CHIR99021 Research Articles

CHIR99021 promotes self-renewal of mouse embryonic stem cells by modulation of protein-encoding gene and long intergenic non-coding RNA expression

Embryonic stem cells (ESCs) can proliferate indefinitely in vitro and differentiate into cells of all three germ layers. These unique properties make them exceptionally valuable for drug discovery and regenerative medicine. However, the practical application of ESCs is limited because it is difficult to derive and culture ESCs. It has been demonstrated that CHIR99021 (CHIR) promotes self-renewal and enhances the derivation efficiency of mouse (m)ESCs. However, the downstream targets of CHIR are not fully understood. In this study, we identified CHIR-regulated genes in mESCs using microarray analysis. Our microarray data demonstrated that CHIR not only influenced the Wnt/β-catenin pathway by stabilizing β-catenin, but also modulated several other pluripotency-related signaling pathways such as TGF-β, Notch and MAPK signaling pathways. More detailed analysis demonstrated that CHIR inhibited Nodal signaling, while activating bone morphogenetic protein signaling in mESCs. In addition, we found that pluripotency-maintaining transcription factors were up-regulated by CHIR, while several developmental-related genes were down-regulated. Furthermore, we found that CHIR altered the expression of epigenetic regulatory genes and long intergenic non-coding RNAs. Quantitative real-time PCR results were consistent with microarray data, suggesting that CHIR alters the expression pattern of protein-encoding genes (especially transcription factors), epigenetic regulatory genes and non-coding RNAs to establish a relatively stable pluripotency-maintaining network.

Enhanced generation of human embryonic stem cells from single blastomeres of fair and poor-quality cleavage embryos via inhibition of glycogen synthase kinase β and Rho-associated kinase signaling

Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)? Inhibition of glycogen synthase kinase β (GSK3β) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs. Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs. In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3β inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH + Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study. Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives. We found that 3 μM CH was the only SM that was capable of directing SBs from fair and poor-quality 6-8-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved hESC generation efficiency by up to 31%. By using of Kenpaullone as an alternative to CH, we confirmed the involvement of GSK3 inhibition in hESC derivation from SBs. Interestingly, by treatment of 4-cell embryos, these SMs could enhance the derivation efficiency of SB-derived hESC lines up to 73% and the maximum number of hESC lines from SBs of one embryo was achieved in this state. The low quality of the embryos used in this study most likely had an effect on hESC generation. Furthermore, although we attempted to minimize any differences in inter-embryo quality, we cannot exclude the possibility that small differences in starting quality between embryos may have contributed to the differences observed, other than the addition of SMs. This approach would allow the establishment of autogeneic or allogeneic matched cells from embryos fertilized in vitro without destroying them. This study was financially supported by the National Elite Foundation and the Royan Institute for Stem Cell Biology and Technology. The authors have no conflict of interest to declare.

Expression Pattern of Pluripotency Markers in Rabbit Embryoblast

The aim of our work was to analyse the expression pattern of embryonic stem cell specificmarkers in rabbit embryos and epiblast cells. There are well-known genes that direct pluripotency inthe mouse, such as Oct4, Nanog, but their expression pattern during the rabbit embryonicdevelopment is not known yet. Using culture medium supplemented with two inhibitors [(2i:PD032590-(PD), CHIR99021-(CH); embryonic stem cell (ESC) lines could be derived efficientlyfrom mouse and rat embryos. We cultured the rabbit embryos from eight-cell-stage in RDH, RDH+2iculture medium until the blastocyst stage. The expression level of epiblast specific Oct4 and Nanog,trophoblast specific Cdx2 and hypoblast specific Gata4 and Gata6 were examined in blastocysts andattached embryoblasts. The expression level of Oct4 and Nanog was significantly higher inblastocysts, cultured in RDH+2i culture medium, but there was no significant difference in CDX2,GATA4, GATA6 expression on mRNA and protein level, compared to embryos cultured in RDHmedium. We hope that we can maintain the pluripotency of the epiblast derived stem cells in 2icontaining medium during long term cultivation.

