Cartilage Engineering from Ovine Umbilical Cord Blood Mesenchymal Progenitor Cells
http://www.100md.com
《干细胞学杂志》
a Children’s Hospital Boston,
b Massachusetts General Hospital,
c Harvard Medical School Center for Minimally Invasive Surgery, Boston, Massachusetts, USA
Key Words. Cord blood cells ? Differentiation ? Fetal stem cells ? In vitro differentiation ? Stem/progenitor cell ? Stromal cells ? TGF-?1 ? Tissue regeneration
Correspondence: Dario O. Fauza, M.D., Children’s Hospital Boston, Department of Surgery, Fegan 3, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. Telephone: 617-919-2966; Fax: 617-730-0910; e-mail: dario.fauza@childrens.harvard.edu
ABSTRACT
Congenital anomalies always entail variable degrees of loss and/or malformation of tissues or organs. Treatment of the most severe cases is often limited by the scarce availability of normal grafts, especially at birth. Autologous grafting is frequently not an option in newborns due to donor-site size limitations. In addition, the well-known severe donor shortage observed in nearly all areas of transplantation is even more critical during the neonatal period. Prosthetic materials, on the other hand, may lead to infection, recurrence of the defect, and growth limitations. Recently, a novel concept in perinatal surgery was introduced, involving minimally invasive harvest of fetal tissue, which is then engineered in vitro in parallel to the remainder of gestation, so that an infant with a prenatally diagnosed birth defect can benefit from having autologous, expanded tissue readily available for surgical implantation in the neonatal period .
Severe tracheal malformations and chest wall deformities are two examples in which engineered autologous cartilage readily available at birth, or even for prenatal repair, would be extremely beneficial, if not life-saving. We have previously shown in large animal models that tracheoplasty and chest wall reconstruction using cartilage engineered either from fetal chondrocytes or bone marrow mesenchymal cells may be viable alternatives for the treatment of these birth defects . One of the limitations of these previous studies is that neither cell source (fetal auricular biopsy or bone marrow aspiration) is easily accessible. This study was aimed at determining whether three-dimensional (3D) cartilage could be engineered from a more accessible cell source, namely umbilical cord blood (CB), and at comparing it with both engineered fetal cartilage and native tissue.
MATERIALS AND METHODS
Cell Morphology
Isolated, adherent CB cells displayed fibroblast-like morphology in culture (Fig. 1). Sparsely distributed colonies were visible in the original culture after 7–10 days in growth medium. By 14–21 days, these colonies extended toward each other to approximately 80% confluence.
Figure 1. Phase-contrast micrograph of fetal mesenchymal progenitor cells isolated from umbilical cord blood (original magnification x20).
Construct Analysis
Histology ? On H&E, CB constructs displayed no evidence of chondrogenic differentiation after 4 weeks in the bioreactor (Fig. 2). After 8 weeks, cell lacunae and basophilic staining were present in the extracellular matrix, in a morphological pattern compatible with ongoing chondrogenesis (Fig. 2). After 12 weeks, CB constructs exhibited evident chondrogenic differentiation by both standard and matrix-specific staining, displaying characteristics of hyaline cartilage, both grossly and microscopically (Figs. 2, 3). At that time point, each construct also contained a multicellular peripheral layer of flattened, elongated, fibroblast-like cells similar to normal perichondrium (Fig. 2).
Figure 2. Time-dependent changes in three-dimensional umbilical cord blood mesenchymal progenitor cell constructs under defined chondrogenic medium in a bioreactor. (A): At 4 weeks, without any evidence of chondrogenic differentiation. (B): At 8 weeks, with preliminary evidence of cartilage formation, including cell lacunae and basophilic matrix staining. (C): At 12 weeks, with clear cartilage-like morphology. (D): At 12 weeks, showing a peripheral multicellular layer of flattened, elongated, fibroblast-like cells, analogous to perichondrium. (E): Native fetal hyaline cartilage from the trachea. All hematoxylin and eosin, original magnification x20.
