Contribution of Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells to Chimeras through Injection and Coculture of Embryos

Blastocyst injection and morula aggregation are commonly used to evaluate stem cell pluripotency based on chimeric contribution of the stem cells. To assess the protocols for generating chimeras from stem cells, 8-cell mouse embryos were either injected or cocultured with mouse embryonic stem cells and induced pluripotent stem cells, respectively. Although a significantly higher chimera rate resulted from blastocyst injection, the highest germline contribution resulted from injection of 8-cell embryos with embryonic stem cells. The fully agouti colored chimeras were generated from both injection and coculture of 8-cell embryos with embryonic stem cells. Additionally, microsatellite DNA screening showed that the fully agouti colored chimeras were fully embryonic stem cell derived mice. Unlike embryonic stem cells, the mouse chimeras were only generated from injection of 8-cell embryos with induced pluripotent stem cells and none of these showed germline transmission. The results indicated that injection of 8-cell embryos is the most efficient method for assessing stem cell pluripotency and generating induced pluripotent stem cell chimeras, embryonic stem cell chimeras with germline transmission, and fully mouse embryonic stem cell derived mice.


Introduction
Chimeric mice are important tools for investigating embryonic development as they can provide insights into the function of a specific gene, they can trace the origin of the cell lineage, and they can assess the potential of cells. The first chimeric mouse was produced in 1961 [1] through the aggregation of two 8-cell embryos. In later years, chimeric mice were created by microinjections of dissociated innercell-mass cells (ICMs) into the cavity of a blastocyst [2]. Since then, the techniques and protocols have been modified and improved [3][4][5][6] such that chimeric mice have been produced successfully using embryos aggregated or injected with ICM cells [2], embryonal carcinoma cells (ECCs) [7], embryonic stem cells (ESCs) [8], embryonic germ cells (EGCs) [9], somatic cell nuclear transfer-derived embryonic stem cells (ntESCs) [10], induced pluripotent stem cells (iPSCs) [11,12], spermatogonial stem cells (SSCs) [13,14], extraembryonic endoderm (XEN) cells [15], and epiblast stem cells (EpiSCs) [16,17]. Chimeras are a mixture of cells derived from both the donor cells and those of the recipient embryo. Since it is extremely difficult to determine the chimeric contribution of donor cells in particular tissues, chimeras are usually identified by means of coat coloration.
Conventional injection of ESCs into a blastocyst is the most popular method of producing chimeras and those generated are partially derived from the ESCs. Well sandwich aggregation is also a highly stable and reproducible method for generating germline transmitted chimeras, but two embryos are required to effect the procedure [6,18]. Although microinjection will also produce good germline transmitted chimeras, specialized equipment is needed. For these reasons, the coculture method was developed to produce chimeric mice [3,4,19] even though it is far less efficient than the microinjection and well sandwich aggregation techniques. However, an improved method for producing chimeras with 2 Stem Cells International a high degree of chimerism and germline transmission which utilized coculture of denuded mouse embryos and ES cells in Eppendorf tubes was reported [20]. Such aggregation in Eppendorf tubes can cause embryo adhesion and form 2-or 3-embryo clusters mixed with ESCs.
Recently, 8-cell stage embryos were used to generate fully ESC-derived mice in a laser- [21] and piezo-assisted micromanipulation system [22]. The authors determined that this 8-cell method is effective for both inbred ES cells, such as C57BL/6 and 129, and hybrid ESCs to generate fully ESC-derived mice; thus, F0-generation mice enable immediate phenotyping [22]. ESCs can adhere to blastomeres and migrate into the ICM after microinjection or aggregation. Nevertheless, the mechanism of ESC migration in chimeric embryos remains unclear [23][24][25] and how they can completely replace the ICM of an embryo and develop into an ESC-derived mouse demands investigation. Hence, in the present study we evaluated ESC development potential and protocols for chimera generation in the production of fully ESC-derived mice. While ESCs are recognized as pluripotent stem cells, we also tested the chimeric contributions of iPSCs on chimera generation by microinjection and coculture in the well-of-the-well (WOW) system.
Mouse ESCs and iPSCs were then frozen in FBS plus 10% dimethyl sulfoxide (DMSO). For cell injection and aggregation, thawed cells were cultured without feeder cells on a 0.1% gelatin-treated 12-well cell culture plate (Corning) and used within 4 days. Before experiments, cells were trypsinized with 0.025% trypsin-EDTA (Gibco) and resuspended in ESCmaintaining medium.

