Reduced Osteogenic Differentiation Potential In Vivo in Acute Myeloid Leukaemia Patients Correlates with Decreased BMP4 Expression in Mesenchymal Stromal Cells

Abstract

The osteogenic differentiation potential of mesenchymal stromal cells (hMSCs) is an essential process for the haematopoiesis and the maintenance of haematopoietic stem cells (HSCs). Therefore, the aim of this work was to evaluate this potential in hMSCs from AML patients (hMSCs-AML) and whether it is associated with BMP4 expression. The results showed that bone formation potential in vivo was reduced in hMSCs-AML compared to hMSCs from healthy donors (hMSCs-HD). Moreover, the fact that hMSCs-AML were not able to develop supportive haematopoietic cells or to differentiate into osteocytes suggests possible changes in the bone marrow microenvironment. Furthermore, the expression of BMP4 was decreased, indicating a lack of gene expression committed to the osteogenic lineage. Overall, these alterations could be associated with changes in the maintenance of HSCs, the leukaemic transformation process and the development of AML.

Keywords: Acute myeloid leukaemia (AML); BMP4 gene expression; Mesenchymal stromal cells (hMSCs); Osteogenic differentiation potential.

Fig. 1

hMSC multipotency capacity in vitro. (A, B) Undifferentiated hMSCs-HD and hMSCs-AML (100× magnification). (C, D) Adipogenic differentiation of hMSCs-HD and hMSCs-AML. The accumulation of neutral lipid vacuoles stained with Oil Red O indicates cell differentiation (20× magnification). (E, F) Osteogenic differentiation of hMSC-HDs and hMSCs-AML. Calcium deposition stained with Alizarin Red indicates cell differentiation (20× magnification). (G) BMP4 is downregulated in hMSCs-AML after 21 days of osteogenic induction. To verify BMP4 expression, we used RT-qPCR to determine changes in the mRNA expression obtained from hMSC-AML and hMSC-HD cultures. Data normalization was performed using the endogenous genes B2M and GAPDH. The bars indicate the mean mRNA expression levels (±stan-dard deviation). *p<0.01. hMSCs-HD: me-senchymal stromal cells derived from healthy donors; hMSCs-AML: mesenchymal stromal cells derived from AML patients.

Fig. 2

In vivo osteogenic potential of BMSCs. In vivo transplantation assays were performed by combining hMSCs with HA/TCP followed by subcutaneous transplantation into immunocompromised mice. (A, B) H&E staining. (A) Implants from hMSC-HD cultures and (B) hMSC-AML cul-tures. hMSCs-HD formed ectopic ossicles that were sometimes populated by host haematopoietic marrow (aste-risk). The arrowheads indicate osteo-cytes. (C∼H) Micro-CT analysis. (C) Bone tissue formed from hMSCs-HD and (D) hMSCs-AML from the 3D reconstruction of implants. For better visualization of the bone tissue for-med (red), part of the HA/TCP (grey) was removed. (H) Tissue mineral density of implants formed from hMSCs-HD and hMSCs-AML. (E) Analysis of bone volume, (F) the relationship between bone volume and tissue volume, (G) and bone tissue thickness in implants formed from hMSCs-HD and hMSCs-AML. (I∼L) Human origin of the woven bone by immu-nohistochemical analysis. Expression of BMP4 within the woven bone from (I) hMSCs-HD and (J) hMSCs-AML. Expression of Osterix within the woven bone from (K) hMSCs-HD and (L) hMSCs-AML. HA/TCP=hydroxyapatite/tricalcium phosphate.