Review Article
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In Utero Stem Cell Transplantation for Radical Treatment of Osteogenesis Imperfecta: Perspectives and Controversies Sherif Abd-Elkarim Mohammed Shazly, MBBCh, MSc1
1 Woman&s Health Center, Assiut University, Assiut, Egypt
Am J Perinatol 2014;31:829–836.
Abstract
Keywords
► genetic diseases ► mesenchymal stem cells ► fetal therapy ► skeletal anomalies
Osteogenesis imperfecta (OI) is a lethal hereditary connective tissue disease that affects the synthesis of type I collagen. Current treatment options including surgical, physical, and medical treatment help to reduce pain, deformities, and rate of bone fracture. However, these choices are insufficient and are associated with many adverse effects. The development of stem cell therapy allows scientists to consider this option for radical treatment of many genetic diseases including OI. In utero stem cell transplantation provides a better opportunity for early prenatal intervention while the fetus is preimmune and before any permanent damage occurs. Few animal and human trials for treatment of OI have been published, and the results were promising but still controversial. Our objective is to review the available evidence and discuss the points of controversy including the parameters of treatment success and postnatal predictors of long-term treatment outcome.
General View Osteogenesis Imperfecta: The Challenging Disease Osteogenesis imperfecta (OI) is a generalized hereditary connective tissue disease affecting the synthesis of type I collagen, which is the primary component of extracellular matrix of bone and skin as well as other connective tissues including ligaments, sclera, teeth, and ear.1,2 The incidence of OI worldwide is about 1/20,000. The autosomal dominant forms represent approximately 90% of cases and occur equally among races, while the autosomal recessive forms occur predominantly in certain ethnic groups.3 The clinical presentation of OI ranges from mild forms with normal lifespan to perinatal lethal forms. The disease is usually presented by a triad of fragile bones (brittle bone disease), blue sclera, and early deafness, it may also be presented by joint hypermotility, dentinogenesis imperfect, and skin hyperlaxity.1,4,5 The most accepted classification of OI is the Sillence classification, which was published in 1978. This classification is based on the mode of inheritance, clinical, and
received August 1, 2013 accepted after revision November 9, 2013 published online December 17, 2013
Address for correspondence Sherif A. Shazly, MBBCh, MSc, Woman&s Health Center, Assiut University, Postal code 71111, Assiut, Egypt (e-mail: [email protected]).
radiological criteria of the disease. It classifies OI into four major autosomal dominant types that are due to mutations in the genes responsible type I collagen synthesis (COL1A1 and COL1A2 genes).4,6,7 However, the four types vary in their severity; Sillence type I is the mildest form followed by types IV, III, and II, which is a perinatally lethal form.8 This classification has been expanded several times; first in 2004 (Montereal modifications) when two other types were added; types V and VI. Type V, an autosomal dominant form, is clinically, radiologically, and histologically distinct, while type VI, an autosomal recessive form, is only histologically distinct. Their molecular changes and responsible genes are still unknown.9–12 In 2006, 2007, and 2011, Morello et al, Cabral et al, and Pyott et al described types VII, VIII, and IX, respectively. The three types are autosomal recessive and are due to defects in a protein complex that is responsible for prolyl-3-hydroxylation of type I collagen. There are three proteins in this complex; cartilage-associated protein (encoded by CRTAP gene) prolyl-3 hydroxylase (encoded by LEPRE1 gene), and cyclophilin B (encoded by PPIB gene).
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DOI http://dx.doi.org/ 10.1055/s-0033-1363501. ISSN 0735-1631.
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Mariam Taher Mohamed Amin1
IUSCT for Treatment of OI
Amin, Shazly
These three genes are defective in types VII, VIII, and IX, respectively.7,13–15 Other recently discovered types of OI include type X which is caused by SERPINH1 gene mutation that encodes collagen-binding protein 2 or heat-shock protein 47, type XI which is caused by FKBP10 mutation that interferes with trimeric procollagen molecule secretion, XII which is caused by SP7 gene mutation that encodes for a major regulator of bone differentiation, and type XIII which is caused by mutations in the bone morphogenetic protein 1 gene (BMP1). All the four types also express an autosomal recessive pattern of inheritance.16 OI is currently a noncurable disease. Available treatment options aim to reduce deformity, relive pain, promote normal function, and improve the quality of life. These options include physiotherapy, rehabilitation, orthopedic surgery, and medical treatment.3,17,18 Orthopedic interventions provide some improvement of mobility and some reduction of skeletal deformities.5 Medical treatment, either approved or still under study, aims to improve bone strength.2 However, most of medical treatment options are controversial and their outcomes are not so impressive. For instance, anabolic agents including parathyroid hormones and growth hormones increase bone turnover and this makes bone deformities more likely to occur. Parathyroid hormone is contraindicated in childhood because of its suspected risk of osteosarcoma.19 Bisphosphonate is another option that acts by inhibition of osteoclast function; its main effect is to increase cortical thickness, the number of trabeculae and to improve pain and muscle strength.4,20,21 Denosumab, a receptor activator of nuclear factor kappa B ligand (RANKL) inhibition, is a recently developed medical option that achieves comparable results to bisphosphonate on laboratory animals. However, long-term outcome has not yet been established for both drugs.22
Fig. 1 Types of stem cells.
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However, these options do not eradicate the problem because bone still contains the defective collagen. Recently, the great evolution of molecular science and genetic studies has motivated researchers to design an earlier and more efficient treatment approach. Radical treatment using stem cells and gene targeting therapy to correct the defective collagen has become an interesting option.
