Page 46 - 2018_12-Haematologica-web

X. Liu et al.
that overexpression of Twist1 in MSC enhances the capac- ity to maintain human CD34+ cells in long-term culture-ini- tiating cell assays through increasing Cxcl12 expression.20 However, the effects of TWIST1 on multiple niche ele- ments and its modulation of normal HSC maintenance and leukemia progression in vivo have not been functionally characterized so far.
To explore this issue, we generated a murine model of a Twist1-deficient microenvironment. We showed that the major niche cellular components and factors changed remarkably upon Twist1 deletion, causing severe dysfunc- tion of normal HSC. Nevertheless, these alterations of the BM microenvironment promoted MLL-AF9 oncogene- induced AML progression in mouse transplantation mod- els, not only pointing to TWIST1 as an instructive signal modulating the stem cell niche, but also emphasizing the importance of the niche for AML development.
Methods
Mice
Twist1flox/flox mice were purchased from Mutant Mouse Regional Resource Centers. ER-Cre mice were a gift from Professor Weiping Yuan. C57BL/6 and B6.SJL mice were purchased from the animal facility of State Key Laboratory of Experimental Hematology. Twist1flox/flox mice were crossed with ER-Cre mice to generate ER-Cre;Twist1fl/fl and ER-Cre;Twist1+/+ mice. Eight- to 12- week old mice were used. Cre expression was induced by daily intraperitoneal injection of tamoxifen (75 mg/kg of total body weight in corn oil; Sigma-Aldrich, St. Louis, MO, USA) for 5 days. All animal procedures complied with the animal care guidelines approved by the Institutional Animal Care and Use Committees of the State Key Laboratory of Experimental Hematology.
Transplantation assays
For non-competitive BM transplantation, to create the chimeras described in Online Supplementary Figure S1A, 2x106 whole BM cells from B6.SJL (CD45.1) mice were transplanted into ER- Cre;Twist1+/+ or ER-Cre;Twist1fl/fl (CD45.2) recipients that were lethally irradiated (9.5 Gy from a Cesium source, 4-24 h before transplantation). Sixteen weeks later, tamoxifen was injected to induce Twist1 deletion. For competitive transplantation, 300 BM long-term HSC (CD45.1) from tamoxifen-treated ER-Cre;Twist1+/+ or ER-Cre;Twist1fl/fl chimeric mice were mixed with 2x105 congenic BM support cells and injected into lethally irradiated CD45.2 recipients. For the MLL-AF9 AML model, 5x105 GFP+ leukemic cells were transplanted into Twist1-deleted or control chimeric recipients.
Flow cytometry analysis and cell sorting
The BM cell suspensions were flushed from femora and tibiae. Spleen cells were pestled by the plug of a 10 mL syringe. The cells were then filtered through a 74 mm nylon mesh. For flow cyto- metric analysis of stromal cells, BM was flushed using phosphate- buffered saline with 2% bovine serum, the bones were minced with scissors, then the plugs were digested in 1 mg/mL collage- nase I (OLC) or IV (MSC and EC) (Sigma-Aldrich) dissolved in Hank’s balanced salt solution with 10% fetal bovine serum for 90 min (collagenase I) or 30 min (collagenase IV) at 37°C. The disso- ciated cells were collected and kept on ice. Cells were incubated with conjugated antibodies. Stained cells were analyzed with FACS LSR II or sorted with a FACS Aria II instrument (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed by FlowJo software.
Statistical analysis
The significance of differences between two groups was deter- mined using unpaired two-tailed Student t tests. Data are present- ed as means ± standard deviations. Overall survival curves were plotted according to the Kaplan-Meier method with the log-rank test applied for comparisons. *P<0.05, **P<0.01, ***P<0.001.
Details of other experimental procedures are given in the Online Supplementary Methods.
Results
Microenvironmental Twist1 deficiency leads
to decreased numbers of mesenchymal stem cells and mature osteoblasts, an increased proportion of endothelial cells, and altered expression of cell factor genes
To explore the role of TWIST1 in the BM niche and its regulation of HSC, we generated microenvironment Twist1-deleted and control chimeric mice according to the method described by Schreck and Saez.21,22 In brief, 2x106 BM cells from B6.SJL wild-type (WT) mice (CD45.1) were transplanted into ER-Cre;Twist1fl/fl and ER-Cre;Twist1+/+ recip- ients (CD45.2) (Online Supplementary Figure S1A). Sixteen weeks later, about 90% of the cells in the peripheral blood of recipients were donor-derived cells (Online Supplementary Figure S1B). Tamoxifen was then injected intraperitoneally for 5 days to induce Twist1 deletion. Two weeks after the last injection, mRNA detection demon- strated that Twist1 had been knocked out in all the MSC, OLC, and EC isolated from Twist1Δ/Δ mice with similar knockout levels (Online Supplementary Figure S1C), while the expression of Twist2 was almost unchanged (data not shown).
To define components of the Twist1-deleted BM microenvironment that may be altered, stromal popula- tions and extracellular factors were assessed in Twist1- deleted and control chimeric mice. We observed that con- ditional deletion of Twist1 led to a significant decrease in the number of MSC (CD140a+CD51+CD45/Ter119/CD31- )23 in the BM compared with that in control mice, as deter- mined by flow cytometry (Figure 1A). The decrease in MSC number was further confirmed by a fibroblastic colony-forming unit assay (Online Supplementary Figure S2A). Furthermore, Twist1-deleted MSC showed a decrease in proliferative cells and an increase in apoptotic cells (Online Supplementary Figure S2B,C), indicating the mecha- nism underlying the reduced number of MSC.
Twist1 deficiency resulted in a marked increase in the fre- quency of Sca-1-/CD166+ stromal cells (Figure 1B), which include immature and mature OLC.24 Meanwhile, the expression of osteoblastic differentiation genes Runx2, Ogn and Gpnmb14,23 was significantly upregulated in Twist1- deleted MSC (Online Supplementary Figure S2D). To assess the ability of MSC to differentiate into the osteoblastic lin- eage, we induced osteoblast differentiation in MSC and found that Twist1 deletion clearly increased alkaline phos- phatase activity and matrix mineralization (Online Supplementary Figure S2E). These results establish that Twist1 deficiency enhanced MSC commitment toward osteoblasts. However, expression of the mature osteoblast marker, Bglap, was downregulated (Online Supplementary Figure S2F). In addition, micro-computed tomography analysis also revealed a significant decrease in mature osteoblasts in Twist1-deleted mice, which was reflected by
1970
haematologica | 2018; 103(12)