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Case Orthop J. Author manuscript; available in PMC 2016 January 21. Published in final edited form as: Case Orthop J. 2012 ; 9(1): 60–65.
MESENCHYMAL STROMAL CELLS AND THEIR ORTHOPAEDIC APPLICATIONS Lindsay A. Bashur, PhD and Guang Zhou, PhD Department of Orthopaedics, Case Western Reserve University
Introduction Author Manuscript Author Manuscript
Stem cells have emerged as a promising alternative to traditional surgical procedures for the regeneration and repair of skeletal tissues resulting from common and rare skeletal diseases and trauma. Skeletal tissues undergo continuous remodeling and regeneration during skeletal development and normal adult life, and in response to injury or disease. Therefore, in the field of orthopaedic regenerative medicine, which seeks to repair, replace, or regenerate tissues and organs damaged by injury or disease, many clues can be drawn from skeletal development. Similar to skeletal development, skeletal healing requires mesenchymal stromal (or stem) cells (MSCs). In this review, we aim to provide a glimpse of the recent progress made in this rapidly developing field and the great promise MSCs hold for future orthopaedic applications. We summarize the application of MSCs in regenerative medicine for the treatment of skeletal diseases, including osteogenesis imperfecta, fibrous dysplasia, osteoarthritis, and fracture healing. We also discuss the recent discovery of induced pluripotent stem (iPS) cells and their potential orthopaedic applications as a renewable source of skeletal cells.
Embryonic Skeletal Development
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During embryonic development, the mesodermal layer contains multipotent mesenchymal progenitor cells that give rise to bone, cartilage, fat, muscle, and other tissues1, 2. Skeletal formation then occurs through either intramembranous ossification or endochondral ossification. During intramembranous ossification, which mostly occurs in flat bones, mesenchymal cells directly differentiate into osteoblasts and produce bone matrix. For endochondral ossification, which occurs in the majority of the skeleton, the undifferentiated mesenchymal cells migrate from the lateral plate mesoderm and paraxial mesoderm and form mesenchymal condensations at the locations of future skeletal elements. The mesenchymal cell condensations form a cartilage template, which grows as the chondrocytes proliferate and becomes surrounded by capillaries. The mesenchymal cells around the cartilage template differentiate into osteoblasts and produce a collar of bone around the middle of the cartilage, and the chondrocytes in the center of the cartilage undergo hypertrophy. Eventually, the capillaries invade the bone collar, and the hypertrophic cartilage is replaced by bone. Transcription factors play an essential role in skeletal development by regulating all aspects of the differentiation of chondrocytes and osteoblasts. Chondrocytes and osteoblasts, as well as many other cell types, originate from the same mesenchymal progenitor cells3. A mouse
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genetic cell-lineage tracing study demonstrated that the progenitor cells expressing the transcription factor Sox9 are committed to the skeletal lineage and can differentiate into both chondrocytes and osteoblasts4. Thus, those early Sox9-expressing cells in future skeletal locations are called skeletal precursor cells or osteochondro progenitor cells4, 5. Subsequently, chondrocyte lineage is determined by Sox9 together with Sox5 and Sox62. Chondrocyte hypertrophy is activated by Runx2 and repressed by Sox9, whereas osteoblast differentiation is promoted by Runx2 and its downstream effector Osterix.
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Sox9 and Runx2 are master transcription factors of skeletogenesis because they regulate many other genes involved in the development of the skeleton, and are required for proper skeletal formation. More tellingly, mutations in SOX9 and RUNX2 both cause human skeletal diseases. Heterozygous mutations in the human SOX9 gene cause campomelic dysplasia, a very severe form of generalized chondrodysplasia2. Campomelic dysplasia is characterized by bowing of the long bones and malformations in all cartilage, and can result in infant death at or soon after birth due to respiratory distress. Heterozygous mutations in the RUNX2 gene are associated with human cleidocranial dysplasia2. Individuals with cleidocranial dysplasia have underdeveloped or absent collarbones, causing their shoulders to be close together in front of the body. The other features of cleidocranial dysplasia include the delayed closing of fontanels, short stature, and dental abnormalities6.
Stem Cells
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Stem cells are unspecialized cells that are defined by their self-renewal and differentiation capacity7–10. Stem cells can be broadly categorized into two major classes: embryonic and adult stem cells. Embryonic stem cells (ESCs) are isolated from the inner cell mass of the blastocyst, can self-renew indefinitely, and are pluripotent cells with the potential of differentiating into cell types from all three germ layers: endoderm, ectoderm, and mesoderm8. However, many factors have limited their use in clinical applications, including ethical concerns, immunological incompatibilities, potential for malignant tumor growth, heterogeneous differentiation, and an insufficient understanding of and control over ESC differentiation.
