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In Vivo Generation of Neural Tumors from Neoplastic Pluripotent Stem Cells Models Early Human Pediatric Brain Tumor Formation



From Stem Cells
By Stuart P. Atkinson

Several recent studies (Ben-Porath et al, Somervaille et al and Wong et al) have identified ESC-like gene expression patterns in a variety of malignant tumors suggesting a molecular relationship between aggressive cancers and pluripotency. However, the molecular determinants of such a link have yet to be uncovered. Now using normal human embryonic stem cells (hESCs) and transformed hESCs (t-hESCs), which have acquired features of neoplastic progression, including enhanced self-renewal, proliferation and tumor-initiating cell capacity, and aberrant neural lineage specification (Werbowetski-Ogilvie TE et al. 2009), researchers from the group of Mickie Bhatia at the McMaster University, Hamilton, Ontario, have shown that derived neural progenitors from the transformed cells possess brain tumor-initiating cell capacity, thereby providing a model system to investigate initiation and progression of primitive human neural cancers, such as medulloblastoma that are difficult to assess using somatic sources (Werbowetski-Ogilvie et al 2012).

Neural precursor differentiation of hESCs and t-hESCs was undertaken using neurosphere formation and adherent culture, with NES and A2B5 expressed at similar levels in hESC- and t-hESC-derived neural precursors (N-hESC and N-t-hESC). SSEA3 was only detected in adherent N-t-hESCs over the first 4 days of differentiation and N-t-hESCs showed a three fold increase in number at 5 days compared to N-hESC in both culture conditions, with cell in the neurospheres showing an increased level of clonogenic self-renewal. Cells were then differentiated toward neurons and oligodendrocytes following EGF and bFGF withdrawal. In contrast to N-hESCs from adherent cultures, N-t-hESCs formed aggregates or small neural-like rosettes showing a reduction in the frequency of βIII tubulin+ neuronal-like cells and an increase in frequency and total number of OLIG4+ oligodendrocyte-like cells. Neurosphere differentiation also led to an increased frequency of βIII tubulin+ and OLIG4+ cells in the N-t-hESCs, but total number was lower compared to hESCs suggesting a differentiation defect in the transformed cells. Astrocytic differentiation through BMP4 exposure demonstrated a complete lack of GFAP+ astrocytes in the N-t-hESC adherent culture, and the frequency and number in neurosphere-derived cells was significantly reduced.

The tumourigenic capacity of the N-hESCs and N-t-hESCs were next evaluated through intracerebral transplantation into NOD-SCID mice. While no physical changes were observed in mice injected with N-hESCs, those injected with N-t-hESCs showed physical deterioration at 4 weeks and upon inspection of the brain, presented with large masses on the ventral surface of the brain, which were shown to be 60-80% neuroectoderm defined by the presence of neural rosettes, as well as vascularised, infiltrative lesions in the brain stem and cerebellar regions. The ventral masses also showed evidence of undifferentiated pluripotent cells through immunohistochemical staining for OCT4, indicating that the t-hESCs retain a pluripotent program even after time in differentiation promoting conditions.   Further analysis of NES+ transplanted cells from N-hESCs and N-t-hESCs, found levels of SSEA3 and OCT4 below threshold levels required for teratoma formation in normal hESCs, although levels in two of the N-t-hESC lines were potentially high enough to initiate teratoma formation. Further, when these cells were replated in self-renewing conditions, N-t-hESCs only were able to form TRA-1-60+ pluripotent colonies.

In addition to the ventral teratomas caused by N-t-hESC-derived cell transplantation, N-t-hESCs also extensively infiltrated the cerebellum and along the ventricles in 4 of the seven mice. Large tumours found in the cerebellum and cortex had a high nuclear-to-cytoplasmic ratio, disorganized appearance, showed the presence of multiple neural rosettes and several blood vessels were identified in tumors lining the ventricles. While the tumours were negative for OCT4, intense βIII tubulin staining for neuron-like cells, as well as a high Ki67 proliferative labelling index (30-40%) was also observed and based on these findings, the samples were independently identified as medulloblastoma or primitive neuroectodermal tumor (PNET)-like tumors.

The medulloblastoma properties of these tumours were further proved through global gene expression profiling. N-t-hESCs showed a medulloblastoma molecular profile with dysregulation of several transcripts already associated with this malignant brain tumor (Pax6, Pax5, Otx2 and FoxG1). Further analysis showed the medulloblastoma to have strong Wnt-pathway dysregulation. To analyse how the pluripotent program may be affecting tumourigenesis, concurrent expression of SSEA3 with the neural precursor cell surface markers A2B5 in N-t-hESCs that were intracerebrally injected into NOD SCID mice. These cells were 99.98%-99.99% SSEA3, indicating a pure population of cells but comparisons of gene expression data sets found that these SSEA3- N-t-hESC-derived cells exhibited dysregulation of 50 ESC-enriched genes, of which 44 were upregulated, including LIN28. Purified populations of A2B5+/SSEA3 compared to A2B5/SSEA3 t-hESCs showed upregulation of pluripotent (Lin28A, Dppa4, and Oct4) and neural/medulloblastoma transcripts and a higher level of neurosphere formation in the A2B5+ population.

Overall, these results suggest that N-t-hESCs exhibit enhanced cell proliferation and self-renewal as well as retention of the pluripotent state and cells from both adherent and neurosphere cultures display aberrant differentiation features, particularly toward the astrocytic lineage. The retention of the pluripotent program leads to enhanced tumourigenic properties allowing teratoma tumor formation and infiltration upon intracerebral transplantation. These infiltrating cells cause tumours with features typically associated with medulloblastoma and (PNET)-like tumors which may be mediated by the enrichment of hESC-associated gene expression. These data alongside the homogenous nature of the N-t-hESCs suggests their potential value as a medulloblastoma model system.



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