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T, B, and NK cell counts were determined in months + 1, + 2, + 3, and + 6 post-transplantation. Lymphocyte subsets in peripheral blood were quantified using Trucount Tubes (Becton Dickinson, Albertslund, Denmark) together with the following panel of conjugated monoclonal antibodies and analyzed on a FC500 flow cytometer (Beckman Coulter, Copenhagen, Denmark): CD3-PerCP, CD3-FITC, CD4-FITC, CD8-PE, CD45-PerCP, CD16/56-PE, CD20-FITC, and CD19-PE (Becton Dickinson). CD3⁺ T cells, CD3⁺CD4⁺ T cells, and CD3⁺CD8⁺ T cells were determined. NK cells were differentiated by CD3⁻CD45⁺CD16⁺CD56⁺ phenotype. The following B cell phenotypes were distinguished: total B cells (CD45⁺CD19⁺), mature B cells (CD45⁺CD19⁺CD20⁺), and immature B cells (CD45⁺CD19⁺CD20⁻). Data of these immune cell populations have been published previously in a different context [46 (link)].

Sudemycin D6 was tested for in vivo efficacy in an adoptive transfer mouse model of CLL, as described by Herman et al [45 (link)] with modifications. Primary cells from CLL patients (50 × 10⁶ per mice; percentage of tumor cells: 93–99%) were intravenously inoculated through tail vein in six- to eight-week old NSG mice (Charles River Laboratories International, Inc), according to a protocol approved by the animal testing ethical committee of the University of Barcelona (Barcelona, Spain). Six CLL cases (n = 3 SF3B1-mutated, n = 3 SF3B1-unmutated) were injected in the mice. Each CLL case was injected in 4 mice (n = 2 control; n = 2 sudemycin). Mice were treated intravenously with sudemycin D6 (14 mg/kg) or vehicle (10% (2-hydroxypropyl)-β-cyclodextrin in PBS) during 4 days and sacrificed after 5 days of inoculation. Then, spleen and PB samples were recovered and processed as previously described [45 (link)]. Cell suspensions were washed, resuspended with 0.5% BSA in PBS and blocked with 10% mouse serum. Samples were incubated with the following anti-human antibodies: CD45-Pacific Blue (PB; Life Technologies), CD19-PE and CD5-FITC (Becton Dickinson) together with PI to assess cell viability. The abundance of CLL cells was counted on an Attune cytometer as the number of viable CLL cells (CD45⁺, CD19⁺ and CD5⁺) per organ (spleen) or per μL (PB).

Flow cytometric analysis was performed to identify the surface marker expressions of IL10-MSCs. The cells were collected by enzymatic digestion, and 1 × 10⁶ cells were incubated with CD45-FITC, CD34-FITC, CD90-FITC, CD44-PE, CD19-PE, CD105-PE, CD73-PE, and HLA-DR-PE antibodies (BD, San Diego, CA, USA) for 15 min, respectively. After washing with PBS three times, a FACScan (BD FACS Aria™; NJ, USA) and Flow Jo V10 software were used to identify and analyze the surface marker expressions of the cells.

Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium (Ficoll-Hypaque density gradient, Axis-shield, Germany) from 10 ml fresh heparinized blood sample. And then PBMCs were incubated with 1 μM CpG-B ODN2006 (In vivoGen, San Diego, CA, USA) for 96 h at 37°C. PMA (3 ng/ml) and ionomycin (100 ng/ml) were added during the last 4h in the presence of 10 μg/ml brefeldin A (all from Sigma, St Louis, MO, USA). Cells were then surface stained for the markers CD19 Pacific Blue-A, and stained intracellularly with anti-IL-10 APC. So we could assess the ability and the frequency of IL-10 production from purified B cells (14 (link)). All antibodies including CD19-PE and IL-10-APC used in flow cytometry were ordered from BD Pharmingen (BD Pharmingen, San Diego, CA, USA). PBMCs were acquired on a FACS Canton flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). FlowJo 7.6.1 (Tree Star Inc., Ashland, OR, USA) was used as the flow analysis software.

Flow cytometry analysis for hematopoietic and non-hematopoietic cell surface markers was performed on freshly isolated mononuclear cells and on adherent cell populations at p0 and p1. Cells were incubated with single or combinations of the following mouse monoclonal anti-human antibodies in PBS containing 1% fetal calf serum for 30 min at 4 °C: CD45-Alexa 488 (1:20, Beckman Coulter, Nyon, Switzerland), CD45-APC (1:5), CD73-PE-Cy7 (1:40), CD105-PE (1:40), CD90-BV421 (1:13), CD146-PeCy7 (1:20), HLA-DR-PE (1:5), CD19-PE (1:5), CD11b-PE (1:5, all BD Bioscience), CD44-APC (1:10), CD34-PE (1:10, both Miltenyi Biotec, Bergisch-Gladbach, Germany), NG2-PE (1:10), PDGF-rβ-Alexa 700 (1:20, both R&D Systems). Unstained isotype (IgG1-PeCy7 (1:20, BD Bioscience)) and single antibody controls were included. Flow cytometry was performed using a BD Aria III, and a minimum of 25,000 events were acquired for each sample, and data were analyzed using BD FACS Diva 6.1.3. Appropriate compensation settings and gating were applied in order to account for cellular debris, cell doublets, spectral overlap, and autofluorescence.

To identify surface marker expression of HUCMSCs and bFGF-HUCMSCs, the cells were collected and 1 × 10⁶ cells were incubated with antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE), CD34-FITC, CD45-FITC, CD90-FITC, CD19-PE, CD44-PE, CD73-PE, CD105-PE, and HLA-DR-PE (BD, San Diego, CA, USA), in the dark for 15 min. After washing with PBS three times, a FACScan (BD FACSAria™; NJ, USA) and FlowJo V10 software were used to analyze the phenotype of cells.