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Adult mesenchymal stem cells (MSCs) are multipotent undifferentiated cells that are capable of self-renewal and responsible for adult tissue regeneration7–10. Bone marrow stromal cells were initially described by Alexander Friedenstein in 196811. Friedenstein and colleagues isolated bone marrow-derived fibroblast-like cells based on their ability to adhere to plastic substrates and form colony units12. They also showed that these cells had osteogenic and adipogenic potential13. Marshall Urist showed that demineralized bone or its extracts induced the differentiation of mesenchymal cells into cartilage and bone when implanted into subcutaneous or intramuscular sites in human and animal models14. Similar work was performed independently by others15 and confirmed the existence of multipotent progenitor cells in adult tissues. In the 1970s, Caplan and colleagues reported the dissociation of embryonic stage 24 chick limb bud mesenchymal progenitor cells and their subsequent differentiation into bone, cartilage, muscle, and other mesenchymal tissues16, 17. During the early 1980s, they showed that exposing embryonic chick limb bud cultures to extracts of demineralized bone can induce BMP-mediated chondrogenic differentiation18–20.
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MSCs are identified in vitro largely based on three specific characteristics8–10. The first is the ability to adhere to plastic culture dishes and to form fibroblast-like colonies. The second characteristic is the ability to differentiate into various specialized cell lineages, such as osteoblasts, adipocytes, and chondrocytes, and some other cell types under certain culture conditions. The third is the expression of defined cell surface markers. The molecular characterization of MSCs is challenging because they do not uniquely express any specific molecule. In addition, cell surface marker expression can vary with different isolation techniques, tissue origins and species, and culture conditions. Thus, the International Society of Cellular Therapy (ISCT) proposed that MSCs must be positive for cell surface markers CD73, CD90, and CD105 and lack expression of typical hematopoietic lineage markers, including CD34, CD45, CD14, CD11b, CD19, CD79a, and HLA-DR [21]. MSCs predominantly reside in the bone marrow compartment, but they have also been described in other tissues such as fat and muscle. They represent a highly heterogeneous population of immature cells of still poorly defined physical, phenotypic, and functional properties, especially in vivo. Nonetheless, at least in mouse models, a subpopulation of bone marrow MSCs are dynamic and critical participants in bone maintenance and regeneration22. MSCs have begun to be utilized in clinical trials for tissue repair and regeneration worldwide, albeit sometimes under very poor or no regulations.
MSCs in Skeletal Disease Models and Treatment Osteogenesis Imperfecta
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Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders in which a genetic defect in type I collagen of osteoblasts causes osteopenia, multiple fractures, severe bony deformities, and shortened stature10, 23. The clinical phenotype of OI ranges from normal life expectancy with mild bone fragility to osteopenia to death. The treatment options are limited for OI as a genetic disease. Many studies have shown that bisphosphate therapy in OI increased bone density and strength, and reduced incidence of fractures10, 23. However, bisphosphates can have potential adverse skeletal effects in children, which is a concern for long-term treatment.
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Stem cells are a promising alternative treatment for OI. A study by Pereira et al. showed that when MSCs from wild-type mice were infused into transgenic mice that exhibited an OI phenotype, the wild-type MSCs contributed to the continual renewal of cells in a number of nonhematopoietic tissues in the mutant mice24. Horwitz et al. performed clinical trials to transplant allogeneic bone marrow into children with severe OI25. The results showed an increase in total body mineral content, increase in bone growth, and reduction in fracture rates after three months. In their next study, Horwitz et al. transplanted MSCs into children that had previously undergone bone marrow transplantation for OI26. The results demonstrated the engraftment of donor MSCs in the recipient bone and an increase in bone velocity. Panaroni et al. evaluated the intrauterine transplantation of adult bone marrow into a knockin murine model for OI27. They showed that the adult bone marrow donor cells can engraft into hematopoietic and nonhematopoietic tissues and further differentiate to trabecular and cortical bone cells, synthesizing up to 20% of all type I collagen in the host bone, and also rescuing the perinatal lethality of mice with dominant OI.
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Gene therapy in combination with stem cells also offers a promising approach to treating OI. Chamberlain et al. used adeno-associated virus (AAV) vectors to target and inactivate mutant COL1A1 genes28 and mutant COL1A2 genes29 in the MSCs from OI individuals. Their results, largely from ex vivo cultures, showed that the targeted-MSCs produced normal type I procollagen and formed bone, thus demonstrating their therapeutic potential. The future goal is to return the corrected stem cells to the OI patients from whom the MSCs were harvested to improve their bone quality. Fibrous Dysplasia
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Fibrous dysplasia (FD) is a skeletal disease caused by activating missense mutations in the GNAS gene, which encodes the α subunit of the stimulatory G protein (Gs)30, 31. FD is a crippling, occasionally lethal disease, where normal bone and bone marrow is replaced by abnormal, fibrous bone tissue. There is currently no cure for FD. Surgery is used to treat fractures and deformities, but is often unsuccessful. Piersanti et al. have explored two roles of MSCs in FD32. First, they were able to stably transfer the FD genotype and phenotype to normal human skeletal progenitor cells to model the mutation effects32. Second, they used MSCs as a tool for the treatment of FD through cell replacement or gene correction in MSCs33. Here, they silenced the mutated allele only in skeletal progenitors. For both goals, they used lentiviral transduction technology to effectively and efficiently target a dominant gain-of-function mutation in GNAS32, 33. Therefore, Piersanti et al. have demonstrated the feasibility of a gene therapy approach for treating FD, through the reversal of the disease phenotype at the cellular level, based on the selective knockdown of the dominant gain-of-function Gsα mutations in skeletal stem cells.
