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Stem Cell-Derived Chondrocytes for Regenerative Medicine
Stem Cell-Derived Chondrocytes for Regenerative Medicine J. Kramer, F. Böhrnsen, P. Schlenke, and J. Rohwedel ABSTRACT The regenerative capacity of cartilage is limited. Transplantation methods used to treat cartilage lesions are based mainly on primary cultures of chondrocytes, which dedifferentiate during cultivation in vitro and lose their functional properties. Stem cells are considered as an alternative source to generate cells for two reasons: first, they can almost indefinitely divide in culture, and second, they are able to differentiate into various mature cell types. Herein, we asked the question whether chondrocytes could be differentiated from mouse embryonic stem (ES) cells to a state suitable for regenerative use. When cultivated as embryoid bodies (EBs), murine ES cells differentiate into mesenchymal progenitor cells, which progressively develop into mature, hypertrophic chondrocytes and transdifferentiate into calcifying cells recapitulating all of the cellular processes of chondrogenesis. Chondrocytes isolated from EBs exhibit a high regenerative capacity. They dedifferentiate initially in culture, but later reexpress stable characteristics of mature chondrocytes. However, in cultures of chondrocytes isolated from EBs, additional mesenchymal cell types can be observed. Mesenchymal stem (MS) cells from bone marrow have already been used in tissue engineering settings. We compared the chondrogenic differentiation of MS and ES cells.
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ESIONS in joint cartilage tissue often are replaced by fibrous connective tissue or show a total lack of repair resulting in arthrosis. Several attempts have been undertaken to develop tissue-engineered grafts based on supportive carriers, such as fibrin, alginate, and poly-alpha-hydroxy acids.1 However, these have not resulted in regeneration and complete functionality. Furthermore, difficulties in obtaining and shaping autologous cartilage for reconstruction of craniofacial features have spurred interest in tissue engineering as a means to generate cell-based implants for plastic and maxillofacial surgery.2– 4 However, all these methods suffer from the use of primary cultures of chondrocytes that have been shown to dedifferentiate rapidly in culture and lose their functional properties.5 In addition, adult chondrocytes show restricted proliferative capacity in culture, resulting in a limited amount of cells which are insufficient for regenerative strategies. An alternative method is the use of cells differentiated in vitro from either mesenchymal stem (MS) or embryonic stem (ES) cells. Undifferentiated stem cells possess the ability to proliferate extensively in culture. Stem cell-derived chondrocytes may have the advantage to achieve a stable phenotype compared with chondrogenic cells derived from primary cultures.6,7 Herein, we have examined the advantages and disadvan0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.02.023 762
tages for regenerative medicine of MS and ES cell-derived chondrocytes. MATERIALS AND METHODS In vitro chondrogenesis by MS cells is typically performed in micromass body (MMB) cultures in the presence of transforming growth factor (TGF).8,9 Osteogenic differentiation of MS cells was induced by application to monolayer cultures of osteogenic medium, which contained dexamethasone, -glycerolphosphate, and ascorbic acid.10 Cultivated bone marrow-derived murine MS cells were differentiated following these protocols.11 The differentiation of murine ES cells via embryoid bodies (EBs) was performed using established protocols.7,12,13 ES cell-derived chondrocytes were isolated using a microdissector.14 Chondrogenesis was characterized From the Department of Medical Molecular Biology (J.K., F.B., J.R.), University of Lübeck; and Medical Department 1, Division of Nephrology & Transplantation Unit, University Clinics of Schleswig-Holstein, Campus Lübeck, Lübeck, Germany. This work was supported by Intermed Service GmbH&CoKG (Geesthacht, Germany). Address reprint requests to Dr Jan Kramer, Medical Department 1, Division of Nephrology & Transplantation unit, University Clinics of Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. E-mail: Jan_Kramer@ gmx.de © 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 762–765 (2006)
STEM CELL-DERIVED CHONDROCYTES by histochemical staining with alcian blue and immunostaining using an antibody against collagen II coupled with scleraxis or aggrecan in situ hybridization.7,12 Osteogenic differentiation was documented by immunostaining for bone sialo protein as well as by staining with alizarin red or for alkaline phosphatase.7,13
RESULTS
We demonstrated that ES cells differentiated spontaneously into mature chondrocytes via mesenchymal cells. Initially, these chondrocyte progenitors were organized in streaks in the EB outgrowths, expressing scleraxis, a characteristic marker for precartilaginous tissue (Fig 1A). Later, the mesenchymal cells aggregated forming condensations (Fig 1B). The onset of collagen type II expression was demonstrated
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by immunostaining, indicating early chondrogenic differentiation of cells within the condensations. During further EB cultivation, cartilage nodule formation was observed in the EB outgrowths (Fig 1C). The chondrogenic cells showed a rounded morphology with increased expression of collagen type II. Moreover, we observed strong expression of the cartilage-specific proteoglycan aggrecan (Fig 1D). The cartilage nodules were surrounded by a dense fibrillar network of extracellular matrix molecules. Finally, osteomineralization within the nodules was documented by alizarin red staining for calcium deposits (Fig 1E). In contrast, MS cells did not differentiate into chondrogenic cells in monolayer culture either spontaneously or after induction with specific stimuli like TGF-beta3 (Fig 1F).
