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Schiavone ML, Crisafulli L, Camisaschi C, De Simone G, Liberati FR, Palagano E, Rucci N, Ficara F, Sobacchi C. Rankl genetic deficiency and functional blockade undermine skeletal stem and progenitor cell differentiation. Stem Cell Res Ther 2024; 15:203. [PMID: 38971808 PMCID: PMC11227705 DOI: 10.1186/s13287-024-03803-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2024] [Accepted: 06/16/2024] [Indexed: 07/08/2024] Open
Abstract
BACKGROUND Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. METHODS To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl-/-) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). RESULTS We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl-/- mice as compared to wild-type. Rankl-/- mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl-/- SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. CONCLUSIONS We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.
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Affiliation(s)
- M L Schiavone
- IRCCS Humanitas Research Hospital, via Manzoni 56, Rozzano, Milan, 20089, Italy
| | - L Crisafulli
- IRCCS Humanitas Research Hospital, via Manzoni 56, Rozzano, Milan, 20089, Italy
- Institute for Genetic and Biomedical Research, Milan Unit, CNR, via Fantoli 16/15, Milan, 20138, Italy
| | - C Camisaschi
- Flow Cytometry Core, IRCCS Humanitas Research Hospital, via Manzoni 56, Rozzano, Milan, 20089, Italy
| | - G De Simone
- Flow Cytometry Core, IRCCS Humanitas Research Hospital, via Manzoni 56, Rozzano, Milan, 20089, Italy
| | - F R Liberati
- IRCCS Humanitas Research Hospital, via Manzoni 56, Rozzano, Milan, 20089, Italy
| | - E Palagano
- Institute of Biosciences and Bioresources, CNR, via Madonna Del Piano 10, Sesto Fiorentino, 50019, FI, Italy
| | - N Rucci
- Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, Via Vetoio - Coppito 2, L'Aquila, 67100, Italy
| | - F Ficara
- IRCCS Humanitas Research Hospital, via Manzoni 56, Rozzano, Milan, 20089, Italy
- Institute for Genetic and Biomedical Research, Milan Unit, CNR, via Fantoli 16/15, Milan, 20138, Italy
| | - Cristina Sobacchi
- IRCCS Humanitas Research Hospital, via Manzoni 56, Rozzano, Milan, 20089, Italy.
- Institute for Genetic and Biomedical Research, Milan Unit, CNR, via Fantoli 16/15, Milan, 20138, Italy.
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Maurizi A. Experimental therapies for osteopetrosis. Bone 2022; 165:116567. [PMID: 36152941 DOI: 10.1016/j.bone.2022.116567] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/03/2022] [Revised: 09/13/2022] [Accepted: 09/20/2022] [Indexed: 11/17/2022]
Abstract
The medical treatment of osteopetrosis is an ongoing clinical problem. There are no effective and safer therapeutic approaches for all its forms. However, recent discoveries concerning the etiology and the pathogenesis of osteopetrosis, the development of dedicated cellular and animal models, and the advent of new technologies are paving the way for the development of targeted and safer therapies for both lethal and milder osteopetrosis. This review summarizes the huge effort and successes made by researchers to identify and develop new experimental approaches with this objective, such as the use of non-genotoxic myeloablation, gene correction of inducible Pluripotent Stem Cells (iPSCs), lentiviral-based gene therapy, protein replacement, prenatal treatment, osteoclast precursors transplantation and RNA Interference.
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Affiliation(s)
- Antonio Maurizi
- Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy.