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Osteoarthritis Osteoarthritis (OA), the most common type of arthritis, is a chronic degenerative joint disease characterized by progressive cartilage deterioration34, 35. The symptoms of OA include joint pain, impairment of movement, and local inflammation. Current treatments relieve pain temporarily with nonsteroidal anti-inflammatory drugs, steroids, hyaluronic acid, or surgery, but eventually fail over time. OA is associated with progressive inflammation; thus, MSCs are a promising candidate for cell therapy because they have antiinflammatory and immunosuppressive properties36. In addition, MSCs have shown the ability to migrate and engraft onto multiple musculoskeletal tissues, especially at sites of injury, and undergo site-specific differentiation, then influence the microenvironment to aid in the regeneration of cartilage.
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Recent efforts have focused on the use of direct intra-articular injection of MSCs as a therapy for OA. Lee et al. intra-articularly injected MSCs suspended in HA for the treatment of cartilage defects in the medial femoral condyle of adult minipigs37. At 6 and 12 weeks postoperatively, the MSC-treated groups showed improved cartilage healing compared with the controls. Horie et al. investigated the efficiency of meniscal regeneration in rat massive meniscal defects using intra-articular MSCs38. Their results showed that the MSCs adhered to the lesion, differentiated into meniscal cells directly, and promoted meniscal regeneration. MSCs have also been genetically modified for OA cell therapy to release therapeutic
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proteins that can interact with the injured cartilage. In one study by Matsumoto et al., when the MSCs expressing bone morphogenetic protein (BMP)-4, in combination with MSCs expressing the vascular endothelial growth factor antagonist soluble Flt-1, were intraarticularly injected into an immunodeficient rat model for OA, the quality and the persistence of regenerated articular cartilage were significantly improved39. Recently, controlled growth factor release from microspheres incorporated in MSC sheets has been shown to enhance cartilage tissue formation40. Furthermore, the over-expression of SOX9, the master regulator of chondrogenesis, can also increase MSC-mediated cartilage repair in an animal model41.
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Wakitani et al. reported the clinical use of transplanted bone marrow-derived MSCs seeded onto type I collagen-containing hydrogels to repair cartilage defects in the medial femoral condyle of human knees with OA42. Forty-two weeks after transplantation, the arthroscopic and histological grading scores were better in the MSC-transplanted group than in the cellfree control group. In another clinical study, by Davatchi et al., four patients ages 54–65 years old with moderate to severe knee OA received intra-articular injections of autologous bone marrow-derived MSCs43. After 6 months, three patients showed improvement in walking time and pain scores. All patients showed improvement in the number of stairs they could climb and pain relief. A recent study reported the discovery of a small molecule, kartogenin, which can promote the differentiation of MSCs into chondrocytes44. Johnson et al. showed that kartogenin can upregulate chondrocyte-specific gene expression in vitro, and significantly increase cartilage thickness in an OA mouse model. Bone Fractures
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Normal bone fracture healing involves a complex network of signaling events in response to injury. Undifferentiated MSCs are recruited to the injury site where, with the aid of regulatory cytokines, e.g. BMPs, they proliferate, differentiate into chondrocytes and osteoblasts, and form bone, thus repairing the defect. Failure to heal properly results in either delayed union, or nonunion if fractures fail to heal after 8–9 months10, 23. While fractures are a common clinical occurrence, the treatment of nonunions remains a challenge. MSCs are promising candidates for bone regeneration due to their ability to migrate to injured tissues and undergo osteogenic and chondrogenic differentiation.
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Numerous studies have successfully used MSCs for fracture repair and bone regeneration in animal models. Granero-Molto et al. demonstrated that MSCs migrated towards the fracture site in a mouse model, and contributed to fracture healing by expressing BMP-2 and modulating the injury-related inflammatory response45. Bone repair often requires a scaffold for structural and mechanical support. Kadiyala et al. expanded bone marrow-derived MSCs in culture, seeded them onto ceramic cylinders, and implanted the constructs into 8 mm segmental defects in rat femurs46. The results showed new bone formation after 8 weeks. Several other studies have also been performed using different scaffold materials in combination with MSCs to achieve bone regeneration7, 47. Gene therapy has also been explored for the use in fracture healing and bone regeneration. Musgrave et al. showed that MSCs can be used to deliver BMP-2 ex vivo48. The delivery of BMP-2 stimulates the MSCs at the injury site to differentiate and form bone. Burastero et al. Case Orthop J. Author manuscript; available in PMC 2016 January 21.
