Human-Induced Pluripotent Stem Cells Produced Under Xeno-Free Conditions

Abstract

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. While cell differentiation protocols have been successfully developed, and animal models of human disease have proved that these cells have the potential to treat human diseases and conditions produced as a consequence of aging, degeneration, injury, and birth defects, logistical issues still remain unsolved and hamper the possibility of testing these cells in human clinical trials. Among them is the widely spread use of animal products for the generation and culture of iPSCs. We report here a xeno-free iPSC generation system that addresses all the steps of iPSCs production including the isolation and culture of adult skin fibroblasts, and iPSCs generation, expansion, and maintenance. iPSCs generated with a polycistronic lentiviral vector under xeno-free conditions displayed markers of pluripotency and gave rise to embryoid bodies (EBs) displaying indicators of the 3 primary germ layers. Xeno-free iPSCs injected into nude mice produced classic teratomas, and teratoma explants cultured under conditions favoring fibroblastic cells gave rise to cells morphologically indistinguishable from input cells. Protocols here described will facilitate the implementation of new cellular therapies for preclinical and clinical studies, potentially reducing the regulatory burden without compromising the differentiation potential of the cells.

Experimental reprogramming of murine embryonic fibroblasts towards induced pluripotent stem cells using a single polycistronic vector

“…In addition, ESC-like cell clusters were evaluated for their ability of form teratomas in vivo. Data from our study reveal that the potent cells could be differentiated into many types of cells derived from the three embryonic germ layers, which was similar to conclusions from previous studies (Carey et al, 2009;Ross et al, 2010).…”

Generation of Leukemia Inhibitory Factor and Basic Fibroblast Growth Factor-Dependent Induced Pluripotent Stem Cells from Canine Adult Somatic Cells

“…The testis was minced and incubated in trypsin (Gibco, Carlsbad, CA) at 37°C for 1 h. Then, shredded tissues were spun down, minced again, and subsequently cultured with fibroblast medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum) at 37°C with 5% CO 2 [24]. We replaced the culture medium every 24 h. All ciPSCs were generated from CTFs older than passage 2.…”

“…We produced and concentrated recombinant OKSIM lentivirus, as previously described [24,25]. Canine fibroblasts were assessed for infection efficiency with recombinant lentivirus using a pSIN-EF1a-YFP reporter gene.…”

“…The immunocytochemistry assay protocol was mostly as described in previous reports [24][25][26] RNA extraction and quantitative reverse transcription polymerase chain reaction analysis RNA was isolated and purified using the NucleoSpin RNA XS Total RNA Isolation Kit (Macherey-Nagle, Bethlehem, PA), following the manufacturer's instructions. We performed the reverse transcription polymerase chain reactions (RT-PCRs) as previously described [24,25].…”

“…Here, we report the production of iPSCs from adult canine cells using a method like that described for human and mouse iPSCs [11,24,25]. We systematically show the degree of pluripotency of the generated lines, explore their capacity for stable maintenance, and assay their ability to form embryoid bodies (EBs) and to differentiate into multiple cell lineages.…”