16 research outputs found

    Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions

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    <div><p>Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×10<sup>6</sup>±0.3×10<sup>6</sup> floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.</p> </div

    Functional assays for M1/M2 macrophages derived from pluripotent stem cells.

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    <p>(A) Flow cytometric analysis of M1/M2 macrophages derived from pluripotent stem cells. (B) The levels of IL-12p70 and IL-10 in supernatants of culture medium with M1/M2 macrophages derived from pluripotent stem cells 24 hours after LPS stimulation. The data of KhES1-derived cells are shown as representative.</p

    Functional assays for monocytes derived from pluripotent stem cells.

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    <p>(A) The levels of IL-6 and TNFα in supernatants of PS-Mo culture medium 4 hours after LPS stimulation. The levels of IL-1β were measured 4 hours after LPS stimulation with/without an additional 30-minute ATP stimulation. (B) Flow cytometric analysis of CX3CR1 on PS-Mo. (C) Chemotaxis assay of PS-Mo for CX3CL1 (fractalkine) using a trans-well migration assay. After the addition of CX3CL1 into either the bottom or top of the trans-well chamber, PS-Mo were applied and incubated for 5 hours at 37°C. (D) Antigen uptake was evaluated in monocytes, immature DCs and mature DCs derived from pluripotent stem cells by examining the fluorescence intensity of Alexa fluor 488-conjugated ovalbumin 45 minute after incubation at 37°C (black). Control samples (white) were kept on ice. (B–D) The data of KhES1-derived cells are shown as representative. PS-Mo: monocyte derived from pluripotent stem cells.</p

    Phenotype analysis and gene expression pattern of monocytic lineage cells derived from pluripotent stem cells.

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    <p>(A) Flow cytometric analysis of monocytic lineage cells derived sequentially from pluripotent stem cells. An analysis of adherent cells on day 6 and supernatant cells on day 13 and 18 is shown. (B) May-Giemsa staining of CD14<sup>+</sup> monocyte-like cells derived from KhES1 on day 16 (left) and primary human monocytes (right). (C) Esterase staining for CD14<sup>+</sup> monocyte-like cells derived from KhES1 on day 16. (D) The percentage of CD14<sup>+</sup> cells within the total floating cells derived from KhES1/iPS-201B7 was evaluated from day 13 to day 28. (E) May-Giemsa staining (left) and phase contrast image (right) of mature DCs derived from pluripotent stem cells. (F) Flow cytometric analysis of immature/mature DCs derived from pluripotent stem cells. (G) Phase contrast image and flow cytometric analysis of macrophages derived from pluripotent stem cells.(H) RT-PCR analysis of monocytic lineage cells derived from KhES1/iPS-201B7 clones for expression of monocytic lineage marker genes (<i>PU.1, c-MAF, TLR4, CCL17</i> and <i>CCL18</i>). Peripheral blood monocytes and peripheral blood monocyte-derived mature DCs were used as positive controls.(A–C, E–G) The data from KhES1-derived cells are shown as representative.</p

    Functional assays for dendritic cells derived from pluripotent stem cells.

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    <p>(A) Flow cytometric analysis of immature/mature DCs derived from pluripotent stem cells. (B) The levels of IL-10 and TNFα in supernatants of culture medium with PS-DCs 24 hours after LPS stimulation. (C) The proliferation of allogeneic naïve T cells (1×10<sup>5</sup> cells per well) co-cultured with 40 Gy-irradiated stimulator cells for 3 days was evaluated. The proliferation of naïve T cells in the last 16 hours was measured by <sup>3</sup>H-thymidine uptake. (A–C) The data of KhES1-derived cells are shown as representative.</p

    Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines

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    <div><p>Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated <i>in vitro</i> BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, <i>IKZF1</i> deletion, and biallelic loss of <i>CDKN2A</i> were associated with favorable response. Even in Ph-negative ALL cell lines, <i>IKZF1</i> deletion and bilallelic loss of <i>CDKN2A</i> were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19) ALL cell lines, and <i>IKZF1</i> deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19) ALL cell line to CFZ. These observations suggested that <i>IKZF1</i> deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.</p></div

    Anti-leukemic activity of bortezomib.

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    <p>(A) Dose-response curve of bortezomib sensitivity in representative cell lines. The vertical axis indicates % viability in alamarBlue cell viability assay and the horizontal axis indicates log concentration of bortezomib (nM). Phenotype, deletion of <i>IKZF1</i> and <i>CDKN2A</i>, and IC50 of each cell is indicated. Abbreviations: del, deletion; -, no deletion; hm, homozygous deletion; ht, heterozygous deletion; nt, not tested. (B) Induction of apoptotic cell death by bortezomib. Three cell lines (KOPN34, YCUB2 and Reh) were cultured in the presence or absence of 20 nM of bortezomib for 17 hours, and analyzed with Annexin V-binding (horizontal axis) and 7AAD-staining (vertical axis) using flow cytometry. The percentages of living cells (Annexin V-negative/7AAD-negative) and early (Annexin V-positive/ 7AAD-negative) and late (Annexin V-positive/ 7AAD-positive) apoptotic cells are indicated. (C) Induction of CHOP and NOXA expression. Highly sensitive cell lines (RS4;11 and Reh) and moderately sensitive cell lines (KOPN1 and YCUB2) were cultured in the presence or absence of bortezomib at 20 nM for eight hours, and immunoblotting was performed using Tubulin as an internal control. (D) Bortezomib sensitivity in 79 BCP-ALL, 9 T-ALL, and two MM cell lines. The vertical axis indicates the IC50 value for bortezomib. The <i>p</i> values determined by a Mann-Whitney test and boxplots are indicated.</p

    Anti-leukemic activity and cross-resistance between carfilzomib and four representative chemotherapeutic agents in BCP-ALL cell lines.