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found that the combination of MSCs with BMP-7 on a scaffold resulted in a better osteoinductive graft than either MSCs or BMP-7 alone49.
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Human studies have been reported in which autologous bone marrow-derived MSCs were expanded in vitro, then loaded onto 100% hydroxyapatite macroporous ceramic scaffolds50. The constructs were then implanted into four patients with diaphyseal segmental defects that ranged from 3 to 28.3 cm3 in one tibia, one humerus, and two ulna fractures, respectively. Good integration of the implanted constructs with the preexisting bone was maintained, and no major adverse reactions were observed. All patients experienced recovery of limb function, and good integration was maintained at the last follow-up (6–7 years after surgery). Hernigou et al. treated 60 cases of atrophic non-union of the tibia with the percutaneous injection of concentrated bone marrow51. They reported a high union rate of 43/60, and a correlation between the number and concentration of colony-forming units in the graft and the volume of mineralized callus at 4 months. Quarto et al. treated critical-sized segmental defects in the long bones of three patients with size-matching hydroxyapatite scaffolds loaded with expanded autologous MSC cultures52. They reported that graft integration was detected as early as 2 months after surgery and all three patients regained function of their limbs with no adverse effects.
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iPS Cells: An Alternative Stem Cell Source Induced pluripotent stem (iPS) cells have recently been discovered as an alternative to ESCs. Takahashi et al. generated iPS cells by transfecting mouse somatic cells with pluripotent transcription factors Oct3/4, c-Myc, Sox2, and K1f453. They reported that these iPS cells were highly similar to ESCs in their morphology, proliferation, gene expression, and in vitro differentiation53. This landmark study has been cited more than 3700 times, reflecting the growing interest and wide application of its findings. Different groups subsequently replicated their findings in human cells. To date, iPS cells have been derived at increased efficiency from easily accessible human cell types such as dermal fibroblasts, blood cells, and keratinocytes54. To eliminate the potential risk of virally induced tumor formation associated with the early iPS cell derivation procedure, intense efforts are underway to develop virus-free and/or vector-free iPS cell protocols. More importantly, various patient-specific iPS cells have been generated to serve as novel models to study disease pathogenesis and to screen for new drugs55. However, in order for iPS cells to be used in clinical settings, many remaining obstacles must be overcome, such as low reprogramming efficiency and tumorigenicity with iPS cells.
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Efforts have also been underway to convert somatic cells directly into differentiated cell types, including chondrocytes. Indeed, the combination of c-Myc, Klf4, and Sox9 can reprogram adult dermal fibroblasts directly into chondrocytes and form hyaline cartilage in mice56. Although SOX9 is required for converting iPS cells and other progenitor cells to chondrocytes, it should be borne in mind that SOX9 expression has to be tightly controlled to avoid unintended effects. Our group has recently showed that ectopic Sox9 expression in differentiated osteoblasts can lead to severe osteopenia, altered bone mechanical properties, and impaired MSC functions in mice57. Furthermore, in addition to cartilage development, SOX9 is also critical for the development of other organs such as the central nervous system, pancreas, prostate, intestine, skin, pituitary, heart, kidney, and liver. Overexpression of SOX9 has been associated with the tumorigenesis of colorectal cancer, breast
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cancer, and prostate cancer. Thus, a more comprehensive understanding of the regulation of SOX9 itself is critical to generate abundant chondrocytes with minimal side effects for orthopaedic applications.
Conclusions
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MSCs and iPS cells can both develop into multiple tissues including bone and cartilage. They hold great promise for orthopaedic applications such as generating specific disease models and developing novel therapies. There have been great advances in MSC technology to treat skeletal diseases since the discovery of MSCs several decades ago. Our knowledge about the role of progenitor cells in skeletal development has enabled us to better understand the role of MSCs in adult skeletal disease and injury. However, MSC transplantation has its risks that cannot be overlooked when developing MSC therapy. For example, ex vivo amplifications of MSCs via extensive passages may lead to malignant transformation with cytogenetic aberrations and sarcoma formation. Thus more research, such as understanding the signaling pathways involved in musculoskeletal tissue regeneration, is critical for developing MSC therapy. The recent development of iPS cell technology is a versatile alternative to MSCs. The first human clinical trial using iPS cells to repair diseased retinas is scheduled to start in 2013 at the RIKEN Center for Developmental Biology in Kobe, Japan. However, for iPS cells, additional research and animal trials are also necessary to establish their safety and efficacy before clinical application. In summary, a better understanding of the molecular basis of pluripotency, cellular reprogramming, and lineage-specific differentiation of MSCs and iPS cells is essential to realize their clinical potential in regenerative medicine in the future.
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Acknowledgments We thank Valerie Schmedlen for editorial assistance.
