Toward Ovine iPSCs

Introduction

Most cells in the mammalian body are committed to progress along a certain lineage. Some are precursor cells that can differentiate into a few cell types, but the majority are fully differentiated with a defined function. Stem cells are different in that they have the ability to produce cells of many types.

The goal of deriving cells capable of both self-renewal while retaining the ability to differentiate into all cells (pluripotential) of the mammalian body has only been reliably achieved for rodents. The journey to achieve this for commercial livestock animals is still being traveled, with progress in porcine cells leading the way. Initially efforts attempted to emulate the success of the mouse embryonic stem cell (ESC), with the only progress over the last 30 years being the derivation of rat ESCs (Buehr et al., 2008). However, the relatively recent, spectacular development of induced pluripotent stem cell (iPSC) technology holds much promise that this journey can now be traveled faster; but whether this new technology will finally deliver reproducibly robust pluripotential cells for livestock is something still tantalizingly out of reach.

Pluripotency

Stem cells are defined as undifferentiated cells presenting two main characteristics—self-renewal and the ability to differentiate in other cell types (Chambers and Tomlinson, 2009). Self-renewal is the ability of the cells to go through numerous cycles of division while maintaining the undifferentiated state. Many different types of stem cells are actively pursued by the scientific community, with the hardest to isolate being the ESC. ESCs are pluripotential stem cells in that they can develop into any cell deriving from the three germ layers (endoderm, mesoderm, ectoderm) but are not able to generate extraembryonic tissues (e.g., cells from the epiblast). Such pluripotency reflects a permissive state, a state that can respond to the multitude of stimuli that direct the formation of all types of cells in the animal.

Pluripotency is a powerful biological state. Initially, the goal was to isolate pluripotent cells and prove their pluripotency through differentiation into the three extraembryonic lineages as determined by expression of lineage markers. Two differentiation assays are often applied. The first in vitro involves outgrowths from embryoid bodies (EBs), and the second is the formation of teratomas in nude mice. It is becoming recognized that both assays provide the same amount of information, therefore questioning the widespread use of the in vivo assay. But to be truly pluripotential, the final assay of germ line transmission is required. To date, this final criterion has only been met for rodent ESCs and iPSCs, with one group reporting success with pig iPSCs (West et al., 2011). For rodent species where robust ESCs and iPSCs exist, the focus is now on the fine detail of organization that exists in the nucleus, drilling down to the precise transcriptional and epigenetic marks that reflect self-renewal and the changes associated with escape from this state toward differentiation. This is being paralleled for research into human ESCs and iPSCs. The livestock field is still far behind this work.

A Revolution in the Field—iPSCs

After early reported successes in the isolation of pluripotent livestock cells, which remains enigmatic given the lack of others able to repeat this work, no real progress was achieved until the spectacular work from the Yamanaka lab (Takahashi and Yamanaka, 2006). The first demonstration of the ability to produce a pluripotent stem cell from a somatic cell simply by the addition of four factors—OCT4, SOX2, KLF4, and c-MYC—is now heralded as one of the most significant advances in cell biology—ever. It builds on the mass of ESC work largely performed in rodent species, early work on reversibility of cell specialization in frogs, and the huge technical achievement of animal cloning, the latter accomplished by Ian Wilmut and Keith Campbell. Although the debate continues about the relative significance of induced pluripotency against embryo-derived pluripotency as an experimental tool for the biologist, these cell states are a wonderful resource.

After first being demonstrated for mice, human and rat iPSCs were quickly established. However, the journey toward livestock iPSCs has been slower and is largely still ongoing. The greatest success and research effort has been for the pig. Leading the way is the work by Bhanu Telugu, Steve Stice, Mike Roberts, and colleagues (Telugu et al., 2010; Telugu et al., 2012; West et al., 2011). Injection of porcine iPSCs into blastocysts initially resulted in the production of chimeric pigs with a high level of chimerism and then subsequently germ line transmission of iPSC-derived cells (West et al., 2011). Neither has yet been achieved for another farm animal species. Nevertheless, some progress has been achieved for other farm animal species, including cattle (Sumer et al., 2012), horse (Breton et al., 2013), and sheep (Bao et al., 2011; Li et al., 2011; Liu et al., 2012; Sartori et al., 2012).

Ovine iPSCs

Ovine iPSC have been produced. However, a critical criterion of being able to contribute to all cells types including the germ line remains to be achieved for ovine iPSCs. Nevertheless, several groups have derived ovine iPSC cells in that they have the ability to differentiate into cells of the three germ lineages—endoderm, mesoderm, and ectoderm—and as such can be termed pluripotent and thus can have the label iPSC attached to them.