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3
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Vacher J. OSTM1 pleiotropic roles from osteopetrosis to neurodegeneration. Bone 2022; 163:116505. [PMID: 35902071 DOI: 10.1016/j.bone.2022.116505] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Revised: 07/18/2022] [Accepted: 07/20/2022] [Indexed: 11/28/2022]
Abstract
Autosomal recessive osteopetroses (ARO) are rare genetic skeletal disorders of high clinical and molecular heterogeneity with an estimated frequency of 1:250,000 worldwide. The manifestations are diverse and although individually rare, the various forms contribute to the prevalence of a significant number of affected individuals with considerable morbidity and mortality. Among the ARO classification, the most severe form is the autosomal recessive-5 (OPTB5) osteopetrosis (OMIM 259720) that results from homozygous mutation in the OSTM1 gene (607649). OSTM1 mutations account for approximately 5 % of instances of autosomal recessive osteopetrosis and lead to a highly debilitating form of the disease in infancy and death within the first few years of life (Sobacchi et al., 2013) [1].
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Affiliation(s)
- Jean Vacher
- Institut de Recherches Cliniques de Montréal (IRCM), 110 avenue des Pins Ouest, Montréal, Québec H2W 1R7, Canada; Département de Médecine, Université de Montréal, Montréal, Québec, Canada.
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Shi C, Pan L, Hu Z. Experimental and clinical progress of in utero hematopoietic cell transplantation therapy for congenital disorders. Front Pharmacol 2022; 13:851375. [PMID: 36120324 PMCID: PMC9478511 DOI: 10.3389/fphar.2022.851375] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2022] [Accepted: 08/08/2022] [Indexed: 11/13/2022] Open
Abstract
In utero hematopoietic cell transplantation (IUHCT) is considered a potentially efficient therapeutic approach with relatively few side effects, compared to adult hematopoietic cell transplantation, for various hematological genetic disorders. The principle of IUHCT has been extensively studied in rodent models and in some large animals with close evolutionary similarities to human beings. However, IUHCT has only been used to rebuild human T cell immunity in certain patients with inherent immunodeficiencies. This review will first summarize the animal models utilized for IUHCT investigations and describe the associated outcomes. Recent advances and potential barriers for successful IUHCT are discussed, followed by possible strategies to overcome these barriers experimentally. Lastly, we will outline the progress made towards utilizing IUHCT to treat inherent disorders for patients, list out associated limitations and propose feasible means to promote the efficacy of IUHCT clinically.
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Affiliation(s)
- Chunyu Shi
- National-Local Joint Engineering Laboratory of Animal Models for Human Diseases, The First Hospital of Jilin University, Changchun, China
- Department of Gastrointestinal Colorectal and Anal Surgery, China-Japan Union Hospital of Jilin University, Changchun, China
| | - Lu Pan
- Department of Pediatric Immunology, Allergy and Rheumatology, The First Hospital of Jilin University, Changchun, China
| | - Zheng Hu
- National-Local Joint Engineering Laboratory of Animal Models for Human Diseases, The First Hospital of Jilin University, Changchun, China
- *Correspondence: Zheng Hu,
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5
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Penna S, Villa A, Capo V. Autosomal recessive osteopetrosis: mechanisms and treatments. Dis Model Mech 2021; 14:261835. [PMID: 33970241 PMCID: PMC8188884 DOI: 10.1242/dmm.048940] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Autosomal recessive osteopetrosis (ARO) is a severe inherited bone disease characterized by defective osteoclast resorption or differentiation. Clinical manifestations include dense and brittle bones, anemia and progressive nerve compression, which hamper the quality of patients' lives and cause death in the first 10 years of age. This Review describes the pathogenesis of ARO and highlights the strengths and weaknesses of the current standard of care, namely hematopoietic stem cell transplantation (HSCT). Despite an improvement in the overall survival and outcomes of HSCT, transplant-related morbidity and the pre-existence of neurological symptoms significantly limit the success of HSCT, while the availability of human leukocyte antigen (HLA)-matched donors still remains an open issue. Novel therapeutic approaches are needed for ARO patients, especially for those that cannot benefit from HSCT. Here, we review preclinical and proof-of-concept studies, such as gene therapy, systematic administration of deficient protein, in utero HSCT and gene editing. Summary: Autosomal recessive osteopetrosis is a heterogeneous and rare bone disease for which effective treatments are still lacking for many patients. Here, we review the literature on clinical, preclinical and proof-of-concept studies.