Stem Cells: Types and Promising Features Stem cells are undifferentiated cells that have a unique ability of self-renewal (making copies of themselves) and differentiation (making specialized progenitor cells).23 Stem cells are divided into embryonic stem cells (ESCs) and adult stem cells (ASCs) (►Fig. 1). ESCs originate from the inner cell mass of the blastocyst (5 days after fertilization). These cells can be isolate in in vitro fertilization laboratories, and they can be differentiated into all types of tissues (i.e., pluripotent cells).24 Although, ESCs can be expanded in vitro and are an excellent source for all tissues, their use is restricted due to ethical issues. To avoid these ethical considerations, researchers identified factors that could induce adult cells (e.g., fibroblasts and fat cells) to behave like ESCs; these cells are so called “induced pluripotent stem cells.”25,26 On the other hand, ASCs are present in almost all body tissues. The first isolated ASCs were hemopiotic stem cells (HSCs). They are found in the bone marrow and they can be differentiated into a variety of blood elements. Thereafter, researchers were able to isolate mesenchymal stem cells (MSCs), neural stem cells, gut stem cells, and cardiac stem cells.23,24,27,28 Each of these groups can produce a variety of specialized cells, hence the name multipotent cells. MSCs can be isolated from many tissues including bone marrow, periosteum, trabecular bone, lungs, fetal liver cells, umbilical cord blood, synovial membranes, and
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Table 1 Criteria of mesenchymal stem cells • Ability to expand in vitro • Low immunogenicity • Suppression of T-lymphocyte proliferation and activation • Chemotaxis properties that guide the cells to the site of injury • Secretion of paracrine factors that modify the microenvironment of the injured tissue
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route provides better cell uptake. However, the risk of complications is higher than the intraperitoneal route.33 In spite of its potential complications during stem cell delivery, IUSCT seems to be a promising competitor to the traditional postnatal stem cell transplantation. The advantages of IUSCT are related to the following features: (1) being smaller than a newborn, the fetus is not in need for a large dose of stem cells.33 (2) The fetus is considered preimmune in the early second trimester.32 (3) The cellular environment of the fetus; cells are in the stage of proliferation and migration to their anatomical sites.33 The first successful IUSCT trial in human was achieved using hemopiotic stem cells in bare lymphocyte syndrome and was reported by Westgren and Tourain.33,34
MSCs and OI: How Far Have We Reached? IUSCT and MSCs adipose tissues. These cells can be differentiated into osteoblasts, chondrocytes, and adipocytes.23,29 MSCs have many features that support their potential role in stem cell therapy (►Table 1). They expand in vitro under normal culture conditions and accordingly, a small amount of bone marrow aspirate will be sufficient for transplantation. Moreover, they are characterized by their low immunogenicity and their ability to suppress T- lymphocyte proliferation and activation. Therefore, they are suitable for allogenic transplantation. These cells can home the site of injury through their chemotaxis properties and they secrete paracrine factors that modify the microenvironment of injured tissue.23,30 Recently, MSCs have been implemented in gene therapy; they are utilized as cellular vehicles for proteinproducing genes after being genetically modified in vitro. Several techniques are available to introduce exogenous DNA into MSCs before transplantation. Eventually, MSCs deliver new genes that serve in tissue regeneration therapies.31 MSCs may be delivered directly to the affected tissues or through infusion. These cells may be delivered in utero to correct some hereditable skeletal diseases either alone or in combination with gene therapy.23
In Utero Stem Cell Transplantation: Rationale and Expectations Nowadays, the substantial development of prenatal diagnosis techniques allows early in utero diagnosis of genetic diseases. Accordingly, early prenatal therapeutic interventions have become justified. In utero stem cell transplantation (IUSCT) is a recent therapeutic intervention that has been experimentally implicated in the management of different hematological, metabolic, and immunological disorders.32 IUSCT is a process of delivery of stem cells to a genetically affected fetus to provide the greatest opportunity to correct a genetic defect while the fetus is still in the developmental stage before any permanent damage occurs. Practically, there are two routes available for IUSCT; the intravascular “through umbilical vessels” and the intraperitoneal routes. Both approaches should be done under ultrasound guidance. The intravascular
Recent IUSCT studies that involved MSC delivery to animal models demonstrated a variable degree of engraftment of these cells in bone; these cells then developed and differentiated into osteogenic cells that improved the biological criteria of the diseased bone.35–37 Based on these results, IUSCT was expected to play a rule in the treatment of skeletal defects including OI. Unlike HSCs, MSCs are characterized by their low immunogenicity. Accordingly, MSC transplantation to immunocompetent fetuses does not require immune tolerance (which occurs in early second trimester) or myeloablasion.32,36,38 This means that MSC transplantation can be considered even in late pregnancy.
MSCs: Human Trials The objectives of IUSCT studies for OI were to evaluate the ability of MSCs to engraft in bone, to form collagen, to improve bone matrix and to improve the course of the disease (including incidence of fractures and growth velocity).39,40 The expression “engraftment level” refers to the amount of donor cells, which are incorporated in the recipient bone tissue. This may be assessed by certain markers in the donor cells, for example, enhanced green fluorescent protein (eGFP)41 or simply by identifying expressed genes if donor cells are of male origin and the recipient is a female.40 Based on theoretical concepts and promising animal trials, the first human trial was conducted by Westgren et al in 2003; a female fetus with OI received fetal MSCs of male origin at the 29th week of gestation. Bone biopsy at the age of 8 months revealed good bone histology, 5% of cells were of male origin. After 1 year of follow-up, no fractures were reported.39 A second trial was reported by Le Blanc et al in 2005, a female fetus received fetal MSCs of male origin at the 32nd weeks of gestation. Bone biopsy, taken at the age of 9 months, showed that 7% of bone cells were of male origin, the authors also reported no lymphocytic proliferation against donor cells. At the age of 4 months, bisphosphonate therapy was initiated. The patient was followed up for 2 years during which three fractures were reported.40 Because human trials were few in number and their results were controversial, researchers considered more animal trials to American Journal of Perinatology
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IUSCT for Treatment of OI
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recruit large samples. Moreover, animal studies allowed investigators to assess the biochemical, immunological, and genetic impact of stem cell therapy on bone conveniently.