Figure 3. Matrix-specific stainings of three-dimensional cartilage engineered from umbilical cord blood mesenchymal progenitor cells. (A): Safranin O (for glycosaminoglycans). (B): Toluidine blue (for glycosaminoglycans). (C): Immunohistochemical staining for type I collagen, showing only peripheral positivity. (D): Immunohistochemical staining for type II collagen, showing positivity throughout the matrix. Original magnification x20.
Further qualitative analysis of the extracellular matrix showed that CB constructs stained positively for the GAG-specific stains safranin O and toluidine blue, also in a pattern comparable with hyaline cartilage (Fig. 3). Immunostaining for type II collagen showed its presence throughout the constructs (Fig. 3). On the other hand, immunostaining for type I collagen revealed positivity only on the periphery of the constructs, in the perichondrium-like layer (Fig. 3). Native fetal hyaline cartilage specimens were essentially negative for type I collagen. Type X collagen was not detected in any of the constructs, whereas minimal staining was present at the border of endochondral ossification in the native specimens.
Quantitative Matrix Analysis ? There was a significant time-dependent increase in the levels of GAG and type II collagen (C-II) but not of EL in CB constructs (Fig. 4). The following quantitative analyses refer to CB constructs at the 12-week time point. There were no significant differences in GAG and C-II levels between CB and fetal chondrocyte constructs, which, however, had higher EL levels. Compared with native fetal cartilage (both hyaline and elastic), C-II and EL levels were, respectively, similar and lower in the CB constructs. Native hyaline cartilage had higher GAG levels than CB constructs, which showed GAG contents comparable with those of native elastic cartilage. There were no differences in the results from each of the four donors within each time point.
Figure 4. Quantitative extracellular matrix comparisons of different forms of engineered and native fetal cartilages. (A): Umbilical CB mesenchymal progenitor cell constructs (CB constructs) at different time points. (B): CB constructs versus chondrocyte-derived constructs. (C): CB constructs versus native elastic and hyaline cartilages. *Significant difference between the groups (p < .01). Abbreviations: C-II, type II collagen; CB, cord blood; GAG, glycosaminoglycan.
DISCUSSION
This study was funded by a grant from the Harvard Center of Minimally Invasive Surgery and by the Children’s Hospital Boston Surgical Foundation.
REFERENCES
Fauza DO, Fishman SJ, Mehegan K et al. Videofetoscopically assisted fetal tissue engineering: bladder augmentation. J Pediatr Surg 1998;33:7–12.
Fauza DO. Fetal tissue engineering. In: Lanza R, Langer R, Vacanti JP, eds. Principles of Tissue Engineering. San Diego: Academic Press, 2000:353–368.
Fuchs JR, Terada S, Ochoa ER et al. Fetal tissue engineering: in utero tracheal augmentation in an ovine model. J Pediatr Surg 2002;37:1000–1006.
Fuchs JR, Hannouche D, Terada S et al. Fetal tracheal augmentation with cartilage engineered from bone marrow-derived mesenchymal progenitor cells. J Pediatr Surg 2003;38:984–987.
Fuchs JR, Terada S, Hannouche D et al. Fetal tissue engineering: chest wall reconstruction. J Pediatr Surg 2003;38:1188–1193.
Yoo JU, Barthel TS, Nishimura K et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998;80:1745–1757.
Niyibizi C, Sagarrigo Visconti C, Gibson G et al. Identification and immunolocalization of type X collagen at the ligament-bone interface. Biochem Biophys Res Commun 1996;222:584–589.
Kim YJ, Sah RL, Doong JY et al. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168–176.
Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173–177.
Madsen K, von der Mark K, van Menxel M et al. Analysis of collagen types synthesized by rabbit ear cartilage chondrocytes in vivo and in vitro. Biochem J 1984;221:189–196.
Brown AN, Kim BS, Alsberg E et al. Combining chondrocytes and smooth muscle cells to engineer hybrid soft tissue constructs. Tissue Eng 2000;6:297–305.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Liechty KW, MacKenzie TC, Shaaban AF et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000;6:1282–1286.
Almeida-Porada G, Flake AW, Glimp HA et al. Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Exp Hematol 1999;27:1569–1575.
Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313.
