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BOR - Papers in Press, published online ahead of print May 5, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029413
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BIOLOGY OF REPRODUCTION 71, 1766–1771 (2004)
DOI: 10.1095/biolreprod.104.029413
© 2004 by the Society for the Study of Reproduction, Inc.

Minireview

Progress with Nonhuman Primate Embryonic Stem Cells1

Don P. Wolf2,3, Hung-Chih Kuo3, K.-Y. Francis Pau3, and Linda Lester4

Division of Reproductive Sciences,3 Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon 97006 Department of Medicine,4 Oregon Health and Science University, Portland, Oregon 97201


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 ISOLATION AND CHARACTERIZATION
 CURRENT RESEARCH FOCUS
 GENETIC STRATEGIES IN ES...
 SAFETY, EFFICACY, AND...
 REFERENCES
 
Embryonic stem cells hold potential in the fields of regenerative medicine, developmental biology, tissue regeneration, disease pathogenicity, and drug discovery. Embryonic stem (ES) cell lines are now available in primates, including man, rhesus, and cynomologous monkeys. Monkey ES cells serve as invaluable clinically relevant models for studies that can't be conducted in humans because of practical or ethical limitations, or in rodents because of differences in physiology and anatomy. Here, we review the current status of nonhuman primate research with ES cells, beginning with a description of their isolation, characterization, and availability. Substantial limitations still plague the use of primate ES cells, such as their required growth on feeder layers, poor cloning efficiency, and restricted availability. The ability to produce homogenous populations of both undifferentiated as well as differentiated phenotypes is an important challenge, and genetic approaches to achieving these objectives are discussed. Finally, safety, efficiency, and feasibility issues relating to the transplantation of ES-derived cells are considered.

assisted reproductive technology, developmental biology, diabetes, differentiation, early development, embryo, monkey, primate, regenerative medicine, stem cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
ISOLATION AND CHARACTERIZATION
 CURRENT RESEARCH FOCUS
 GENETIC STRATEGIES IN ES...
 SAFETY, EFFICACY, AND...
 REFERENCES
 
Nonhuman primates (NHP), specifically Old World Macaques, such as rhesus and cynomolgus monkeys, are extremely valuable animal models because of their widespread use in biomedical research, their close phylogenetic relationship to man, and hence their clinical relevance (Table 1). While cell-based therapies can and do benefit from experimentation in rodent models, potential applications of human embryonic stem (ES) cells in regenerative medicine must be pioneered in nonhuman primate species before or in parallel with human research, for scientific and ethical reasons. This is particularly relevant for neural degenerative disease applications, where the mouse is, for the most part, inadequate as a transplantation or disease model. Thus, we envision nonhuman primate research as a prelude to human cell-based therapeutic applications, in studies of primate development where ethical issues limit human experimentation, in drug discovery, and in ex vivo studies of disease processes. Additionally, it is clear that stem cells are extremely important as research subjects in and by themselves, as the mechanisms underlying cellular differentiation, expansion, and self-renewal can be studied along with differentiated tissue development and regeneration in vitro. This minireview will emphasize progress achieved since the report of Thomson and Marshall in 1998 [1].


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  		TABLE 1. Advantages and disadvantages to the NHP model


   ISOLATION AND CHARACTERIZATION
TOP
 ABSTRACT
 INTRODUCTION
 ISOLATION AND CHARACTERIZATION
CURRENT RESEARCH FOCUS
 GENETIC STRATEGIES IN ES...
 SAFETY, EFFICACY, AND...
 REFERENCES
 
ES cells have been successfully isolated from nonhuman primate embryos [2–5], including marmosets and cynomolgus and rhesus macaques. Eight rhesus monkey ES cell lines, derived from in vivo flushed blastocysts are available, four male and four female, all with a diploid set of 42 chromosomes [1]. Additionally, 10 rhesus monkey ES cell lines derived from in vitro-produced blastocysts, ORMES-1–10, are under development by the authors at the Oregon National Primate Research Center. In the cynomolgus macaque, four ES cell lines have been derived from embryos [4] and one line from a parthenogenetic blastocyst [5].

The conventional approach to ES cell derivation involves immunosurgery at the expanded blastocyst stage, thereby separating inner cell mass from trophectoderm [1]. Primate ES cells are routinely cultured on feeder cells (e.g., mouse embryonic fibroblasts mitotically inactivated by chemical exposure or gamma irradiation) because the molecular pathway and the key molecules required to maintain pluripotency are unknown. The use of feeder cells greatly limits the scaled-up production of undifferentiated primate ES cell populations and the potential contamination by mouse cells is a safety concern when considering transplantation. Moreover, for clinical applications in patients, the development of alternative isolation approaches that circumvent the use of feeder cells or avoid embryo destruction are a prerequisite. In this regard, activation of the canonical Wnt signaling pathway has recently been reported as sufficient to maintain self-renewal of both human and mouse ES cells [6].

