Int J Stem Cells.  2022 May;15(2):227-232. 10.15283/ijsc21138.

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

• Azevedo PL
• Dias RB
• Nogueira LP
• Maradei S
• Bigni R
• Aragao JSR
• Abdelhay E
• Binato R

• KMID: 2529837
• DOI: http://doi.org/10.15283/ijsc21138

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.

Keyword

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

Figure

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  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.

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  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.

Reference

References

1. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE. 1994; A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 367:645–648. DOI: 10.1038/367645a0. PMID: 7509044.

2. Shlush LI, Mitchell A, Heisler L, Abelson S, Ng SWK, Trotman-Grant A, Medeiros JJF, Rao-Bhatia A, Jaciw-Zurakowsky I, Marke R, McLeod JL, Doedens M, Bader G, Voisin V, Xu C, McPherson JD, Hudson TJ, Wang JCY, Minden MD, Dick JE. 2017; Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature. 547:104–108. DOI: 10.1038/nature22993. PMID: 28658204.

3. Ladikou EE, Sivaloganathan H, Pepper A, Chevassut T. 2020; Acute myeloid leukaemia in its niche: the bone marrow microenvironment in acute myeloid leukaemia. Curr Oncol Rep. 22:27. DOI: 10.1007/s11912-020-0885-0. PMID: 32048054. PMCID: PMC7012995.

4. Binato R, de Almeida Oliveira NC, Du Rocher B, Abdelhay E. 2015; The molecular signature of AML mesenchymal stromal cells reveals candidate genes related to the leukemogenic process. Cancer Lett. 369:134–143. DOI: 10.1016/j.canlet.2015.08.006. PMID: 26279521.

5. Geyh S, Rodríguez-Paredes M, Jäger P, Khandanpour C, Cadeddu RP, Gutekunst J, Wilk CM, Fenk R, Zilkens C, Hermsen D, Germing U, Kobbe G, Lyko F, Haas R, Schroeder T. 2016; Functional inhibition of mesenchymal stromal cells in acute myeloid leukemia. Leukemia. 30:683–691. DOI: 10.1038/leu.2015.325. PMID: 26601782.

6. Diaz de la Guardia R, Lopez-Millan B, Lavoie JR, Bueno C, Castaño J, Gómez-Casares M, Vives S, Palomo L, Juan M, Delgado J, Blanco ML, Nomdedeu J, Chaparro A, Fuster JL, Anguita E, Rosu-Myles M, Menéndez P. 2017; Detailed characterization of mesenchymal stem/stromal cells from a large cohort of AML patients demonstrates a definitive link to treatment outcomes. Stem Cell Reports. 8:1573–1586. DOI: 10.1016/j.stemcr.2017.04.019. PMID: 28528702. PMCID: PMC5470078.

7. Goldman DC, Bailey AS, Pfaffle DL, Al Masri A, Christian JL, Fleming WH. 2009; BMP4 regulates the hematopoietic stem cell niche. Blood. 114:4393–4401. DOI: 10.1182/blood-2009-02-206433. PMID: 19759357. PMCID: PMC2777124.

8. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. 2006; Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8:315–317. DOI: 10.1080/14653240600855905. PMID: 16923606.

9. Azevedo PL, Oliveira NCA, Corrêa S, Castelo-Branco MTL, Abdelhay E, Binato R. 2019; Canonical WNT signaling pathway is altered in mesenchymal stromal cells from acute myeloid leukemia patients and is implicated in BMP4 down-regulation. Transl Oncol. 12:614–625. DOI: 10.1016/j.tranon.2019.01.003. PMID: 30703678. PMCID: PMC6350721.

10. Dias RB, Guimarães JAM, Cury MB, Rocha LR, da Costa ES, Nogueira LP, Hochman-Mendez C, Fortuna-Costa A, Silva AKF, Cunha KS, de Souza SAL, Duarte MEL, Sartore RC, Bonfim DC. 2019; The manufacture of GMP-grade bone marrow stromal cells with validated in vivo bone-forming potential in an orthopedic clinical center in Brazil. Stem Cells Int. 2019:2608482. DOI: 10.1155/2019/2608482. PMID: 31781235. PMCID: PMC6875385. PMID: 4391de146b3c4e978a2ebfd0737c7c7a.