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Affiliation(s)
- Sara Penna
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan 20132, Italy.,Translational and Molecular Medicine (DIMET), University of Milano-Bicocca, Monza 20900, Italy
| | - Anna Villa
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan 20132, Italy.,Institute of Genetic and Biomedical Research, Milan Unit, National Research Council, Milan 20090, Italy
| | - Valentina Capo
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan 20132, Italy.,Institute of Genetic and Biomedical Research, Milan Unit, National Research Council, Milan 20090, Italy
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Generation of an immunodeficient mouse model of tcirg1-deficient autosomal recessive osteopetrosis. Bone Rep 2020; 12:100242. [PMID: 31938717 PMCID: PMC6953598 DOI: 10.1016/j.bonr.2020.100242] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Revised: 12/23/2019] [Accepted: 01/04/2020] [Indexed: 01/16/2023] Open
Abstract
Background Autosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the TCIRG1 gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected ex vivo through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated in vivo because a relevant animal model for xenotransplant is missing. Methods We generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells. Results We demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34+ cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred. Conclusion Our work paves the way to generating an improved xenograft model for in vivo evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies.
Ex vivo corrected autologous HSCs might cure Autosomal Recessive Osteopetrosis (ARO). There is no animal model to prove in vivo functional rescue of corrected human cells. NSG oc/oc mice display osteoclast-rich cell-autonomous ARO and immunodeficiency. Human CD34+ cell-transplanted NSG oc/oc mice show human cell chimerism in the BM. Further improvements will allow in vivo evaluating corrected patient-derived cells.
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Penna S, Capo V, Palagano E, Sobacchi C, Villa A. One Disease, Many Genes: Implications for the Treatment of Osteopetroses. Front Endocrinol (Lausanne) 2019; 10:85. [PMID: 30837952 PMCID: PMC6389615 DOI: 10.3389/fendo.2019.00085] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/14/2018] [Accepted: 01/31/2019] [Indexed: 11/23/2022] Open
Abstract
Osteopetrosis is a condition characterized by increased bone mass due to defects in osteoclast function or formation. In the last decades, the molecular dissection of osteopetrosis has unveiled a plethora of molecular players responsible for different forms of the disease, some of which present also primary neurodegeneration that severely limits the therapy. Hematopoietic stem cell transplantation can cure the majority of them when performed in the first months of life, highlighting the relevance of an early molecular diagnosis. However, clinical management of these patients is constrained by the severity of the disease and lack of a bone marrow niche that may delay immune reconstitution. Based on osteopetrosis genetic heterogeneity and disease severity, personalized therapies are required for patients that are not candidate to bone marrow transplantation. This review briefly describes the genetics of osteopetrosis, its clinical heterogeneity, current therapy and innovative approaches undergoing preclinical evaluation.
