Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue-culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with Inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI-supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. SNP genotyping with Illumina HumanOmniExpressExome-array version 8v1-2_A, encompassing >964.000 single nucleotide polymorphism (SNP)-markers, were performed by the SNP&SEQ technology Platform in Uppsala (www.genotyping.se), according to the manufacturer’s instructions. Each of the four hPS cell lines (i.e. H181, H207, HUES1 and K2C) were analyzed in three separate SNP-array experiments for the 12 samples described here. A first analysis was performed after 2-5 passages of growth for initial reference (4 samples), and subsequently, two analyses were performed per cell line after 16-21 passages in E8:VN or E8:IαI culture (8 samples). The CN-calls of the autosomal chromosomes were illustrated using the Nexus-software.