Production of mice using iPS cells and tetraploid complementation.
ABSTRACT Induced pluripotent stem cells (iPSCs) are considered to be an attractive alternative to embryonic stem cells (ESCs) and may provide great potential for clinical applications in regenerative medicine. Although possessing characteristics similar to ESCs, the true pluripotency of these newly studied iPSCs was not known because none of the previously developed iPSCs passed the tetraploid complementation assay, which is regarded as the most stringent test for pluripotency. We have recently shown that by modifying some of the culture conditions for inducing iPSCs, we were able to generate cell lines of high pluripotency, resulting in the production of live-born, fertile animals through tetraploid complementation. In this paper, we describe details of our methods of generating iPS cell lines and subsequently producing full-term live animals through the tetraploid complementation assay; the procedure can be completed within 2 months.
SourceAvailable from: Yue Yu[Show abstract] [Hide abstract]
ABSTRACT: Tens of millions of patients are affected by liver disease worldwide. Many of these patients can benefit from therapy involving hepatocyte transplantation. Liver transplantation is presently the only proven treatment for many medically refractory liver diseases including end-stage liver failure and inherited metabolic liver disease. However, the shortage in transplantable livers prevents over 40% of listed patients per year from receiving a liver transplant; many of these patients die before receiving an organ offer or become too sick to transplant. Therefore, new therapies are needed to supplement whole-organ liver transplantation and reduce mortality on waiting lists worldwide. Furthermore, the remarkable regenerative capacity of hepatocytes in vivo is exemplified by the increasing number of innovative cell-based therapies and animal models of human liver disorders. Induced pluripotent stem cells (iPSCs) have similar properties to those of embryonic stem cells (ESCs) but bypass the ethical concerns of embryo destruction. Therefore, generation of hepatocyte-like cells (HLCs) using iPSC technology may be beneficial for the treatment of severe liver diseases, screening of drug toxicities, basic research of several hepatocytic disorders, and liver transplantation. Here we briefly summarize the growing number of potential applications of iPSCs for treatment of liver disease.11/2014; 7(1). DOI:10.3727/215517914X680056
[Show abstract] [Hide abstract]
ABSTRACT: Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3' end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.PLoS ONE 10/2014; 9(10):e108682. DOI:10.1371/journal.pone.0108682 · 3.53 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Objectives Recently, pluripotency of induced pluripotent stem (iPS) cells has been displayed after producing adult mice, in tetraploid complementation assays. These studies lead us to the last piece of the puzzle for reprogramming somatic cells into fully pluripotent cells which function as embryonic stem cells in most applications. However, in all of previous studies, skin fibroblasts were used as the starting population for reprogramming, raising questions as to whether the pluripotency of the iPS cells was dependent on the particular starting cell type.Materials and methodsOur iPS cell lines were prepared from murine adipose stem cells (ASCs). Their multi-potency was first tested by teratoma formation in nude mice. Then, tetraploid complementation was performed to generate progeny from them.ResultsWe succeeded to the birth of viable and fertile adult mice derived entirely from reprogrammed ASC, indicating cell types other than fibroblasts can also be restored to the embryonic level of pluripotency.Conclusions We also directed differentiation of iPS cells into chondrocytes, thus adipose-derived iPS cells can be used as models to study chondrogenic differentiation and cartilage regeneration.Cell Proliferation 11/2014; 48(1). DOI:10.1111/cpr.12152 · 3.28 Impact Factor