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We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

Molecular and embryology studies have demonstrated that mouse pre-implantation embryo development is a process of progressive cell fate determination. At the time of implantation, three cell lineages are present in the developing blastocyst: the trophectoderm (TE), the epiblast (Epi) and the primitive endoderm (PrE). From these early embryo cells, trophoblast stem (TS) cells, embryonic stem (ES) cells and extra-embryonic endoderm stem (XEN) cells can be derived. Recently, we derived stem cells with blastomere-like features from mouse cleavage-stage embryos, which we named expanded-potential stem cells (EPSCs). Here, we provide detailed protocols that describe how to establish EPSCs from single eight-cell-stage blastomeres or whole eight-cell pre-implantation mouse embryos, or by conversion of mouse ES cells or induced pluripotent stem (iPS) cells reprogrammed from fibroblasts. It takes 2–3 weeks to derive EPSCs from each cell source. The EPSCs derived from these protocols can differentiate into all embryonic and extra-embryonic lineages when implanted into chimeras. Furthermore, bona fide TS and XEN cell lines can be derived from EPSCs in vitro.

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In heavy-chain deposition disease truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC)–deficient (L−/−) mice. In L−/− animals, the known functions of LC, providing part of the antigen-binding site of an antibody and securing progression of B-cell development, may not be required. Here, we show a novel function of LC in preventing antibody aggregation. L−/− mice produce truncated HC naturally, constant region (C)γ and Cα lack CH1, and Cμ is without CH1 or CH1 and CH2. Most plasma cells found in these mice are CD138+ Mott cells, filled with RBs, formed by aggregation of HCs of different isotypes. The importance of LC in preventing HC aggregation is evident in knock-in mice, expressing Cμ without CH1 and CH2, which only develop an abundance of RBs when LC is absent. These results reveal that preventing antibody aggregation is a major function of LC, important for understanding the physiology of heavy-chain deposition disease, and in general recognizing the mechanisms, which initiate protein conformational diseases.

Ig are multifunctional molecules with distinct properties assigned to individual domains. To assess the importance of IgM domain assembly in B cell development we generated two transgenic mouse lines with truncated μH chains by homologous integration of the neomycin resistance gene (neor) into exons Cμ1 and Cμ2. Upon DNA rearrangement shortened μH chain transcripts, VH–D–JH–Cμ3–Cμ4, are produced independent of the transcriptional orientation and termination signals provided by neor. The truncated μH chain of ~52 kDa associates non-covalently with the L chain to form a monovalent HL heterodimer. Surface IgM is assembled into a defective BCR complex which has lost important signalling capacity. In immunizations with T-dependent and T-independent antigens, specific IgM antibodies cannot be detected, whilst IgG responses remain normal. B cell development in the bone marrow is characterized by an increase in early B cells, but a decrease of B220+ cells from the stage when μH chain rearrangement is completed. The peritoneal lymphocyte population has elevated levels of CD5+ B cells and their expansion may be the result of a negative feedback mechanism. The results show that antigenic stimulation is compromised by truncated monovalent IgM and that this deficit in stimulation leads to reduced levels of conventional B-2 lymphocytes, but dramatically increased levels of B-1 cells.

This study was designed to produce cloned goats from cumulus cells. Cloning donor nuclei were from cumulus cells either freshly isolated or cultured in vitro. Enucleated oocytes were either injected with cumulus cell nuclei without piezo-driven manipulator (injection method) or fused with cumulus cells (fusion method). The survival rate of cloned embryos, obtained by injection, was higher than that derived from fusion (62.7 and 45.9%, respectively). Two cloned goats were derived by fusion with in vitro cultured cumulus cells without starvation, but died shortly after natural birth, from respiratory difficulties. Their birth weights (2.23 kg and 2.03 kg) were within the normal range (2.0-2.7 kg) and postmortem analysis revealed no morphological abnormalities. The third cloned goat, derived by injection of nuclei from freshly isolated cumulus cells, weighed 3.3 kg at birth, and was 37% overweight compared with the average weight of the same species. This goat is healthy and well as this paper is being prepared. Nested PCR-RFLP analysis confirmed that all the cloned goats were derived from the donor cells.

Transgenic mice carrying a 380-kb region of the human immunoglobulin (Ig) λ light (L) chain locus in germline configuration were created. The introduced translocus on a yeast artificial chromosome (YAC) accommodates the most proximal Igλ variable region (V) gene cluster, including 15 Vλ genes that contribute to >60% of λ L chains in humans, all Jλ-Cλ segments, and the 3′ enhancer. HuIgλYAC mice were bred with animals in which mouse Igκ production was silenced by gene targeting. In the κ−/− background, human Igλ was expressed by ∼84% of splenic B cells. A striking result was that human Igλ was also produced at high levels in mice with normal κ locus. Analysis of bone marrow cells showed that human Igλ and mouse Igκ were expressed at similar levels throughout B cell development, suggesting that the Igλ translocus and the endogenous κ locus rearrange independently and with equal efficiency at the same developmental stage. This is further supported by the finding that in hybridomas expressing human Igλ the endogenous L chain loci were in germline configuration. The presence of somatic hypermutation in the human Vλ genes indicated that the Igλ-expressing cells function normally. The finding that human λ genes can be utilized with similar efficiency in mice and humans implies that L chain expression is critically dependent on the configuration of the locus.

Expression studies of multigene families, such as the immunoglobulin (Ig) loci, are difficult because of their large size and the necessity to introduce germline configured regions into an animal. Antibody diversity from Ig gene miniloci is limited by the number of variable (V) region genes and the need for distal regulatory elements to control expression. Here, we show germline transfer into mice of a 1300 kb human Igκ light chain locus on a yeast artificial chromosome that resulted in early DNA rearrangement and highly efficient human light chain expression. The human locus was assembled from a 300 kb authentic region using contig extension by addition of cosmid multimers to supplement the variable gene cluster. This resulted in the addition of about 100 V region genes in germline configuration from different families. In transgenic animals with Igκ disruption, this large human κ locus replaced the endogenous locus, and subsequent down‐regulation of Igλ light chain contribution led to a dominant expression of the rearranged human genes. Contrary to expectation, rather than providing a solely selective advantage for ensuring repertoire formation controlled by the sheer number of introduced genes, the λ/κ ratio in serum appears to be the result of competition for early surface Ig expression maintained in the developing B cell.—Zou, X., Xian, J., Davies, N. P., Popov, A. V., Brüggemann, M. Dominant expression of a 1.3 Mb human Igκ locus replacing mouse light chain production. FASEB J.10, 1227‐1232 (1996)