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Case Orthop J. Author manuscript; available in PMC 2016 January 21. Published in final edited form as: Case Orthop J. 2012 ; 9(1): 60–65.
MESENCHYMAL STROMAL CELLS AND THEIR ORTHOPAEDIC APPLICATIONS Lindsay A. Bashur, PhD and Guang Zhou, PhD Department of Orthopaedics, Case Western Reserve University
Introduction Author Manuscript Author Manuscript
Stem cells have emerged as a promising alternative to traditional surgical procedures for the regeneration and repair of skeletal tissues resulting from common and rare skeletal diseases and trauma. Skeletal tissues undergo continuous remodeling and regeneration during skeletal development and normal adult life, and in response to injury or disease. Therefore, in the field of orthopaedic regenerative medicine, which seeks to repair, replace, or regenerate tissues and organs damaged by injury or disease, many clues can be drawn from skeletal development. Similar to skeletal development, skeletal healing requires mesenchymal stromal (or stem) cells (MSCs). In this review, we aim to provide a glimpse of the recent progress made in this rapidly developing field and the great promise MSCs hold for future orthopaedic applications. We summarize the application of MSCs in regenerative medicine for the treatment of skeletal diseases, including osteogenesis imperfecta, fibrous dysplasia, osteoarthritis, and fracture healing. We also discuss the recent discovery of induced pluripotent stem (iPS) cells and their potential orthopaedic applications as a renewable source of skeletal cells.
Embryonic Skeletal Development
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During embryonic development, the mesodermal layer contains multipotent mesenchymal progenitor cells that give rise to bone, cartilage, fat, muscle, and other tissues1, 2. Skeletal formation then occurs through either intramembranous ossification or endochondral ossification. During intramembranous ossification, which mostly occurs in flat bones, mesenchymal cells directly differentiate into osteoblasts and produce bone matrix. For endochondral ossification, which occurs in the majority of the skeleton, the undifferentiated mesenchymal cells migrate from the lateral plate mesoderm and paraxial mesoderm and form mesenchymal condensations at the locations of future skeletal elements. The mesenchymal cell condensations form a cartilage template, which grows as the chondrocytes proliferate and becomes surrounded by capillaries. The mesenchymal cells around the cartilage template differentiate into osteoblasts and produce a collar of bone around the middle of the cartilage, and the chondrocytes in the center of the cartilage undergo hypertrophy. Eventually, the capillaries invade the bone collar, and the hypertrophic cartilage is replaced by bone. Transcription factors play an essential role in skeletal development by regulating all aspects of the differentiation of chondrocytes and osteoblasts. Chondrocytes and osteoblasts, as well as many other cell types, originate from the same mesenchymal progenitor cells3. A mouse
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genetic cell-lineage tracing study demonstrated that the progenitor cells expressing the transcription factor Sox9 are committed to the skeletal lineage and can differentiate into both chondrocytes and osteoblasts4. Thus, those early Sox9-expressing cells in future skeletal locations are called skeletal precursor cells or osteochondro progenitor cells4, 5. Subsequently, chondrocyte lineage is determined by Sox9 together with Sox5 and Sox62. Chondrocyte hypertrophy is activated by Runx2 and repressed by Sox9, whereas osteoblast differentiation is promoted by Runx2 and its downstream effector Osterix.
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Sox9 and Runx2 are master transcription factors of skeletogenesis because they regulate many other genes involved in the development of the skeleton, and are required for proper skeletal formation. More tellingly, mutations in SOX9 and RUNX2 both cause human skeletal diseases. Heterozygous mutations in the human SOX9 gene cause campomelic dysplasia, a very severe form of generalized chondrodysplasia2. Campomelic dysplasia is characterized by bowing of the long bones and malformations in all cartilage, and can result in infant death at or soon after birth due to respiratory distress. Heterozygous mutations in the RUNX2 gene are associated with human cleidocranial dysplasia2. Individuals with cleidocranial dysplasia have underdeveloped or absent collarbones, causing their shoulders to be close together in front of the body. The other features of cleidocranial dysplasia include the delayed closing of fontanels, short stature, and dental abnormalities6.
Stem Cells
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Stem cells are unspecialized cells that are defined by their self-renewal and differentiation capacity7–10. Stem cells can be broadly categorized into two major classes: embryonic and adult stem cells. Embryonic stem cells (ESCs) are isolated from the inner cell mass of the blastocyst, can self-renew indefinitely, and are pluripotent cells with the potential of differentiating into cell types from all three germ layers: endoderm, ectoderm, and mesoderm8. However, many factors have limited their use in clinical applications, including ethical concerns, immunological incompatibilities, potential for malignant tumor growth, heterogeneous differentiation, and an insufficient understanding of and control over ESC differentiation.