Noth U, Tuli R, Osyczka AM et al. In vitro engineered cartilage constructs produced by press-coating biodegradable polymer with human mesenchymal stem cells. Tissue Eng 2002;8:131–144.
In ’t Anker PS, Scherjon SA, Kleijburg-van der Keur C et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003;102:1548–1549.
Campagnoli C, Roberts IA, Kumar S et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001;98:2396–2402.
Lee OK, Kuo TK, Chen WM et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 2004;103:1669–1675.
Zvaifler NJ, Marinova-Mutafchieva L, Adams G et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res 2000;2:477–488.
Barry F, Boynton RE, Liu B et al. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res 2001;268:189–200.
Kojima K, Ignotz RA, Kushibiki T et al. Tissue-engineered trachea from sheep marrow stromal cells with transforming growth factor beta2 released from biodegradable microspheres in a nude rat recipient. J Thorac Cardiovasc Surg 2004;128:147–153.
Indrawattana N, Chen G, Tadokoro M et al. Growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem Biophys Res Commun 2004;320:914–919.
Albuquerque CA, Nijland MJ, Ross MG. Human and ovine amniotic fluid composition differences: implications for fluid dynamics. J Matern Fetal Med 1999;8:123–129.
Tongsong T, Wanapirak C, Kunavikatikul C et al. Fetal loss rate associated with cordocentesis at midgestation. Am J Obstet Gynecol 2001;184:719–723.
Abdel-Fattah SA, Bartha JL, Kyle PM et al. Safety of fetal blood sampling by cordocentesis in fetuses with single umbilical arteries. Prenat Diagn 2004;24:605–608.
Wexler SA, Donaldson C, Denning-Kendall P et al. Adult bone marrow is a rich source of human mesenchymal "stem" cells but umbilical cord and mobilized adult blood are not. Br J Haematol 2003;121:368–374.
Bianchi DW. Fetal cells in the mother: from genetic diagnosis to diseases associated with fetal cell microchimerism. Eur J Obstet Gynecol Reprod Biol 2000;92:103–108.
Bianchi DW, Simpson JL, Jackson LG et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn 2002;22:609–615.(Julie R. Fuchsa,b,c, Didi)
b Massachusetts General Hospital,
c Harvard Medical School Center for Minimally Invasive Surgery, Boston, Massachusetts, USA
Key Words. Cord blood cells ? Differentiation ? Fetal stem cells ? In vitro differentiation ? Stem/progenitor cell ? Stromal cells ? TGF-?1 ? Tissue regeneration
Correspondence: Dario O. Fauza, M.D., Children’s Hospital Boston, Department of Surgery, Fegan 3, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. Telephone: 617-919-2966; Fax: 617-730-0910; e-mail: dario.fauza@childrens.harvard.edu
ABSTRACT
Congenital anomalies always entail variable degrees of loss and/or malformation of tissues or organs. Treatment of the most severe cases is often limited by the scarce availability of normal grafts, especially at birth. Autologous grafting is frequently not an option in newborns due to donor-site size limitations. In addition, the well-known severe donor shortage observed in nearly all areas of transplantation is even more critical during the neonatal period. Prosthetic materials, on the other hand, may lead to infection, recurrence of the defect, and growth limitations. Recently, a novel concept in perinatal surgery was introduced, involving minimally invasive harvest of fetal tissue, which is then engineered in vitro in parallel to the remainder of gestation, so that an infant with a prenatally diagnosed birth defect can benefit from having autologous, expanded tissue readily available for surgical implantation in the neonatal period .
Severe tracheal malformations and chest wall deformities are two examples in which engineered autologous cartilage readily available at birth, or even for prenatal repair, would be extremely beneficial, if not life-saving. We have previously shown in large animal models that tracheoplasty and chest wall reconstruction using cartilage engineered either from fetal chondrocytes or bone marrow mesenchymal cells may be viable alternatives for the treatment of these birth defects . One of the limitations of these previous studies is that neither cell source (fetal auricular biopsy or bone marrow aspiration) is easily accessible. This study was aimed at determining whether three-dimensional (3D) cartilage could be engineered from a more accessible cell source, namely umbilical cord blood (CB), and at comparing it with both engineered fetal cartilage and native tissue.