The first two reports of ovine iPSCs appeared in January, 2011, from Chinese groups. The work of Li and collegaues worked with ovine fetal fibroblasts and tetracycline (Tet)-inducible lentiviruses carrying the four Yamanaka factors (Li et al., 2011). Colonies started to appear after 14 days and after a further 14 days started to take on a human ESC-like appearance. Colony appearance required expression of the four factors and removing doxycycline led to nearly all colonies losing alkaline phosphatase (AP) staining. Culture in fetal bovine serum (FBS) medium gave more AP-staining colonies than knockout serum replacement (KSR) medium, implying fibroblast growth factor (FGF) was beneficial in this regard. With all four exogenous reprogamming factors being expressed, these cells were positive for endogenous Oct4, Sox2, Nanog, and SSEA-4, whereas the same colonies were negative for endogenous SSEA-1, SSEA-3, Tra-1-60, and Tra-1-81. Antibody staining indicated that ectoderm (βIII-tubulin), mesoderm (desmin), and ectoderm (cytokeratin) could be identified in cell outgrowth from EBs, whereas histology indicated the presence of cells from all three germ layers in teratomas. In parallel, Bao and colleagues reported ovine iPSCs derived from neonatal sheep fibroblasts using a similar Tet-inducible lentivirus system for the four Yamanaka factors supplemented with Nanog, Lin28, SV40 large T, and human telomerase reverse transcriptase (hTERT) (Bao et al., 2011). These cells were cultured in Dulbecco's modified Eagle medium (DMEM) plus KSR medium, with colonies appearing between 11 and 20 days posttransduction. Visually resembling mouse ESCs, these ovine iPSCs stained positive for AP, Oct4, Sox2, Nanog, SSEA-1, Tra1-60, and Tra-1-81. Again cells displaying markers or morphology for all three germ layers were identified in EBs and teratomas. The implication in this work is that the introduced genes were active throughout.

Subsequently work from an Australian group derived ovine iPSCs from fetal fibroblasts using retroviral vectors carrying the four Yamanaka factors and culturing in DMEM plus FGF (Liu et al., 2012). The cells expressed Oct4 and Sox2 and silenced all transgenes, with all three lineages identified in EB outgrowths and teratomas. This group showed that these cells could contribute to the inner cell mass of preimplantation embryos. We have also reported the production of ovine fetal iPSCs (Sartori et al., 2012). In our hands, ovine fibroblasts were reprogrammed through the introduction of the four Yamanaka factors using a standard retroviral vector and culture in DMEM plus FGF. By 14 days of culture, human ESC-like colonies had appeared (Fig. 1). We observed partial silencing of the transduced factors, with Oct4 never truly silenced. These cells were positive for AP and Nanog, but we did not see robust staining for either SSEA-1 or SSEA-4. As in the first two reports, we observed expression of marker genes associated with all three lineages from EB outgrowths and in teratomas. We evaluated the ability of our ovine iPSCs to contribute to production of chimeric animals. A few 3-week-old fetuses and lambs were identified as positive for the Oct-4 transgene by PCR; however, the level of contribution was extremely low. Whether this reflected partial reprogramming or some other influence on our cells we do not know.

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FIG. 1.

Selection of ovine iPSCs grown in KO-DMEM, KO serum replacement and human basic growth factor as described in Sartori et al. (2012).

One hope for livestock stem cells is that they could function as better donor cells for somatic cell nuclear transfer (SCNT) (Telugu et al, 2010). We know that treating ovine somatic cells with oocyte extracts can improve reprogramming and result in improved birth rates (Rathbone et al., 2013). Several lines of argument imply that a stem cell could accomplish similar improvements. Given that our cells could persist in a chimeric animal and had passed to some degree all the standard tests for pluripotency, we were encouraged to push the cells further. We teamed up with Keith Campbell at Nottingham, who was working on improving reprogramming since the birth of Dolly, to see if our ovine iPSCs could be used as donors for SCNT. Very sadly, this project will now not be seen through to its conclusion.

Going Forward with Ovine iPSCs

In summary, several groups have produced ovine iPSCs, but there is no consensus on the method and, not surprisingly, because of this there is no consensus on what properties these cells have—morphology and pluripotency markers (in comparison to what is known for human and rodent iPSCs). Why is it so difficult to isolate robust ovine pluripotential cells? Why is it difficult to isolate ungulate pluripotential cells in general? There are many possibilities, all well described in the literature (Gandolfi et al., 2011; Malaver-Ortega et al., 2012; Telugu et al., 2010). To overcome these issues, we need multiple approaches—the journey continues—and there is good reason for this. Beyond the fundamental desire to increase our knowledge of reprogramming and pluripotency, ovine iPSCs offer numerous opportunities for studying other aspects of stem cell and developmental biology. The commonality (or not) of key pathways will be uncovered (Telugu et al., 2012; Thomson et al., 2012). There is also the hope that animal stem cell systems will enable acceleration of translation of new cell therapies, enabling biosafety aspects to be evaluated in an animal model. This may well focus on the pig, and for certain applications sheep may well offer benefits. Initial aims to use such cells for genetic engineering remain, but given the success afforded by DNA editors (Carlson et al., 2012) perhaps less so. How do we get to robust ovine iPSCs? First, we need to improve our understanding of core pluripotency factors and how these factors can be harnessed to reprogram cells. A good starting point will be to understand what Nanog (Chambers and Tomlinson, 2009) does in livestock (Sumer et al., 2012).