Functional activity of NK cells derived from co-culture with MS-5/SP-HOXB4.

<p>(A) The functional activity of cells was assessed in the presence of IL-12 and against K562 target cells; the NK-cell activity was evaluated by the mobilisation of the CD107a antigen on the cell surface and the intra-cytoplasmic expression of IFNγ. Cells were gated on CD56⁺ cells (not shown). (B) Cytotoxic activity of NK effector cells against target K562 cells. a) Target K562 cells were labelled by CFSE. b) Percentage of 7AAD⁺ death cells among the CFSE⁺ target cells at various Effector-Target (E/T) ratios (one experiment out of two). c) Percentage of cytotoxicity (<i>i.e</i> percentage of 7AAD⁺ death cells among the CFSE⁺ target cells) according to the ratio E/T (dotted line and bold line represent two independent experiments).</p

NK-cell culture procedure of hESC-derived hematopoietic precursor cells.

<p>Schematic representation of the three steps of NK-cells differentiation from hESCs. hEBs derived from the H1 hESC cell line were dissociated then co-cultured with MS-5/SP-HOXB4 cells or MS-5/EGFP cells as control during 2 weeks. Then, the cells derived from the first step of co-culture were submitted to a second step of 3-week co-culture with unmodified MS-5 cells, in permissive conditions for NK-cell differentiation in presence of SCF, IL-2 and IL-15. NK-cell culture differentiation was conducted directly with un-co-cultured hEB-derived cells as control. (B) Analysis of the presence of the HOXB4 protein within hEB-derived cells co-cultured with either MS-5/SP-HOXB4 (dark line) or MS-5/EGFP (dotted line) stromal cell lines. Data are from one experiment out of two. Gray histogram corresponds to isotypic control. Abbreviations : cy, cytoplasmic.</p

Analysis of NK differentiation potential and NK progenitor cell expansion mediated by HOXB4.

<p>(A) Percentages of NK cells (CD56⁺CD3⁻CD19⁻) among nucleated cells collected at the end of 5 weeks of co-culture. Cells derived from the 2 weeks primary co-cultures with MS-5/SP-HOXB4 or MS-5/EGFP were then plated on unmodified MS-5 cells in conditions known to promote NK-cell differentiation and maintained during three weeks. At the end of the culture period, cells were analyzed by FACS for the expression of CD56 and CD3/CD19 markers. Un-co-cultured hEB cells were directly cultured under NK-cell differentiation conditions for 3 weeks (n = 5, *<i>p</i><0.05, **<i>p</i><0.01). (B) Fold increase of total NK cells. NK cells were derived from total cells isolated from the primary 2-week co-cultures of hEB-derived cells with either MS-5/SP-HOXB4 or MS-5/EGFP control and then cultured under NK-cell differentiation condition for three weeks. NK cells were then numbered. Bars represent fold amplifications relative to day-0 control (un-co-cultured hEBs) (designated as 100%) (n = 5, *<i>p</i><0,05).</p

Analysis of hematopoietic progenitor cells among hEB-derived cells.

<p>(A) Phenotypic analysis of hEB-derived cells. Cells derived from un-co-cultured hEBs or from hEB cells co-cultured with MS-5/SP-HOXB4 and MS-5/EGFP, were analysed by FACS according to CD34, CD43, CD45 and CD45RA antigen expression (one representative experiment out of three). (B) Percentages of cells co-expressing CD34 and CD43, CD45 and CD45RA among total nucleated cells collected at day 14 after EB cells co-cultured with MS-5/SP-HOXB4 or MS-5/EGFP or directly obtained from un-co-cultured hEBs (n = 3, *<i>p</i><0.05). (C) Relative expansion of total CD34⁺ cells. Total CD34⁺ cells were derived from hEB-derived cells co-culture with MS-5/SP-HOXB4 or MS-5/EGFP. Bar represents fold amplification relative to MS-5/EGFP control (designated as 100%) from three independent experiments.</p

Expression of inhibitory and activating receptors and of the cytotoxic arsenal of NK cells derived from co-culture with MS-5/SP-HOXB4.

<p>NK cells were obtained from NK progenitors derived from hEB cells co-cultured with MS-5/SP-HOXB4. Cells were FACS analyzed for surface expression of CD56, CD16, CD94, the mix of CD158a, h, CD158b1/b2, j, CD158e1/e2 and CD158i (referred as CD158) and of CD159, CD335, CD336 and CD337. For CD159, CD94, CD335, CD336 and CD337 expression, cells were gated on CD56⁺ cells (not shown). NK cells obtained using the HOXB4 co-culture model were also analyzed for the intra-cytoplasmic expression of Perforin, Granzyme-A and Granzyme-B. Cells were gated on CD56⁺ cells (not shown). Data are from one experiment out of two.</p