Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel mesenchymal stem cell-based regimen for acute myeloid leukemia differentiation therapy

doi: 10.1016/j.apsb.2023.05.007

Figure Lengend Snippet: MSC promotes AML cell differentiation by EVs. (A) Electron microscopy image of EVs isolated from the conditional medium of MSC. Scale bar = 200 nm. (B) Western blotting for the expression of Alix, Flotillin-1, Annexin V and NE in MSC, supernatant and EVs which were extracted from MSC conditional medium, respectively. Data are quantified on the right. (C) Confocal microscopy images of U937 cells treated with EVs derived from CFSE-labelled MSC for 2 h. 100 × magnification; scale bar = 20 μm; (D) U937 cells were cultured with EVs (10, 20, and 30 μg) for 48 h. The myeloid differentiation markers CD11b and CD14 in U937 cells were analyzed by flow cytometry ( n = 3). (E) U937 cells were cultured with MSC in the presence of EVs trafficking inhibitor Y27632 (10, 20, and 40 μmol/L) for 48 h. The myeloid differentiation markers CD11b and CD14 in U937 cells were analyzed by flow cytometry ( n = 3). (F) MSC EVs (30 μg) were added to stimulate U937 cells. Western blotting was applied to detect the levels of phosphorylation of p38 and STAT3 in U937 cells. Data are quantified on the right. (G) U937 cells were treated by the p38 MAPK inhibitor SB239063 (SB) (10 μmol/L) and/or the STAT3 inhibitor Stattic (5 μmol/L) followed by incubating with MSC for 48 h. The percentage of CD14 + CD11b + cells was determined by flow cytometry. (H, I) Flow cytometry analysis of the percentage of leukemic cells (CD45 positive cells) in peripheral blood (PB) (I) and bone marrow (BM) (J) in vehicle, MSC-treated and MSC-EVs-treated mice after 25 days of transplantation ( n = 4 or 5 each group). (J, K) Flow cytometry analysis of the expression of the differentiation marker CD14 in PB (K) and BM (L) derived leukemic cells ( n = 4 or 5 each group). (L) Fluorescent images of the AML cells infiltration in the spleen as observed via immunofluorescence with anti-CD45 antibody (green). Nuclei are stained with DAPI (blue). 10 × magnification; scale bar = 100 μm. Data are expressed as the mean ± SEM. Data were analyzed by Student's t test (E, F) and one-way ANOVA followed by Tukey's test (D, G–K). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: AML cells (3 × 10 5 ) in complete medium were added to the MSC culture with or without 1 α ,25-dihydroxyvitamin D3 (1,25D3) (MCE), Y27632 (MCE), SB239063 (MCE) or Stattic (Sigma–Aldrich).

Techniques: Cell Differentiation, Electron Microscopy, Isolation, Western Blot, Expressing, Confocal Microscopy, Derivative Assay, Cell Culture, Flow Cytometry, Phospho-proteomics, Transplantation Assay, Marker, Immunofluorescence, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel mesenchymal stem cell-based regimen for acute myeloid leukemia differentiation therapy

doi: 10.1016/j.apsb.2023.05.007

Figure Lengend Snippet: VDR modulates the production and release of NE carried EVs. (A) Luciferase reporter assay evaluating the transcriptional activity of VDR, following treatment with 1,25D3 ( n = 6). (B) Relative NE protein level in DMSO- or 1,25D3-treated MSC was evaluated by Western blotting ( n = 3). (C) Confocal microscopy analysis of the NE expression (Alexa Fluor 488) in DMSO- or 1,25D3-treated MSC. Data are quantified on the right. 100× magnification; scale bar = 20 μm. (D) The NE expressions in MSC & shcon or MSC & shVDR treated by U937 cells with or without 1,25D3 were assessed by RT-qPCR ( n = 3). (E) The NE expression in MSC OE-con or MSC OE-VDR treated by U937 cells with or without 1,25D3 was assessed by RT-qPCR ( n = 3). (F) MSC was cultured in the presence or absence of 1,25D3. The expressions of Alix, Flotillin-1, NE in MSC-EVs were detected by Western blotting ( n = 3). Data are quantified on the right. (G) EVs isolated from 1,25D3-primed MSC (pre-EVs) were added to treat U937 cells. The myeloid differentiation markers CD11b and CD14 in U937 cells were analyzed by flow cytometry. ( n = 3) (H) The numbers of EVs existed in MSC & shcon or MSC & shVDR medium were assessed by flow cytometry ( n = 3). (I) U937 cells were treated by MSC or MSC VDR-KO in the presence with or without 1,25D3. The expressions of CD14 and CD11b in U937 cells were evaluated by flow cytometry ( n = 3). All statistical data in this figure are expressed as the mean ± SEM. Data were analyzed by Student's t test (B, C), two-way ANOVA followed by Bonferroni's test (D, E, F, I) and one-way ANOVA followed by Tukey's test (B, C, H). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: AML cells (3 × 10 5 ) in complete medium were added to the MSC culture with or without 1 α ,25-dihydroxyvitamin D3 (1,25D3) (MCE), Y27632 (MCE), SB239063 (MCE) or Stattic (Sigma–Aldrich).