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Adult mesenchymal stem cells (MSCs) are multipotent undifferentiated cells that are capable of self-renewal and responsible for adult tissue regeneration7–10. Bone marrow stromal cells were initially described by Alexander Friedenstein in 196811. Friedenstein and colleagues isolated bone marrow-derived fibroblast-like cells based on their ability to adhere to plastic substrates and form colony units12. They also showed that these cells had osteogenic and adipogenic potential13. Marshall Urist showed that demineralized bone or its extracts induced the differentiation of mesenchymal cells into cartilage and bone when implanted into subcutaneous or intramuscular sites in human and animal models14. Similar work was performed independently by others15 and confirmed the existence of multipotent progenitor cells in adult tissues. In the 1970s, Caplan and colleagues reported the dissociation of embryonic stage 24 chick limb bud mesenchymal progenitor cells and their subsequent differentiation into bone, cartilage, muscle, and other mesenchymal tissues16, 17. During the early 1980s, they showed that exposing embryonic chick limb bud cultures to extracts of demineralized bone can induce BMP-mediated chondrogenic differentiation18–20.
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MSCs are identified in vitro largely based on three specific characteristics8–10. The first is the ability to adhere to plastic culture dishes and to form fibroblast-like colonies. The second characteristic is the ability to differentiate into various specialized cell lineages, such as osteoblasts, adipocytes, and chondrocytes, and some other cell types under certain culture conditions. The third is the expression of defined cell surface markers. The molecular characterization of MSCs is challenging because they do not uniquely express any specific molecule. In addition, cell surface marker expression can vary with different isolation techniques, tissue origins and species, and culture conditions. Thus, the International Society of Cellular Therapy (ISCT) proposed that MSCs must be positive for cell surface markers CD73, CD90, and CD105 and lack expression of typical hematopoietic lineage markers, including CD34, CD45, CD14, CD11b, CD19, CD79a, and HLA-DR [21]. MSCs predominantly reside in the bone marrow compartment, but they have also been described in other tissues such as fat and muscle. They represent a highly heterogeneous population of immature cells of still poorly defined physical, phenotypic, and functional properties, especially in vivo. Nonetheless, at least in mouse models, a subpopulation of bone marrow MSCs are dynamic and critical participants in bone maintenance and regeneration22. MSCs have begun to be utilized in clinical trials for tissue repair and regeneration worldwide, albeit sometimes under very poor or no regulations.
MSCs in Skeletal Disease Models and Treatment Osteogenesis Imperfecta
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Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders in which a genetic defect in type I collagen of osteoblasts causes osteopenia, multiple fractures, severe bony deformities, and shortened stature10, 23. The clinical phenotype of OI ranges from normal life expectancy with mild bone fragility to osteopenia to death. The treatment options are limited for OI as a genetic disease. Many studies have shown that bisphosphate therapy in OI increased bone density and strength, and reduced incidence of fractures10, 23. However, bisphosphates can have potential adverse skeletal effects in children, which is a concern for long-term treatment.
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Stem cells are a promising alternative treatment for OI. A study by Pereira et al. showed that when MSCs from wild-type mice were infused into transgenic mice that exhibited an OI phenotype, the wild-type MSCs contributed to the continual renewal of cells in a number of nonhematopoietic tissues in the mutant mice24. Horwitz et al. performed clinical trials to transplant allogeneic bone marrow into children with severe OI25. The results showed an increase in total body mineral content, increase in bone growth, and reduction in fracture rates after three months. In their next study, Horwitz et al. transplanted MSCs into children that had previously undergone bone marrow transplantation for OI26. The results demonstrated the engraftment of donor MSCs in the recipient bone and an increase in bone velocity. Panaroni et al. evaluated the intrauterine transplantation of adult bone marrow into a knockin murine model for OI27. They showed that the adult bone marrow donor cells can engraft into hematopoietic and nonhematopoietic tissues and further differentiate to trabecular and cortical bone cells, synthesizing up to 20% of all type I collagen in the host bone, and also rescuing the perinatal lethality of mice with dominant OI.
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Gene therapy in combination with stem cells also offers a promising approach to treating OI. Chamberlain et al. used adeno-associated virus (AAV) vectors to target and inactivate mutant COL1A1 genes28 and mutant COL1A2 genes29 in the MSCs from OI individuals. Their results, largely from ex vivo cultures, showed that the targeted-MSCs produced normal type I procollagen and formed bone, thus demonstrating their therapeutic potential. The future goal is to return the corrected stem cells to the OI patients from whom the MSCs were harvested to improve their bone quality. Fibrous Dysplasia
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Fibrous dysplasia (FD) is a skeletal disease caused by activating missense mutations in the GNAS gene, which encodes the α subunit of the stimulatory G protein (Gs)30, 31. FD is a crippling, occasionally lethal disease, where normal bone and bone marrow is replaced by abnormal, fibrous bone tissue. There is currently no cure for FD. Surgery is used to treat fractures and deformities, but is often unsuccessful. Piersanti et al. have explored two roles of MSCs in FD32. First, they were able to stably transfer the FD genotype and phenotype to normal human skeletal progenitor cells to model the mutation effects32. Second, they used MSCs as a tool for the treatment of FD through cell replacement or gene correction in MSCs33. Here, they silenced the mutated allele only in skeletal progenitors. For both goals, they used lentiviral transduction technology to effectively and efficiently target a dominant gain-of-function mutation in GNAS32, 33. Therefore, Piersanti et al. have demonstrated the feasibility of a gene therapy approach for treating FD, through the reversal of the disease phenotype at the cellular level, based on the selective knockdown of the dominant gain-of-function Gsα mutations in skeletal stem cells.