11. Object Research Systems. 2020. Dragonfly 2020 [Internet]. Object Research Systems;Montreal: Available from: http://www.theobjects.com/dragonfly/. cited 2020 Jul 20.

12. Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Müller R. 2010; Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res. 25:1468–1486. DOI: 10.1002/jbmr.141. PMID: 20533309.

13. Robey PG, Kuznetsov SA, Riminucci M, Bianco P. 2014; Bone marrow stromal cell assays: in vitro and in vivo. Methods Mol Biol. 1130:279–293. DOI: 10.1007/978-1-62703-989-5_21. PMID: 24482181. PMCID: PMC4152770.

14. Pievani A, Donsante S, Tomasoni C, Corsi A, Dazzi F, Biondi A, Riminucci M, Serafini M. 2021; Acute myeloid leukemia shapes the bone marrow stromal niche in vivo. Haematologica. 106:865–870. DOI: 10.3324/haematol.2020.247205. PMID: 32381570. PMCID: PMC7928008.

15. Frisch BJ, Ashton JM, Xing L, Becker MW, Jordan CT, Calvi LM. 2012; Functional inhibition of osteoblastic cells in an in vivo mouse model of myeloid leukemia. Blood. 119:540–550. DOI: 10.1182/blood-2011-04-348151. PMID: 21957195. PMCID: PMC3384480.

16. Thomas S, Jaganathan BG. 2021; Signaling network regulating osteogenesis in mesenchymal stem cells. J Cell Commun Signal. doi: 10.1007/s12079-021-00635-1. [Epub ahead of print]. DOI: 10.1007/s12079-021-00635-1. PMID: 34236594. PMCID: PMC8688675.

17. Komori T. 2006; Regulation of osteoblast differentiation by transcription factors. J Cell Biochem. 99:1233–1239. DOI: 10.1002/jcb.20958. PMID: 16795049.

18. Krause DS, Scadden DT. 2015; A hostel for the hostile: the bone marrow niche in hematologic neoplasms. Haematologica. 100:1376–1387. DOI: 10.3324/haematol.2014.113852. PMID: 26521296. PMCID: PMC4825315.

19. Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I, Tagliafico E, Ferrari S, Robey PG, Riminucci M, Bianco P. 2007; Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell. 131:324–336. DOI: 10.1016/j.cell.2007.08.025. PMID: 17956733.

20. Chandran P, Le Y, Li Y, Sabloff M, Mehic J, Rosu-Myles M, Allan DS. 2015; Mesenchymal stromal cells from patients with acute myeloid leukemia have altered capacity to expand differentiated hematopoietic progenitors. Leuk Res. 39:486–493. DOI: 10.1016/j.leukres.2015.01.013. PMID: 25703353.

21. Urist MR. 1965; Bone: formation by autoinduction. Science. 150:893–899. DOI: 10.1126/science.150.3698.893. PMID: 5319761. PMCID: PMC5808255.

22. James AW. 2013; Review of signaling pathways governing MSC osteogenic and adipogenic differentiation. Scientifica (Cairo). 2013:684736. DOI: 10.1155/2013/684736. PMID: 24416618. PMCID: PMC3874981.

23. Okamoto M, Murai J, Yoshikawa H, Tsumaki N. 2006; Bone morphogenetic proteins in bone stimulate osteoclasts and osteoblasts during bone development. J Bone Miner Res. 21:1022–1033. DOI: 10.1359/jbmr.060411. PMID: 16813523.

24. Bandyopadhyay A, Tsuji K, Cox K, Harfe BD, Rosen V, Tabin CJ. 2006; Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet. 2:e216. DOI: 10.1371/journal.pgen.0020216. PMID: 17194222. PMCID: PMC1713256.

25. Zhang J, Li L. 2005; BMP signaling and stem cell regulation. Dev Biol. 284:1–11. DOI: 10.1016/j.ydbio.2005.05.009. PMID: 15963490.