Stem cells with multilineage potential derived from porcine skin Paul W. Dyce, 1 Hai Zhu, Jesse Craig, 2 and Julang Li * Department of Animal and Poultry Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 21 January 2004 Abstract Stem cells from farm animals are valuable cell models for the study of development, differentiation, and are potential efficient donors for nuclear transfer. Here we report the isolation and characterization of stem cells from porcine skin. These porcine skin- originated sphere (PSOS) cells expressed the neural progenitor marker, nestin, as well as genes that are critical for pluripotency such as Oct4 and Stat3. The PSOS cells proliferated actively in vitro and retained normal karyotype after long-term culture. When cultured in defined medium, they generated cells with characteristics of neurons and astrocytes. A subpopulation of cells differ- entiated into adipocyte-like cells when cultured in 10% fetal bovine serum. Clonal study demonstrated that PSOS exhibited clonal- generating capability. Clonal populations from individual stem cells could form neuron-, astrocyte-, and adipocyte-like cells upon inducted differentiation. Our findings represent the first report of skin-originated stem cells isolated from non-rodent animals. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Stem cell; Porcine; Differentiation; Isolation; Skin; Neural cells In mammals, the epidermis of skin is continually re- furbished every day. The turnover time of the epidermis is about 60 days in human and 7 days in mice [1,2]. This rapid remodeling is maintained by stem cells that are capable of self-renewal and supplying differentiated cells in a constant manner. Stem cells within the hair follicle [3,4], sebaceous gland, and epidermis [5] have been demonstrated to be involved in cutaneous renewal dur- ing postnatal life. Moreover, multipotent stem cells that can form neural and adipose cells have also been iso- lated from the dermis of mouse skin [6]. These findings indicate that multiple classes of stem cells with different differentiation potentials are present in the skin, making skin a valuable source of stem cells for the study of development and differentiation. Despite many successes in the isolation and characterization of stem cells from various tissues of rodents and humans, there have been few studies on stem cells from non-rodent animals. This limits the advancement of stem cell biology and its ultimate translation to human clinical applications. Stem cells derived from livestock animals may also offer solutions for more efficient nuclear transfer (animal cloning). The current extremely low success rate of live births from nuclear transfer is thought to be due to the reprogramming failure of the transferred somatic nu- cleus to a state equivalent to that of a early embryo in a confined time window by the oocyte [7]. It was thus hypothesized that the genome of undifferentiated stem cells may be more easily reprogrammed to resemble the genome of the zygote, which may make stem cells more efficient donors for nuclear transfer [7,8]. In this report, we describe the isolation of multipotent stem cells from fetal porcine skin. Clonal populations from individual stem cells can proliferate and differentiate to form cells with characteristics of neurons, astrocytes, and adipo- cytes. Our findings represent the first report of skin- originated stem cells isolated from non-rodent animals. Materials and methods Cell isolation and culture. Cells were isolated using a previously described method [6] with modifications. Briefly, fetuses were * Corresponding author. Fax: +1-519-836-9873. E-mail address: jli@uoguelph.ca (J. Li). 1 Recipient of Ontario Graduate Scholarship in Science and Technology. 2 Recipient of Natural Sciences and Engineering Research Council (NSERC) studentship. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.093 Biochemical and Biophysical Research Communications 316 (2004) 651–658 BBRC www.elsevier.com/locate/ybbrc