MATERIALS AND METHODS
Cell Morphology
Isolated, adherent CB cells displayed fibroblast-like morphology in culture (Fig. 1). Sparsely distributed colonies were visible in the original culture after 7–10 days in growth medium. By 14–21 days, these colonies extended toward each other to approximately 80% confluence.
Figure 1. Phase-contrast micrograph of fetal mesenchymal progenitor cells isolated from umbilical cord blood (original magnification x20).
Construct Analysis
Histology ? On H&E, CB constructs displayed no evidence of chondrogenic differentiation after 4 weeks in the bioreactor (Fig. 2). After 8 weeks, cell lacunae and basophilic staining were present in the extracellular matrix, in a morphological pattern compatible with ongoing chondrogenesis (Fig. 2). After 12 weeks, CB constructs exhibited evident chondrogenic differentiation by both standard and matrix-specific staining, displaying characteristics of hyaline cartilage, both grossly and microscopically (Figs. 2, 3). At that time point, each construct also contained a multicellular peripheral layer of flattened, elongated, fibroblast-like cells similar to normal perichondrium (Fig. 2).
Figure 2. Time-dependent changes in three-dimensional umbilical cord blood mesenchymal progenitor cell constructs under defined chondrogenic medium in a bioreactor. (A): At 4 weeks, without any evidence of chondrogenic differentiation. (B): At 8 weeks, with preliminary evidence of cartilage formation, including cell lacunae and basophilic matrix staining. (C): At 12 weeks, with clear cartilage-like morphology. (D): At 12 weeks, showing a peripheral multicellular layer of flattened, elongated, fibroblast-like cells, analogous to perichondrium. (E): Native fetal hyaline cartilage from the trachea. All hematoxylin and eosin, original magnification x20.
Figure 3. Matrix-specific stainings of three-dimensional cartilage engineered from umbilical cord blood mesenchymal progenitor cells. (A): Safranin O (for glycosaminoglycans). (B): Toluidine blue (for glycosaminoglycans). (C): Immunohistochemical staining for type I collagen, showing only peripheral positivity. (D): Immunohistochemical staining for type II collagen, showing positivity throughout the matrix. Original magnification x20.
Further qualitative analysis of the extracellular matrix showed that CB constructs stained positively for the GAG-specific stains safranin O and toluidine blue, also in a pattern comparable with hyaline cartilage (Fig. 3). Immunostaining for type II collagen showed its presence throughout the constructs (Fig. 3). On the other hand, immunostaining for type I collagen revealed positivity only on the periphery of the constructs, in the perichondrium-like layer (Fig. 3). Native fetal hyaline cartilage specimens were essentially negative for type I collagen. Type X collagen was not detected in any of the constructs, whereas minimal staining was present at the border of endochondral ossification in the native specimens.
Quantitative Matrix Analysis ? There was a significant time-dependent increase in the levels of GAG and type II collagen (C-II) but not of EL in CB constructs (Fig. 4). The following quantitative analyses refer to CB constructs at the 12-week time point. There were no significant differences in GAG and C-II levels between CB and fetal chondrocyte constructs, which, however, had higher EL levels. Compared with native fetal cartilage (both hyaline and elastic), C-II and EL levels were, respectively, similar and lower in the CB constructs. Native hyaline cartilage had higher GAG levels than CB constructs, which showed GAG contents comparable with those of native elastic cartilage. There were no differences in the results from each of the four donors within each time point.
Figure 4. Quantitative extracellular matrix comparisons of different forms of engineered and native fetal cartilages. (A): Umbilical CB mesenchymal progenitor cell constructs (CB constructs) at different time points. (B): CB constructs versus chondrocyte-derived constructs. (C): CB constructs versus native elastic and hyaline cartilages. *Significant difference between the groups (p < .01). Abbreviations: C-II, type II collagen; CB, cord blood; GAG, glycosaminoglycan.
DISCUSSION
This study was funded by a grant from the Harvard Center of Minimally Invasive Surgery and by the Children’s Hospital Boston Surgical Foundation.