MSCs: Animal Trials Animal Models There are many animal models that are identified to study OI including murine, canine, feline, bovine, and ovine models.42 However, murine models, either engineered or spontaneously occurring, are usually used. Murine models include Mov-13 mouse, Brittle II mouse, Oim mouse, and Brittle IV mouse; these four models are used to study the first four types of OI, respectively.42,43 The principles of engineering of these four models are described briefly in ►Table 2.
Animal Studies The first animal trial was conducted by Guillot et al in 2008 using the Oim mouse model. The engraftment and differentiation of stems cells were detected by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). qRTPCR evaluates gene expression of human osteocalcin (OC), osteopontin (OP), osteoprotegerin (OPG), bone morphogenic protein (BMP2), and osterix (OSX). The authors reported decreased incidence of fractures, improvement of bone strength, and increased bone engraftment at sites of bone formation.44 The second trial was conducted by Panaroni et al in 2009 using heterozygous Brtl IV and wild type (WT) mice. Both WT and Brtl IV mice received e-GFP expressed bone marrow cells from transgenic mice. The authors reported equal survival rate at the age of 2 months in both genotypes. The untransplanted BrtlIV mice had a survival rate two times lower than WT mice. Engraftment of the e-GFP þ cells in bone was detected by fluorescence and confocal microscope; it was the same level in both models. The mineral content and cortical thickness assessed by peripheral quantitative computed tomography and Microcomputed tomography (MicroCT) showed significant increase in mineral content and density and cortical thickness in both models. The average
matrix mineralization near host and donor osteocytes in BrtlIV mice was comparable. However, the mineral density around donor osteocytes was more homogeneous and the matrix was well organized. Improvement of bone mechanics (stiffness, yield force, and ultimate force) was also reported.41 The third trial was conducted by Vanleene et al in 2011; the authors transplanted fetal MSCs into Oim/Oim model (untransplanted Oim/Oim and WT mice were used as a control group). The donor cells engrafted in bone differentiated into osteoblasts and produced the COL1a2 chain protein which was absent. The presence of normal collagen resulted in decreased hydroxyproline content, which was formed by the abnormal collagen chains. Bone matrix was stiffer in transplanted mice but did not reach the stiffness recognized in WT mice. There was no difference between transplanted and untransplanted mice regarding stiffness, yield force, and ultimate force. However, the ratio of plastic/total work to fracture indicated that the treated bone required more force to be fractured. Bone, examined by MicroCT, showed no significant difference in cortical thickness and cross-section area between transplanted and untransplanted mice, both had thinner cortex than WT mice.45
Discussion: Authors’ Insight In general, the number of available trials was not enough to suggest a recommendation or conclusion (►Table 3). IUSCT in treatment of OI is still a matter of controversy in many aspects. The most recognizable point is the wide variety of responses to this intervention. This should be carefully analyzed before going further. Only two published human trials were available in the literature. Both Westgren et al and Le Blanc et al revealed good but not similar outcomes.39,40 Westgren et al reported no fractures during 1 year of follow-up while Le Blanc et al reported three fractures in 2 years in spite of receiving bisphosphonate treatment beginning from the 4th postnatal month. This discrepancy cannot be explained by sex (both were females), gestational age (both were treated late in gestation), severity (both had intrauterine fractures), type
Table 2 Animal models for osteogenesis imperfecta Animal model
Engineering
Corresponding type
Mov-13 mouse
This model is engineered by integration of Moloney leukemia virus at the 5′ end of the proα1 (I) gene
This heterozygous model is used to for research on type I OI
Brittle II mouse (BrtlII)
The model is produced by cre/lox recombination system, which results in a lethal murine knock-in model
This model is used for research on type II OI
Oim mouse
This model emerges from a spontaneous frame shift mutation of COL1A2 gene, which causes subsequent alteration in C-propeptide formation
This model is used for research on type III OI
Brittle IV mouse (BrtlIV)
This model is produced by cre/lox recombination system, which results in a nonlethal knock-in murine model
This model is used for research on type IV OI
Abbreviation: OI, osteogenesis imperfect.
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Female fetus
Oim/Oim mice
BrtlIV/WT mice
Le Blanc et al (2005)
Guillot et al (2008)
Panaroni et al (2009)
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32 Oim (20 mice were not treated and 12 mice received fetal MSCs) þ 15 WT mice
169 (82 BrtlIV mice and 87 WT mice)
77 (48 mice not received MSCs and 29 received)
1
1
Sample size
Human fetal blood stem cells
E13.5–E15
E13.5–E14.5
E13.5–E15
Human fetal blood cells
Bone marrow cells expressing e-GFP from transgenic mice
32nd wk of gestation
29th wk of gestation
Time of delivery of MSCs
Human fetal MSCs of male origin
Human fetal MSCs of male origin
Source of MSCs
Intraperitoneal
Intrahepatic
Intraperitoneal
Intravascular
Intravascular
Route of MSCs delivery
5%
2%
5%
7%
5%
Engraftment levels
content and bone histology
trabeculae
thickness
Increased survival rate/decreased lethality Increased cortical thickness Improvement of matrix quality Improvement of bone mechanics (stiffness, yield force and ultimate force)
Reduction in femoral fractures (84%) Decreased hydroxyproline content in bone Increased bone matrix stiffness Decreased bone brittleness No changes in bone morphology “cortical and trabecular thickness” • Protein-mineral structure of bones contained more mature apatite crystal in females
• • • • •
• • • •
remodeling (growth plates and fracture sites)
• More donor cells at the sites of bone
• Two-thirds reduction in fracture incidence • Increased bone length, strengths and
• Normal psychomotor development • Increased growth velocity
• Good arrangement of matrix and
• Decreased fracture incidence
• Improvement of bone mineral
Results
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Abbreviations: E, embryonic day; e-GFP, enhanced green fluorescent protein; IUCT, in utero stem cell transplantation; MSCs; mesenchymal stem cells; OI, osteogenesis imperfecta; WT, wild type.
