Derivation and characterization of embryonic stem cells lines derived from transgenic Fischer 344 and Dark Agouti rats

Abstract

Rat embryonic stem cell (ESC) lines are not widely available, and there are only 2 lines available for distribution. Here, ESC lines were derived and characterized from Fischer 344 (F344) rats that express marker transgenes either β-galactosidase or human placental alkaline phosphatase (AP), nontransgenic F344 rats, and from Dark Agouti (DA) rats. The ESC lines were maintained in an undifferentiated state as characterized by colony morphology, expression of Oct4, Nanog, Sox-2, Cdx2, and Stella, staining for AP, and stage-specific embryonic antigen-1. Pluripotency was demonstrated in vitro by differentiation to embryoid bodies, followed by embryonic monsters. The Cdx2 expression by ESCs was unexpected and was confirmed via reverse transcriptase-polymerase chain reaction, immunocytochemistry. Pluripotency of ESCs was demonstrated in vivo by production of teratoma after an injection into F344 nontransgenic rats, and by an injection of male DA ESCs into F344 or Sprague-Dawley rat blastocysts and the generation of chimeric rats and germline contribution. ESCs from both F344 and DA contributed to chimeric rats, and one DA ESC line was proved to be germline competent. ESC sublines were created by transfection with a plasmid expressing enhanced green fluorescent protein (eGFP) under the control of a beta actin promoter and cytomegalovirus enhancer (pCX-eGFP) or by transfection with a plasmid expressing GFP under the control of a 3.1 kb portion of the rat Oct4 promoter (pN1-Oct4-GFP). In pN1-Oct4-GFP sublines, GFP gene expression and fluorescence were shown to be correlated with endogenous Oct4 gene expression. Therefore, these new ESC lines may be useful for tissue engineering and transplantation studies or for optimizing culture conditions required for self-renewal and differentiation of rat ESCs. While they made chimeric rats, further work is needed to confirm whether the transgenic F344 rat ESCs described here are germline-competent ESCs.

FIG. 1.

Generation and characterization of rat embryonic stem cells (ESCs) from transgenic F344 and nontransgenic Dark Agouti (DA) rats. (A, B) Rat ESCs and primitive endoderm-like (XEN-like) cells in a mixed culture. After collapse of the blastocyst, the inner cell mass starts expanding. Phase-bright colonies with ESC morphology are found on or adjacent to XEN-like cells (A). The ESCs are positive for alkaline phosphatase (AP, shown in A) and Oct4 staining (B), and the XEN-like cells are negative for both Oct4 and AP. (C) Both XEN and ESCs can be expanded, frozen/thawed, and maintained in the 2i medium. To enrich ESCs, the colony is manually selected, dissociated, and expanded (see C). (D) RT-PCR characterization of pluripotency transcription factors expression by ESCs. Lanes from left to right: Oct4, Sox2, Nanog, PBGD, and water only (control). Block 1 (left): Positive-control ESCs (supplied by Dr. Qilong Ying, University of Southern California). Block 2: ESCs derived from a β-galactosidase (β-gal) transgenic rat (line 3). At left: Dppa3/Stella expression by ESCs. Lanes labeled by rat ESC lines (lines are listed in Table 1). Lines 2 and 3 were derived using 2i medium with 15% heat-inactivated fetal bovine serum (CHIR99021, GSK-3β inhibitor and PD0325901, MEK inhibitor) and leukemia inhibitory factor (LIF). Line 1 was derived using human basic fibroblast growth factor (bFGF) and LIF and 15% heat-inactivated fetal bovine serum. Lane labeled USC represents positive control rat ESCs from Qilong Ying (University of Southern California). Lane labeled W, water only (control). Lane labeled L is a 100 bp ladder. (E) Oct4 and Nanog immunocytochemical staining. Top: ESCs derived from a F344 β-gal transgenic rat (line 3) at passage 4 stained for Oct4 (green), or Nanog (red). Bottom: Secondary antibody only controls. Panels from left to right: Normarski illumination shows colony size and morphology, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, blue) staining for DNA, immunofluorescence micrograph for Oct4 (green), and at the far right, immunofluorescent staining for Nanog (red). Lower panel shows ESCs stained with secondary antibody only. (F) SSEA1 immunocytochemical staining. ESC derived from a F344 β-gal transgenic rat (line 2) at passage 12 stained for SSEA1 (red). From LEFT to RIGHT: Normarski illumination, DAPI nuclear stain (blue), SSEA1 (red), and at the far right, merged DAPI and SSEA1 staining. In all images, scale bar is 100 μm. PCR, polymerase chain reaction; SSEA1, stage-specific embryonic antigen-1; GSK, glycogen synthase kinase; MEK, mitogen-activated protein kinase; F344, Fischer 344; USC, University of Southern California; 2i, 2 inhibitor. Color images available online at www.liebertonline.com/scd

FIG. 2.