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Osteoarthritis Osteoarthritis (OA), the most common type of arthritis, is a chronic degenerative joint disease characterized by progressive cartilage deterioration34, 35. The symptoms of OA include joint pain, impairment of movement, and local inflammation. Current treatments relieve pain temporarily with nonsteroidal anti-inflammatory drugs, steroids, hyaluronic acid, or surgery, but eventually fail over time. OA is associated with progressive inflammation; thus, MSCs are a promising candidate for cell therapy because they have antiinflammatory and immunosuppressive properties36. In addition, MSCs have shown the ability to migrate and engraft onto multiple musculoskeletal tissues, especially at sites of injury, and undergo site-specific differentiation, then influence the microenvironment to aid in the regeneration of cartilage.
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Recent efforts have focused on the use of direct intra-articular injection of MSCs as a therapy for OA. Lee et al. intra-articularly injected MSCs suspended in HA for the treatment of cartilage defects in the medial femoral condyle of adult minipigs37. At 6 and 12 weeks postoperatively, the MSC-treated groups showed improved cartilage healing compared with the controls. Horie et al. investigated the efficiency of meniscal regeneration in rat massive meniscal defects using intra-articular MSCs38. Their results showed that the MSCs adhered to the lesion, differentiated into meniscal cells directly, and promoted meniscal regeneration. MSCs have also been genetically modified for OA cell therapy to release therapeutic
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proteins that can interact with the injured cartilage. In one study by Matsumoto et al., when the MSCs expressing bone morphogenetic protein (BMP)-4, in combination with MSCs expressing the vascular endothelial growth factor antagonist soluble Flt-1, were intraarticularly injected into an immunodeficient rat model for OA, the quality and the persistence of regenerated articular cartilage were significantly improved39. Recently, controlled growth factor release from microspheres incorporated in MSC sheets has been shown to enhance cartilage tissue formation40. Furthermore, the over-expression of SOX9, the master regulator of chondrogenesis, can also increase MSC-mediated cartilage repair in an animal model41.
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Wakitani et al. reported the clinical use of transplanted bone marrow-derived MSCs seeded onto type I collagen-containing hydrogels to repair cartilage defects in the medial femoral condyle of human knees with OA42. Forty-two weeks after transplantation, the arthroscopic and histological grading scores were better in the MSC-transplanted group than in the cellfree control group. In another clinical study, by Davatchi et al., four patients ages 54–65 years old with moderate to severe knee OA received intra-articular injections of autologous bone marrow-derived MSCs43. After 6 months, three patients showed improvement in walking time and pain scores. All patients showed improvement in the number of stairs they could climb and pain relief. A recent study reported the discovery of a small molecule, kartogenin, which can promote the differentiation of MSCs into chondrocytes44. Johnson et al. showed that kartogenin can upregulate chondrocyte-specific gene expression in vitro, and significantly increase cartilage thickness in an OA mouse model. Bone Fractures
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Normal bone fracture healing involves a complex network of signaling events in response to injury. Undifferentiated MSCs are recruited to the injury site where, with the aid of regulatory cytokines, e.g. BMPs, they proliferate, differentiate into chondrocytes and osteoblasts, and form bone, thus repairing the defect. Failure to heal properly results in either delayed union, or nonunion if fractures fail to heal after 8–9 months10, 23. While fractures are a common clinical occurrence, the treatment of nonunions remains a challenge. MSCs are promising candidates for bone regeneration due to their ability to migrate to injured tissues and undergo osteogenic and chondrogenic differentiation.
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Numerous studies have successfully used MSCs for fracture repair and bone regeneration in animal models. Granero-Molto et al. demonstrated that MSCs migrated towards the fracture site in a mouse model, and contributed to fracture healing by expressing BMP-2 and modulating the injury-related inflammatory response45. Bone repair often requires a scaffold for structural and mechanical support. Kadiyala et al. expanded bone marrow-derived MSCs in culture, seeded them onto ceramic cylinders, and implanted the constructs into 8 mm segmental defects in rat femurs46. The results showed new bone formation after 8 weeks. Several other studies have also been performed using different scaffold materials in combination with MSCs to achieve bone regeneration7, 47. Gene therapy has also been explored for the use in fracture healing and bone regeneration. Musgrave et al. showed that MSCs can be used to deliver BMP-2 ex vivo48. The delivery of BMP-2 stimulates the MSCs at the injury site to differentiate and form bone. Burastero et al. Case Orthop J. Author manuscript; available in PMC 2016 January 21.