Oim/Oim mice and WT mice
Female fetus
Westgren et al (2003)
Vanleene et al (2011)
Model/fetal sex
Author
Table 3 Summary of human and animal IUSCT trials in the treatment of OI
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of stem cells (both were treated with fetal MSCs), or route of stem cell delivery (both were delivered intravenously). Surprisingly, the level of engraftment did not explain this discrepancy but was even higher in the second case (7 vs. 5%). Furthermore, the degree of improvement reported by Le Blanc et al could not be definitely attributed to IUSCT because the patient started an adjuvant medical treatment. A last variable that may explain this discrepancy was the type of OI itself. The second patient was diagnosed with OI type II, which is the most severe form of the disease, and this may partially explain the less favorable outcome reported in this case. Unfortunately, the type of OI was not reported in the first case and accordingly, we cannot confirm this explanation. We still need to understand this discrepancy to evaluate the prognosis of IUSCT and the factors that determine patient eligibility for IUSCT. Westgren suggested that the difference might be explained by the type of stem cells he used (adult versus fetal MSCs).33 All failed cases including unpublished cases of Porta and Chile used adult MSCs. However, this does not explain our first dilemma because both human cases were treated with fetal MSCs. Therefore, we may consider the type of OI as a possible predictor of treatment outcome. Unlike human trials, animal trials showed uniformly promising outcomes. Unfortunately, these trials involved only two animal models (Oim/Oim and BrtlIV) that were used to present types III and IV of the disease, respectively. The level of engraftment was comparable to that of human studies. In both types of studies, engraftment level was low (2–7%) and accordingly, engraftment alone cannot explain the degree of improvement. This conclusion was not limited to in utero studies; Jones et al conducted a postnatal (day 2–3) animal trial on Oim/WT mice. Their aim was to enhance bone engraftment by priming human fetal MSCs with stromal driving factor 1, which increases the expression of CXCR4 on the cell surface. This subsequently enhanced chemotaxis and engraftment in bone and bone marrow of both mice. However, the authors reported there was no significant reduction in the incidence of bone fractures despite the increase in engraftment level.46 The results were comparable in the three animal studies regardless of mice gender, type, and source of stem cells, route of stem cell delivery and type of OI. No clear predictors of improvement could be identified in animal studies. Unfortunately, the most lethal type II OI was not investigated (unlike human trials). Researchers may need to consider the type of OI as a predictor of success in further studies. While Vanleene et al 2011 suggested more favorable outcome in female mice, patient sex should be also more investigated. Another point of controversy was how postnatal assessment following IUSCT can predict treatment success and favorable outcomes. Histological and biochemical tests have been more applicable in animal studies. The aim was to understand how treatment may benefit the patients and what histological or biochemical markers may predict long-term outcome. Again, reduced fracture rate was observed in the three animal experiments but increased cortical thickness was observed only in the first two experiments. Surprisingly, this difference was not associated with significantly different outcomes. Accordingly, American Journal of Perinatology
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it seems that bone mechanics (including bone stiffness) were more important than bone thickness. Bone mechanics are not as simple to assess as cortical thickness and according to these results, considering cortical thickness, which may even be assessed in utero, may be misleading. Unfortunately, all human and animal studies lack long-term follow-up. Therefore, our evaluation of distant outcome is lacking. Finally, it is essential to understand the hazards of this approach to weigh them against potential benefits. Unfortunately, the limited number of cases recruited in IUSCT studies makes it difficult to reveal these hazards. However, it is apparent that the potential risks of IUSCT are generally attributed to the procedure, by which stem cells are delivered to the fetus. Accordingly, the risks of abortion, fetomaternal hemorrhage, and infection are expected. In one IUSCT study, the risk of abortion involved two out of six fetuses.47 However, it is difficult to determine whether abortion was attributed to the procedure itself, to the severity of the disease or to the presence of possible lethal genetic associations. Moreover, there is an additional risk of fetal immunological response against transplanted cells if cells are introduced after 12 weeks of gestation.48 This is a general complication of IUSCT that is fortunately not expected in fetuses treated with MSCs because of their low immunogenicity.
Conclusion In conclusion, IUSCT is a promising future option for treatment of OI. However, more studies are needed to evaluate the exact efficacy of this intervention and the possible predictors of success to improve patient selection. The ideal type of stem cells (adult vs. fetal MSCs) needs to be investigated. The results need to be correlated carefully to different variables including sex and type of OI. Long-term outcomes should be investigated and different histological and biochemical investigations should be correlated with treatment outcome. This helps to determine clear parameters for future prognosis of this future therapeutic option.
Acknowledgments The authors would like to thank all the members of the mentor–student research link pilot project (a part of the Healthcare Reform Egypt; HRE) to which this research project belongs. They appreciate their great effort to develop a strong research system and to improve the skills of young researchers in Egypt. This represents a fundamental part of the national medical development goals of this organization.
Conflict of Interest The authors have no conflict of interest.
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IUSCT for Treatment of OI
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repairs bone and reduces fractures in osteogenesis imperfecta mice. Blood 2008;111(3):1717–1725 45 Vanleene M, Saldanha Z, Cloyd KL, et al. Transplantation of human fetal blood stem cells in the osteogenesis imperfecta mouse leads to improvement in multiscale tissue properties. Blood 2011; 117(3):1053–1060 46 Jones GN, Moschidou D, Lay K, et al. Upregulating CXCR4 in human fetal mesenchymal stem cells enhances engraftment and bone
mechanics in a mouse model of osteogenesis imperfecta. Stem Cells Transl Med 2012;1(1):70–78 47 Touraine JL. In utero transplantation of fetal liver stem cells into human fetuses. J Hematother 1996;5(2):195–199 48 Surbek DV, Tercanli S, Holzgreve W. Transabdominal first trimester embryofetoscopy as a potential approach to early in utero stem cell transplantation and gene therapy. Ultrasound Obstet Gynecol 2000;15(4):302–307
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In Utero Stem Cell Transplantation for Radical Treatment of Osteogenesis Imperfecta: Perspectives and Controversies Sherif Abd-Elkarim Mohammed Shazly, MBBCh, MSc1
1 Woman&s Health Center, Assiut University, Assiut, Egypt
Am J Perinatol 2014;31:829–836.
