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Objective: CD14+ monocyte cell lines can differentiate into an osteoclast (OC)-like lineage. However, the identification of human cell lines with stem cell characteristics, capable of differentiating into OCs, would provide a tool for the study of the molecular mechanisms regulating their commitment, differentiation, and function. Since the human acute myeloid leukemia cell line MUTZ-3 contains both CD34+ stem cell and CD14+ cell populations, we investigated the capacity of the stem/progenitor CD34+ population to differentiate into functional OCs. Materials and Methods: Sorted MUTZ-3-CD34+ and MUTZ-3-CD14+ cells were cultured in presence of M-CSF, RANK-L, and TNF-α to generate OCs. Differentiation was evaluated by TRAP staining and RT-PCR, which assessed the expression of c-fms, RANK, MMP-9, CATK, TRAP, and CTR in -CD34+OC and -CD14+OC cells. Resorption pit formation was also evaluated. CD34, CD14, M-CSF-R, RANK, and CTR expression was assessed by FACS analysis. Results: MUTZ-3-CD34+ differentiated into OCs, displaying the full range of differentiation markers; MMP-9, CATK, TRAP, and RANK mRNA were detected from day 3 of culture, whereas CTR from day 12. Stimulated MUTZ-3-CD34+ generated functional osteoclasts that formed extensive resorption lacunae on both mineralized surface and bone slices. Surprisingly, in both sorted populations we identified a population M-CSF-R+/RANK+ that at the same time co-expressed CD14 and CD34. Conclusions: These findings demonstrate that MUTZ-3 cells constitute an invaluable model to study the expression pattern in different developmental stages of commitment and differentiation. Importantly, the data indicate that the CD14+CD34+M-CSF-R+RANK+ population represents an intermediate stage of differentiation from CD34 precursors and monocytes to osteoclast. [Copyright &y& Elsevier]

Abstract: The identity of biochemical players which underpin the commitment of CD34+ hematopoietic stem cells to immunogenic or tolerogenic dendritic cells is largely unknown. To explore this issue, we employed a previously established cell-based system amenable to shift dendritic cell differentiation from the immunogenic into the tolerogenic pathway upon supplementation with a conventional cytokine cocktail containing thrombopoietin (TPO) and IL-16. We show that stringent regulation of cathepsins S and D, two proteases involved in antigen presentation, is crucial to engage cell commitment to either route. In response to TPO+IL-16-dependent signaling, both cathepsins undergo earlier maturation and down-regulation. Additionally, cystatin C orchestrates cathepsin S expression through a tight but reversible interaction that, based on a screen of adult stem cells from disparate origins, CD14+ cells, primary fibroblasts and the MCF7 cell line, appears unique to CD34+ stem cells from peripheral and cord blood. As shown by CD4+ T cell proliferation in mixed-lymphocyte reactions, cell commitment to either pathway is disrupted upon cathepsin knockdown by RNAi. Surprisingly, similar effects were also observed upon gene overexpression, which prompts atypically accelerated maturation of cathepsins S and D in cells of the immunogenic pathway, similar to the tolerogenic route. Furthermore, RNAi studies revealed that cystatin C is a proteolytic target of cathepsin D and has a direct, causal impact on cell differentiation. Together, these findings uncover a novel biochemical cluster that is subject to time-controlled and rigorously balanced expression to mediate specific stem cell commitment at the crossroads towards tolerance or immunity. [Copyright &y& Elsevier]