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found that the combination of MSCs with BMP-7 on a scaffold resulted in a better osteoinductive graft than either MSCs or BMP-7 alone49.
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Human studies have been reported in which autologous bone marrow-derived MSCs were expanded in vitro, then loaded onto 100% hydroxyapatite macroporous ceramic scaffolds50. The constructs were then implanted into four patients with diaphyseal segmental defects that ranged from 3 to 28.3 cm3 in one tibia, one humerus, and two ulna fractures, respectively. Good integration of the implanted constructs with the preexisting bone was maintained, and no major adverse reactions were observed. All patients experienced recovery of limb function, and good integration was maintained at the last follow-up (6–7 years after surgery). Hernigou et al. treated 60 cases of atrophic non-union of the tibia with the percutaneous injection of concentrated bone marrow51. They reported a high union rate of 43/60, and a correlation between the number and concentration of colony-forming units in the graft and the volume of mineralized callus at 4 months. Quarto et al. treated critical-sized segmental defects in the long bones of three patients with size-matching hydroxyapatite scaffolds loaded with expanded autologous MSC cultures52. They reported that graft integration was detected as early as 2 months after surgery and all three patients regained function of their limbs with no adverse effects.
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iPS Cells: An Alternative Stem Cell Source Induced pluripotent stem (iPS) cells have recently been discovered as an alternative to ESCs. Takahashi et al. generated iPS cells by transfecting mouse somatic cells with pluripotent transcription factors Oct3/4, c-Myc, Sox2, and K1f453. They reported that these iPS cells were highly similar to ESCs in their morphology, proliferation, gene expression, and in vitro differentiation53. This landmark study has been cited more than 3700 times, reflecting the growing interest and wide application of its findings. Different groups subsequently replicated their findings in human cells. To date, iPS cells have been derived at increased efficiency from easily accessible human cell types such as dermal fibroblasts, blood cells, and keratinocytes54. To eliminate the potential risk of virally induced tumor formation associated with the early iPS cell derivation procedure, intense efforts are underway to develop virus-free and/or vector-free iPS cell protocols. More importantly, various patient-specific iPS cells have been generated to serve as novel models to study disease pathogenesis and to screen for new drugs55. However, in order for iPS cells to be used in clinical settings, many remaining obstacles must be overcome, such as low reprogramming efficiency and tumorigenicity with iPS cells.
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Efforts have also been underway to convert somatic cells directly into differentiated cell types, including chondrocytes. Indeed, the combination of c-Myc, Klf4, and Sox9 can reprogram adult dermal fibroblasts directly into chondrocytes and form hyaline cartilage in mice56. Although SOX9 is required for converting iPS cells and other progenitor cells to chondrocytes, it should be borne in mind that SOX9 expression has to be tightly controlled to avoid unintended effects. Our group has recently showed that ectopic Sox9 expression in differentiated osteoblasts can lead to severe osteopenia, altered bone mechanical properties, and impaired MSC functions in mice57. Furthermore, in addition to cartilage development, SOX9 is also critical for the development of other organs such as the central nervous system, pancreas, prostate, intestine, skin, pituitary, heart, kidney, and liver. Overexpression of SOX9 has been associated with the tumorigenesis of colorectal cancer, breast
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cancer, and prostate cancer. Thus, a more comprehensive understanding of the regulation of SOX9 itself is critical to generate abundant chondrocytes with minimal side effects for orthopaedic applications.
Conclusions
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MSCs and iPS cells can both develop into multiple tissues including bone and cartilage. They hold great promise for orthopaedic applications such as generating specific disease models and developing novel therapies. There have been great advances in MSC technology to treat skeletal diseases since the discovery of MSCs several decades ago. Our knowledge about the role of progenitor cells in skeletal development has enabled us to better understand the role of MSCs in adult skeletal disease and injury. However, MSC transplantation has its risks that cannot be overlooked when developing MSC therapy. For example, ex vivo amplifications of MSCs via extensive passages may lead to malignant transformation with cytogenetic aberrations and sarcoma formation. Thus more research, such as understanding the signaling pathways involved in musculoskeletal tissue regeneration, is critical for developing MSC therapy. The recent development of iPS cell technology is a versatile alternative to MSCs. The first human clinical trial using iPS cells to repair diseased retinas is scheduled to start in 2013 at the RIKEN Center for Developmental Biology in Kobe, Japan. However, for iPS cells, additional research and animal trials are also necessary to establish their safety and efficacy before clinical application. In summary, a better understanding of the molecular basis of pluripotency, cellular reprogramming, and lineage-specific differentiation of MSCs and iPS cells is essential to realize their clinical potential in regenerative medicine in the future.
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Acknowledgments We thank Valerie Schmedlen for editorial assistance.
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