The Tumorigenicity of Mouse Embryonic Stem Cells and In Vitro Differentiated Neuronal Cells Is Controlled by the Recipients' Immune Response

Embryonic stem (ES) cells have the potential to differentiate into all cell types and are considered as a valuable source of cells for transplantation therapies. A critical issue, however, is the risk of teratoma formation after transplantation. The effect of the immune response on the tumorigenicity of transplanted cells is poorly understood. We have systematically compared the tumorigenicity of mouse ES cells and in vitro differentiated neuronal cells in various recipients. Subcutaneous injection of 1×106 ES or differentiated cells into syngeneic or allogeneic immunodeficient mice resulted in teratomas in about 95% of the recipients. Both cell types did not give rise to tumors in immunocompetent allogeneic mice or xenogeneic rats. However, in 61% of cyclosporine A-treated rats teratomas developed after injection of differentiated cells. Undifferentiated ES cells did not give rise to tumors in these rats. ES cells turned out to be highly susceptible to killing by rat natural killer (NK) cells due to the expression of ligands of the activating NK receptor NKG2D on ES cells. These ligands were down-regulated on differentiated cells. The activity of NK cells which is not suppressed by cyclosporine A might contribute to the prevention of teratomas after injection of ES cells but not after inoculation of differentiated cells. These findings clearly point to the importance of the immune response in this process. Interestingly, the differentiated cells must contain a tumorigenic cell population that is not present among ES cells and which might be resistant to NK cell-mediated killing

Lysis of ES and <i>in vitro</i> differentiated cells by NK cells derived from naïve LOU/c rats and expression analysis of MHC class I molecules and NKG2D ligands.

<p>(A) Mean of specific lysis and standard deviation (SD) of triplicates of ES or <i>in vitro</i> differentiated cells at three effector∶target (E∶T) ratios. Effector cells were lymphocytes obtained from spleens of 10 individual LOU/c rats by density gradient centrifugation on Biocoll. Results obtained with lymphocytes from female rats are indicated by open symbols and from male rats by filled symbols. (B) The mean specific lysis and SD of triplicates of ES, YAC-1, and RMA target cells at three effector∶target (E∶T) ratios by freshly isolated NK cells or NK cell-depleted splenocytes from naïve LOU/c rats are shown. The NK cell enriched fraction contained 86% and the NK cell depleted fraction 3% NKR-P1A-positive cells as determined by flow cytometry. The results are representative for 3 independent experiments. (C) The mean specific lysis and SD of triplicates of ES and YAC-1 cells at three effector∶target (E∶T) ratios by freshly isolated (open symbols) or 3 days <i>in vitro</i> with 1000 U/ml IL-2 stimulated (closed symbols) mouse and rat NK cells are shown. The NK cell-enriched fractions contained more than 80% NK cells as determined by flow cytometry. The results shown are representative for 3 independent experiments. (D) The expression of MHC class I molecules on ES and differentiated cells was analyzed by flow cytometry using anti-H2K^b and anti-H2D^b Abs (full lines). The stainings with the isotype control are shown by the dotted lines. RMA cells (H2^b) served as positive control for these antibodies. NKG2D ligands were stained with a recombinant mouse NKG2D-Fc chimeric protein and a FITC-conjugated goat anti-human IgG antibody as secondary reagent (full lines). Stainings with the secondary reagent only are shown by the dotted lines. YAC-1 cells (H2^a) served as positive controls for these stainings. The results shown are representative for more than 3 independent experiments. (E) ES and as positive control YAC-1 cells were stained with the NKG2D-Fc chimeric protein and with mAbs specific for the NKG2D ligands H60, MULT-1, and RAE-1. The mean+SD of the proportion of positive cells and the mean fluorescence intensity+SD determined in 6 independent experiments is shown. (F) The mean inhibition+SD of specific lysis of ES and YAC-1 cells by soluble mouse NKG2D is shown as determined in 3 experiments. The mean of specific lysis of the target cells by rat NK cells at an effector to target ratio of 20∶1 was determined as described above and adjusted to 100%. For inhibition of lysis a soluble mouse NKG2D protein was added to the test at a concentration of 3 µg/ml. The relative lysis of the target cells exposed to NKG2D was calculated. (G) The mean inhibition+SD of specific lysis of ES cells by soluble mouse NKG2D and various inhibitory antibodies against NKG2D ligands is shown. The mean of specific lysis of the target cells by IL-2 activated mouse NK cells at an effector to target ratio of 10∶1 was determined as described above and adjusted to 100%. For inhibition of lysis a soluble mouse NKG2D protein or the indicated mAbs were added to the test at a concentration of 3 µg/ml. The relative lysis of the target cells exposed to NKG2D or the mAbs was calculated.</p

Tumor growth in CsA-treated 129Sv mice and LOU/c rats after injection of ES cells or <i>in vitro</i> differentiated cells.