Fig 1. Stem cell-derived chondrocytes. Embryonic stem (ES) cells recapitulate cellular events of chondrogenesis and differentiate spontaneously via mesenchymal progenitors, which are initially organized in streak-like cell clusters (A), and later form precartilaginous condensations (B), into mature chondrocytes (C,D). Mesenchymal cells express the marker molecule scleraxis (green) as revealed by in situ hybridization. The mesenchymal condensations foreshadow the cartilage anlagen in the ES cell cultures as indicated by immunostainings showing the onset of collagen type II expression (B; red). ES cell-derived chondrocytes are detected within cartilage nodules and show a typical round-shaped morphology (C). During this mature stage of chondrogenesis, collagen type II expression increases and expression of scleraxis becomes down-regulated. In addition, the combination of immunostaining for collagen II (red) with in situ hybridization for aggrecan (green) demonstrates expression of this cartilage-specific proteoglycan (D). Finally, osteogenic differentiation and osteomineralization within the nodules are indicated by alizarin red staining (E). In contrast to ES cells, mesenchymal stem (MS) cells in monolayer culture (F) do not spontaneously differentiate into the chondrogenic lineage. Only micromass body 3-dimensional (3-d) cultivation and additional application of specific growth factors like transforming growth factor (TGF)-beta3 result in MS cell-derived chondrogenesis. Cartilage-specific proteoglycans are stained with alcian blue (G). Collagen type II expression (green) is demonstrated by immunostaining (H). MS cell differentiation into osteocytes has to be induced by dexamethasone, -glycerolphosphate, and ascorbic acid. Immunostaining for bone sialo protein (I) and alkaline phosphatase staining (J) demonstrates osteocyte differentiation. The cell nuclei are stained with DAPI (blue; H,I). Bars ⫽ 100 m.
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MS cells had to be mechanically induced, to form cellular aggregates, so-called MMBs eg, by centrifugation. These 3-dimensional cultures are critical for cellular interactions, which play an important role in the chondrogenic differentiation of MS cells.8,9 After induction with TGF-beta3, alcian blue staining for cartilage-specific proteoglycans (Fig 1G) as well as immunostaining for collagen type II (Fig 1H) showed chondrogenic differentiation within the MMBs. Osteomineralization was not observed within these MMB cultures (data not shown). However, in monolayer cultures osteogenic differentiation was observed after induction with dexamethasone, -glycerolphosphate, and ascorbic acid, as demonstrated by immunostaining for bone sialo protein (Fig 1I) and alkaline phosphatase staining (Fig 1J). DISCUSSION
Based on their origin from the inner cell mass of the blastocyst, ES cells are pluripotent. If murine ES cells are cultivated via EBs, they spontaneously differentiate into various cell types representing all three germ layers. Cellular processes of embryogenesis are mimicked during EB cultivation. For example, we recently demonstrated that cellular events of enchondral ossification were closely recapitulated during ES cell differentiation in vitro.6,12,14,15 ES cells cultivated via EBs spontaneously differentiated into chondrogenic cells.12 Initially, prechondrogenic mesenchymal condensations were found within the EBs. Cells in these cellular aggregates expressed characteristic precartilaginous mesenchymal molecules like scleraxis, Sox5 and Sox6,14 as well as N-cadherin. The cells also bound peanutagglutinin.16 Ultrastructural analysis showed a typical morphology of these cells, resembling early chondrocyte progenitors in vivo.14 Later the cells formed cartilage