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Affiliation(s)
- Sara Penna
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), San Raffaele Hospital, Milan, Italy
- Translational and Molecular Medicine (DIMET), University of Milano-Bicocca, Monza, Italy
| | - Valentina Capo
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), San Raffaele Hospital, Milan, Italy
| | - Eleonora Palagano
- The National Research Council (CNR) Institute for Genetic and Biomedical Research (IRGB)- CNR-IRGB, Milan Unit, Milan, Italy
- Humanitas Research Hospital, Rozzano, Italy
| | - Cristina Sobacchi
- The National Research Council (CNR) Institute for Genetic and Biomedical Research (IRGB)- CNR-IRGB, Milan Unit, Milan, Italy
- Humanitas Research Hospital, Rozzano, Italy
| | - Anna Villa
- San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), San Raffaele Hospital, Milan, Italy
- The National Research Council (CNR) Institute for Genetic and Biomedical Research (IRGB)- CNR-IRGB, Milan Unit, Milan, Italy
- *Correspondence: Anna Villa
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8
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Blair HC, Soboloff J, Robinson LJ, Tourkova IL, Larrouture QC, Witt MR, Holaskova I, Schafer R, Elliott M, Hirsch R, Barnett JB. Suppression of arthritis-induced bone erosion by a CRAC channel antagonist. RMD Open 2016; 2:e000093. [PMID: 26819750 PMCID: PMC4716559 DOI: 10.1136/rmdopen-2015-000093] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2015] [Revised: 11/10/2015] [Accepted: 12/10/2015] [Indexed: 12/22/2022] Open
Abstract
OBJECTIVE We have shown in vitro and in vivo that osteoclast maturation requires calcium-release activated calcium (CRAC) channels. In inflammatory arthritis, osteoclasts mediate severe and debilitating bone erosion. In the current study, we assess the value of CRAC channels as a therapeutic target to suppress bone erosion in acute inflammatory arthritis. METHODS Collagen-induced arthritis (CIA) was induced in mice. The CRAC channel inhibitor 3,4-dichloropropionaniline (DCPA) and a placebo was administered 1 day prior to collagen II booster to induce arthritis. Effects on swelling, inflammatory cell invasion in joints, serum cytokines and bone erosion were measured. RESULTS Assays, by blinded observers, of arthritis severity showed that DCPA, 21 mg/kg/day, suppressed arthritis development over 3 weeks. Bone and cartilage damage in sections of animal feet was reduced approximately 50%; overall swelling of joints was reduced by a similar amount. Effects on bone density by µCT showed clear separation in DCPA-treated CIA animals from CIA without treatment, while differences between controls without CIA and CIA treated with DCPA differed by small amounts and in most cases were not statistically different. Response was not related to anticollagen titres. There were no adverse effects in the treated group on animal weight or activity, consistent with low toxicity. The effect was maximal 12-17 days after collagen booster, during the rapid appearance of arthritis in untreated CIA. At 20 days after treatment (day 40), differences in arthritis score were reduced and tumour necrosis factor α, interleukin (IL)-1, or IL-6 in the serum of the animals were similar in treated and untreated animals. CONCLUSIONS DCPA, a novel inhibitor of CRAC channels, suppresses bone erosion associated with acute arthritis in mice and might represent a new treatment modality for acute arthrits.
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Affiliation(s)
- Harry C Blair
- Departments of Pathology and of Cell Biology, The Pittsburgh VA Medical Center and the University of Pittsburgh, Pittsburgh, USA
| | - Jonathan Soboloff
- Fels Institute for Cancer Research and Molecular Biology and the Department of Medical Genetics & Molecular Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
| | - Lisa J Robinson
- Departments of Pathology and of Microbiology, Immunology & Cell Biology, West Virginia University School of Medicine, Morgantown, West Virginia, USA
| | - Irina L Tourkova
- Departments of Pathology and of Cell Biology, The Pittsburgh VA Medical Center and the University of Pittsburgh, Pittsburgh, USA
| | - Quitterie C Larrouture
- Departments of Pathology and of Cell Biology, The Pittsburgh VA Medical Center and the University of Pittsburgh, Pittsburgh, USA
| | - Michelle R Witt
- Departments of Pathology and of Cell Biology, The Pittsburgh VA Medical Center and the University of Pittsburgh, Pittsburgh, USA
| | - Ida Holaskova
- Department of Microbiology, Immunology & Cell Biology, and the Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown, West Virginia, USA