Abstract
Keywords
► genetic diseases ► mesenchymal stem cells ► fetal therapy ► skeletal anomalies
Osteogenesis imperfecta (OI) is a lethal hereditary connective tissue disease that affects the synthesis of type I collagen. Current treatment options including surgical, physical, and medical treatment help to reduce pain, deformities, and rate of bone fracture. However, these choices are insufficient and are associated with many adverse effects. The development of stem cell therapy allows scientists to consider this option for radical treatment of many genetic diseases including OI. In utero stem cell transplantation provides a better opportunity for early prenatal intervention while the fetus is preimmune and before any permanent damage occurs. Few animal and human trials for treatment of OI have been published, and the results were promising but still controversial. Our objective is to review the available evidence and discuss the points of controversy including the parameters of treatment success and postnatal predictors of long-term treatment outcome.
General View Osteogenesis Imperfecta: The Challenging Disease Osteogenesis imperfecta (OI) is a generalized hereditary connective tissue disease affecting the synthesis of type I collagen, which is the primary component of extracellular matrix of bone and skin as well as other connective tissues including ligaments, sclera, teeth, and ear.1,2 The incidence of OI worldwide is about 1/20,000. The autosomal dominant forms represent approximately 90% of cases and occur equally among races, while the autosomal recessive forms occur predominantly in certain ethnic groups.3 The clinical presentation of OI ranges from mild forms with normal lifespan to perinatal lethal forms. The disease is usually presented by a triad of fragile bones (brittle bone disease), blue sclera, and early deafness, it may also be presented by joint hypermotility, dentinogenesis imperfect, and skin hyperlaxity.1,4,5 The most accepted classification of OI is the Sillence classification, which was published in 1978. This classification is based on the mode of inheritance, clinical, and
received August 1, 2013 accepted after revision November 9, 2013 published online December 17, 2013
Address for correspondence Sherif A. Shazly, MBBCh, MSc, Woman&s Health Center, Assiut University, Postal code 71111, Assiut, Egypt (e-mail: [email protected]).
radiological criteria of the disease. It classifies OI into four major autosomal dominant types that are due to mutations in the genes responsible type I collagen synthesis (COL1A1 and COL1A2 genes).4,6,7 However, the four types vary in their severity; Sillence type I is the mildest form followed by types IV, III, and II, which is a perinatally lethal form.8 This classification has been expanded several times; first in 2004 (Montereal modifications) when two other types were added; types V and VI. Type V, an autosomal dominant form, is clinically, radiologically, and histologically distinct, while type VI, an autosomal recessive form, is only histologically distinct. Their molecular changes and responsible genes are still unknown.9–12 In 2006, 2007, and 2011, Morello et al, Cabral et al, and Pyott et al described types VII, VIII, and IX, respectively. The three types are autosomal recessive and are due to defects in a protein complex that is responsible for prolyl-3-hydroxylation of type I collagen. There are three proteins in this complex; cartilage-associated protein (encoded by CRTAP gene) prolyl-3 hydroxylase (encoded by LEPRE1 gene), and cyclophilin B (encoded by PPIB gene).
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DOI http://dx.doi.org/ 10.1055/s-0033-1363501. ISSN 0735-1631.
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Mariam Taher Mohamed Amin1
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These three genes are defective in types VII, VIII, and IX, respectively.7,13–15 Other recently discovered types of OI include type X which is caused by SERPINH1 gene mutation that encodes collagen-binding protein 2 or heat-shock protein 47, type XI which is caused by FKBP10 mutation that interferes with trimeric procollagen molecule secretion, XII which is caused by SP7 gene mutation that encodes for a major regulator of bone differentiation, and type XIII which is caused by mutations in the bone morphogenetic protein 1 gene (BMP1). All the four types also express an autosomal recessive pattern of inheritance.16 OI is currently a noncurable disease. Available treatment options aim to reduce deformity, relive pain, promote normal function, and improve the quality of life. These options include physiotherapy, rehabilitation, orthopedic surgery, and medical treatment.3,17,18 Orthopedic interventions provide some improvement of mobility and some reduction of skeletal deformities.5 Medical treatment, either approved or still under study, aims to improve bone strength.2 However, most of medical treatment options are controversial and their outcomes are not so impressive. For instance, anabolic agents including parathyroid hormones and growth hormones increase bone turnover and this makes bone deformities more likely to occur. Parathyroid hormone is contraindicated in childhood because of its suspected risk of osteosarcoma.19 Bisphosphonate is another option that acts by inhibition of osteoclast function; its main effect is to increase cortical thickness, the number of trabeculae and to improve pain and muscle strength.4,20,21 Denosumab, a receptor activator of nuclear factor kappa B ligand (RANKL) inhibition, is a recently developed medical option that achieves comparable results to bisphosphonate on laboratory animals. However, long-term outcome has not yet been established for both drugs.22
Fig. 1 Types of stem cells.
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However, these options do not eradicate the problem because bone still contains the defective collagen. Recently, the great evolution of molecular science and genetic studies has motivated researchers to design an earlier and more efficient treatment approach. Radical treatment using stem cells and gene targeting therapy to correct the defective collagen has become an interesting option.