nodules and expressed type II collagen. In addition, we observed the expression of aggrecan, the major proteoglycan of cartilage tissue, and oligomeric cartilage matrix protein12 within these nodules. Ultrastructural analysis confirmed that mature ES cell-derived chondrocytes produced large amounts of collagen and proteoglycans, closely resembling their embryonic counterparts.14 Finally, the ES cell-derived chondrocytes expressed type X collagen indicating hypertrophy and transdifferentiation into calcifying elements of cells producing osteocalcin, bone sialo protein, and alkaline phosphatase.7,13 Osteomineralization is demonstrated by alizarin red staining. These observations document that all cellular stages of enchondral ossification can be analyzed using the ES cell model system. Beside this process of endochondral ossification, the direct differentiation of precursors into osteoblasts may be detected in EB outgrowths, corresponding to the in vivo process of intramembranous ossification.6 Chondrocytes derived from ES cells may have the advantage of a stable phenotype compared with the progressively dedifferentiating chondrogenic cells derived from primary cultures.5 For example, chondrocytes isolated by microdissection from EBs initially dedifferentiated in culture, but later redifferentiated into mature chondrocytes
KRAMER, BÖHRNSEN, SCHLENKE ET AL
indicating their plasticity.6 ES cell-derived chondrocytes may be helpful tools for cellular replacement therapies to treat cartilage lesions, especially once human ES cell lines are established.17 However, chondrocytes isolated from murine EBs, which have been investigated by clonal analysis, are capable of transdifferentiation into other mesenchymal cell types, especially of the adipogenic lineage.6 Therefore, it is indispensable to develop further selection strategies for therapeutic application of ES cell-derived cells. It is particularly important to eliminate undifferentiated ES cells from the chondrocyte cultures to avoid teratoma formation after transplantation into cartilage defects.18 ES cells have advantages compared with adult stem cells like MS cells: until now ES cells show the greatest proliferation among stem cells, an observation that ensures generation of sufficient amounts of cells for therapeutic applications. MS cells obviously have a more restricted proliferative capacity. In addition, ES cells show an outstanding capacity to differentiate into chondrocytes, as indicated by their inherent spontaneous differentiation. However, various ES cell lines showed a variable degree of chondrogenic differentiation in vitro15; it may be necessary to perform cell line selection. Compared with ES cells, the generation of adult stem cell lines is not associated with an ethical dilemma. MS cells are easily accessible from human bone marrow, eg, by iliac crest biopsy, or from other sources like adipogenic tissue.19 Moreover, MS cells are autologous, while ES cells are allogenic stem cells. In contrast to ES cells, MS cells do not spontaneously differentiate into chondrocytes or osteocytes in vitro. Rather, they have to be induced using complex induction media. However, MS cells have already been used for therapeutic applications for cartilage defects,20 although protocols for standardized, optimized differentiation of MS cells have still not been established.21 A detailed comparison of MS and ES differentiation is required to develop such cultivation protocols.22 Transplantation experiments in animal models using both cell types may also lead the way to the ideal chondrogenic cell (progenitor vs mature chondrocyte) for regenerative strategies of joint diseases. ACKNOWLEDGMENT The skillful technical assistance of A. Eirich and M. Dose is gratefully acknowledged.