Cdx2 staining of rat embryonic stem cells and rat blastocysts. (A) RT-PCR of Cdx2. From left to right: (L) 100 bp Ladder, Lane 1: ESC line 2 at passage 6, Lane 2: ESC line 3 at passage 6, Lane 3: ESC Control from Qilong Ying, Lane 4: Positive Control TS #1, Lane 5: Positive Control TS #2 supplied by Dr. Michael Soares and Mohammed Rumi at University of Kansas Medical Center, Lane 6: ESC line 1 at passage 10, Lane 7: Negative Control mouse D3 ESCs obtained from ATCC, Lane 8: Negative Control (water). (B) Semi-quantitative RT-PCR for Cdx2. 18 rat ESC lines derived here and two rat ESC control supplied by Dr. Qilong Ying express Cdx2 gene at levels higher than negative control cells lines: rat adult tissues and rat embryonic fibroblasts (REFs) and rat umbilical cord mesenchymal stromal cells (RUCs). The positive control cells, rat trophoblast stem cells (TS) express Cdx2 at a higher level than all rat ESCs lines except one euploid rat ESC line that was 60% tetraploid. The delta threshold count (delta Ct) was averaged from two technical replicates and normalized using the Ct value of PBGD. The data from all lines is shown in Table 3. (C) Immunocytochemistry for Cdx2 and Nanog. Left panels are DAPI staining of nuclei. Middle panels are immunofluorescence for Cdx2. Right panels are immunofluorescence for Nanog. As seen in the middle panels, while the staining intensity of Cdx2 varies throughout the colonies, it is more intense at the edges of the colony. While Cdx2 staining varies all Nanog staining cells also had some degree of Cdx2 staining. There was consistent overlap of Cdx2 and Nanog staining. (D) Immunocytochemistry for Cdx2 and Nanog in 4.5 days post coitus rat blastocysts. As seen in the bottom left panel, the inner cell mass contains Cdx2 positively stained nuclei (arrow), albeit at lower staining intensity than in trophectoderm cells. Additionally, Cdx2 staining was observed in the cytoplasm of inner cell mass cells (arrowhead). XEN and TS cell pellets were supplied by Drs. Michael Soares and Mohammed Rumi, University of Kansas Medical Center; rat ESC control cell pellet was supplied by Dr. Qilong Ying, University of Southern California (USC). In C the calibration bar is 50 μm; in D the calibration bar is 25 μm. Color images available online at www.liebertonline.com/scd

FIG. 3.

Characterization of rat ESCs derived from transgenic rats. (A–C) Detection of transgenes genes in ESCs derived from transgenic F344 rats. (A) X-gal histochemistry (X-gal Histochem) to detect β-gal in ESCs (line 2, passage 11) derived from β-gal transgenic rat. (B) Immunocytochemical staining for β-gal protein (β-gal Immuno, red) in rat ESC line 2 counterstained with DAPI for DNA (blue). Scale bar is 100 μm. (C) PCR on genomic DNA extracted from rat ESC lines 61, 62, 1, 2, and 3, to detect human placental AP (left) or β-gal transgene expression (β-gal, middle and right). ESC lines 61 and 61 were derived from the human placental AP transgenic rats and lines 1, 2, and 3 were derived from the β-gal transgenic rats. Lane W, water (negative control). (D) Embryoid bodies (EBs) derived from ESCs. Left: EBs formed using the hanging-drop method for 8 days. Right: EBs formed using suspension culture method for 8 days. EBs were derived from line 2 (β-gal F344 ESCs) at passage 12. Scale bar 100 μm. (E) RT-PCR characterization of pluripotency transcription factor expression in EBs. EBs cultured in the presence of bone morphogenetic protein 4 (BMP4) for 5 or 10 days show progressive down-regulation of Oct4 (lane1), and Nanog (lane 2) and not the control gene (PBGD, lane 3). Top: ESCs maintained in 2i medium to prevent differentiation (undifferentiated control). Middle and bottom: EBs maintained in medium with BMP4 for 5 days (middle) or 10 days (bottom). (F) ESCs derived from transgenic F344 rats form teratoma after injection into parental strain F344 rats. ESCs from Line 1 were injected subcutaneously into parental strain F344 rats. Ten weeks later, a tumor was palpated at the injection site (tumor in situ, left). The tumor was isolated, collected, and histologically processed by the KSU veterinary medicine diagnostic laboratory. Hematoxylin and eosin staining (right) of paraffin sections showed hair follicles, sweat glands, adipose tissue, skeletal muscle and hyaline cartilage (right at top), and respiratory epithelium (right, bottom). Thus, ESC-derived tumors formed tissues from all 3 germ layers. Color images available online at www.liebertonline.com/scd

FIG. 4.