Stem Cells: Types and Promising Features Stem cells are undifferentiated cells that have a unique ability of self-renewal (making copies of themselves) and differentiation (making specialized progenitor cells).23 Stem cells are divided into embryonic stem cells (ESCs) and adult stem cells (ASCs) (►Fig. 1). ESCs originate from the inner cell mass of the blastocyst (5 days after fertilization). These cells can be isolate in in vitro fertilization laboratories, and they can be differentiated into all types of tissues (i.e., pluripotent cells).24 Although, ESCs can be expanded in vitro and are an excellent source for all tissues, their use is restricted due to ethical issues. To avoid these ethical considerations, researchers identified factors that could induce adult cells (e.g., fibroblasts and fat cells) to behave like ESCs; these cells are so called “induced pluripotent stem cells.”25,26 On the other hand, ASCs are present in almost all body tissues. The first isolated ASCs were hemopiotic stem cells (HSCs). They are found in the bone marrow and they can be differentiated into a variety of blood elements. Thereafter, researchers were able to isolate mesenchymal stem cells (MSCs), neural stem cells, gut stem cells, and cardiac stem cells.23,24,27,28 Each of these groups can produce a variety of specialized cells, hence the name multipotent cells. MSCs can be isolated from many tissues including bone marrow, periosteum, trabecular bone, lungs, fetal liver cells, umbilical cord blood, synovial membranes, and
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Table 1 Criteria of mesenchymal stem cells • Ability to expand in vitro • Low immunogenicity • Suppression of T-lymphocyte proliferation and activation • Chemotaxis properties that guide the cells to the site of injury • Secretion of paracrine factors that modify the microenvironment of the injured tissue
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route provides better cell uptake. However, the risk of complications is higher than the intraperitoneal route.33 In spite of its potential complications during stem cell delivery, IUSCT seems to be a promising competitor to the traditional postnatal stem cell transplantation. The advantages of IUSCT are related to the following features: (1) being smaller than a newborn, the fetus is not in need for a large dose of stem cells.33 (2) The fetus is considered preimmune in the early second trimester.32 (3) The cellular environment of the fetus; cells are in the stage of proliferation and migration to their anatomical sites.33 The first successful IUSCT trial in human was achieved using hemopiotic stem cells in bare lymphocyte syndrome and was reported by Westgren and Tourain.33,34
MSCs and OI: How Far Have We Reached? IUSCT and MSCs adipose tissues. These cells can be differentiated into osteoblasts, chondrocytes, and adipocytes.23,29 MSCs have many features that support their potential role in stem cell therapy (►Table 1). They expand in vitro under normal culture conditions and accordingly, a small amount of bone marrow aspirate will be sufficient for transplantation. Moreover, they are characterized by their low immunogenicity and their ability to suppress T- lymphocyte proliferation and activation. Therefore, they are suitable for allogenic transplantation. These cells can home the site of injury through their chemotaxis properties and they secrete paracrine factors that modify the microenvironment of injured tissue.23,30 Recently, MSCs have been implemented in gene therapy; they are utilized as cellular vehicles for proteinproducing genes after being genetically modified in vitro. Several techniques are available to introduce exogenous DNA into MSCs before transplantation. Eventually, MSCs deliver new genes that serve in tissue regeneration therapies.31 MSCs may be delivered directly to the affected tissues or through infusion. These cells may be delivered in utero to correct some hereditable skeletal diseases either alone or in combination with gene therapy.23
In Utero Stem Cell Transplantation: Rationale and Expectations Nowadays, the substantial development of prenatal diagnosis techniques allows early in utero diagnosis of genetic diseases. Accordingly, early prenatal therapeutic interventions have become justified. In utero stem cell transplantation (IUSCT) is a recent therapeutic intervention that has been experimentally implicated in the management of different hematological, metabolic, and immunological disorders.32 IUSCT is a process of delivery of stem cells to a genetically affected fetus to provide the greatest opportunity to correct a genetic defect while the fetus is still in the developmental stage before any permanent damage occurs. Practically, there are two routes available for IUSCT; the intravascular “through umbilical vessels” and the intraperitoneal routes. Both approaches should be done under ultrasound guidance. The intravascular
Recent IUSCT studies that involved MSC delivery to animal models demonstrated a variable degree of engraftment of these cells in bone; these cells then developed and differentiated into osteogenic cells that improved the biological criteria of the diseased bone.35–37 Based on these results, IUSCT was expected to play a rule in the treatment of skeletal defects including OI. Unlike HSCs, MSCs are characterized by their low immunogenicity. Accordingly, MSC transplantation to immunocompetent fetuses does not require immune tolerance (which occurs in early second trimester) or myeloablasion.32,36,38 This means that MSC transplantation can be considered even in late pregnancy.
MSCs: Human Trials The objectives of IUSCT studies for OI were to evaluate the ability of MSCs to engraft in bone, to form collagen, to improve bone matrix and to improve the course of the disease (including incidence of fractures and growth velocity).39,40 The expression “engraftment level” refers to the amount of donor cells, which are incorporated in the recipient bone tissue. This may be assessed by certain markers in the donor cells, for example, enhanced green fluorescent protein (eGFP)41 or simply by identifying expressed genes if donor cells are of male origin and the recipient is a female.40 Based on theoretical concepts and promising animal trials, the first human trial was conducted by Westgren et al in 2003; a female fetus with OI received fetal MSCs of male origin at the 29th week of gestation. Bone biopsy at the age of 8 months revealed good bone histology, 5% of cells were of male origin. After 1 year of follow-up, no fractures were reported.39 A second trial was reported by Le Blanc et al in 2005, a female fetus received fetal MSCs of male origin at the 32nd weeks of gestation. Bone biopsy, taken at the age of 9 months, showed that 7% of bone cells were of male origin, the authors also reported no lymphocytic proliferation against donor cells. At the age of 4 months, bisphosphonate therapy was initiated. The patient was followed up for 2 years during which three fractures were reported.40 Because human trials were few in number and their results were controversial, researchers considered more animal trials to American Journal of Perinatology
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recruit large samples. Moreover, animal studies allowed investigators to assess the biochemical, immunological, and genetic impact of stem cell therapy on bone conveniently.
