Rat iPSCs Research Articles

Derivation and characterization of rat iPS cells using a mouse STEMCCA reprogramming vector

Rats are considered an important animal model system by having many similarities to humans in terms of physiological processes of the cardiovascular, metabolic and neurological systems. Induced pluripotent stem cell (iPSC) technology has great potential to advance in the field of regenerative medicine by generating patient‐specific stem cell for treatment or in vitro disease modeling. Here, we derived rat iPSC's from Fischer 344 embryonic fibroblasts using a mouse STEMCCA cre‐excisable reprogramming vector expressing Oct‐4, Klf4, Sox‐2 and c‐Myc. Once rat ESC‐like colonies were derived, we removed the transgene vector and demonstrated that one of the clones tested showed a normal karyotype by G‐banding. The iPSC colonies demonstrate a morphology that is similar to rat embryonic stem cells (ESCs), are positively stained for pluripotency markers such as alkaline phosphatase, Oct‐4, Nanog, Sox‐2 and SSEA‐1. The rat iPSCs have been maintained more than 27 passages. Using a custom‐designed microarray, we compared the gene expression profile of rat iPSCs to rat ESCs and there was good similarity between the two with the exception of Cdx2 expression which is expressed in rat ESCs. We plan to further characterize the rat iPSCs to determine whether they can undergo neural differentiation in vitro and whether the micro RNA expression of rat iPSCs is similar to rat ESCs.

Differentiation of rat iPS cells and ES cells into granulosa cell-like cells <italic>in vitro</italic>

Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarian follicles before 40 years of age, representing one major cause of female infertility. Stem cells provide the possibility of a potential treatment for POF. In this study, rat embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were co-cultured with granulosa cells (GCs) to differentiate to GC-like cells. The level of estradiol (E2) analyzed by radioimmunoassay showed that the E2 concentration of the culture supernatant of co-cultured rat iPSCs and ESCs increased in a time-dependent manner, compared with the GCs group that has an opposite trend. The expression of follicle-stimulating hormone receptor (FSHR) was confirmed by immunostaining. These results indicated that rat iPSCs and ESCs were effectively induced to GC-like cells through indirect cell-to-cell contact. Real-time polymerase chain reaction was performed to analyze the expression level of marker genes in POF, including BMP15, FMR1, FSHR, INHA, AMH, NOBOX, FOXO3, EIF2B, FIGLA, and GDF9. The BMP15, FSHR, INHA, AMH, NOBOX, and GDF9 genes were significantly up-regulated in iPSCs and ESCs co-cultured with GCs in comparison with cells that were not co-cultured. Thus, here we demonstrated an available method to differentiate rat iPSCs and ESCs into GC-like cells in vitro for the possible cell therapy of POF.

Proof of pluripotency of rat iPSCs missing

We found the article (1) by Drs. G. Li, Y. Chunxu andL. Guisheng, published in February 2013 issue of CellProliferation, to raise several questions and we wouldwelcome a number of points of clarification from theauthors. In the article, the authors describe reprogram-ming Fawn Hood Hypertensive (FHH) rat embryonic fi-broblasts (REFs), using a piggyBac transposon andtransposase, to introduce genes Oct4, Sox2, Klf4, Myc,(OSKM) or Oct4, Sox2, Klf4, Myc and Lin28 (OSKML),which are linked by sequences encoding 2A peptidesand driven by the CAG promoter, to produce inducedpluripotent stem cells (iPSCs). While the authors claimto have produced iPSCs, their data did not show germ-line competence of the reprogrammed cells. For that rea-son, we think, it is premature to call these rat cellsiPSCs.The authors did not report whether rat or mouseOSKM or OSKML sequences were used in their vector.While they did not report reprogramming efficiency,they showed five small and one large colony in one field(Fig. 1c), which was seen on day 10. This would sug-gest high reprogramming efficiency and reprogramming,that occurred rapidly. In Fig. 1d, readers are unable tosee alkaline phosphatase (AP) staining, and we ask:what proportion of the colonies stained for AP?In their iPSC characterization, immunocytochemicalstaining of SSEA-1, Oct4, Nanog and Sox2 was shownin Fig. 2; however, readers are unable to verify co-local-ization of these transcription factors in Fig. 2. Stainingin Fig. 2 appears to be from colonies selected to expressthe pluripotency genes. We note that phase contrast andnuclear staining micrographs were not provided. Readersare unable to confirm whether an entire colony stainedfor transcription factors Oct4, Nanog and Sox2 and mosttroubling, the Oct4 staining appears to be cytoplasmicand not nuclear, for many cells.As mentioned above, readers were not told whetherrat or mouse OSKM or OSKML sequences were usedin their piggyBac transposon vector for reprogramming,thus, we were unsure how to interpret the authors’ claimthat endogenous pluripotency markers were expressed(Fig. 2b). How did the authors confirm transfection ofthe piggyBac vector? In Fig. 2b, the authors reportedRT-PCR analysis of endogenous pluripotency markerKlf4, but it was not shown. From information provided,we are able to verify that Nanog was induced, but weare uncertain whether Oct4 and Sox2 expression was theresult of the exogenous vector or of expression ofendogenous genes.According to the Materials and methods section,the authors here injected FHH iPSCs into blastocystscollected from FHH X SD F1 rats. In the Results sec-tion, however, they indicated using FHBN14 X SDblastocysts. Using a GOOGLE Search, FHBN14 ratswere not found. Did they mean FHH-Chr 14BN/Mcwi,for example, FHH rats containing the Brown Norwaychromosome 14? We were confused by Fig. 4b and4d, which showed FHH rat iPSC ‘chimeras’ with adark coat colour. The FHH rat is white with a ‘lighttan’ coloured hood. Would the authors explain why the‘chimeras’ had dark coat colour? Figure 4e does notmake sense either. If we assume that the authors usedFHH-Chr 14BN X SD blastocysts and injected FHHiPSCs, microsatellite analysis would distinguish chima-eras made of FHH iPSCs in the FHH-Chr 14BNxSDF1 host. On the other hand, if they used FHH x SDblastocysts, they would be unable to use microsatelliteanalysis to identify chimaera. If FHH-Chr14BNxSD F1blastocysts were transferred, however, they could nothave an SD littermate (as indicated in the Fig. 4).Finally, according to the Rat Genome Database (http://www.rgd.mcw.edu/rgdweb/report/marker/main.html?id=36520), microsatellite D14Rat7 differentiatesbetween BN rats (174), SD (166) and FHH (142). Werequest clarification from the authors on these points,please.From our experience with embryo transfer (2), webelieve, it is unlikely that blastocysts transferred intothe uterus of 0.5 days post-coitus (dpc) pseudopregnantfemales would implant and be carried to term. Weinvite the authors to comment on their rationale forembryo transfer into 0.5 dpc females. Do the authorsbelieve that blastocyst transfer into 0.5 dpc pseudopreg-nant females is more efficient than transfer into3.5 dpc pseudopregnant females (as we and othershave performed)?In summary, we cannot agree with the authors’ con-clusion that pluripotency of their iPSCs has been dem-

