
Although acute myeloid leukaemia (AML) is a heterogeneous disease, it is known to have a unique origin from the malignant transformation of normal haematopoietic stem cells (HSCs) into leukaemic stem cells (LSCs) (1). However, what leads to this transformation is still unclear. Several studies suggest that HSCs undergo mutation(s), which gives rise to LSCs. However, not all LSCs present these mutations (2). In this context, changes in signalling in the bone marrow (BM) microenvironment, specifically in mesenchymal stromal cell (hMSC) signalling, could promote malignant transformation (3).
hMSCs play a key role, as they provide essential signals for maintaining and regulating HSCs (3). Various studies, including work from our group, have shown that hMSCs derived from AML patients (hMSCs-AML) are molecularly and functionally altered and that the
In this sense, the aim of this study was to evaluate whether the osteogenic differentiation potential
BM-derived samples were collected from adult patients with AML at diagnosis (without any treatment) and from adult healthy donors (HDs) registered at the National Cancer Institute (Rio de Janeiro, Brazil). All samples were obtained in accordance with the guidelines of the local Ethics Committee and the Declaration of Helsinki. This study was approved by the INCA Ethics Committee (CAAE 06281419.0.0000.5274), and all participants signed informed consent forms.
hMSCs at passage 3 derived from BM samples were isolated and cultured as previously described (4) and were characterized as defined by the International Society for Cellular Therapy (8).
mRNA was extracted from hMSCs at the end of the process of inducing osteogenic differentiation
The subcutaneous xenotransplantation assay with AML-hMSCs and HD-hMSCs was performed as previously reported (10). All animal procedures were performed following the guidelines of the Institutional Animal Care and Use Committee (010/2020).
The implants obtained were processed for histology and immunohistochemistry analyses as previously reported (10). Rabbit anti-OSX antibody (sc-393325, Santa Cruz Biotechnology, USA) diluted 1:100 and goat anti-BMP-2/4 antibody (sc-6267, Santa Cruz Biotechnology, USA) diluted 1:100 were used.
MicroCT acquisition was performed as described in Dias (10). The deep learning segmentation tool available on DragonFly (11) was used to separate the newly formed bone from hydroxyapatite. Quantification was performed according to previously described methods (12).
All experiments were carried out in triplicate, and the data are expressed as the mean±standard error of the mean. The data were compared using unpaired Mann–Whitney tests, and a p-value<0.05 was considered statistically significant. Statistical analysis was performed, and graphical representations were created using GraphPad PrismTM software (GraphPad Software Inc.).
All hMSCs used in this study were confirmed by the minimum criteria established by Dominici et al., 2006 (8) (Fig. 1). We observed a reduction in the osteogenic differentiation potential of hMSCs-AML
Histological examination of the implants revealed that only hMSC-HD cultures formed ectopic ossicles with similar trabecular bone architecture and were able to support haematopoietic stroma (Fig. 2A and 2B). This reconstituted marrow stroma was filled with haematopoietic cells, which is an indicator of the multipotent capacity of hMSCs.
In micro-CT-based 3D reconstruction, bone neoformation was identified in the hMSC-HD (Fig. 2C) and hMSC-AML implants (Fig. 2D); moreover, we observed that the quality (mineral density) of the newly formed tissue was similar under both conditions (Fig. 2H). However, quantitative reductions in the bone volume formed, its thickness, and the relationship between bone volume formed and the volume of the tissue analysed were observed in the implants formed from hMSCs-AML compared to hMSCs-HD (Fig. 2E∼G). Thus, these results showed that hMSCs-AML maintained their
These results corroborate those of Alice Pievani and colleagues, who observed significant alterations in mature bone formation from hMSCs derived from paediatric AML patients (14), and Frisch and coworkers, where a reduction in mineralized bone tissue formation was described after
This potential of hMSCs to differentiate into osteoblasts in BM is an essential process for normal haematopoiesis and for the maintenance of HSCs. The process occurs from commitment towards the osteogenic lineage, osteoprogenitor cell proliferation, osteoblast maturation and bone matrix mineralization, driven by a complex network of cytokines, hormones, and growth factors (16, 17). Changes in the regulation of this process, associated with lower production of osteoblasts, may result in altered bone deposition and alter the maintenance of HSCs. It is believed that reduced bone deposition can promote the exit of quiescent HSCs from the endosteal niche, associated with an increase in the number of circulating blasts in BM (18).
Interestingly, we also observed that hMSCs-HD were able to develop a supportive haematopoietic stroma and reconstruct an
In addition,
Finally, osteoblast differentiation is a multistep process in which hMSCs differentiate into osteoblast lineage cells, including osteocytes. Osterix (OSX) is an osteoblast-spe-cific transcription factor that activates a repertoire of genes during the differentiation of preosteoblasts into mature osteoblasts and osteocytes (16). Similar to
In conclusion, the current study showed a reduction in osteogenic differentiation potential
Supplementary data including one table can be found with this article online at https://doi.org/10.15283/ijsc21138.
ijsc-15-2-227-supple.pdfThis work was financially supported by FAPERJ (E-26/202.874/2015) and CNPq (308121/2018-0).
The authors have no conflicting financial interest.
PLA performed all experiments, drafted the manuscript, participated in the study design and contributed intellectual content; RBD performed xenotransplantation, histology and immunohistochemistry assays; LPN performed X-ray microtomography (micro-CT) and morphometric quantification; SM, RBigni and JSRA provided and classified all patient samples used in this study; EA participated in the study design, provided financial support and contributed intellectual content and RB conceived the study and its design and coordination, provided financial support, drafted the manuscript and contributed intellectual content. All authors read and approved the final manuscript.
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