Collection of Mouse
Embryos. KM albino female mice 6-8 weeks old were superovulated by intraperitoneal injection of 5 IU eCG and 5 IU hCG at 17:00, 48 h apart. At the time of the hCG injection, the females were mated by KM males. Vaginal plugs were checked the following morning when the time was defined as day 0.5 post coitum (dpc). Females with vaginal plugs were killed by cervical dislocation at 2.5 and 3.5 dpc for collection of, respectively, 8-cell embryos and blastocysts. The oviducts or uteri were removed and transferred into 20 mM HEPES-buffered KSOM (95 mM NaCl, 2.5 mM KCl, 0.35 mM KH 2 PO 4 , 0.2 mM MgSO 4 ⋅7H 2 O, 0.2 mM D-glucose, 10 mM Na-lactate, 4 mM NaHCO 3 , 0.2 mM Na pyruvate, 1.71 mM CaCl⋅2H 2 O, 1.0 mM glutamine, 0.01 mM Na 2 EDTA⋅2H 2 O, and 1% penicillin and streptomycin) [30] supplemented with 3.0 mg/mL bovine serum albumin (BSA) in 35 mm Petri dishes (Corning). They were then flushed with a fine bore needle attached to a 1 mL syringe [6]. The 8-cell embryos were retrieved and cultured in 4-well dishes (Nunc) in KSOM modified with 25 mM NaHCO 3 and supplemented with 1.0 mg/mL BSA, 0.5% MEM nonessential amino acids (Gibco), and 0.5% essential amino acids (Gibco) at 37 ∘ C in a humidified atmosphere containing 5% CO 2 before being used subsequently in experiments. Blastocysts were used immediately for ESC injection.

Generation of Mouse Chimeras by Injection of Embryos with ESCs and iPSCs.
Approximately 10-15 cells were aspirated into the injection pipette and injected gently into the blastocoel cavity using a piezo-assisted micromanipulator attached to an inverted microscope [22,31]. The injected embryos were cultured to enable reexpansion of the blastocoel cavity and then transferred to the uteri of pseudopregnant KM mice at 2.5 dpc [6].
In a similar manner to the injection of blastocysts, 8cell embryos were injected with cells placed carefully into the perivitelline space under the zona pellucida. The injected embryos were cultured overnight; blastocysts that developed were transferred to the uteri of pseudopregnant KM mice at 2.5 dpc [6]. Chimeras were confirmed by the coat color pattern of the pups at birth.  [32]. Approximately 100 cells were selected and transferred into each well of the system to coculture them with the embryo overnight. The resulting blastocysts were transferred to the uteri of pseudopregnant KM mice at 2.5 dpc [6]. Again, chimeras were identified by coat color of the pups at birth.

Embryo Transfer.
Embryo transfer recipients were prepared by pairing mature KM female mice with vasectomized KM males overnight. Vaginal plugs were examined the following morning and plugged mice were used as pseudopregnant recipients for embryo transfer. They were anesthetized by intraperitoneal injection of Avertin (0.3 mg/g body weight) and 10-15 blastocysts were transferred into the tip of each uterine horn.

Microsatellite DNA Analysis of Fully ESC-Derived Mice.
Chimeric pups were initially identified by coat color, and the full agouti-coated mice were selected for microsatellite DNA analysis [33]. Sequences of microsatellite marker primers were obtained from the Mouse Genome Informatics website (The Jackson Laboratory, http://www.informatics.jax.org) [34,35] (Table 1). Genomic DNA was extracted from tail biopsies recovered from the chimeric mice and from recipient and donor ESCs using a DNeasy Blood & Tissue Kit (Qiagen). Microsatellite DNAs were amplified by polymerase chain reaction (PCR) using a Type-it Microsatellite PCR Kit (Qiagen). The PCR reaction was performed in a 25 L volume containing 1 g DNA, 10 mol/L primers, and 12.5 L double strength master mix. Amplification was performed in 96-well microtiter plates and the PCR conditions were initialized at 94 ∘ C for 30-60 sec: 25-40 cycles of denaturation at 94 ∘ C for 30-60 sec, annealing at 55 ∘ C for 30-60 sec, and extension at 72 ∘ C for 1 min, followed by a final extension step at 72 ∘ C for 5-10 min (Table 1). PCR products were diluted (1 : 1) in loading buffer and electrophoresed in 12% polyacrylamide gels at 150 V for 4 h [36]. The resulting gels were then silver stained, scanned, and photographed [37]. Briefly, the gel was fixed and stained for 5 min in a solution containing 5% ethanol, 1% HNO 3 , and 0.1% AgNO 3 and then washed in water. DNA bonds were developed for 8 min in developer containing 1.3% NaOH, 0.65% NaCO 3 , and 0.4% formaldehyde. Development was then stopped by addition for 1 min of 5% ethanol with 1% HNO 3 . The gels were stored in water for photography.

Transmission Screening of Mouse Chimeras.
Some chimeras and fully ESC-derived mice were selected and mated with KM mice. The germline transmission competence of ESCs and iPSCs was determined by the coat colors of the resulting F1 pups.

Statistical Analysis.
Data expressed as percent-ages were analyzed using the chi-squared test (http://statpages.org/ ctab2x2.html). A value less than 0.05 between two groups within the same column was considered to indicate significance.

