Generating porcine chimeras using inner cell mass cells and parthenogenetic preimplantation embryos

Abstract

Background: The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. METHODOLOGY/SIGNIFICANT PRINCIPAL FINDINGS: In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4-8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4-8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively.

Conclusion/significance: Our findings indicate that the aggregation method using parthenogenetic morulae or 4-8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.

Figure 1. Generalized scheme for the production of chimeric porcine blastocysts and fetuses by the aggregation method.

For in vitro analysis of the chimeric blastocyst formation, donor ICMs were isolated from parthenogenetic blastocysts derived from IVM oocytes. Isolated ICMs stained with DiI were aggregated with blastomeres isolated from parthenogenetic host embryos in a microwell made on the bottom of a culture dish. For in vivo analysis of chimeric fetus formation, the donor ICMs were isolated from blastocysts fertilized in vitro by transgenic boar sperm carrying the fluorescent huKO gene. ICMs of the IVF blastocysts were similarly aggregated with the parthenogenetic host embryos as the DiI-stained ICMs, and the resultant blastocysts were transferred to recipient pigs to obtain chimeric fetuses.

Figure 2. Production of chimeric blastocysts with donor ICM and parthenogenetic host embryos.

(A, D) A donor ICM (stained with Dil) aggregated with host blastomeres isolated from parthenogenetic embryos at the morula (A) or 4–8 cell stage (D). (B, E) Bright field images of chimeric blastocysts developed from the aggregated embryos. (C, F) Confocal fluorescence images of chimeric blastocysts showing DiI fluorescence in ICMs. Single confocal sections of fluorescence were overlaid on the bright field images. (G-I) Parthenogenetic host morulae injected with DiI-stained donor ICM (G) and resultant chimeric blastocysts (H, I). Arrow heads, ICM. Scale bars = 50 µm.

Figure 3. Production of chimeric blastocysts by blastomere aggregation.

(A, D, G) Aggregation of donor (DiI-stained) and host blastomeres between synchronous (A, D) and asynchronous (G) embryonic stages. (B, E, H) Chimeric blastocysts developed from the aggregated blastomeres. (C, F, I) Confocal fluorescence images of the chimeric blastocysts showing DiI fluorescence in ICMs. Single confocal sections of fluorescence were overlaid on the bright field images. Arrow heads, ICM. Scale bars = 50 µm.

Figure 4. Chimeric fetuses produced by aggregation of the ICM carrying huKO transgene and parthenogenetic host embryos.

(A, B) Morphological appearance of the chimeric blastocysts before embryo transfer. (C–K) Chimeric fetuses (day 18) showing huKO fluorescence derived from the donor ICM cells (C, D, F, G, I, J) and immunohistochemical images showing proportion of the donor-derived (huKO-positive) cells in the tissue of chimeric fetuses (E, H, K). (L, M, N) A day-19 fetus developed from an embryo fertilized in vitro with the huKO transgenic boar sperm as a positive control, showing the systemic expression of huKO (M, N). (O, P, Q) A non-chimeric fetus (day 22) developed from the aggregates of two parthenogenetic embryos as a negative control.