Morphologic and Gene Expression Criteria for Identifying Human Induced Pluripotent Stem Cells

<div><p>Induced pluripotent stem (iPS) cells can be generated from somatic cells by the forced expression of four factors, Oct3/4, Sox2, Klf4, and c-Myc. While a great variety of colonies grow during induction, only a few of them develop into iPS cells. Researchers currently use visual observation to identify iPS cells and select colonies resembling embryonic stem (ES) cells, and there are no established objective criteria. Therefore, we exhaustively analyzed the morphology and gene expression of all the colonies generated from human fibroblasts after transfection with four retroviral vectors encoding individual factors (192 and 203 colonies in two experiments) and with a single polycistronic retroviral vector encoding all four factors (199 and 192 colonies in two experiments). Here we demonstrate that the morphologic features of emerged colonies can be categorized based on six parameters, and all generated colonies that could be passaged were classified into seven subtypes in colonies transfected with four retroviral vectors and six subtypes with a single polycistronic retroviral vector, both including iPS cell colonies. The essential qualifications for iPS cells were: cells with a single nucleolus; nucleus to nucleolus (N/Nls) ratio ∼2.19: cell size ∼43.5 µm²: a nucleus to cytoplasm (N/C) ratio ∼0.87: cell density in a colony ∼5900 cells/mm²: and number of cell layer single. Most importantly, gene expression analysis revealed for the first time that endogenous Sox2 and Cdx2 were expressed specifically in iPS cells, whereas Oct3/4 and Nanog, popularly used markers for identifying iPS cells, are expressed in colonies other than iPS cells, suggesting that Sox2 and Cdx2 are reliable markers for identifying iPS cells. Our findings indicate that morphologic parameters and the expression of endogenous Sox2 and Cdx2 can be used to accurately identify iPS cells.</p> </div

Characterization of iPS cell colony G.

<p>(A–D) Immunocytochemistry for (A) Oct3/4, (B) Nanog, (C) Sox2, (D) TRA-1-81 in iPS cell colony G. Scale bar = 100 µm. (E, F) EBs generated from colony G were plated on gelatin coated dishes containing DMEM/F12 medium supplemented with 20% knockout serum replacement. After 10 days, cell differentiation was confirmed by immunocytochemistry for mesodermal (smooth muscle actin; SMA) (E), endodermal (alpha-fetoprotein; alpha-FP) (E) and ectodermal markers (neurofilament; NF) (F). Scale bar = 100 µm. (G) RT-PCR of differentiation markers in undifferentiated iPS cell colony G (Undifferentiation) and embryoid bodies derived from iPS cell colony G. Differentiation). (H–K) Hematoxylin and eosin staining of teratoma formed by transplantation of iPS cell colony G into immunodeficient mice testis. (H), Low magnification of the formed teratoma (12 weeks after injection). Endodermal (I), mesodermal (J) and ectodermal (K) tissue were observed in the teratoma.</p

RT-PCR and Q-PCR of typical examples in each colonies H∼L and iPS colony G and human ES cells.

<p>(A) RT-PCR analysis examined the expression of endogenous Oct3/4, Sox2, Nanog, Klf4, c-Myc, as well as FoxD3, Rex1, Dnmt3b, Abcg2 and Cdx2. Beta-actin was used as an internal control. (B) Q-PCR data for expression of endogenous Oct3/4, endogenous Sox2, Nanog, endogenous Klf4, endogenous c-Myc, FoxD3, Rex1, Dnmt3b, Abcg2 and Cdx2.</p

Morphometric analysis in colonies generated from human fibroblasts by using a single polycistronic Oct3/4-Klf4-Sox2-c-Myc-GFP expressing viral vector.

<p>(H–L, G) Photograph and parameters of colonies H∼L and G. Graphs shows parameters of each classified colony, including that of iPS cell colony G. The numerical value in the graph indicate the ratio to the maximum value (setting 100 for maximum value) in each parameter. Scale bars = 100 µm.</p

Classification of morphologic characteristics in colonies generated from human fibroblasts using a single polycistronic viral vector.

<p>Each replicate represented 2×10⁵ GFP positive cells seeded onto a 60-mm dish containing feeder cells and cultured in Primate ES cell medium for thirty days.</p

Classification of morphologic characteristics of colonies generated from human fibroblasts using four retroviral vectors encoding Oct3/4, Sox2, Klf4, and c-Myc.

