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Ex vivo maintenance and expansion and subsequent transplantation of adult stem cells are indispensable for successful cell therapy of self-renewing tissues, such as the epidermis and cornea epithelium. Adult stem cells maintain their stem cell properties throughout cell culture. After transplantation, they permanently engraft, self-renew, and properly produce functional progenies, which results in long-term therapeutic success (De Luca et al., 2006; Barrandon et al., 2012). Human epidermal keratinocyte stem cells (holoclones; Barrandon and Green, 1987b) can be cultivated under suitable conditions (Rheinwald and Green, 1975), and a single holoclone can generate a progeny large enough to entirely reconstitute the epidermis of an adult human for a lifetime (Rochat et al., 1994; Mathor et al., 1996). This has enabled the autologous transplantation of cultured keratinocytes onto patients with extensive burns (Gallico et al., 1984; Pellegrini et al., 1999; Ronfard et al., 2000) and genetic disorders (Mavilio et al., 2006; De Rosa et al., 2014), and the successful application of human stem cells for regenerative medicine (De Luca et al., 2006; Green, 2008; Barrandon et al., 2012; Fuchs, 2012). A human keratinocyte culture also contains other clonogenic keratinocytes with restricted growth capabilities (Barrandon and Green, 1987b): progenitor cells (meroclones), which can regenerate an epidermis for a short duration, and transient amplifying cells (paraclones) that cannot regenerate an epidermis at all. Holoclones are eventually converted into meroclones or paraclones during serial cultivation (Barrandon et al., 2012; Rochat et al., 2012), and the loss of holoclones hinders successful transplantation (Rama et al., 2010; Pellegrini et al., 2013). Hence, for regenerative medicine, the determination of number of holoclones in a keratinocyte culture is the best criteria to assess quality (Rama et al., 2010; Barrandon et al., 2012; Rochat et al., 2012; Pellegrini et al., 2013). However, holoclones have been discriminated from meroclones and paraclones by ex post clonal analysis (Barrandon and Green, 1987b) and expression of transcription factor p63 (Pellegrini et al., 2001). A holoclone assay takes 19 d to perform (Barrandon and Green, 1987b; Barrandon et al., 2012), and that has limited its usefulness for regenerative medicine applications. Expression of cell surface proteins including 1, 2, 4, 6, 1, and 4 integrin subunits, transferin receptor, Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that 6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation. Cell motion predicts human epidermal stemness