Sebastian T. Balbach

Production of cloned mice by somatic cellnuclear transfer

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains <5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cells genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in ≥3 months.

Subsets of cloned mouse embryos and their non-random relationship to development and nuclear reprogramming

An important question in oocyte-mediated nuclear reprogramming is whether gene expression of the donor nucleus changes randomly or follows a pattern. Since cloned embryos are very heterogeneous and arrest frequently during preimplantation development, a random scenario is generally accepted. In the present study, we resolve the heterogeneity of cumulus cell-derived mouse clones by recognizing structured subsets, and we analyze their relationship to reprogramming of donor nuclei. We utilize live cell imaging of the Oct4 promoter-driven GFP transgene to resolve the populations of cloned and ICSI-fertilized morulae, and we sort them both into three subsets based on different GFP expression. Functionally, subsets of cloned but not ICSI morulae form blastocysts and ES cells proportional to Oct4-GFP expression. Regulatively, the subsets of cloned morulae are characterized by small differences of transcript level for the pluripotency-associated genes Oct4, Nanog and Sox2. Small differences of the level of select mRNAs across subsets suggest a uniform rather than random course of reprogramming from the morula stage on. Since these small differences correspond with substantial differences in developmental competence, we propose that developmental potential of clones relates to levels of gene expression in a different way than fertilized embryos.

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.

Somatic cell nuclear reprogramming of mouse oocytes endures beyond reproductive decline

The mammalian oocyte has the unique feature of supporting fertilization and normal development, while capable of reprogramming nuclei of somatic cells toward pluripotency, and occasionally even totipotency. While oocyte quality is known to decay with somatic aging, it is not a given that different biological functions decay concurrently. In this study, we tested whether oocyte’s reprogramming ability decreases with aging. We show that oocytes isolated from mice aged beyond the usual reproductive age (climacteric) yield ooplasts that retain reprogramming capacity after somatic nuclear transfer (SCNT), giving rise to higher blastocysts rates compared to young donors ooplasts. Despite the differences in transcriptome between climacteric and young ooplasts, gene expression profiles of SCNT blastocysts were very similar. Importantly, embryonic stem cell lines with capacity to differentiate into tissues from all germ layers were derived from SCNT blastocysts obtained from climacteric ooplasts. Although apoptosis‐related genes were down‐regulated in climacteric ooplasts, and reprogramming by transcription factors (direct‐induced pluripotency) benefits from the inhibition of p53‐mediated apoptosis, reprogramming capacity of young ooplasts was not improved by blocking p53. However, more outgrowths were derived from SCNT blastocysts developed in the presence of a p53 inhibitor, indicating a beneficial effect on trophectoderm function. Results strongly suggest that oocyte‐induced reprogramming outcome is determined by the availability and balance of intrinsic pro‐ and anti‐reprogramming factors tightly regulated and even improved throughout aging, leading to the proposal that oocytes can still be a resource for somatic reprogramming when they cease to be considered safe for sexual reproduction.

Reprogramming of two somatic nuclei in the same ooplasm leads to pluripotent embryonic stem cells

The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (somatic cell nuclear transfer [SCNT]) in a process termed “reprogramming.” This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT‐ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency‐associated gene Oct4 in fertilized embryos, derivative tNT‐ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes, and endodermal cells; in vivo, tNT‐ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, for example, in the quantitative expression of gastrulation‐associated genes. In conclusion, we have shown that the oocytes reprogramming capacity is in excess of a single nucleus and that double nucleus‐transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion‐mediated reprogramming and from fetal and adult hepatocyte‐derived induced pluripotent stem cells, we have now accomplished it with enucleated oocytes. Stem Cells 2013;31:2343–2353

Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism.

Live Embryo Imaging to Follow Cell Cycle and Chromosomes Stability After Nuclear Transfer

Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

Observing and Manipulating Pluripotency in Normal and Cloned Mouse Embryos

The mouse ooplasm is the ideal platform to study and compare induced and natural pluripotency because it can support both, after somatic cell nuclear transfer (cloning) and after fertilization, respectively. The amount of pluripotency induced after cloning is variable but always limited compared to fertilization. It can be visualized conveniently if the nucleus donor cells carry a green fluorescent protein (GFP) reporter under control of the pluripotency-associated gene Oct4 promoter. Thus we produced cloned and fertilized mouse embryos transgenic for Oct4-GFP (GOF18-∆PE-EGFP). We also developed and validated a live cell imaging method, whereby we resolve and selectively pick cloned embryos that hold distinct amounts of induced pluripotency as predicted by GFP intensity and measured by embryonic stem cell derivation. Currently we are developing a microinjection method to change the level of Oct4 without modifying the genome of the embryo. Here we discuss our findings in relation to the epigenetic reprogramming of the nucleus transplant and to cell fate decisions in the cloned or fertilized mouse embryo.

Proteomic Approach to the Reprogramming Machinery of the Mouse Oocyte

The mature oocyte is the only cell of the body that can incorporate either sperm or somatic nuclei and express developmental totipotency. In order to prepare for these tasks, the oocyte stockpiles dedicated molecules during oogenesis. If one aims to solve the special properties of oocytes, one should consider that information on the transcriptomic level is of limited use, as ultimately proteins are the main functional units of any given cell type. Therefore, an in-depth characterization of the oocyte proteome is a promising approach to advance our understanding of reprogramming and germ cell biology. To date, limitations on oocyte numbers and proteomic technology have impeded this task, and the search for reprogramming factors has instead been conducted in embryonic stem cells (ESCs). Here we highlight the importance of proteomics in the field of mouse oocyte-mediated reprogramming. We present data on the proteome of mouse metaphase II oocytes and ESCs to a depth of 3699 and 4723 proteins, respectively. Active reprogramming molecules should reside in the nucleus, act on chromatin, and function as enzymes. Following these criteria, we analyzed and filtered the shared proteome of oocytes and ESCs for proteins matching the gene ontology (GO) terms nuclear localization, chromatin modification, and catalytic activity. As a result, we identified 28 oocyte proteins that match the criteria of our multilevel approach to screen for putative active reprogramming factors and thus advance the definition of the “reprogrammome.”