<p>Each replicate represented 2×10⁵ transduced cells seeded onto a 60-mm dish containing feeder cells and cultured in Primate ES cell medium for thirty days.</p

Transplantation efficacy demonstrated by the percentage of human nuclei in the transplanted muscles.

<p>The number of cells of human origin was divided by the number of total nuclei stained by DAPI. The result also appears in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051638#pone-0051638-g004" target="_blank">Figures 4C and 4D</a>. SD: standard deviation. ND: not detected. *, ** indicate the cell numbers transplanted at the site: * 5.0×10⁵ cells/site, ** 1.0×10⁵ cells/site. †: One mouse transplanted with 253G4-derived cells died accidentally before analysis. NS: statistically not significant.</p

Schematic presentation of the differentiation protocol.

<p>Two different differentiation protocols were compared (Left: embryoid body (EB) culture, Right: Dissociation culture) and were exactly the same until the first 21 (7+14) days of culture. On the left side, EBs continued to be incubated in serum-containing medium without specific manipulation until the end of culture. On the right side, EBs and their outgrowth cells were dissociated and seeded onto collagen type I-coated tissue culture plates in serum-containing medium. The medium was changed to serum-free ITS medium on day 49 (7+14+28). In some experiments, the cells were harvested and used as donor cells for the transplantation assay at this time point.</p

Characterization and differentiation of the derived myogenic mesenchymal cells.

<p>(A) Morphology of the derived myogenic mesenchymal progenitors 2 days (day 7+14+2) and 4 weeks (day 7+14+28) after replating. Homogeneous spindle-shaped fibroblastic cells were observed. (B) Surface marker analysis of myogenic mesenchymal progenitors. Representative data from KhES1 differentiation are shown. Note that CD56 in addition to mesenchymal markers CD73, CD105, CD166, and CD29 was exclusively expressed. (C) Changes in the expression of myogenic markers were analyzed by immunofluorescence. The number of Cy3-positive nuclei was divided by the total number of nuclei stained by DAPI. The expression of myogenic progenitor markers decreased after exposure to serum-free medium, whereas the number of MYOG-positive cells substantially increased after serum deprivation. (D) Changes in the number of MYOG-positive nuclei were observed up to 3 weeks after serum deprivation. hES/iPS-derived myofibers tended to detach from tissue culture plates during long-term culture in serum-free medium. (E) Serum deprivation increased the number of skeletal myosin-positive fibers and MYOG-positive nuclei for more than 2 weeks. KhES1 was used in this figure. (F) Multinucleated myofibers denoted by MYOG myogenin-positive nuclei aligned in skeletal myosin-positive fibers. (G) Morphology of mature myofibers, which were stained with skeletal myosin, MYOG, and dystrophin, from both KhES1 and 253G4 cells. Skeletal myosin was visualized with fluorescein isothiocyanate (FITC) (Green), myogenin was visualized with Cy3 (red), and nuclei were counterstained with DAPI (blue). Scale bars  =  (C, E) 100 µm, (D) 50 µm.</p

Engraftment of myogenic progenitors in damaged muscles of immunodeficient mice.

<p>(A) Human nuclei labeled with human-specific lamin A/C localized mainly inside muscle fibers surrounded by laminin. (B) Muscle reconstruction by transplanted human cells was demonstrated by the detection of human-specific laminin-alpha 2. (C) The proportion of myofibers containing human nuclei at 4, 12, and 24 weeks after transplantation. (D) The proportion of myofibers containing human nuclei in reinjured (3+1 weeks) and in non-reinjured mice (4 weeks) at 4 weeks after transplantation. In C and D, data are presented as the mean ± standard deviation. (E) Distribution of the transplanted cells at 24 weeks after transplantation. Typical central nuclei of human origin were observed (outlined arrowheads). Some human cells located within the lamina rara beneath the basal lamina, indicating engraftment of the transplanted cells into a satellite cell compartment (white arrowhead). (F) Triple-staining for human Lamin A/C, PAX7, and pan-Laminin clearly demonstrated the existence of PAX7-positive human nuclei indicating the transplanted cells engrafted as satellite cells (white arrowhead). Human lamin A/C-negative host satellite cells were also detected (outlined arrowhead). Laminin was stained by a polyclonal antibody that recognizes both human and murine laminin, and was subsequently visualized with fluorescein isothiocyanate (FITC) (Green); human lamin A/C and human-specific laminin, with Cy3 (red). Nuclei were counterstained with DAPI (blue). Scale bars  =  (A) 100 µm, (B) and (E) 50 µm.</p