REFERENCES 1. Sittinger M, Perka C, Schultz O, et al: Joint cartilage regeneration by tissue engineering. Z Rheumatol 58:130, 1999 2. Bonassar LJ, Vacanti CA: Tissue engineering: the first decade and beyond. J Cell Biochem Suppl 30 –31:297, 1998 3. Britt JC, Park SS: Autogenous tissue-engineered cartilage: evaluation as an implant material. Arch Otolaryngol Head Neck Surg 124:671, 1998 4. Puelacher WC, Wisser J, Vacanti CA, et al: Temporomandibular joint disc replacement made by tissue-engineered growth of cartilage. J Oral Maxillofac Surg 52:1172, 1994 5. von der Mark K, Gauss V, von der Mark H, et al: Relationship between cell shape and type of collagen synthesised as
STEM CELL-DERIVED CHONDROCYTES chondrocytes lose their cartilage phenotype in culture. Nature 267:531, 1977 6. Hegert C, Kramer J, Hargus G, et al: Plasticity of hypertrophic chondrocytes differentiated from mouse embryonic stem cells. J Cell Sci 115:4617, 2002 7. Kramer J, Hegert C, Rohwedel J: In vitro differentiation of mouse ES cells: bone and cartilage. Methods Enzymol 365:251, 2003 8. Mackay AM, Beck SC, Murphy JM, et al: Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 4:415, 1998 9. Yoo JU, Barthel TS, Nishimura K, et al: The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Jt Surg Am 80:1745, 1998 10. Jaiswal N, Haynesworth SE, Caplan AI, et al: Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64:295, 1997 11. Kramer J, Böhrnsen F, Lindner U, et al: In vivo matrixguided human mesenchymal stem cells. Cell Mol Life Sci (in press) 12. Kramer J, Hegert C, Guan K, et al: Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech Dev 92:193, 2000 13. Kramer J, Hegert C, Hargus G, et al: Chondrocytes derived from mouse embryonic stem cells. Cytotechnology 41:177, 2003
765 14. Kramer J, Klinger M, Kruse C, et al: Ultrastructural analysis of mouse embryonic stem cell-derived chondrocytes. Anat Embryol (Berl) 210:175, 2005 15. Kramer J, Hegert C, Hargus G, et al: Mouse ES cell lines show a variable degree of chondrogenic differentiation in vitro. Cell Biol Int 29:139, 2005 16. Hargus G, Kist R, Kramer J, et al: Loss of Sox9 function results in defective chondrocyte differentiation of mouse embryonic stem cells in vitro (submitted) 17. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al: Embryonic stem cell lines derived from human blastocysts. Science 282:1145, 1998 18. Wakitani S, Takaoka K, Hattori T, et al: Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology (Oxford) 42:162, 2003 19. Zuk PA, Zhu M, Ashjian P, et al: Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13:4279, 2002 20. Redman SN, Oldfield SF, Archer CW: Current strategies for articular cartilage repair. Eur Cell Mater 9:23, 2005 21. Gregory CA, Ylostalo J, Prockop DJ: Adult bone marrow stem/progenitor cells (MSCs) are preconditioned by microenvironmental “niches” in culture: a two-stage hypothesis for regulation of MSC fate. Sci STKE 294:e37, 2005 22. Vats A, Bielby RC, Tolley NS, et al: Stem cells. Lancet 366:592, 2005
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ESIONS in joint cartilage tissue often are replaced by fibrous connective tissue or show a total lack of repair resulting in arthrosis. Several attempts have been undertaken to develop tissue-engineered grafts based on supportive carriers, such as fibrin, alginate, and poly-alpha-hydroxy acids.1 However, these have not resulted in regeneration and complete functionality. Furthermore, difficulties in obtaining and shaping autologous cartilage for reconstruction of craniofacial features have spurred interest in tissue engineering as a means to generate cell-based implants for plastic and maxillofacial surgery.2– 4 However, all these methods suffer from the use of primary cultures of chondrocytes that have been shown to dedifferentiate rapidly in culture and lose their functional properties.5 In addition, adult chondrocytes show restricted proliferative capacity in culture, resulting in a limited amount of cells which are insufficient for regenerative strategies. An alternative method is the use of cells differentiated in vitro from either mesenchymal stem (MS) or embryonic stem (ES) cells. Undifferentiated stem cells possess the ability to proliferate extensively in culture. Stem cell-derived chondrocytes may have the advantage to achieve a stable phenotype compared with chondrogenic cells derived from primary cultures.6,7 Herein, we have examined the advantages and disadvan0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.02.023 762