The characteristics of primate embryonic stem cells as previously itemized [1] include 1) derivation from the pre- or periimplantation embryo, 2) capacity for prolonged undifferentiated proliferation, 3) ability to differentiate after prolonged culture to form derivatives of all three embryonic germ layers, and 4) maintenance of a normal karyotype through undifferentiated culture. For the routine determination of pluripotency in primate ES cells, a unique repertoire of molecular markers can be applied, including cell surface antigens detected by antibodies, such as stage-specific embryonic antigens (SSEA-3 and -4), and glycoproteins (TRA-1-60 and TRA-1-81), enzymatic activities, such as alkaline phosphatase and telomerase, and cloned markers that are rapidly downregulated upon differentiation, including Oct-4 and Rex1 (Table 2 [7–13]). While none of these markers alone carries absolute predictive value for pluripotency, their presence as a group is highly informative and ongoing efforts are focused on setting standards that clearly carry relevance to all primate ES cells [7]. It is important to note that the karyotype of undifferentiated cell populations should be evaluated periodically or certainly when changes in morphology or growth characteristics are detected.


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  		TABLE 2. A marker panel for identifying undifferentiated primate ES cells

In addition to in vitro characterization, an assessment of ES cell behavior in vivo is normally undertaken, involving teratoma formation after transplantation into severely compromised immunodeficient mice [1]. This approach is cumbersome and qualitative, providing a yes or no answer with regard to differentiation into cellular phenotypes representing all three embryonic germ layers. Indeed, the full developmental potential of ES cells can best be determined by measuring their ability to contribute to embryonic, fetal, and adult cell lineages in chimeric animals. Initial successes have been reported following in utero transplantation of peripheral blood stem cells [14] or embryonic stem cells [15]. We have established that membrane-labeled ES cells, when injected into four- or eight-cell-stage embryos, propagate and integrate into the blastocyst as a first step in producing chimeric rhesus monkeys [16]. The availability of green fluorescent protein (GFP) positive cell lines in rhesus (Thomson, personal communication) and cynomolgus monkeys [17] allows the accurate tracking of ES cell progeny in chimeric animals, an extremely important activity that obviously could not be undertaken in humans for ethical reasons.

With the development of directed differentiation protocols for ES cells in vitro, a quantitative approach can now be considered whereby ES cell progeny commitment to the three major embryonic germ layer lineages is determined. It is well established that undifferentiated monkey ES cells can be directed to differentiate into multiple lineages, including cardiomyocytes, muscle cells, pancreatic islet-like cells, liver cells, hematopoietic cells, neurons, and glial cells [1–5, 18–22]. We are employing serotonergic phenotypes [20] to represent the ectodermal lineage, insulin-expressing, islet-like phenotypes [21] for the endodermal lineage and contracting cardiomyocytes [22] for the mesodermal lineage. These lineage-specific phenotypes can be derived by differentiating monkey ES cells first into embryoid bodies (EBs; Fig. 1B), then into nestin-positive progenitor cells (Fig. 1C). When subjected to differentiation protocols, nestin progenitor cells yield enriched populations of serotonergic phenotypes (Fig. 1D) or insulin and C-peptide-positive cells (Fig. 1E). The presence of foci of contracting cardiomyocytes in EBs has also been quantitated (Fig. 1F).



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  		FIG. 1. Micrographs of monkey ES cells and ES cell-derived progeny. Monkey ES cells can be incorporated with a reporter gene or differentiated into multiple phenotypes. A) Monkey ES cell colony expressing transgenic green fluorescence protein (GFP). The GFP signal was identified by a specific anti-GFP antibody as shown in red. Cell nuclei were counterstained by DAPI, which appeared as white-blue as the result of color overlay with the red GFP cells. The scale bar in this panel is equivalent to 100 µm. B) Monkey ES cell-derived pluripotent embryoid bodies (EBs) in suspension (scale bar = 100 µm). C) Nestin-positive progenitor cells (shown in red) derived from EBs. D, E) Lineage-specific phenotypes derived from nestin-positive progenitor cells: the ectodermal lineage is represented by serotonergic neuronal phenotypes expressing tryptophan hydroxylase (D, shown in green), the endodermal lineage is represented by insulin-producing cells (E, shown in green), and the mesodermal lineage is represented by contracting cardiomyocytes expressing tropomyosin1 (F, shown in green). Cell nuclei counterstained with DAPI are shown in blue. Scale bars are equivalent to 40 µm in panels C–F


