Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines

International audienceHematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process

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

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

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

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

<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 several iPS cell lines.

<p>Twelve different iPS cell lines were assayed by the EB method. (A) Comparative analysis of total number of CFC. iPS cell lines derived by lentiviral vectors (<i>OCT4</i>, <i>SOX2</i>, <i>LIN28</i> and <i>NANOG</i>) in light blue, (<i>OCT4</i> and <i>SOX2</i>) in dark blue, retroviral vectors (<i>OCT4</i>, <i>SOX2</i>, <i>KLF4</i> and <i>C-MYC</i>) in red and by Sendai virus (<i>OCT4</i>, <i>SOX2</i>, <i>KLF4 and C-MYC</i>) in violet, n = 3, 3, 3, 6, 3, 3, 3, 12, 6, 4, 3 and 3, for PB3, PB4, PB5, PB7, PB8, PB9 PB10, PB11, PB13, PB17, PB30 and PB33 cell lines respectively. (B) Types of CFC obtained. Some iPS lines show different colony subtypes, PB7, PB10, PB11, PB13, PB17, PB30 and PB33 cell lines n = 6, 3, 12, 6, 4, 3 and 3, respectively, unlike PB3 (n = 3) and PB4 (n = 3) which differentiate almost exclusively into the myeloid lineage. (C) Flow cytometric analysis of the percentage of CD34+ cells in EB-derived cells, n = 3, 3, 3, 6, 3, 12, 6, 4, 3 and 3, for PB3, PB4, PB5, PB7, PB10, PB11, PB13, PB17, PB30 and PB33 cell lines respectively. (D) EB-derived cells from PB7, PB11 and PB13 co-expressed CD34/CD45, CD34/CD43 and CD45/CD43 hematopoietic markers (n = 3, 3 and 4 respectively), unlike PB5 (n = 3), which do not express these markers. (E) Representative FACS analysis for EB-derived cells from PB11 iPS cell line co-expressed CD34/CD45, CD34/CD43 and CD45/CD43 hematopoietic markers.</p

Human engraftment of NOG mice transplanted with ES or iPS cell lines.

<p>EB cells were injected directly into the femur of non-lethally irradiated NOG mice. (A) Representative FACS analysis for detection of human CD45+ cells in the blood and BM of transplanted mice. Percentage of human CD45+ cells in blood at 4 weeks (top) and in injected femur at 12 weeks post-transplant (bottom): H9, iPS-MSC-H9, SA01, iPS-MSC-SA01 and control mouse transplanted with human cord blood CD34+ cells and isotype control. (B) Quantitative RT-PCR analysis for <i>HoxA3</i> and <i>RUNX1c</i> expression in CD34+ EB derived cells from ES and iPS-MSC-ES cell lines, compared to CD34+ human fetal liver (n = 2).</p

Primer sequences.

<p>Primer sequences.</p

Primer sequences.

<p>Primer sequences.</p

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

<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