Osteopetrosis is a rare inherited disease characterized by impaired osteoclast activity causing defective bone resorption and bone marrow aplasia. It is fatal in early childhood unless hematopoietic stem cell transplantation is performed. But, the transplant course is complicated with engraftment failure. Recently, osteoclasts have been described as the potential regulators of hematopoietic stem cell (HSC) niche. Here we investigated the alterations in the HSC and mesenchymal stromal cell (MSC) components of osteopetrotic niche and their interactions to mimic the stem cell dynamics/trafficking in the BM niche after HSC transplantation. Induced pluripotent stem cells were generated from peripheral blood mononuclear cells of patients with osteopetrosis carrying TCIRG1 mutation. iPSC lines were differentiated into hematopoietic and myeloid progenitors, then into osteoclasts using a step-wise protocol. We first demonstrated a shift toward monocyte-macrophages lineage regarding hematopoietic differentiation potential of osteopetrotic iPSC-derived hematopoietic progenitors (HPCs) and phenotypically normal and functionally defective osteoclast formation. The expression of the genes involved in HSC homing and maintenance (Sdf-1, Jagged-1, Kit-L, and Opn) in osteopetrotic MSCs recovered significantly after coculture with healthy HPCs. Similarly, the restoration of phenotype, impaired differentiation, and migratory potential of osteopetrotic iHPCs were observed upon interaction with healthy MSCs. Our results establish significant alterations in both MSC and HPC compartments of the osteopetrotic niche, and support the impact of functionally impaired osteoclasts in defective niche formation.
Background
Patients with osteopetrosis present with defective bone resorption caused by the lack of osteoclast activity and hematopoietic alterations, but their bone marrow hematopoietic stem/progenitor cell and osteoclast contents might be different. Osteoclasts recently have been described as the main regulators of HSCs niche, however, their exact role remains controversial due to the use of different models and conditions. Investigation of their role in hematopoietic stem cell niche formation and maintenance in osteopetrosis patients would provide critical information about the mechanisms of altered hematopoiesis. We used patient-derived induced pluripotent stem cells (iPSCs) to model osteoclast defect and hematopoietic niche compartments in vitro.
Methods
iPSCs were generated from peripheral blood mononuclear cells of patients carrying TCIRG1 mutation. iPSC lines were differentiated first into hematopoietic stem cells-(HSCs), and then into myeloid progenitors and osteoclasts using a step-wise protocol. Then, we established different co-culture conditions with bone marrow-derived hMSCs and iHSCs of osteopetrosis patients as an in vitro hematopoietic niche model to evaluate the interactions between osteopetrotic-HSCs and bone marrow-derived MSCs as osteogenic progenitor cells.
Results
We first demonstrated myeloid-skewed hematopoietic differentiation potential of osteopetrotic iPSC-derived hematopoietic progenitors and phenotypically normal and functionally defective osteoclast formation. Upon co-culture with healthy iHSCs, the expression of the genes involved in HSC homing and maintenance (Ang-1, Sdf-1, Jagged-1, N-Cadherine, Kit-L, Opn) in osteopetrotic MSCs which revealed impaired osteogeneic differentiation, as well as their attraction ability over HSCs recovered significantly. Similar change in the phenotype of osteopetrotic iHSCs occured when they interacted with healthy MSCs.
Conclusion
Our results establish significant alterations in both MSC and HSC compartments of the hematopoietic niche in osteopetrosis patients, which are restored with normal MSC activity supporting the role of defective osteoclasts in all these processes.
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