tages for regenerative medicine of MS and ES cell-derived chondrocytes. MATERIALS AND METHODS In vitro chondrogenesis by MS cells is typically performed in micromass body (MMB) cultures in the presence of transforming growth factor (TGF).8,9 Osteogenic differentiation of MS cells was induced by application to monolayer cultures of osteogenic medium, which contained dexamethasone, -glycerolphosphate, and ascorbic acid.10 Cultivated bone marrow-derived murine MS cells were differentiated following these protocols.11 The differentiation of murine ES cells via embryoid bodies (EBs) was performed using established protocols.7,12,13 ES cell-derived chondrocytes were isolated using a microdissector.14 Chondrogenesis was characterized From the Department of Medical Molecular Biology (J.K., F.B., J.R.), University of Lübeck; and Medical Department 1, Division of Nephrology & Transplantation Unit, University Clinics of Schleswig-Holstein, Campus Lübeck, Lübeck, Germany. This work was supported by Intermed Service GmbH&CoKG (Geesthacht, Germany). Address reprint requests to Dr Jan Kramer, Medical Department 1, Division of Nephrology & Transplantation unit, University Clinics of Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. E-mail: Jan_Kramer@ gmx.de © 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 762–765 (2006)
STEM CELL-DERIVED CHONDROCYTES by histochemical staining with alcian blue and immunostaining using an antibody against collagen II coupled with scleraxis or aggrecan in situ hybridization.7,12 Osteogenic differentiation was documented by immunostaining for bone sialo protein as well as by staining with alizarin red or for alkaline phosphatase.7,13
RESULTS
We demonstrated that ES cells differentiated spontaneously into mature chondrocytes via mesenchymal cells. Initially, these chondrocyte progenitors were organized in streaks in the EB outgrowths, expressing scleraxis, a characteristic marker for precartilaginous tissue (Fig 1A). Later, the mesenchymal cells aggregated forming condensations (Fig 1B). The onset of collagen type II expression was demonstrated
763
by immunostaining, indicating early chondrogenic differentiation of cells within the condensations. During further EB cultivation, cartilage nodule formation was observed in the EB outgrowths (Fig 1C). The chondrogenic cells showed a rounded morphology with increased expression of collagen type II. Moreover, we observed strong expression of the cartilage-specific proteoglycan aggrecan (Fig 1D). The cartilage nodules were surrounded by a dense fibrillar network of extracellular matrix molecules. Finally, osteomineralization within the nodules was documented by alizarin red staining for calcium deposits (Fig 1E). In contrast, MS cells did not differentiate into chondrogenic cells in monolayer culture either spontaneously or after induction with specific stimuli like TGF-beta3 (Fig 1F).
Fig 1. Stem cell-derived chondrocytes. Embryonic stem (ES) cells recapitulate cellular events of chondrogenesis and differentiate spontaneously via mesenchymal progenitors, which are initially organized in streak-like cell clusters (A), and later form precartilaginous condensations (B), into mature chondrocytes (C,D). Mesenchymal cells express the marker molecule scleraxis (green) as revealed by in situ hybridization. The mesenchymal condensations foreshadow the cartilage anlagen in the ES cell cultures as indicated by immunostainings showing the onset of collagen type II expression (B; red). ES cell-derived chondrocytes are detected within cartilage nodules and show a typical round-shaped morphology (C). During this mature stage of chondrogenesis, collagen type II expression increases and expression of scleraxis becomes down-regulated. In addition, the combination of immunostaining for collagen II (red) with in situ hybridization for aggrecan (green) demonstrates expression of this cartilage-specific proteoglycan (D). Finally, osteogenic differentiation and osteomineralization within the nodules are indicated by alizarin red staining (E). In contrast to ES cells, mesenchymal stem (MS) cells in monolayer culture (F) do not spontaneously differentiate into the chondrogenic lineage. Only micromass body 3-dimensional (3-d) cultivation and additional application of specific growth factors like transforming growth factor (TGF)-beta3 result in MS cell-derived chondrogenesis. Cartilage-specific proteoglycans are stained with alcian blue (G). Collagen type II expression (green) is demonstrated by immunostaining (H). MS cell differentiation into osteocytes has to be induced by dexamethasone, -glycerolphosphate, and ascorbic acid. Immunostaining for bone sialo protein (I) and alkaline phosphatase staining (J) demonstrates osteocyte differentiation. The cell nuclei are stained with DAPI (blue; H,I). Bars ⫽ 100 m.
