scientificprotocols authored about 7 years ago
Authors: Jun Wu, Daiji Okamura & Juan Carlos Izpisua Belmonte
This protocol describes a method to graft conventional primed pluripotent stem cells (PSCs) and region-selective PSCs to different locations of post-implantation mouse epiblast followed by in vitro embryo culture. Compared to conventionally used teratoma formation assay, epiblast grafting offers a more relevant in vivo test for evaluating the primed PSCs’ pluripotency and uniquely serves to distinguish different flavors of primed PSCs that harbor distinct spatial properties. Moreover, the observation that grafted human rsESCs could enter into early mouse embryogenesis provides a novel way of studying human development.
Two temporally distinct pluripotent stem cells (PSCs) have been derived in mouse: “naïve” embryonic stem cells (ESCs) (1,2) and “primed” epiblast stem cells (EpiSCs) (3,4). They differ from each other in molecular signatures and phenotypic properties (5). As embryo development is a dynamic process not only in time but also in space, it remains unknown whether pluripotent states with distinct regional properties may exist. Through optimization of culture parameters using mouse epiblast explants, we derived with high efficiency a novel pluripotent cell type sharing features of the posterior-proximal (PP) epiblasts of post-implantation mouse embryos. We designated this new cell type as region-selective EpiSCs, or rsEpiSCs. Mouse rsEpiSCs have high cloning efficiency and faster growth kinetics and differ from EpiSCs at global transcriptomic, epigenomic and metabolic levels.
Conventional human ESCs exist in a primed pluripotent state similar to mouse EpiSCs due to their similar colony morphology, signaling dependency, poor cloning efficiency and epigenetic features. We tested mouse rsEpiSCs-based culture on human ESCs and found human ESCs could self-renew while maintaining karyotypic stability after long-term culture (the cells were thus named human rsESCs after treatment). Moreover, human rsESCs share similar molecular characteristics with mouse rsEpiSCs.
We analyzed the grafting properties of distinct primed pluripotent cells in isolated non-intact and non-viable mouse E7.5 epiblasts. mEpiSCs were previously reported to efficiently incorporate and differentiate in anterior, posterior as well as distal part of the E7.5 mouse epiblasts (6-8). In contrast, mouse rsEpiSCs only selectively colonize and give rise to derivatives of the three embryonic germ layers after being grafted to the posterior pole of isolated non-intact and non-viable E7.5 mouse embryos. Surprisingly, unlike mouse EpiSCs, we found that after grafting conventional human ESCs could hardly survive inside E7.5 mouse epiblasts regardless of the location. Interestingly, just like mouse rsEpiSCs, human rsESCs could efficiently integrate into the posterior region of isolated non-intact and non-viable mouse E7.5 epiblast and differentiated into three embryonic germ lineages after 36 h in vitro embryo culture. These results indicate that we have captured a spatially distinct primed pluripotent state that confers ex-vivo intra- and inter-species chimeric competency.
This protocol describes in detail of isolation and preparation of non-intact and non-viable mouse E7.5 embryos for grafting. The epiblast grafting and in vitro embryo culture procedures will also be elaborated.
Embryo dissection medium:
Embryo culture medium:
To prepare F12 (N2), mix Ham’s F12 Nutrient Mix, GlutaMAX Supplement (Life Technologies, 31765-035) with N2 Supplement (Life Technologies, 17502-048) at a ratio of 100:1. Embryo culture medium is prepared by mixing Rat serum (Harlan, B.4520) at a ratio of 1:1 with F12 (N2).
Preparation and isolation of E7.5 mouse embryos
All mice used in this study are housed in a modern, air-conditioned facility and maintained according to NIH guidelines.
Preparation of non-intact and non-viable mouse embryos for grafting
Epiblast grafting
For mouse, after grafting conventional EpiSCs can be incorporated efficiently in the distal and posterior regions and to a lesser extent the anterior region of E7.5 epiblast, similarly to published reports (6-8). In contrast, rsEpiSCs only integrated efficiently in the posterior epiblast and they poorly integrated in the anterior region and not in the distal region. After 36 h of embryo culture posteriorly grafted rsEpiSCs could proliferate and differentiate into the three germ layers in chimeric embryos.
For human, conventional ESCs could not efficiently integrate and proliferate inside mouse epiblasts regardless grafting locations. In contrast, human rsESCs efficiently integrated, proliferated and differentiated into three embryonic germ layers when grafted in the posterior region of the E7.5 mouse epiblasts and cultured in vitro for 36 h, consistent with mouse rsEpiSCs.
Figure 1: Grafting PSCs or rsPSCs to non-intact and non-viable E7.5 mouse embryos
Left, an illustration of an intact E7.5 mouse embryo with different embryonic structures indicated; Right, non-intact and non-viable E7.5 mouse embryo prepared before cell injections. A, Anterior; P, Posterior; D, Distal.
An alternative pluripotent state confers interspecies chimaeric competency, Jun Wu, Daiji Okamura, Mo Li, Keiichiro Suzuki, Chongyuan Luo, Li Ma, Yupeng He, Zhongwei Li, Chris Benner, Isao Tamura, Marie N. Krause, Joseph R. Nery, Tingting Du, Zhuzhu Zhang, Tomoaki Hishida, Yuta Takahashi, Emi Aizawa, Na Young Kim, Jeronimo Lajara, Pedro Guillen, Josep M. Campistol, Concepcion Rodriguez Esteban, Pablo J. Ross, Alan Saghatelian, Bing Ren, Joseph R. Ecker, and Juan Carlos Izpisua Belmonte, Nature doi:10.1038/nature14413
Jun Wu & Juan Carlos Izpisua Belmonte, The Salk Institute for Biological Studies
Correspondence to: Juan Carlos Izpisua Belmonte ([email protected])
Source: Protocol Exchange (2015) doi:10.1038/protex.2015.042. Originally published online 18 May 2015.