MSCs: Animal Trials Animal Models There are many animal models that are identified to study OI including murine, canine, feline, bovine, and ovine models.42 However, murine models, either engineered or spontaneously occurring, are usually used. Murine models include Mov-13 mouse, Brittle II mouse, Oim mouse, and Brittle IV mouse; these four models are used to study the first four types of OI, respectively.42,43 The principles of engineering of these four models are described briefly in ►Table 2.
Animal Studies The first animal trial was conducted by Guillot et al in 2008 using the Oim mouse model. The engraftment and differentiation of stems cells were detected by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). qRTPCR evaluates gene expression of human osteocalcin (OC), osteopontin (OP), osteoprotegerin (OPG), bone morphogenic protein (BMP2), and osterix (OSX). The authors reported decreased incidence of fractures, improvement of bone strength, and increased bone engraftment at sites of bone formation.44 The second trial was conducted by Panaroni et al in 2009 using heterozygous Brtl IV and wild type (WT) mice. Both WT and Brtl IV mice received e-GFP expressed bone marrow cells from transgenic mice. The authors reported equal survival rate at the age of 2 months in both genotypes. The untransplanted BrtlIV mice had a survival rate two times lower than WT mice. Engraftment of the e-GFP þ cells in bone was detected by fluorescence and confocal microscope; it was the same level in both models. The mineral content and cortical thickness assessed by peripheral quantitative computed tomography and Microcomputed tomography (MicroCT) showed significant increase in mineral content and density and cortical thickness in both models. The average
matrix mineralization near host and donor osteocytes in BrtlIV mice was comparable. However, the mineral density around donor osteocytes was more homogeneous and the matrix was well organized. Improvement of bone mechanics (stiffness, yield force, and ultimate force) was also reported.41 The third trial was conducted by Vanleene et al in 2011; the authors transplanted fetal MSCs into Oim/Oim model (untransplanted Oim/Oim and WT mice were used as a control group). The donor cells engrafted in bone differentiated into osteoblasts and produced the COL1a2 chain protein which was absent. The presence of normal collagen resulted in decreased hydroxyproline content, which was formed by the abnormal collagen chains. Bone matrix was stiffer in transplanted mice but did not reach the stiffness recognized in WT mice. There was no difference between transplanted and untransplanted mice regarding stiffness, yield force, and ultimate force. However, the ratio of plastic/total work to fracture indicated that the treated bone required more force to be fractured. Bone, examined by MicroCT, showed no significant difference in cortical thickness and cross-section area between transplanted and untransplanted mice, both had thinner cortex than WT mice.45
Discussion: Authors’ Insight In general, the number of available trials was not enough to suggest a recommendation or conclusion (►Table 3). IUSCT in treatment of OI is still a matter of controversy in many aspects. The most recognizable point is the wide variety of responses to this intervention. This should be carefully analyzed before going further. Only two published human trials were available in the literature. Both Westgren et al and Le Blanc et al revealed good but not similar outcomes.39,40 Westgren et al reported no fractures during 1 year of follow-up while Le Blanc et al reported three fractures in 2 years in spite of receiving bisphosphonate treatment beginning from the 4th postnatal month. This discrepancy cannot be explained by sex (both were females), gestational age (both were treated late in gestation), severity (both had intrauterine fractures), type
Table 2 Animal models for osteogenesis imperfecta Animal model
Engineering
Corresponding type
Mov-13 mouse
This model is engineered by integration of Moloney leukemia virus at the 5′ end of the proα1 (I) gene
This heterozygous model is used to for research on type I OI
Brittle II mouse (BrtlII)
The model is produced by cre/lox recombination system, which results in a lethal murine knock-in model
This model is used for research on type II OI
Oim mouse
This model emerges from a spontaneous frame shift mutation of COL1A2 gene, which causes subsequent alteration in C-propeptide formation
This model is used for research on type III OI
Brittle IV mouse (BrtlIV)
This model is produced by cre/lox recombination system, which results in a nonlethal knock-in murine model
This model is used for research on type IV OI
Abbreviation: OI, osteogenesis imperfect.
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Female fetus
Oim/Oim mice
BrtlIV/WT mice
Le Blanc et al (2005)
Guillot et al (2008)
Panaroni et al (2009)
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32 Oim (20 mice were not treated and 12 mice received fetal MSCs) þ 15 WT mice
169 (82 BrtlIV mice and 87 WT mice)
77 (48 mice not received MSCs and 29 received)
1
1
Sample size
Human fetal blood stem cells
E13.5–E15
E13.5–E14.5
E13.5–E15
Human fetal blood cells
Bone marrow cells expressing e-GFP from transgenic mice
32nd wk of gestation
29th wk of gestation
Time of delivery of MSCs
Human fetal MSCs of male origin
Human fetal MSCs of male origin
Source of MSCs
Intraperitoneal
Intrahepatic
Intraperitoneal
Intravascular
Intravascular
Route of MSCs delivery
5%
2%
5%
7%
5%
Engraftment levels
content and bone histology
trabeculae
thickness
Increased survival rate/decreased lethality Increased cortical thickness Improvement of matrix quality Improvement of bone mechanics (stiffness, yield force and ultimate force)
Reduction in femoral fractures (84%) Decreased hydroxyproline content in bone Increased bone matrix stiffness Decreased bone brittleness No changes in bone morphology “cortical and trabecular thickness” • Protein-mineral structure of bones contained more mature apatite crystal in females
• • • • •
• • • •
remodeling (growth plates and fracture sites)
• More donor cells at the sites of bone
• Two-thirds reduction in fracture incidence • Increased bone length, strengths and
• Normal psychomotor development • Increased growth velocity
• Good arrangement of matrix and
• Decreased fracture incidence
• Improvement of bone mineral
Results
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Abbreviations: E, embryonic day; e-GFP, enhanced green fluorescent protein; IUCT, in utero stem cell transplantation; MSCs; mesenchymal stem cells; OI, osteogenesis imperfecta; WT, wild type.
