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Müller cells are the major supportive and protective glial cells in the retina with important functions in histogenesis and synaptogenesis during development, and in maintenance of mature neurons as they show to secrete various cytokines and manifest potentials of self‐renewal and transdifferentiation into retinal neurons following injury in the vertebrate retinas. The swine retina has a visual streak structure similar to the human macular where cone photoreceptors are highly concentrated, thereby can serve as a better model for studying retinal diseases and for formulating cell‐based therapeutics than the rodent retinas. Like most differentiated somatic mammalian cells, the isolated swine and human Müller glia become senescent over passages in culture, which restricts their potential application in basic and clinic researches. Here, we demonstrate that the senescence of swine and human Müller cells is caused by telomere attrition upon multiplications in vitro; and the senescent cells can be rejuvenated by sphere suspension culture. We also provide evidence that sphere‐induced extension of telomeres in swine and human Müller glia is achieved by alternative lengthening of telomeres or/and by telomerase activation.  S tem  C ells   2017;35:1579–1591

Sphere‐Induced Rejuvenation of Swine and Human Müller Glia Is Primarily Caused by Telomere Elongation