Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7-related osteopetrosis.

Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl- /H+ -exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

CRISPR-Cas9-Edited Tacrolimus-Resistant Antiviral T Cells for Advanced Adoptive Immunotherapy in Transplant Recipients.

Viral infections, such as with cytomegalovirus (CMV), remain a major risk factor for mortality and morbidity of transplant recipients because of their requirement for lifelong immunosuppression (IS). Antiviral drugs often cause toxicity and sometimes fail to control disease. Thus, regeneration of the antiviral immune response by adoptive antiviral T cell therapy is an attractive alternative. Our recent data, however, show only short-term efficacy in some solid organ recipients, possibly because of malfunction in transferred T cells caused by ongoing IS. We developed a vector-free clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based good manufacturing practice (GMP)-compliant protocol that efficiently targets and knocks out the gene for the adaptor protein FK506-binding protein 12 (FKBP12), required for the immunosuppressive function of tacrolimus. This was achieved by transient delivery of ribonucleoprotein complexes into CMV-specific T cells by electroporation. We confirmed the tacrolimus resistance of our gene-edited T cell products in vitro and demonstrated performance comparable with non-tacrolimus-treated unmodified T cells. The alternative calcineurin inhibitor cyclosporine A can be administered as a safety switch to shut down tacrolimus-resistant T cell activity in case of adverse effects. Furthermore, we performed safety assessments as a prerequisite for translation to first-in-human applications.

Adult Osteosclerotic Metaphyseal Dysplasia With Progressive Osteonecrosis of the Jaws and Abnormal Bone Resorption Pattern Due to a LRRK1 Splice Site Mutation.

Osteosclerotic metaphyseal dysplasia (OSMD) is a rare autosomal recessive sclerosing skeletal dysplasia. We report on a 34-year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of the tubular bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment over a period of 11 years. An iliac crest bone biopsy revealed the presence of hypermineralized cartilage remnants, large multinucleated osteoclasts with abnormal morphology, and inadequate bone resorption typical for osteoclast-rich osteopetrosis. After exclusion of mutations in TCIRG1 and CLCN7 we performed trio-based exome sequencing. The novel homozygous splice-site mutation c.261G>A in the gene LRRK1 was found and co-segregated with the phenotype in the family. cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33). Osteoclasts differentiated from the patient's peripheral blood monocytes were extremely large. Instead of resorption pits these cells were only capable of superficial erosion. Phosphorylation of L-plastin at position Ser5 was strongly reduced in patient-derived osteoclasts showing a loss of function of the mutated LRRK1 kinase protein. Our analysis indicates a strong overlap of LRRK1-related OSMD with other forms of intermediate osteopetrosis, but an exceptional abnormality of osteoclast resorption. Like in other osteoclast pathologies an increased risk for progressive osteonecrosis of the jaws should be considered in OSMD, an intermediate form of osteopetrosis. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Generation of a human induced pluripotent stem cell line (BIHi002-A) from a patient with CLCN7-related infantile malignant autosomal recessive osteopetrosis.

Autosomal recessive osteopetrosis (ARO) is a genetic bone disease that can be caused by mutations in the CLCN7 gene preventing osteoclast-mediated bone resorption. We generated a human induced pluripotent stem cell (hiPSC) line, BIHi002-A, from peripheral blood mononuclear cells of an ARO patient carrying the CLCN7 mutations c.875G>A and c.1208G>A using Sendai viral vectors. The pluripotent identity of the BIHi002-A line was confirmed by their expression of typical markers for undifferentiated hiPSCs, their capacity to differentiate into cells of the three germ layers and by PluriTest analysis. The BIHi002-A line provides a tool for disease modelling and therapy development.