İnfantil Osteopetrozisli Hasta Uyarılmış Pluripotent Kök Hücreleri ile Osteoklast Defektinin Modellenmesi,Osteoklast ve Hematopoietik Niş İlişkisi

Abstract

Malignant infantile osteopetrosis is an autosomal recessive disorder. The only available treatment is allogeneic hematopoietic stem cell transplantation. The objective of this study was to model hematopoietic niche in ostepetrosis. hiPSC lines were generated from peripheral blood mononuclear cells of three patients carrying TCIRG1. IPSC lines were differentiated first into HSCs, then into myeloid progenitors and osteoclasts using a step-wise protocol. iHSCs-derived osteoclasts were characterized by scanning-electron-microscope(SEM), flow-cytometry, IF-staining and expression of osteoclast-specific molecular markers. Different coculture conditions with bone marrow-derived-hMSCs and iHSCs were set up to study the interaction between osteopetrotic-HSCs and healthy- and/or osteopetrotics-MSCs as an in vitro hematopoietic niche model. After coculture, expression of the genes in MSCs related to HSC kinetics were analyzed and migration assays were done. All iPSC lines were showed typical ESC features with regards to morphology, pluripotency surface markers and pluripotency-associated gene expression profile. All lines were differentiated succesfully into HSCs, myeloid progenitors and osteoclasts. Osteopetrotic-iPSCs-derived osteoclasts were weak positive for Cathepsin K, and TRAP, were showed weak positive actin ring and short podosome formation, and exhibited osteoclast-spesific surface markers, but showed significantly reduced expression of Cathepsin K, Calcitonin-R, and NFATC1compared to controls. Following coculture with healthy-control iHSCs, the expression of Jagged-1,Ang-1,Kit-L,Sdf-1, and Opn increased while overexpression of N-cadherin decreased after coculture in osteopetrotic MSCs. The migration potential of osteopetrotic iHCSs were found impaired compared to control-iHSCs. In conclusion, our results indicate that dysfunctional osteoclasts in osteopetrosis lead to defective HSC niche formation resulting from altered MSC compartment, and abnormal HSC homing besides insufficient marrow space resulting from defective bone resorbtion.