<p>(A) 1×10⁶ ES cells or <i>in vitro</i> differentiated cells were injected subcutaneously at day 0 into syngeneic 129Sv mice (for the numbers of animals see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002622#pone-0002622-t002" target="_blank">Table 2</a>) which were treated daily with CsA (10 mg/kg body weight). The tumor size was recorded every second day until day 100 using linear calipers. The growth of tumors in individual mice is shown. Tumor growth in female hosts is indicated by red lines and male hosts by blue lines. (B) 1×10⁶ cells differentiated <i>in vitro</i> for 14 days were injected subcutaneously into LOU/c rats (n = 18) which were treated daily with CsA (10 mg/kg body weight). The tumor size was recorded every second day until day 100 using linear calipers. The growth of tumors in individual rats is shown. Tumor growth in female hosts is indicated by red and in male hosts by blue lines. One tumor in a female rat was not progressive and remained very small until the end of the experiment. In two male rats a tumor regression occurred.</p

Tumor formation after subcutaneous inoculation of ES or <i>in vitro</i> differentiated cells into various hosts.

<p>ES cells and cells differentiated <i>in vitro</i> for 14 days were injected subcutaneously into the flank of syngeneic or allogeneic mice or xenogeneic rats (1×10⁶ cells in PBS/animal). Some recipients received an immunosuppressive treatment with CsA (10 mg/kg/day). The percentage and number of animals in which tumors were found during autopsy or in which tumors were palpable (at least during 3 consecutive observations) at the side of injection before day 100 after injection is indicated.</p>1<p>in 2 mice (8%) a tumor regression was observed before day 100.</p>2<p>in 2 mice (9%) a tumor regression was observed before day 100.</p>3<p>in 1 mouse (7%) a tumor regression was observed before day 100.</p>4<p>in 2 mice (11%) a tumor regression was observed before day 100.</p>5<p>in 8 female mice (47%) a tumor regression was observed before day 100.</p>6<p>in 2 rats (11%) a tumor regression was observed before day 100.</p

Lysis of ES and <i>in vitro</i> differentiated cells by splenocytes derived from rats 6 weeks after intracerebral grafting of <i>in vitro</i> differentiated cells.

<p>Mean of specific lysis and SD of triplicates of ES cells (A, C, E, G) or <i>in vitro</i> differentiated cells (B, D, F) at three effector∶target (E∶T) ratios. Effector cells were lymphocytes obtained by density gradient centrifugation on Biocoll from spleens of grafted (no. 113–117) or naïve (co1 and co2) DA rats (A, B), grafted (no. 118–122) or naïve (co1 and co2) LEW.1N rats (C, D), and grafted Wistar rats (no. 38, 39, 48, 49, 51, 52, 53, 54, 56, 57). The individual rats are indicated by symbols. (G) Mean of specific lysis and SD of triplicates of ES cells by lymphokine-activated killer (LAK) cells derived from splenocytes of Wistar rats (no. 38, 48, 49, 51, 52, 54, 56) after culture for 4 days in the presence of 100 U/ml IL-2.</p

Immunohistochemical staining identifies neuronal and glial cells in teratomas derived from ES and <i>in vitro</i> differentiated cells.

<p>The tumors were grown in SCID/beige mice after injection of ES cells (A, C) or after injection of <i>in vitro</i> differentiated cells (B, D). The teratomas were stained by immunohistochemistry for the neuronal marker NeuN (A, B) and the glial marker GFAP (C, D) which is mainly found in astrocytes. In the sections shown here, groups of cells positive for NeuN and confluent groups of cells positive for GFAP were found.</p

Tumor formation after subcutaneous inoculation of various numbers of ES cells or <i>in vitro</i> differentiated cells in 129Sv mice.

<p>The indicated number of ES cells and cells differentiated <i>in vitro</i> for 14 days were injected subcutaneously into the flank of 129Sv mice. The percentage and number of animals is indicated in which tumors were found during autopsy at the side of injection before day 100 after injection.</p

Expression of neuronal and glial markers in teratomas grown in 129Sv and SCID/beige mice after injection of ES and differentiated cells.

<p>Teratomas were obtained after injection of ES cells or differentiated (diff.) cells into syngeneic 129Sv mice and immunodeficient SCID/beige mice. Histological sections were stained by immunohistochemistry for the neuronal marker NeuN and the glial marker GFAP. The presence of the markers is indicated semi quantitatively in four categories: − negative, + single positive cells, ++ groups of positive cells, +++ confluent groups of positive cells. Five representative teratomas per group were analyzed.</p>1<p>2 of 5 teratomas were negative for this marker.</p>2<p>3 of 5 teratomas were negative for this marker.</p