Techniques: Luciferase, Reporter Assay, Activity Assay, Western Blot, Confocal Microscopy, Expressing, Quantitative RT-PCR, Cell Culture, Isolation, Flow Cytometry

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel mesenchymal stem cell-based regimen for acute myeloid leukemia differentiation therapy

doi: 10.1016/j.apsb.2023.05.007

Figure Lengend Snippet: VDR activation enhances MSC-mediated pro-differentiation effect on AML cells. (A, B) U937 cells were cultured alone or incubated with MSC in the presence or absence of 1,25D3 (8 nmol/L) for 48 h. The differentiation markers CD14 and CD11b (A), phagocytosis activity (B) in U937 cells were measured by flow cytometry. Fold increase of CD14 and CD11b expressions and phagocytosis beads (mean fluorescence intensity relative to untreated cells) were quantified on the right ( n = 3). (C) Morphological changes were confirmed by Wright–Giemsa staining of cells with or without MSC and 1,25D3 culture. Scale bar = 25 μm. (D) U937 cells were cultured alone or incubated with MSC in the presence or absence of 1,25D3. Flow cytometry analysis of cell cycle of U937 cells ( n = 3). (E, F) The weight index of liver (E) and spleen (F) in different groups ( n = 6). (G, H) Flow cytometry analysis of the percentage of leukemic cells in PB (G) and BM (H) in vehicle, single agent-treated and combination-treated mice after 25 days of transplantation ( n = 6). (I, J) Flow cytometry analysis of the expression of the differentiation marker CD14 in PB (I) and BM (J) derived leukemic cells ( n = 6). (K) Fluorescent images (left) and quantitation (right) of the AML cells infiltration in the spleen as observed via immunofluorescence with anti-CD45 antibody (red). Nuclei are stained with DAPI (blue). Scale bar = 75 μm. A dot depicts data from an individual animal, and all the histograms in this figure show the mean ± SEM. Data were analyzed by two-way ANOVA followed by Bonferroni's test (D) or one-way ANOVA followed by Tukey's test (A, B, E–K). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: AML cells (3 × 10 5 ) in complete medium were added to the MSC culture with or without 1 α ,25-dihydroxyvitamin D3 (1,25D3) (MCE), Y27632 (MCE), SB239063 (MCE) or Stattic (Sigma–Aldrich).

Techniques: Activation Assay, Cell Culture, Incubation, Activity Assay, Flow Cytometry, Staining, Transplantation Assay, Expressing, Marker, Derivative Assay, Quantitation Assay, Immunofluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel mesenchymal stem cell-based regimen for acute myeloid leukemia differentiation therapy

doi: 10.1016/j.apsb.2023.05.007

Figure Lengend Snippet: Sw-22 combined with MSC synergistically played an anti-leukemia effect without hypercalcemia. (A, B) U937 cells were cultured alone or cocultured with MSC in the presence or absence of different doses of sw-22 (2, 4, and 8 μmol/L) for 48 h, 1,25D3 (8 nmol/L) was applied as positive control. The differentiation markers CD14 and CD11b (A) and phagocytosis (B) were measured by flow cytometry ( n = 3). (C) Flow cytometry assessed the numbers of EVs existed in sw-22-treated MSC ( n = 3). (D) Western blotting for the expression of NE in MSC treated by U937 cells combined with different doses of sw-22 (2, 4, and 8 μmol/L). Data are quantified below. (E) MSC & shcon or MSC & shVDR were cultured alone or cocultured with U937 cells in the presence or absence of different doses of sw-22. The expression of NE in MSC & shcon or MSC & shVDR was assessed by RT-qPCR ( n = 3). (F) U937 cells were treated with MSC & shcon or MSC & shVDR in the presence or absence of sw-22 for 48 h. Flow cytometry analyzed the CD14 and CD11b expressions. ( n = 3) (G) Survival curves of AML recipient mice treated with vehicle, sw-22, MSC, sw-22 plus MSC or cytarabine. Mice treated with the combination treatment showed prolonged survival. (H, I) Flow cytometry analysis of the percentage of leukemic cells in PB (H) and BM (I) of vehicle, single agent-treated and combination-treated mice after 25 days of transplantation ( n = 6 or 7 per group). (J) Histologic sections of spleen were stained with hematoxylin–eosin (HE). Scale bar = 100 μm. (K) CD11b + CD14 + cells in primary human cells from AML-M5 patients after combination stimulation. (L) CD11b + CD14 + cells in primary human cells from FAB M4 and M5 patients after single-agent or combination treatment ( n = 9 or 10 per group). (M) Graphical illustration of the interaction between MSC and AML cells. All statistical data in this figure are expressed as the mean ± SEM. Data were analyzed by two-way ANOVA followed by Bonferroni's test (E, F) or one-way ANOVA followed by Tukey's test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: AML cells (3 × 10 5 ) in complete medium were added to the MSC culture with or without 1 α ,25-dihydroxyvitamin D3 (1,25D3) (MCE), Y27632 (MCE), SB239063 (MCE) or Stattic (Sigma–Aldrich).

Techniques: Cell Culture, Positive Control, Flow Cytometry, Western Blot, Expressing, Quantitative RT-PCR, Transplantation Assay, Staining