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MS cells had to be mechanically induced, to form cellular aggregates, so-called MMBs eg, by centrifugation. These 3-dimensional cultures are critical for cellular interactions, which play an important role in the chondrogenic differentiation of MS cells.8,9 After induction with TGF-beta3, alcian blue staining for cartilage-specific proteoglycans (Fig 1G) as well as immunostaining for collagen type II (Fig 1H) showed chondrogenic differentiation within the MMBs. Osteomineralization was not observed within these MMB cultures (data not shown). However, in monolayer cultures osteogenic differentiation was observed after induction with dexamethasone, -glycerolphosphate, and ascorbic acid, as demonstrated by immunostaining for bone sialo protein (Fig 1I) and alkaline phosphatase staining (Fig 1J). DISCUSSION
Based on their origin from the inner cell mass of the blastocyst, ES cells are pluripotent. If murine ES cells are cultivated via EBs, they spontaneously differentiate into various cell types representing all three germ layers. Cellular processes of embryogenesis are mimicked during EB cultivation. For example, we recently demonstrated that cellular events of enchondral ossification were closely recapitulated during ES cell differentiation in vitro.6,12,14,15 ES cells cultivated via EBs spontaneously differentiated into chondrogenic cells.12 Initially, prechondrogenic mesenchymal condensations were found within the EBs. Cells in these cellular aggregates expressed characteristic precartilaginous mesenchymal molecules like scleraxis, Sox5 and Sox6,14 as well as N-cadherin. The cells also bound peanutagglutinin.16 Ultrastructural analysis showed a typical morphology of these cells, resembling early chondrocyte progenitors in vivo.14 Later the cells formed cartilage nodules and expressed type II collagen. In addition, we observed the expression of aggrecan, the major proteoglycan of cartilage tissue, and oligomeric cartilage matrix protein12 within these nodules. Ultrastructural analysis confirmed that mature ES cell-derived chondrocytes produced large amounts of collagen and proteoglycans, closely resembling their embryonic counterparts.14 Finally, the ES cell-derived chondrocytes expressed type X collagen indicating hypertrophy and transdifferentiation into calcifying elements of cells producing osteocalcin, bone sialo protein, and alkaline phosphatase.7,13 Osteomineralization is demonstrated by alizarin red staining. These observations document that all cellular stages of enchondral ossification can be analyzed using the ES cell model system. Beside this process of endochondral ossification, the direct differentiation of precursors into osteoblasts may be detected in EB outgrowths, corresponding to the in vivo process of intramembranous ossification.6 Chondrocytes derived from ES cells may have the advantage of a stable phenotype compared with the progressively dedifferentiating chondrogenic cells derived from primary cultures.5 For example, chondrocytes isolated by microdissection from EBs initially dedifferentiated in culture, but later redifferentiated into mature chondrocytes
KRAMER, BÖHRNSEN, SCHLENKE ET AL
indicating their plasticity.6 ES cell-derived chondrocytes may be helpful tools for cellular replacement therapies to treat cartilage lesions, especially once human ES cell lines are established.17 However, chondrocytes isolated from murine EBs, which have been investigated by clonal analysis, are capable of transdifferentiation into other mesenchymal cell types, especially of the adipogenic lineage.6 Therefore, it is indispensable to develop further selection strategies for therapeutic application of ES cell-derived cells. It is particularly important to eliminate undifferentiated ES cells from the chondrocyte cultures to avoid teratoma formation after transplantation into cartilage defects.18 ES cells have advantages compared with adult stem cells like MS cells: until now ES cells show the greatest proliferation among stem cells, an observation that ensures generation of sufficient amounts of cells for therapeutic applications. MS cells obviously have a more restricted proliferative capacity. In addition, ES cells show an outstanding capacity to differentiate into chondrocytes, as indicated by their inherent spontaneous differentiation. However, various ES cell lines showed a variable degree of chondrogenic differentiation in vitro15; it may be necessary to perform cell line selection. Compared with ES cells, the generation of adult stem cell lines is not associated with an ethical dilemma. MS cells are easily accessible from human bone marrow, eg, by iliac crest biopsy, or from other sources like adipogenic tissue.19 Moreover, MS cells are autologous, while ES cells are allogenic stem cells. In contrast to ES cells, MS cells do not spontaneously differentiate into chondrocytes or osteocytes in vitro. Rather, they have to be induced using complex induction media. However, MS cells have already been used for therapeutic applications for cartilage defects,20 although protocols for standardized, optimized differentiation of MS cells have still not been established.21 A detailed comparison of MS and ES differentiation is required to develop such cultivation protocols.22 Transplantation experiments in animal models using both cell types may also lead the way to the ideal chondrogenic cell (progenitor vs mature chondrocyte) for regenerative strategies of joint diseases. ACKNOWLEDGMENT The skillful technical assistance of A. Eirich and M. Dose is gratefully acknowledged.