| | - Rosana Schafer
- Department of Microbiology, Immunology & Cell Biology, and the Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown, West Virginia, USA
| | - Meenal Elliott
- Department of Microbiology, Immunology & Cell Biology, and the Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown, West Virginia, USA
| | - Raphael Hirsch
- Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City,
| | - John B Barnett
- Department of Microbiology, Immunology & Cell Biology, and the Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown, West Virginia, USA
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9
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Jahan E, Rafiq AM, Otani H. In utero and exo utero fetal surgery on histogenesis of organs in animals. World J Surg Proced 2015; 5:198-207. [DOI: 10.5412/wjsp.v5.i2.198] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2014] [Revised: 01/22/2015] [Accepted: 03/18/2015] [Indexed: 02/06/2023] Open
Abstract
Until recently, fetal surgery was only used for fetuses with very poor prognosis who were likely to die without intervention. With advances in imaging, endoscopic techniques, anesthesia and novel interventions, fetal surgery is becoming a realistic option for conditions with less severe prognoses, where the aim is now to improve quality of life rather than simply allow survival. Until forty years ago, the uterus shielded the fetus from observation and therapy. Rapid changes in the diagnosis and treatment of human fetal anatomical abnormalities are due to improved fetal imaging studies, fetal sampling techniques (e.g., amniocentesis and chorionic villus sampling), and a better understanding of fetal pathophysiology derived from laboratory animals. Fetal therapy is the logical culmination of progress in fetal diagnosis. In other words, the fetus is now a patient. Now-a-days, in utero (IU) and exo utero (EU) surgical methods are popular for experimental analyses of the histogenesis of organ development. Using these surgical methods, developmental anomalies can be created and then repaired. By applying microinjection and/or fetal surgery with these methods, models of developmental anomalies such as neural tube defects, temporomandibular joint defects, hip joint defects, digit amputation, limb and digit development and regeneration, and tooth germ transplantation in the jaw could be created and later observed. After observing different types of anomalies, novel IU and EU surgical techniques would be the best approach for repairing or treating those anomalies or diseases. This review will focus on the rationale for the IU and EU creation of animal models of different organ defects or anomalies and their repair, based on analyses of organ histogenesis and pathologic observations. It will also focus in detail on the surgical techniques of both IU and EU methods.
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Sobacchi C, Schulz A, Coxon FP, Villa A, Helfrich MH. Osteopetrosis: genetics, treatment and new insights into osteoclast function. Nat Rev Endocrinol 2013; 9:522-36. [PMID: 23877423 DOI: 10.1038/nrendo.2013.137] [Citation(s) in RCA: 388] [Impact Index Per Article: 32.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Osteopetrosis is a genetic condition of increased bone mass, which is caused by defects in osteoclast formation and function. Both autosomal recessive and autosomal dominant forms exist, but this Review focuses on autosomal recessive osteopetrosis (ARO), also known as malignant infantile osteopetrosis. The genetic basis of this disease is now largely uncovered: mutations in TCIRG1, CLCN7, OSTM1, SNX10 and PLEKHM1 lead to osteoclast-rich ARO (in which osteoclasts are abundant but have severely impaired resorptive function), whereas mutations in TNFSF11 and TNFRSF11A lead to osteoclast-poor ARO. In osteoclast-rich ARO, impaired endosomal and lysosomal vesicle trafficking results in defective osteoclast ruffled-border formation and, hence, the inability to resorb bone and mineralized cartilage. ARO presents soon after birth and can be fatal if left untreated. However, the disease is heterogeneous in clinical presentation and often misdiagnosed. This article describes the genetics of ARO and discusses the diagnostic role of next-generation sequencing methods. The management of affected patients, including guidelines for the indication of haematopoietic stem cell transplantation (which can provide a cure for many types of ARO), are outlined. Finally, novel treatments, including preclinical data on in utero stem cell treatment, RANKL replacement therapy and denosumab therapy for hypercalcaemia are also discussed.