Generation of Mouse Chimeras Using ESCs. When
ESCs at passage 20-22 were used for chimera production (Figure 1 (Figures 3(a) and 3(b)), and blastocyst injection showed coat color chimerism ( Table 2). The chimeric rate from blastocyst injection was significantly higher than that from 8-cell embryo injection, but there was no significant difference between the 8-cell embryo injection and 8-cell embryo coculture groups. Three of 25 (12.0%) and one of 23 (4.3%) full-colored chimeras were produced by 8-cell embryo injection and 8-cell coculture, respectively (Figure 3(c)). No full-colored chimeras were produced by blastocyst injection.    blastocyst injection groups (Table 3). Most iPSC chimeras were fertile when mated with female KM mice, although no germline transmitting chimeras were produced.

Microsatellite DNA Analysis of Fully ESC-Derived
Mice. Three full agouti-colored chimeras were scanned for microsatellite DNAs to determine whether they were fully ESC-derived mice. This showed that all 3 mice were fully derived from ESCs, but not from KM mouse embryos ( Figure 5).

Discussion
Expression of pluripotency genes, immunocytochemistry of pluripotency markers, embryoid body formation in vitro, and teratoma formation in vivo are generally used to test the pluripotency of stem cells [38,39]. More importantly, however, their pluripotency is best evaluated based according to their contribution of cells in chimeras. Blastocyst injection and morula aggregation are the methods of choice to generate stem cell chimeras [3,6,40]. Our results are similar to other   published reports; more germline transmitted chimeras were produced from the same batch of ESCs by 8-cell embryo injection than by blastocyst injection in our experiments, despite more pups being produced by the latter [41,42]. Eightcell embryo injection is physically more demanding than blastocyst injection due to the small size of the perivitelline space increasing the chances of damage to the blastomeres of the embryo during the injection procedure. In addition, the beginning of processes involved in compaction of mouse 8-cell embryos might be disturbed by injection of ESC. Consequently, both the ICM and the trophectoderm are unable to differentiate into normal cell types at the correct time, thereby resulting in loss of pups. This indicates that compaction of the mouse embryo is important for its further development [43] and that coculture of 8-cell embryos and ESCs is more desirable to generate transmitting chimeras from ESCs. We conclude that the aggregation modified by our method of coculture in WOW is more convenient and efficient for generating ESC chimeras and even fully ESCderived mice compared with that of blastocyst and 8-cell embryo injection [22,44]. ESC chimeras were generated by 8-cell embryo injection, blastocyst injection, and 8-cell embryo coculture in this study. However, iPSC chimeras could be generated only by 8-cell embryo injection, although none of the resulting iPSC chimeras were germline transmitted. Interestingly, implantation nodules were observed when the embryos derived from 8-cell embryos cocultured with iPSCs were transferred to recipient mice but sections of these implantations showed that the fetuses had been replaced by tumorous cells (data not shown). Thus, we presume that the iPSCs, due to their oncogenicity, drove the ICM cells into tumor rather than embryo formation. iPSC chimeras can be generated by injection of 8-cell embryos because fewer iPSCs were injected into the perivitelline space, unlike the situation in 8-cell embryo coculture. Here, a few iPSCs were induced by ICM cells which then developed into normal embryos. These results suggest that the contribution of iPSCs in chimeras does not correspond to their full pluripotency potential [45].
Our study complements the previous report that formation of the cell niche in chimeric embryos is closely associated with the dominant cells and the cell niche determines the fates of stem cells [46]. The ICM cell niche dominates the ESC's niche, but the iPSC niche is dominant to the ICM. Thus, fewer iPSCs similar to ESCs can be induced to form ICM cell-like cells and contribute to chimeras, but large numbers of iPSCs Stem Cells International 7 can induce ICM cells to transform into iPSC-like cells, which ultimately results in tumor formation.
We found that fully ESC-derived mice could be produced by coculturing and injecting 8-cell embryos but not blastocysts and that coculture is a simple and effective method for producing chimeras and fully ESC-derived mice [21,22,47]. This improved coculture technique is more convenient than vial coculture [20]. The finding also indicates that the protocol used has little effect on the generation of fully ESC-derived mice, but the embryonic stage has a marked effect. Consistent with other reports, ESCs can adhere to the surfaces of 8-cell embryos, but not to 2-and 4-cell embryos [25]. Although the developmental mechanism of fully ESCderived mice remains unclear, we presume that ESC clusters tend to form ICM cells due to their tight junctions [48][49][50][51][52] and asymmetry formation derived from variation in cell sizes and shapes [53] between ESCs and blastomeres [51,54] because more fully ESC-derived mice were generated by coculture of 8-cell embryos and ESCs in which ESC clusters aggregated with embryos were formed. Further investigation is required to determine how ES cells completely replace ICMs and develop thereafter into an ESC-derived mouse.
In conclusion, our noninvasive 8-cell embryo coculture is a simple and suitable protocol for generating ESC chimeras and fully ECS-derived mice that can be employed to characterize the totipotency of ESC lines. However, 8cell embryo injection to generate iPSC chimeras is the only suitable method for characterizing chimeric developmental potential of iPSCs. The ability of stem cells to contribute to chimeric animals may not represent the totipotency of stem cells. Transmission of stem cells and fully stem-cell-derived animals should also be considered.