Chimera formation after injection of rat 4.5 dpc blastocysts. (A) ESC line 3 cells were nucleofected with the pCX-eGFP plasmid and expanded to passage 11. These cells were injected into non-eGFP, parental strain F344 4.5 dpc blastocysts. Injected blastocysts were cultured for 10 days on inactivated mouse embryonic fibroblast feeder (MEF) cells after injection (top) or after 14 days (bottom) of culture on inactivated MEFs. The same field is shown in each panel using phase-contrast illumination (left), GFP fluorescence (middle), and a merged image is shown on the right. Scale Bar 100 μm. (B) A single female chimera from a litter of 9 pups after injection of the male line 54.1 into F344 blastocysts. (C) Left: 4 chimera from a litter of 5 pups after injection of male line 53.1 into SD blastocysts. Note that this ESC line demonstrated germline transmission. Note that there is a variation in the degree of chimerism for each ESC line. Middle and right: 4 chimera from 2 litters of 3 pups after injection of male line 52 into SD blastocysts. (D) PCR microsatellite genotyping data after injection of male F344 line into SD blastocysts (lanes 1–4 on left and lines 1–4 on right). Lanes 5–8 are control samples for F344, DA, DA× SD, and SD, respectively. Note that 3 F344× SD chimera are indicated by astrisks in gel on the right. eGFP, enhanced green fluorescent protein; dpc, days post coitum; SD, Sprague-Dawley.

FIG. 5.

Construction and Characterization of Oct4 GFP reporter ESCs. (A, left) Schema of the Oct4 promoter reporter construct. (A, right) Plasmid map for the N1 Oct4 GFP reporter. (B) (1 and 2) ESCs in suspension culture after transfection with the N1 Oct4 GFP reporter vector and G418 selection. Phase contrast (1) and fluorescence images (2) of the same field showing the high degree of GFP fluorescence in undifferentiated ESCs. (3 and 4) In contrast, when the same ESCs are differentiated for 4-6 days by removal of the 2i inhibitors and LIF, the cell morphology indicates that the ESCs differentiated and the GFP fluorescence decreases. Scale bar 100 μm. (C) qRT-PCR analysis of the Oct4 and EGFP transgene expression. Oct4 expression in undifferentiated cells (red bars) and differentiated cells (blue bars). Undifferentiated pCX-EGFP transgenic ESCs (green bars) were used as a positive control for both Oct4 and EGFP expression. Undifferentiated nontransgenic ESCs (pink bars) were used as a positive control for Pou5F1 expression and as a negative control for EGFP expression. The control, housekeeping gene was PBGD. (D) Immunohistochemistry of N1-Oct4-EGFP reporter ESC line in culture. Top, undifferentiated N1-Oct4-GFP reporter ESCs were stained with an antibody to Oct4 (red) to observe the colocalization of eGFP (green) and Oct4 expression. Bottom panel shows the same ESC line after differentiation induced by withdrawal of PD0325901 (MEK inhibitor), CHIR99021 (GSK3β inhibitor) and leukemia inhibitory factor (LIF) for 6 days. 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to counterstain the DNA. The field indicated in the inset is shown at higher magnification on the right. Note that the Oct4 staining is localized to the nucleus, and in contrast the GFP signal is found throughout the cytoplasm (arrows indicates one cell that shows nuclear localization of Oct4 and cytoplasmic staining of GFP). Bottom row: Once the N1-Oct4-EGFP reporter line is differentiated by removal of 2i inhibitors and LIF, the cells change morphology (indicating their differentiation). In addition, the DNA changes to a more heterochromatin state (DAPI staining) and the Oct4 staining is lost. Note that when Oct4 immunofluorescence is decreased, so is GFP fluorescence. Note that some cells continue to express Oct4 and they continue to express GFP (examples indicated by arrows). These data suggest that N1-Oct4-EGFP reporter cells faithfully report Oct4 promoter activity and Oct4 protein expression. Scale bar 100 μm. Color images available online at www.liebertonline.com/scd