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Affiliation(s)
- Cristina Sobacchi
- Unit Of Support/Institute of Genetic and Biomedical Research, Milan Unit, National Research Council, Humanitas Clinical and Research Centre, Via Manzoni 113, 20089 Rozzano, Italy
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11
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Lo Iacono N, Blair HC, Poliani PL, Marrella V, Ficara F, Cassani B, Facchetti F, Fontana E, Guerrini MM, Traggiai E, Schena F, Paulis M, Mantero S, Inforzato A, Valaperta S, Pangrazio A, Crisafulli L, Maina V, Kostenuik P, Vezzoni P, Villa A, Sobacchi C. Osteopetrosis rescue upon RANKL administration to Rankl(-/-) mice: a new therapy for human RANKL-dependent ARO. J Bone Miner Res 2012; 27:2501-10. [PMID: 22836362 DOI: 10.1002/jbmr.1712] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/20/2012] [Revised: 06/15/2012] [Accepted: 07/03/2012] [Indexed: 01/16/2023]
Abstract
In the last decades the molecular basis of monogenic diseases has been largely unraveled, although their treatment has often remained unsatisfactory. Autosomal recessive osteopetrosis (ARO) belongs to the small group of genetic diseases that are usually treated with hematopoietic stem cell transplantation (HSCT). However, this approach is not effective in the recently identified form carrying mutations in the receptor activator of NF-κB ligand (RANKL) gene. In this subset, therapy replacement approach based on RANKL delivery has a strong rationale. Here we demonstrate that the systematic administration of RANKL for 1 month to Rankl(-/-) mice, which closely resemble the human disease, significantly improves the bone phenotype and has beneficial effects on bone marrow, spleen and thymus; major adverse effects arise only when mice are clearly overtreated. Overall, we provide evidence that the pharmacological administration of RANKL represents the appropriate treatment option for RANKL-deficient ARO patients, to be validated in a pilot clinical trial.
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Affiliation(s)
- Nadia Lo Iacono
- UOS/IRGB, Milan Unit, CNR, Milan, Italy; Humanitas Clinical and Research Center, Rozzano, Italy
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12
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Nijagal A, Flake AW, MacKenzie TC. In utero hematopoietic cell transplantation for the treatment of congenital anomalies. Clin Perinatol 2012; 39:301-10. [PMID: 22682381 DOI: 10.1016/j.clp.2012.04.004] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy for the treatment of common hematopoietic disorders and for inducing immune tolerance in the fetus. Although the efficacy of IUHCTx has been demonstrated in multiple small and large animal models, the clinical application of this technique in humans has had limited success. Recent studies in mice have demonstrated that the maternal immune system plays a critical role in limiting engraftment in the fetus. This article reviews the therapeutic rationale of IUHCTx, potential barriers to its applications, and recent experimental strategies to improve its clinical success.
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Affiliation(s)
- Amar Nijagal
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Surgery, 513 Parnassus Avenue, San Francisco, CA 94143-0570, USA
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13
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Henriksen K, Flores C, Thomsen JS, Brüel AM, Thudium CS, Neutzsky-Wulff AV, Langenbach GEJ, Sims N, Askmyr M, Martin TJ, Everts V, Karsdal MA, Richter J. Dissociation of bone resorption and bone formation in adult mice with a non-functional V-ATPase in osteoclasts leads to increased bone strength. PLoS One 2011; 6:e27482. [PMID: 22087326 PMCID: PMC3210177 DOI: 10.1371/journal.pone.0027482] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2011] [Accepted: 10/17/2011] [Indexed: 01/23/2023] Open
Abstract
Osteopetrosis caused by defective acid secretion by the osteoclast, is characterized by defective bone resorption, increased osteoclast numbers, while bone formation is normal or increased. In contrast the bones are of poor quality, despite this uncoupling of formation from resorption.To shed light on the effect of uncoupling in adult mice with respect to bone strength, we transplanted irradiated three-month old normal mice with hematopoietic stem cells from control or oc/oc mice, which have defective acid secretion, and followed them for 12 to 28 weeks.Engraftment levels were assessed by flow cytometry of peripheral blood. Serum samples were collected every six weeks for measurement of bone turnover markers. At termination bones were collected for µCT and mechanical testing. An engraftment level of 98% was obtained. From week 6 until termination bone resorption was significantly reduced, while the osteoclast number was increased when comparing oc/oc to controls. Bone formation was elevated at week 6, normalized at week 12, and reduced onwards. µCT and mechanical analyses of femurs and vertebrae showed increased bone volume and bone strength of cortical and trabecular bone.In conclusion, these data show that attenuation of acid secretion in adult mice leads to uncoupling and improves bone strength.
