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 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

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