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

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

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

Hematopoietic differentiation potential among four ES cell lines.

<p>(A) Protocol schematic for hematopoietic differentiation by EB and OP9 co-cultures methods. ES cell lines are cultured on MEF with bFGF, treated with collagenase IV, broken into small clumps and plated for hematopoiesis induction. Clumps are seeded in EB differentiation media in ultra low attachment plates incubate at 37°C in 5% CO₂ for 16 days, media was change 2–3 times. For OP9 co-culture, clumps are seeded in hematopoiesis differentiation media on OP9 grown to confluence and media changed at days 4, 6 and 8 cultured at 37°C in 5% CO₂ for 13 days. At day 16 (EB) and day 13 (OP9 co-culture), cells were disrupt to single cell suspension and plate for CFC, analyze flow cytometry, Q-RT-PCR, or mouse reconstitution assay analysis. B-F) by the EB method. (B) Number of CFC counted and classified according to morphology. Each assay was performed in triplicate, data is shown as mean ± s.d. of n = 27, 34, 3 and 3 independent experiments for SA01, H1, H9 and CL01 respectively. There is no statistically significant difference for CFC number when comparing SA01 <i>vs</i> H1 and H9 <i>vs</i> CL01 (p>0.05), in contrast to SA01 <i>vs</i> H9, SA01 <i>vs</i> CL01, H1 <i>vs</i> H9, H1 <i>vs</i> CL01 are statistically significant different (*<i>p</i><0.05). (C) Types of CFC (CFU-E, BFU-E, CFU-GM and CFU-GEMM). (D) Flow cytometric analysis of the percentage of CD34+ cells in EB-derived ES cells n = 12, 22, 3 and 3 for SA01, H1, H9 and CL01 respectively. Differences of expression of CD34 for SA01 <i>vs</i> H1, SA01 <i>vs</i> H9, SA01 <i>vs</i> CL01, H1 <i>vs</i> H9, H1 <i>vs</i> CL01 were statistically significant (*p<0.05). (E) EB-derived cells from SA01, H1 and H9 cell lines co-expressed CD34/CD45, CD34/CD43 and CD45/CD43 hematopoietic markers n = 4, 8 and 3, respectively. (F) Representative FACS analysis for EB-derived cells from H1 cell line co-expressed CD34/CD45, CD34/CD43 and CD45/CD43 hematopoietic markers. G-I) on OP9 co-cultures, (G) Number of CFC generated, SA01 n = 6, H1 n = 8, H9 n = 3 and CL01 n = 3, CFC number from SA01 was significantly different from that obtained from the 3 others ES cell lines (*<i>p</i><0.05). (H) Distribution of CFC types. (I) Flow cytometric analysis of the percentage of CD34+ cells after OP9 differentiation, SA01 n = 6, H1 n = 8, H9 n = 7 and CL01 n = 3 were not statistically significant (p>0.05).</p

Human engraftment of NOG mice transplanted with ES or iPS cells.

<p>Human engraftment of NOG mice transplanted with ES or iPS cells.</p

Primer sequences.

<p>Primer sequences.</p

Cell’s origin, reprogrammation method, characterization and hematopoietic analysis of iPS.

<p>Cell’s origin, reprogrammation method, characterization and hematopoietic analysis of iPS.</p

Primer sequences.

<p>Primer sequences.</p