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14
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Cenni E, Scioscia L, Baldini N. Orthopaedic research in italy: state of the art. Int J Immunopathol Pharmacol 2011; 24:157-78. [PMID: 21669157 DOI: 10.1177/03946320110241s230] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
The most significant results in experimental and clinical orthopaedic research in Italy within the last three years have been primarily in major congenital diseases, bone tumors, regenerative medicine, joint replacements, spine, tendons and ligaments. The data presented in the following discussion is comparable with leading international results, highlighting Italian orthopaedic research excellemce as well as its shortcomings.
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Affiliation(s)
- E Cenni
- Istituto Ortopedico Rizzoli, Bologna, Italy
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Migration of cells from the yolk sac to hematopoietic tissues after in utero transplantation of early and mid gestation canine fetuses. Transplantation 2011; 91:723-30. [PMID: 21325997 DOI: 10.1097/tp.0b013e31820c85bc] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
BACKGROUND In utero hematopoietic cell transplantation offers a means of early intervention for the treatment of diseases before birth. Delivery of cells to the yolk sac is a minimally invasive approach that results in low levels of chimerism. However, there is little information on the optimal doses, timing of delivery, and migration of transplanted cells from the yolk sac into the fetus. METHODS Varying cell doses of mesenchymal stromal cells or bone marrow mononuclear cells labeled with fluorescent supraparamagnetic iron oxide nanoparticles and a fluorescent intracellular dye, 5- and 6-([(4-chloromethyl)benzoyl]-amino) tetramethylrhodamine, were transplanted under ultrasound guidance to the yolk sacs of day 25 or day 35 canine fetuses. Ex vivo whole body fluorescence imaging and microscopy of tissue sections were correlated with the presence of iron oxide in injected and control fetuses. RESULTS Day 25 and day 35 recipients showed similar survival rates after injection of cells into yolk sacs, although increased fetal morality was associated with cell doses greater than 10 cells/kg to day 25 fetuses. The fluorescence and iron oxide signals were predominantly localized to the abdominal regions, with no fluorescence visible in yolk sacs. Microscopy of tissues revealed colocalization of fluorophore with iron oxide in donor cells detected in the fetal livers and bone marrow of recipients 7 and 17 days after receiving mesenchymal stromal cells or bone marrow mononuclear cells. CONCLUSIONS These studies demonstrated that cells injected into the yolk sacs of early gestation canine fetuses migrate to recipient hematopoietic tissues. Thus, yolk sac injection offers a safe and effective approach for engraftment of cells to fetal hematopoietic tissues.