Oim/Oim mice and WT mice
Female fetus
Westgren et al (2003)
Vanleene et al (2011)
Model/fetal sex
Author
Table 3 Summary of human and animal IUSCT trials in the treatment of OI
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of stem cells (both were treated with fetal MSCs), or route of stem cell delivery (both were delivered intravenously). Surprisingly, the level of engraftment did not explain this discrepancy but was even higher in the second case (7 vs. 5%). Furthermore, the degree of improvement reported by Le Blanc et al could not be definitely attributed to IUSCT because the patient started an adjuvant medical treatment. A last variable that may explain this discrepancy was the type of OI itself. The second patient was diagnosed with OI type II, which is the most severe form of the disease, and this may partially explain the less favorable outcome reported in this case. Unfortunately, the type of OI was not reported in the first case and accordingly, we cannot confirm this explanation. We still need to understand this discrepancy to evaluate the prognosis of IUSCT and the factors that determine patient eligibility for IUSCT. Westgren suggested that the difference might be explained by the type of stem cells he used (adult versus fetal MSCs).33 All failed cases including unpublished cases of Porta and Chile used adult MSCs. However, this does not explain our first dilemma because both human cases were treated with fetal MSCs. Therefore, we may consider the type of OI as a possible predictor of treatment outcome. Unlike human trials, animal trials showed uniformly promising outcomes. Unfortunately, these trials involved only two animal models (Oim/Oim and BrtlIV) that were used to present types III and IV of the disease, respectively. The level of engraftment was comparable to that of human studies. In both types of studies, engraftment level was low (2–7%) and accordingly, engraftment alone cannot explain the degree of improvement. This conclusion was not limited to in utero studies; Jones et al conducted a postnatal (day 2–3) animal trial on Oim/WT mice. Their aim was to enhance bone engraftment by priming human fetal MSCs with stromal driving factor 1, which increases the expression of CXCR4 on the cell surface. This subsequently enhanced chemotaxis and engraftment in bone and bone marrow of both mice. However, the authors reported there was no significant reduction in the incidence of bone fractures despite the increase in engraftment level.46 The results were comparable in the three animal studies regardless of mice gender, type, and source of stem cells, route of stem cell delivery and type of OI. No clear predictors of improvement could be identified in animal studies. Unfortunately, the most lethal type II OI was not investigated (unlike human trials). Researchers may need to consider the type of OI as a predictor of success in further studies. While Vanleene et al 2011 suggested more favorable outcome in female mice, patient sex should be also more investigated. Another point of controversy was how postnatal assessment following IUSCT can predict treatment success and favorable outcomes. Histological and biochemical tests have been more applicable in animal studies. The aim was to understand how treatment may benefit the patients and what histological or biochemical markers may predict long-term outcome. Again, reduced fracture rate was observed in the three animal experiments but increased cortical thickness was observed only in the first two experiments. Surprisingly, this difference was not associated with significantly different outcomes. Accordingly, American Journal of Perinatology
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it seems that bone mechanics (including bone stiffness) were more important than bone thickness. Bone mechanics are not as simple to assess as cortical thickness and according to these results, considering cortical thickness, which may even be assessed in utero, may be misleading. Unfortunately, all human and animal studies lack long-term follow-up. Therefore, our evaluation of distant outcome is lacking. Finally, it is essential to understand the hazards of this approach to weigh them against potential benefits. Unfortunately, the limited number of cases recruited in IUSCT studies makes it difficult to reveal these hazards. However, it is apparent that the potential risks of IUSCT are generally attributed to the procedure, by which stem cells are delivered to the fetus. Accordingly, the risks of abortion, fetomaternal hemorrhage, and infection are expected. In one IUSCT study, the risk of abortion involved two out of six fetuses.47 However, it is difficult to determine whether abortion was attributed to the procedure itself, to the severity of the disease or to the presence of possible lethal genetic associations. Moreover, there is an additional risk of fetal immunological response against transplanted cells if cells are introduced after 12 weeks of gestation.48 This is a general complication of IUSCT that is fortunately not expected in fetuses treated with MSCs because of their low immunogenicity.
Conclusion In conclusion, IUSCT is a promising future option for treatment of OI. However, more studies are needed to evaluate the exact efficacy of this intervention and the possible predictors of success to improve patient selection. The ideal type of stem cells (adult vs. fetal MSCs) needs to be investigated. The results need to be correlated carefully to different variables including sex and type of OI. Long-term outcomes should be investigated and different histological and biochemical investigations should be correlated with treatment outcome. This helps to determine clear parameters for future prognosis of this future therapeutic option.
Acknowledgments The authors would like to thank all the members of the mentor–student research link pilot project (a part of the Healthcare Reform Egypt; HRE) to which this research project belongs. They appreciate their great effort to develop a strong research system and to improve the skills of young researchers in Egypt. This represents a fundamental part of the national medical development goals of this organization.
Conflict of Interest The authors have no conflict of interest.
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American Journal of Perinatology
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No. 10/2014
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IUSCT for Treatment of OI
IUSCT for Treatment of OI
Amin, Shazly
repairs bone and reduces fractures in osteogenesis imperfecta mice. Blood 2008;111(3):1717–1725 45 Vanleene M, Saldanha Z, Cloyd KL, et al. Transplantation of human fetal blood stem cells in the osteogenesis imperfecta mouse leads to improvement in multiscale tissue properties. Blood 2011; 117(3):1053–1060 46 Jones GN, Moschidou D, Lay K, et al. Upregulating CXCR4 in human fetal mesenchymal stem cells enhances engraftment and bone
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