnifications, ×400; inserts ×100
Figure 2. Relative mRNA expression of ASCs and HUCMs cultured as micromass pellets under chondrogenic conditions. qRT–PCR was performed on day 0 (left data point) and day 14 (right data point). On day 0, expression of COL2A1, AGC1 and COL10A1 was not detectable in ASCs. n = 3 donors
and HUCMs (Figure 2). Notably, COL2A1 expression was below the detection limit in ASCs prior to chondrogenic differentiation but showed approximately 250-fold expression compared to HUCMs on day 14. Similar to this, Wang et al. (2009) demonstrated that BMSCs outperformed HUCMs regarding COL2A1 expression. Although our results of HUCMs show that Copyright 2009 John Wiley & Sons, Ltd.
upregulation of COL2A1 and COL1A1 is in the same range (3- and 3.4-fold), COL1A1 is expressed approximately 105 -fold higher than COL2A1 (Figure 2). This is in agreement with immunohistological data of studies performed by Bailey et al. (2007) and Wang et al. (2009), who reported only minute amounts of collagen II, while collagen I was strongly expressed in HUCMs J Tissue Eng Regen Med 2010; 4: 242–245. DOI: 10.1002/term
How chondrogenic are human umbilical cord matrix cells?
after chondrogenic differentiation. COL9A2 expression was strongly enhanced in both cell types but was induced approximately 10-fold higher in ASCs compared to HUCMs on day 14 (Figure 2). Collagen IX protein is reported to act as an important molecule for the long-term stability of articular cartilage (Hu et al., 2006). COMP, another important marker for chondrogenesis, was also upregulated in both cell types. Expression of COL10A1 was 10-fold upregulated during chondrogenic differentiation of HUCMs, indicating a high osteogenic potential of HUCMs, as previously demonstrated by Eblenkamp et al. (2004). In ASCs, COL10A1 was absent on day 0 but increased to an expression level similar to that of COL2A1 and COL9A2 on day 14. MIA, a well-accepted marker for chondrogenic differentiation (Bosserhoff and Buettner, 2003; Tscheudschilsuren et al., 2006) showed three-fold induction in HUCMs and 150-fold in ASCs (Figure 2). Interestingly, CRTL1 and the transcription factor SOX9 were downregulated in HUCMs during chondrogenic differentiation, while they were strongly upregulated in ASCs. In conclusion, we demonstrate that both cell types, ASCs and HUCMs, possess potential to differentiate towards the chondrogenic lineage. This study provides information about the expression of 10 cartilage-related genes, indicating development of a fibrous/hypertrophic cartilage phenotype during chondrogenic differentiation of HUCMs and ASCs. However, histological data, GAG quantification and qRT–PCR clearly demonstrate that a cartilage-like matrix of higher quality is produced by ASCs compared to HUCMs.
Acknowledgements We wish to express our thanks to Tamara Jagersberger and Mag. Katja Hofer for excellent technical assistance. This work was supported by funding from HIPPOCRATES (NMP3CT-2003-505758), Lorenz Boehler Fonds and EXPERTISSUES (NMP3-CT-2004-500283).
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J Tissue Eng Regen Med 2010; 4: 242–245. DOI: 10.1002/term