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    <p>(A) Association of carfilzomib sensitivity with different types of translocation in BCP-ALL cell lines. The vertical axis indicates the log IC50 value for carfilzomib. Boxplot of each type is indicated. The <i>p</i> value determined by a Mann-Whitney test is indicated when < 0.1. (B) Association of <i>IKZF1</i> deletion with carfilzomib sensitivity in Ph+ALL and Ph-negative BCP-ALL cell lines. The vertical axis indicates the log IC50 value for carfilzomib. The <i>p</i> value determined by a Mann-Whitney test is indicated. (C and D) Cross-resistance between carfilzomib and representative chemotherapeutic agents in 79 BCP-ALL cell lines. The vertical axes indicate the log IC50 values for daunorubicin (C), vincristine (C), L-asparaginase (D), and dexamethasone (D), whereas the horizontal axis indicates that for carfilzomib. Correlation coefficients and <i>p</i> values are shown at the top of each panel.</p

    Effect of overexpression and knockout of P-glycoprotein on carfilzomib sensitivity.

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    <p>(A) Cell surface expression of P-glycoprotein in parental 697 cells and 697R cells. Dotted and solid lines indicate fluorescence intensities of isotype control and anti-P-glycoprotein (P-gp) antibodies in parental cells, respectively. Shade indicates fluorescence intensity of anti-P-glycoprotein antibody in 697R cells. (B) Effects of P-glycoprotein (P-gp) inhibitors on carfilzomib (upper panels) and bortezomib (lower panels) sensitivities in parental 697 cells (left panels) and 697R cells (right panels). The vertical axes indicate % viability determined by alamarBlue cell viability assay and the horizontal axes indicate the concentration of carfilzomib or bortezomib. Blue, red, and green lines indicate dose response curves of carfilzomib or bortezomib alone, carfilzomib or bortezomib in combination with 0.8 μM of nilotinib, and carfilzomib or bortezomib in combination with 5 μM of verapamil, respectively. Mean values in triplicated analyses are indicated, and asterisks are indicated when p value in t-test is <0.01. (C) Induction of apoptotic cell death by carfilzomib in combination with P-glycoprotein (P-gp) inhibitors. Parental 697 cells (left panels) and 697R cells (right panels) were cultured with 20 nM of carfilzomib in the presence or absence of 0.8 μM of nilotinib or 5 μM of verapamil for 18 hours, and analyzed with Annexin V-binding (horizontal axis) and 7AAD-staining (vertical axis) using flow cytometry. The percentages of living cells (Annexin V-negative/7AAD-negative) and early (Annexin V-positive/ 7AAD-negative) and late (Annexin V-positive/ 7AAD-positive) apoptotic cells are indicated. (D) Effect of P-glycoprotein knockout on bortezomib and carfilzomib sensitivities in HALO1 cells. P-glycoprotein-positive HALO1 cells were electroporated with an <i>ABCB1</i>-specific CRISPR/Cas 9 vector that contains cDNA of Cas9 fused to human CD4 cDNA via a 2A peptide sequence. 48 hours after electroporation, CD4-positive cells were harvested using anti-CD4 antibody. Upper middle panel indicates flow cytometric analysis of CD4 expression 3 days after electroporation. Upper left and upper right panels indicate flow cytometric analyses of P-glycoprotein (P-gp) expression in parental and CD4-positive population of HALO1 cells, respectively. Bottom panels indicate two color analysis of P-glycoprotein (P-gp) expression (vertical axis) and Annexin V-binding (horizontal axis) by flow cytometry in the CD4-positive populations of HALO1 cells treated with 20nM of bortezomib and 20nM of carfilzomib for 12 hours. Cell viabilities in P-glycoprotein (P-gp)-positive and negative populations are indicated at the left side of each panel.</p

    Anti-leukemic activity of bortezomib in BCP-ALL cell lines.

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    <p>(A) Association of bortezomib sensitivity with different types of translocation in BCP-ALL cell lines. The vertical axis indicates the IC50 value for bortezomib. Boxplot of each type is indicated. The <i>p</i> value determined by a Mann-Whitney test is indicated when < 0.1. (B-E) Associations of <i>IKZF1</i> deletion (B and D) and homozygous <i>CDKN2A</i> deletion (C and E) with bortezomib sensitivity in BCP-ALL cell liens (B and C) or in Ph-negative BCP-ALL cell lines (D and E). The vertical axis indicates the IC50 value for bortezomib. The <i>p</i> value determined by a Mann-Whitney test and boxplots are indicated.</p
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