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Flores C, de Vries TJ, Moscatelli I, Askmyr M, Schoenmaker T, Langenbach GEJ, Ehinger M, Everts V, Richter J. Nonablative neonatal bone marrow transplantation rapidly reverses severe murine osteopetrosis despite low-level engraftment and lack of selective expansion of the osteoclastic lineage. J Bone Miner Res 2010; 25:2069-77. [PMID: 20568230 DOI: 10.1002/jbmr.90] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Infantile malignant osteopetrosis (IMO) is caused by lack of functional osteoclasts leading to skeletal abnormalities, blindness owing to compression of the optic nerves, bone marrow (BM) failure, and early death. In most patients, TCIRG1, a proton pump subunit essential for bone resorption, is mutated. oc/oc mice represent a model for IMO owing to a deletion in Tcirg1 and die around 4 weeks of age. To determine if hematopoietic stem cell transplantation without prior conditioning can reverse osteopetrosis, neonatal mice were transplanted intravenously with lineage-depleted BM cells. More than 85% of oc/oc mice transplanted with 5 × 10(6) cells survived long term with an engraftment of 3% to 5% in peripheral blood (PB). At 3 weeks, engraftment in the BM was 1% to 2%, but the cellularity had increased 60-fold compared with untreated oc/oc mice, and RANKL and macrophage colony-stimulating factor (M-CSF) expression in the BM was normalized. Histopathology and micro-computed tomography revealed almost complete reversal of osteopetrosis after 4 weeks. In vitro studies showed that bone resorption by osteoclasts from transplanted oc/oc mice was 14% of transplanted controls, and immunofluorescence microscopy revealed that resorption was mainly associated with osteoclasts of donor origin. Lineage analysis of BM, PB, and spleen did not provide any evidence for selective recruitment of cells to the osteoclastic lineage. The vision also was preserved in transplanted oc/oc mice, as determined by a visual tracking drum test. In summary, nonablative neonatal transplantation leading to engraftment of only a small fraction of normal cells rapidly reverses severe osteopetrosis in the oc/oc mouse model.
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Affiliation(s)
- Carmen Flores
- Molecular Medicine and Gene Therapy, Lund Stem Cell Center, University of Lund, Lund, Sweden
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Abstract
Mammalian development has been best characterized using the mouse model. Direct intervention of the postimplantation mouse embryo in utero represents one of many experimental approaches that can be used to probe mammalian embryogenesis. Experimental access to the mouse embryo is difficult, but techniques have been developed to circumvent some of the challenges of operating on the embryo in vivo. Experimental studies have been carried out on postimplantation stage embryos from E8.5 to term, so much of the gestational period is accessible for experimentation. One approach that has helped to enhance embryo accessibility was the development of surgical techniques based on the finding that embryonic development continued normally exo utero. Exo utero development refers to the surgically created condition in which the embryo develops outside of the uterine cavity, yet within the female abdominal cavity and attached, via the placenta, to the uterus. Using this approach it is feasible to carry out precise surgical manipulations of the mouse embryo without compromising embryo viability associated with postsurgery uterine contractions. In this chapter we review technical aspects of both in utero and exo utero surgical approaches and how these surgeries are used in conjunction with other experimental applications.
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Affiliation(s)
- Valérie Ngô-Muller
- CNRS EAC4413, Functional and Adaptative Biology, Physiology of the Gonadotrope Axis, Université Paris Diderot, Paris, France
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Mazzolari E, Forino C, Razza A, Porta F, Villa A, Notarangelo LD. A single-center experience in 20 patients with infantile malignant osteopetrosis. Am J Hematol 2009; 84:473-9. [PMID: 19507210 DOI: 10.1002/ajh.21447] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Infantile malignant osteopetrosis (IMO) includes various genetic disorders that affect osteoclast development and/or function. Genotype-phenotype correlation studies in IMO have been hampered by the rarity and heterogeneity of the disease and by the severity of the clinical course, which often leads to death early in life. We report on the clinical and molecular findings and treatment in 20 consecutive patients (11 males, nine females) with IMO, diagnosed at a single center in the period 1991-2008. Mean age at diagnosis was 3.9 months, and mean follow-up was 66.75 months. Mutations in TCIRG1, OSTM1, ClCN7, and TNFRSF11A genes were detected in nine, three, one, and one patients, respectively. Six patients remain genetically undefined. OSTM1 and ClCN7 mutations were associated with poor neurologic outcome. Among nine patients with TCIRG1 defects, six presented with hypogammaglobulinemia, and one showed primary pulmonary hypertension. Fourteen patients received hematopoietic cell transplantation; of these, nine are alive and eight of them have evidence of osteoclast function. These data may provide a basis for informed decisions regarding the care of patients with IMO.
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Affiliation(s)
- Evelina Mazzolari
- Department of Pediatrics, University of Brescia, 25123 Brescia, Italy.
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