12 research outputs found

    Comparison of caudal vein and retro orbital injections.

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    <p> After classical busulfan pre-conditioning (2x25 mg/Kg), female mice received 1000 non manipulated CD34+ cells by i.v. injection either in the caudal vein or retro orbital sinus. Their bone marrow were analyzed 8 weeks later for <b>A.</b> total huCD45 chimerism, <b>B.</b> huCFU content. Each mouse represented by (●), Median represented by (─).</p

    Impact on engraftment level of time delay between mice pre-conditioning and cell injections.

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    <p>Female mice received 2 doses of busulfan (25mg/Kg each) at 24 hours, 4 days or 7 days intervals. Mice received 1000 non manipulated CD34+ cells by caudal injection and their bone marrow were analyzed 8 weeks later for <b>A.</b> total huCD45 chimerism, <b>B.</b> B lymphoid (▪) versus myeloid (□) cells ration in CD45+ cells, and <b>C.</b> huCFUs content per femur. Each mouse represented by (●), Median represented by (─).</p

    Impact of gender on human cells engraftment.

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    <p>After classical busulfan pre-conditioning (2x25 mg/Kg), female and male mice received 100, 500 or 1000 non manipulated CD34+ cells by i.v. injection in the retro orbital sinus. Their bone marrow were analyzed 8 weeks later for <b>A.</b> total huCD45 chimerism, <b>B.</b> huCFU content. Each mouse represented by ● (female) or ○ (male), Median represented by (─).</p

    Hematopoietic differentiation potential among four ES cell lines.

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    <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% CO<sub>2</sub> 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% CO<sub>2</sub> 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

    Expression of hematopoietic transcription factors for ES and iPS cell lines.

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    <p>Gene expression level of <i>RUNX1</i>, <i>HOXB4</i>, <i>TAL1</i>, <i>PU</i>.<i>1</i>, <i>GATA1</i>, <i>GATA2</i>, <i>GATA3</i>, <i>MPO</i> and <i>IKAROS</i> was evaluated by Q-RT-PCR (A) at pluripotent stage and (B) after differentiation in EB at day 16 and human BM as a positive control of expression hematopoietic genes. Gene expression was normalized relative to the endogenous RNA control human <i>HPRT</i>.</p

    Hematopoietic differentiation potential among different ES cell lines <i>versus</i> iPS-MSC-ES.

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    <p>Total number of CFC obtained from SA01 and H9 cell lines and iPS-MSC-SA01 and iPS-MSC-H9 by the EB differentiation method, n = 3 for each assay (A) Number of CFC, data is shown as mean ± s.d. of 4 independent experiments for each cell line, CFC numbers from iPS-MSC-SA01 <i>vs</i> parental SA01 cell line was significantly higher (*<i>p</i><0.05), unlike iPS-MSC-H9 <i>vs</i> H9 the difference was not significant (<i>p</i>>0.05). (B) Types of CFC from ES and iPS-MSC-ES (n = 4 for each cell line). (C) Flow cytometric analysis of the percentage of CD34+ cells in EB-derived cells from ES and iPS-MSC-ES. Percentage of CD34+ cells for SA01 <i>vs</i> iPS-MSC-SA01 were similar (p>0.05) (n = 5), unlike for H9 <i>vs</i> iPS-MSC-H9 cell lines percentage of CD34+ cells was statistically significant (*p<0.05) (n = 7).</p
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