Ciro Menale

Murine Rankl‐/‐ Mesenchymal Stromal Cells Display an Osteogenic Differentiation Defect Improved by a RANKL‐expressing Lentiviral Vector

Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL‐dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM‐MSC) lines from wild type and Rankl−/− mice, and found that Rankl−/− BM‐MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl−/− BM‐MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM‐MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL‐defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL‐dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics. Stem Cells 2017;35:1365–1377

Soluble Factors on Stage to Direct Mesenchymal Stem Cells Fate

Mesenchymal stem cells (MSCs) are multipotent stromal cells that are identified by in vitro plastic adherence, colony-forming capacity, expression of a panel of surface molecules, and ability to differentiate at least toward osteogenic, adipogenic, and chondrogenic lineages. They also produce trophic factors with immunomodulatory, proangiogenic, and antiapoptotic functions influencing the behavior of neighboring cells. On the other hand, a reciprocal regulation takes place; in fact, MSCs can be isolated from several tissues, and depending on the original microenvironment and the range of stimuli received from there, they can display differences in their essential characteristics. Here, we focus mainly on the bone tissue and how soluble factors, such as growth factors, cytokines, and hormones, present in this microenvironment can orchestrate bone marrow-derived MSCs fate. We also briefly describe the alteration of MSCs behavior in pathological settings such as hematological cancer, bone metastasis, and bone marrow failure syndromes. Overall, the possibility to modulate MSCs plasticity makes them an attractive tool for diverse applications of tissue regeneration in cell therapy. Therefore, the comprehensive understanding of the microenvironment characteristics and components better suited to obtain a specific MSCs response can be extremely useful for clinical use.

Hematopoietic stem cell transplantation corrects osteopetrosis in a child carrying a novel homozygous mutation in the FERMT3 gene

Osteopetrosis (OPT) is a rare skeletal disorder with phenotypic and genotypic heterogeneity: a variety of clinical features besides the bony defect may be present, and at least ten different genes are known to be involved in the disease pathogenesis. In the framework of this heterogeneity, we report the clinical description of a neonate, first child of consanguineous parents, who had osteoclast-rich osteopetrosis and bone marrow failure in early life, but no other usual classical features of infantile malignant OPT, such as visual or hearing impairments. Because of the severe presentation at birth, the patient received Hematopoietic Stem Cell Transplantation (HSCT) at 2months of age with successful outcome. Post-HSCT genetic investigation by means of exome sequencing identified a novel homozygous mutation in the Fermitin Family Member 3 (FERMT3) gene, which was predicted to disrupt the functionality of its protein product kindlin 3. Our report provides information relevant to physicians for recognizing patients with one of the rarest forms of infantile malignant OPT, and clearly demonstrates that HSCT cures kindlin 3 deficiency with severe phenotype.

Genetics of Osteopetrosis

Purpose of ReviewThe term osteopetrosis refers to a group of rare skeletal diseases sharing the hallmark of a generalized increase in bone density owing to a defect in bone resorption. Osteopetrosis is clinically and genetically heterogeneous, and a precise molecular classification is relevant for prognosis and treatment. Here, we review recent data on the pathogenesis of this disorder.Recent FindingsNovel mutations in known genes as well as defects in new genes have been recently reported, further expanding the spectrum of molecular defects leading to osteopetrosis.SummaryExploitation of next-generation sequencing tools is ever spreading, facilitating differential diagnosis. Some complex phenotypes in which osteopetrosis is accompanied by additional clinical features have received a molecular classification, also involving new genes. Moreover, novel types of mutations have been recognized, which for their nature or genomic location are at high risk being neglected. Yet, the causative mutation is unknown in some patients, indicating that the genetics of osteopetrosis still deserves intense research efforts.

Synonymous Mutations Add a Layer of Complexity in the Diagnosis of Human Osteopetrosis

Autosomal recessive osteopetroses (AROs) are rare, genetically heterogeneous skeletal diseases with increased bone density that are often lethal if left untreated. A precise molecular classification is relevant for the patients management, because in some subgroups hematopoietic stem cell transplantation (HSCT), which is the only curative therapy, is contraindicated. In two unrelated ARO patients, the molecular analysis revealed the presence of a synonymous variant in known ARO genes, namely in the TCIRG1 gene in one patient and in the CLCN7 in the other patient, predicted to impact on the splicing process. In the latter case, sequencing of the transcript confirmed the splicing defect, whereas in the former, for whom an RNA sample was not available, the defect was reconstructed in vitro by the minigene technology. These results strongly suggest that these synonymous changes were responsible for the disease in our patients. Our findings are novel with respect to ARO and add to the few reports in literature dealing with different diseases, underlining the importance of cDNA analysis for the correct assessment of exonic changes, even when exome sequencing is performed. In particular, we highlight the possibility that at least in some cases ARO is due to synonymous changes, erroneously considered clinically silent, in the genes already described in literature, and suggest carefully reevaluating the sequencing results of these genes when mutations are not found at a first analysis. In addition, with respect to the CLCN7 gene, we suggest that synonymous variants might also contribute to the large spectrum of severity typical of CLCN7‐dependent osteopetrosis through more subtle, but not negligible, effects on protein availability and functionality.