Inhibition of glycogen synthase kinase-3 promotes efficient derivation of pluripotent stem cells from neonatal mouse testis

Several studies have demonstrated the derivation of multi- or pluripotent stem cells from testicular cells of both newborn and adult mice by a spontaneous conversion process, when these cells are cultured in vitro for an extended time. To obtain a better and robust derivation, we have attempted to identify small molecules (SMs) that induce reprogramming of testicular cells in culture into germline-derived pluripotent stem cells (gPSCs). We tested several SMs based on previous reports that have shown enhancement of establishment of induced pluripotent stem cells or embryonic stem cells (ESCs) on mouse NMRI (outbred strain) and C57BL/6 (inbred strain) testicular cells. After appearance of ESC-like colonies at Day 6, they were passaged on mitotically arrested mouse embryonic fibroblasts in mouse ESC medium in the absence or presence of SMs up to Day 14. The generated cells were characterized using a variety of experimental approaches. The application of several SMs involved in pluripotent reprogramming led to the discovery that CHIR99021 (CHIR), a glycogen synthase kinase-3 (GSK-3) inhibitor, promotes efficient derivation of gPSCs from neonatal mouse NMRI and C57BL/6 testes. The pluripotency of the generated cell lines has been confirmed by in vitro spontaneous and direct differentiation toward cardiac and neural lineages, and formation of chimeras after injection of gPSCs into blastocysts. We have shown that the generated gPSCs could be maintained and expanded under chemically defined serum and feeder-free conditions by inhibition of both the extracellular signal-regulated kinases (Erk1/2) and GSK-3. To our knowledge, this is the first report of a simple and efficient protocol to reprogram gPSCs from testicular cells solely by inhibition of GSK-3 in two strains of mice with different genetic backgrounds. Additionally, this brings us closer to eliminating the need for genetic modification in pluripotent reprogramming. Future studies will determine whether the inhibition of GSK-3 could affect the generation of naïve gPSCs lines in other mammals.

216 EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS ANALOGOUS TO INNER CELL MASS-DERIVED LIF-DEPENDENT MOUSE EMBRYONIC STEM CELLS ESTABLISHED FROM THE DOMESTIC PIG, SUS SCROFA

Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The established piPSC are analogous to FGF2-dependent human (h) ESC and murine “epiblast stem cells,” and are likely to advance swine as a model in biomedical research. Here, we report for the first time, the establishment of LIF-dependent, so called naïve type pluripotent stem cells (1) from the inner cell mass (ICM) of porcine blastocysts by up-regulating the expression of KLF4 and POU5F1; and (2) from umbilical cord mesenchyme (Wharton's jelly) by transduction with OSKM factors and subsequent culture in the presence of LIF-based medium with inhibitors that substitute for low endogenous expression of c-MYC and KLF4 and promote pluripotency. The 2 compounds that have been used in this study are, CHIR99021 (CH), which substitutes c-MYC by inhibiting GSK3B and activating WNT signalling and Kenpaullone (KP), which inhibits both GSK3B and CDK1 and supplants KLF4 function. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression (tet-on) and are ‘floxed’ for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5.5 in vitro produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived stem cell lines are alkaline phosphatase-positive, resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile and expression of pluripotent markers, such as POU5F1, SOX2 and surface marker SSEA1. They are dependent on LIF signalling for maintenance of pluripotency, can be cultured over extended passage (>50) with no senescence. Of importance, the ICM-derived lines have been successful in their ability to form teratomas. The cells could be cultured in feeder free conditions on a synthetic matrix in the presence of chemically defined medium and can be coaxed to differentiate under xeno-free conditions. Currently, the piPSC lines are being investigated for their ability to give rise to teratomas and to produce a live offspring by nuclear transfer. Supported by Addgene Innovation Award, MO Life Sciences Board Grant 00022147 and NIH grant HD21896.