REFERENCES
Fauza DO, Fishman SJ, Mehegan K et al. Videofetoscopically assisted fetal tissue engineering: bladder augmentation. J Pediatr Surg 1998;33:7–12.
Fauza DO. Fetal tissue engineering. In: Lanza R, Langer R, Vacanti JP, eds. Principles of Tissue Engineering. San Diego: Academic Press, 2000:353–368.
Fuchs JR, Terada S, Ochoa ER et al. Fetal tissue engineering: in utero tracheal augmentation in an ovine model. J Pediatr Surg 2002;37:1000–1006.
Fuchs JR, Hannouche D, Terada S et al. Fetal tracheal augmentation with cartilage engineered from bone marrow-derived mesenchymal progenitor cells. J Pediatr Surg 2003;38:984–987.
Fuchs JR, Terada S, Hannouche D et al. Fetal tissue engineering: chest wall reconstruction. J Pediatr Surg 2003;38:1188–1193.
Yoo JU, Barthel TS, Nishimura K et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998;80:1745–1757.
Niyibizi C, Sagarrigo Visconti C, Gibson G et al. Identification and immunolocalization of type X collagen at the ligament-bone interface. Biochem Biophys Res Commun 1996;222:584–589.
Kim YJ, Sah RL, Doong JY et al. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 1988;174:168–176.
Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173–177.
Madsen K, von der Mark K, van Menxel M et al. Analysis of collagen types synthesized by rabbit ear cartilage chondrocytes in vivo and in vitro. Biochem J 1984;221:189–196.
Brown AN, Kim BS, Alsberg E et al. Combining chondrocytes and smooth muscle cells to engineer hybrid soft tissue constructs. Tissue Eng 2000;6:297–305.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Liechty KW, MacKenzie TC, Shaaban AF et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000;6:1282–1286.
Almeida-Porada G, Flake AW, Glimp HA et al. Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Exp Hematol 1999;27:1569–1575.
Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313.
Noth U, Tuli R, Osyczka AM et al. In vitro engineered cartilage constructs produced by press-coating biodegradable polymer with human mesenchymal stem cells. Tissue Eng 2002;8:131–144.
In ’t Anker PS, Scherjon SA, Kleijburg-van der Keur C et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003;102:1548–1549.
Campagnoli C, Roberts IA, Kumar S et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001;98:2396–2402.
Lee OK, Kuo TK, Chen WM et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 2004;103:1669–1675.
Zvaifler NJ, Marinova-Mutafchieva L, Adams G et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res 2000;2:477–488.
Barry F, Boynton RE, Liu B et al. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res 2001;268:189–200.
Kojima K, Ignotz RA, Kushibiki T et al. Tissue-engineered trachea from sheep marrow stromal cells with transforming growth factor beta2 released from biodegradable microspheres in a nude rat recipient. J Thorac Cardiovasc Surg 2004;128:147–153.
Indrawattana N, Chen G, Tadokoro M et al. Growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem Biophys Res Commun 2004;320:914–919.
Albuquerque CA, Nijland MJ, Ross MG. Human and ovine amniotic fluid composition differences: implications for fluid dynamics. J Matern Fetal Med 1999;8:123–129.
Tongsong T, Wanapirak C, Kunavikatikul C et al. Fetal loss rate associated with cordocentesis at midgestation. Am J Obstet Gynecol 2001;184:719–723.
Abdel-Fattah SA, Bartha JL, Kyle PM et al. Safety of fetal blood sampling by cordocentesis in fetuses with single umbilical arteries. Prenat Diagn 2004;24:605–608.
Wexler SA, Donaldson C, Denning-Kendall P et al. Adult bone marrow is a rich source of human mesenchymal "stem" cells but umbilical cord and mobilized adult blood are not. Br J Haematol 2003;121:368–374.
Bianchi DW. Fetal cells in the mother: from genetic diagnosis to diseases associated with fetal cell microchimerism. Eur J Obstet Gynecol Reprod Biol 2000;92:103–108.
Bianchi DW, Simpson JL, Jackson LG et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn 2002;22:609–615.(Julie R. Fuchsa,b,c, Didi)