REFERENCES 1. Sittinger M, Perka C, Schultz O, et al: Joint cartilage regeneration by tissue engineering. Z Rheumatol 58:130, 1999 2. Bonassar LJ, Vacanti CA: Tissue engineering: the first decade and beyond. J Cell Biochem Suppl 30 –31:297, 1998 3. Britt JC, Park SS: Autogenous tissue-engineered cartilage: evaluation as an implant material. Arch Otolaryngol Head Neck Surg 124:671, 1998 4. Puelacher WC, Wisser J, Vacanti CA, et al: Temporomandibular joint disc replacement made by tissue-engineered growth of cartilage. J Oral Maxillofac Surg 52:1172, 1994 5. von der Mark K, Gauss V, von der Mark H, et al: Relationship between cell shape and type of collagen synthesised as
STEM CELL-DERIVED CHONDROCYTES chondrocytes lose their cartilage phenotype in culture. Nature 267:531, 1977 6. Hegert C, Kramer J, Hargus G, et al: Plasticity of hypertrophic chondrocytes differentiated from mouse embryonic stem cells. J Cell Sci 115:4617, 2002 7. Kramer J, Hegert C, Rohwedel J: In vitro differentiation of mouse ES cells: bone and cartilage. Methods Enzymol 365:251, 2003 8. Mackay AM, Beck SC, Murphy JM, et al: Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 4:415, 1998 9. Yoo JU, Barthel TS, Nishimura K, et al: The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Jt Surg Am 80:1745, 1998 10. Jaiswal N, Haynesworth SE, Caplan AI, et al: Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64:295, 1997 11. Kramer J, Böhrnsen F, Lindner U, et al: In vivo matrixguided human mesenchymal stem cells. Cell Mol Life Sci (in press) 12. Kramer J, Hegert C, Guan K, et al: Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech Dev 92:193, 2000 13. Kramer J, Hegert C, Hargus G, et al: Chondrocytes derived from mouse embryonic stem cells. Cytotechnology 41:177, 2003
765 14. Kramer J, Klinger M, Kruse C, et al: Ultrastructural analysis of mouse embryonic stem cell-derived chondrocytes. Anat Embryol (Berl) 210:175, 2005 15. Kramer J, Hegert C, Hargus G, et al: Mouse ES cell lines show a variable degree of chondrogenic differentiation in vitro. Cell Biol Int 29:139, 2005 16. Hargus G, Kist R, Kramer J, et al: Loss of Sox9 function results in defective chondrocyte differentiation of mouse embryonic stem cells in vitro (submitted) 17. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al: Embryonic stem cell lines derived from human blastocysts. Science 282:1145, 1998 18. Wakitani S, Takaoka K, Hattori T, et al: Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology (Oxford) 42:162, 2003 19. Zuk PA, Zhu M, Ashjian P, et al: Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13:4279, 2002 20. Redman SN, Oldfield SF, Archer CW: Current strategies for articular cartilage repair. Eur Cell Mater 9:23, 2005 21. Gregory CA, Ylostalo J, Prockop DJ: Adult bone marrow stem/progenitor cells (MSCs) are preconditioned by microenvironmental “niches” in culture: a two-stage hypothesis for regulation of MSC fate. Sci STKE 294:e37, 2005 22. Vats A, Bielby RC, Tolley NS, et al: Stem cells. Lancet 366:592, 2005