Figure 1.
The hypothesis of miPS differentiation when exposed to normal or malignant niche.
miPS cells should be induced to some kinds of progenitor cells, such as hematopoietic cells and neural stem cells, differentiating into various phenotypes, such as macrophage, monocytes, neural cells, cardiac cells and pancreatic β- cells, when exposed to the normal niche. We hypothesized that CSCs may also be derived from miPS cells only when exposure to a malignant niche.
Figure 2.
Characterization of miPS-LLCcm cells and derived tumor.
(A) Histology of miPS-LLCcm cells derived tumor. The tumor exhibited malignant phenotype with glandular epithelial hyperplasia (asterisk), high nuclear to cytoplasmic ratio, severe nuclear atypia and multiple pathological mitotic figures (arrowheads, inset) (top left); micrometastases (arrow, top right); and hypervascularization indicative of angiogenesis (bottom left) by HE staining. The positive of CD31 (Rat monoclonal antibody, brown) by IHC staining showed multiple vascular vessels in the tumor (bottom right). Scale bars: 100 µm (top left and bottom), 50 µm (top right). (B) Primary culture derived from miPS-LLCcm tumor. The primary culture exhibited stem-like cells (asterisk in top left) expressing GFP (top right) and fibroblast-like cells (arrow in top left) without GFP expression (top right). Spheroid cells grown from the primary culture in suspension (middle left) with GFP expression (middle right). The spheroid cells were placed back in adherent culture maintained stem-like cells (asterisk in bottom left) with GFP expression (bottom right) and fibroblast-like cells (arrow in bottom left) without GFP expression (bottom right). (C) Immunofluorescence staining for Nanog and Oct 3/4 in spheroid cells. Cryosections of spheroid cells were stained with primary antibodies (Rabbit anti-Nanog or Mouse anti-Oct-3/4) followed by anti-Rabbit or anti-mouse secondary antibodies labeled with Alexa fluorophores 555 (red) or 488 (Green). The cells were counterstained with DAPI (blue). Scale bars: 20 µm. (D) The expression levels of p53, MMP-2 and MMP-9 were analyzed by quantitative real-time PCR. miPS+LIF/−MEF, miPS cells cultured with LIF in the medium but without MEFfeeder cells; miPS-LLCcm spheroid, the spheroid cells derived form miPS-LLCcm cells; miPS-LLCcm LMT spheroid, the spheroid cells derived from miPS-LLCcm cells lung metastatic tumor; LLC, Lewis lung carcinoma cells.
Table 1.
Summary of tumorigenic potential of cells derived from miPS cells.
Figure 3.
Characterization of tumor derived from spheroid cells.
(A) Histology of tumor derived from spheroid cells. The tumor showed some glandular structure (asterisks) with multiple pathologic mitotic figures (arrowheads, inset) (left), high mitotic rates (arrowheads in middle), and hypervascularization (right) by HE staining. Scale bars: 100 µm. (B) Lung metastasis after tail vein injection of spheroid cells. Lungs were occupied by metastatic tumor nodules. (C) The metastases showed some glandular structure (asterisks) with multiple pathologic mitotic figures (arrowheads in top left); hypervascularization (top right); invasion into lung parenchymal tissue (bottom left) by HE staining. The expression of GFP (Rabbit polyclonal antibody, brown) was found in these metastatic nodules by IHC staining (bottom right). T, tumor; L, lung tissue. Scale bars: 100 µm. (D) Immunohistochemistry of CK and GFP localization in tumors derived from miPS, miPS-LLCcm and spheroid cells. Serial sections were stained with CK (mouse monoclonal antibody, brown) and GFP (Rabbit polyclonal antibody, brown), and counterstained with hematoxylin. Glandular region were CK positive but GFP negative in the tumors. Scale bars: 100 µm.
Table 2.
Summary of tumorigenic potential of miPS-LLCcm spheroid cells.
Figure 4.
Gene expression in miPS cells, derived cells, MEF cells and tumor tissues.
(A) RT-PCR analysis of embryonic stem cell marker gene expression. (B) RT-PCR analysis of the four miPS transcription factors. The PCR products were the coding regions (Total), endogenous transcripts only (Endo.), and transgene transcripts only (tg). (C) Expression levels of embryonic stem cell marker gene were analyzed by quantitative real-time PCR. (D) Expression levels of the four miPS cell transcription factors were analyzed by quantitative real-time PCR.