Efficient p53 gene targeting by homologous recombination in rat‐induced pluripotent stem cells

To generate rat induced pluripotent stem cells (iPSCs) using the PiggyBac (PB) transposon system, and to explore whether these iPSCs would be amenable to genetic manipulation. The PB transposon system was used to reprogramme rat embryonic fibroblasts (EF) to become iPSCs. Cells were identified with regard to pluripotency and differentiation capacity in vitro and in vivo, population growth characteristics and gene expression; furthermore, targeting vector was electroporated into them. Correct recombination colonies were acquired by positive and negative selection, and then phenotype confirmed by Southern blotting. The rat EF cells were reprogrammed into iPSCs successfully, using the PB transposon system. Cell morphology was found to display characteristics of rat embryonic stem cells (ESCs) and results of immunofluorescence staining and PCR indicated that they expressed pluripotency markers. In vivo and in vitro differentiation experiments proved that the cells could differentiate into all phenotypes from three germ layers, and to form chimaeras with high rat iPSC contribution. After electroporation with p53 targeting vector, approximately (5.44 ± 0.74) × 10(-6) colonies tolerated selection. Southern blotting confirmed that p53 gene was targeted successfully in the colonies. The PB transposon system proved to be an effective method for reprogramming of rat EF cells into iPSCs. The rat iPSCs were amenable to gene targeting mediated by routine homologous recombination.

Generation of Germline-Competent Rat Induced Pluripotent Stem Cells

BackgroundRecent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.Methodology/Principal FindingsWe generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines.Conclusions/SignificanceOur data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.

Intramyocardial Transplantation of Undifferentiated Rat Induced Pluripotent Stem Cells Causes Tumorigenesis in the Heart

BackgroundInduced pluripotent stem cells (iPSCs) are a novel candidate for use in cardiac stem cell therapy. However, their intrinsic tumorigenicity requires further investigation prior to use in a clinical setting. In this study we investigated whether undifferentiated iPSCs are tumorigenic after intramyocardial transplantation into immunocompetent allogeneic recipients.Methodology/Principal FindingsWe transplanted 2×104, 2×105, or 2×106 cells from the established rat iPSC line M13 intramyocardially into intact or infarcted hearts of immunocompetent allogeneic rats. Transplant duration was 2, 4, or 6 weeks. Histological examination with hematoxylin-eosin staining confirmed that undifferentiated rat iPSCs could generate heterogeneous tumors in both intracardiac and extracardiac sites. Furthermore, tumor incidence was independent of cell dose, transplant duration, and the presence or absence of myocardial infarction.Conclusions/SignificanceOur study demonstrates that allogeneic iPSC transplantation in the heart will likely result in in situ tumorigenesis, and that cells leaked from the beating heart are a potential source of tumor spread, underscoring the importance of evaluating the safety of future iPSC therapy for cardiac disease.

Rats, Cats, and Elephants, but Still No Unicorn: Induced Pluripotent Stem Cells from New Species

Two independent studies in this issue of Cell Stem Cell (Liao et al., 2009; Li et al., 2009) derive rat induced pluripotent stem cells (iPSCs). In one report, the method used results in rat and human iPSCs that exhibit phenotypic traits similar to mouse embryonic stem cells.