MSC‐Seeded Biomimetic Scaffolds as a Factory of Soluble RANKL in Rankl‐Deficient Osteopetrosis

Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell‐based therapy for RANKL‐deficient autosomal recessive osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl−/− mesenchymal stromal cells (MSCs) overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three‐dimensional (3D) culture system based on a magnesium‐doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col‐seeded murine MSCs showed improved properties, as compared to two‐dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL‐transduced cells. When implanted subcutaneously in Rankl−/− mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl−/− mice that carried scaffolds with either WT or LVhsRL‐transduced Rankl−/− MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl−/− mice. Stem Cells Translational Medicine 2019;8:22–34

3D Bone Biomimetic Scaffolds for Basic and Translational Studies with Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are recognized as an attractive tool owing to their self-renewal and differentiation capacity, and their ability to secrete bioactive molecules and to regulate the behavior of neighboring cells within different tissues. Accumulating evidence demonstrates that cells prefer three-dimensional (3D) to 2D culture conditions, at least because the former are closer to their natural environment. Thus, for in vitro studies and in vivo utilization, great effort is being dedicated to the optimization of MSC 3D culture systems in view of achieving the intended performance. This implies understanding cell–biomaterial interactions and manipulating the physicochemical characteristics of biomimetic scaffolds to elicit a specific cell behavior. In the bone field, biomimetic scaffolds can be used as 3D structures, where MSCs can be seeded, expanded, and then implanted in vivo for bone repair or bioactive molecules release. Actually, the union of MSCs and biomaterial has been greatly improving the field of tissue regeneration. Here, we will provide some examples of recent advances in basic as well as translational research about MSC-seeded scaffold systems. Overall, the proliferation of tools for a range of applications witnesses a fruitful collaboration among different branches of the scientific community.

Mutations in the Neuroblastoma Amplified Sequence gene in a family affected by Acrofrontofacionasal Dysostosis type 1

Acrofrontofacionasal Dysostosis type 1 (AFFND1) is an extremely rare, autosomal recessive syndrome, comprising facial and skeletal abnormalities, short stature and intellectual disability. We analyzed an Indian family with two affected siblings by exome sequencing and identified a novel homozygous truncating mutation in the Neuroblastoma-Amplified Sequence (NBAS) gene in the patients genome. Mutations in the NBAS gene have recently been associated with different phenotypes mainly involving skeletal formation, liver and cognitive development. The NBAS protein has been implicated in two key cellular processes, namely the non-sense mediated decay and the Golgi-to-Endoplasmic Reticulum retrograde traffic. Both functions were impaired in HEK293T cells overexpressing the truncated NBAS protein, as assessed by Real-Time PCR, Western blot analysis, co-immunoprecipitation, and immunofluorescence analysis. We examined the expression of NBAS protein in mouse embryos at various developmental stages by immunohistochemistry, and detected expression in developing chondrogenic and osteogenic structures of the skeleton as well as in the cortex, hippocampus and cerebellum, which is compatible with a role in bone and brain development. Functional genetics in the zebrafish model showed that depletion of endogenous z-nbas in fish embryos results in defective morphogenesis of chondrogenic cranial skeletal elements. Overall, our data point to a conserved function of NBAS in skeletal morphogenesis during development, support the hypothesis of a causative role of the mutated NBAS gene in the pathogenesis of AFFND1 and extend the spectrum of phenotypes associated with defects in this gene.

631. Rankl Knock-Out Mesenchymal Stromal Cells Have an Unexpected Osteogenic Differentiation Defect Which Is Improved by a RANKL-Expressing Lentiviral Vector

Osteoclast-poor RANKL-dependent Autosomal Recessive Osteopetrosis (ARO) is a rare bone disease characterized by an increase in bone density due to the failure of bone resorption by impaired osteoclast formation. Hematopoietic stem cell transplantation is not an effective therapy for this ARO form, since in bone RANKL is produced mainly by cells of mesenchymal origin. Therefore Mesenchymal Stromal Cells (MSC) transplantation together with a gene-therapy strategy to correct RANKL defect in MSC could represent a possible effective therapy. Of note, whether also MSC, besides the osteoclasts, are affected by RANKL deficiency is unknown. To verify this, we established and characterized bone marrow derived MSC (BM-MSC) lines from the Rankl−/− (KO) mouse model, which recapitulates the human disease, and from wild type (WT) mice. No differences were found between KO and WT MSC in terms of morphology, immunophenotype and proliferation capacity. However, KO MSC displayed a reduced clonogenic potential with a decrease in stemness genes expression. KO MSC were able to normally differentiate towards the adipogenic and chondrogenic lineages, while showed a significantly impaired osteogenic differentiation capacity compared to WT MSC, as demonstrated by reduced Alizarin Red staining (ARS) and expression of osteogenic genes. To confirm that this alteration was due to the lack of functional RANKL, we developed a third generation lentiviral vector expressing human soluble RANKL (hsRL) for the genetic correction of KO MSC. We first investigated lentiviral transduction in 293T cells to optimize transduction efficiency at different multiplicity of infection (MOI) ranging from 1 to 100. hsRL production increased proportionally to the MOI and was stable over time. However, the higher the MOI the higher the cytotoxicity observed. Based on these data, we performed a lentiviral hsRL transduction in KO MSC at 20 and 50 MOI, to define the optimal transduction conditions. After transduction 99.5% of MSC were GFP+. While in Rankl−/− control cells the cytokine was not detected, in corrected cells hsRL production and secretion was measurable and comparable to sRL levels in WT mouse. KO MSC stably expressing hsRL showed an improved osteogenic differentiation capacity compared to untransduced KO MSC, as demonstrated by increased ARS and expression of osteogenic genes. Moreover, the expression of RANK receptor in both MSC suggested an autocrine role of sRL as possible mechanism. Our data suggest that restoration of RANKL production in lentiviral-transduced KO MSC might not only allow osteoclast differentiation in Rankl−/− mice upon transplantation, but also improve the osteogenic differentiation defect of KO MSC.