   CURRENT RESEARCH FOCUS
TOP
 ABSTRACT
 INTRODUCTION
 ISOLATION AND CHARACTERIZATION
 CURRENT RESEARCH FOCUS
GENETIC STRATEGIES IN ES...
 SAFETY, EFFICACY, AND...
 REFERENCES
 
Primate ES cells are relatively cumbersome to maintain and manipulate, requiring considerable technical expertise and attention confounded by their slow growth rate and requirement for culture on feeder layers, typically mouse embryonic fibroblasts. Unlike murine ES cells, leukemia inhibiting factor (LIF) alone is unable to sustain undifferentiated primate ES cell growth, in contrast with murine cells, in which the molecular requirements, including components of the LIF-gp130-STAT3 signaling pathway, have been identified [23]. While undifferentiated monkey ES cells can be maintained on mouse feeder layers for >50 passages, the manual technique employed is labor intensive and incompatible with the scaled-up propagation envisioned for transplantation research. Human ES cell lines have been maintained in an undifferentiated state for many passages in a feeder cell-conditioned medium supplemented with LIF and fibroblast growth factor-2 [24], and our preliminary observations suggest that this method will also work for monkey ES cells. However, primate ES cell colonies grown without feeder cell contracts are typically less adherent and slightly altered in appearance compared with those grown on feeder cells, and there is a greater tendency for cells to spontaneously differentiate [24].

Another limitation, secondary to the need for feeder cell contact perhaps, is the inability to subclone populations of primate ES cells. When cells are completely dissociated, cloning efficiencies may be under 1% and, therefore, procedures requiring ES cell dispersion usually connote the creation of small cell clusters rather than individual cells. Efficient subcloning would allow the generation of homogenous populations of undifferentiated ES cells, the recovery of contaminated cultures, or the elimination of cells with abnormal karyotypes as well as the enrichment of selected ES cells such as GFP+ phenotypes. Related to this subject of poor recovery is the concern that inadvertent selection of undesirable or altered cells occurs during routine handling, culture, or cryopreservation of primate ES cells.

The stability and diversity among different primate ES cell lines must be understood before effective application of ES cell-based therapy. We currently have little insight into the variations that may exist between cell lines. A comparative undertaking is important for several reasons. First, it carries implications to ongoing efforts to establish stem cell banks and it may be possible to derive a primate line with simplified growth requirements, that replicates faster, is more amenable to subcloning, and can be maintained in the absence of feeder layers or conditioned medium. Second, it is possible that the developmental potential of primate ES cell lines will be line specific. This possibility holds significance for any strategy for cell-based therapy, to studies of primate development, or to the use of ES-derived differentiated phenotypes in drug discovery or pathogenicity studies. Finally, because the variability between individual ES cell lines is unknown, investigators from different groups using different lines may not be able to directly compare their results. It is, therefore, prudent if not essential, to generate a relatively large number of ES cell lines and to more completely characterize new and available lines as a robust resource to the scientific community.

Although empirically defined approaches to the efficient maintenance and propagation of undifferentiated primate ES cells may be forthcoming, identifying genes responsible for pluripotency or stemness may ultimately catalyze the process by defining genes/factors critical to the maintenance of the undifferentiated state. By transcriptional profiling several murine stem cells and differentiated cell populations, a set of stemness genes has been identified [25– 27], although concern exists over inconsistencies in currently available results [28]. A limitation of all transcriptional profiling relates to the homogeneity of the cell types examined, and it is not possible to exclude the presence of contaminating cell types in undifferentiated primate ES cell preparations. Moreover, the approach is limited to the genes represented on the microarray and monkey microarrays are not yet even available. However, in the absence of monkey microarrays, cross-species reactivity with human arrays has been reported, with the recognition that some monkey genes will be missed by the human array [29]. Fortunately, efforts are ongoing to develop a rhesus monkey microarray (Dr. Robert Norgren, personal communication: http://rhesusgenechip.unomaha.edu).

Gene-expression profiling is also a powerful and necessary way of defining ES cell-derived, differentiated cell populations. Thus, in addition to immunocytochemistry, the ontogeny of specific gene expression during differentiation lends important information to phenotype identification. For instance, in the pancreatic islet cell paradigm, genes such as Oct-4 should disappear as differentiation begins and markers associated with early neuronal or islet cell development such as NeuroD, NGN3, and PDX1 (pancreatic duodenal homeobox) and nestin should appear. As differentiation proceeds, intermediate and mature islet cell markers should become detectable, including PDX1, glucagons, insulin, NKX6.1, and GLP-1 (glucagons-like peptide) receptor at the expense of NeuroD and nestin. Of course, additional markers of other lineages may be present; amylase and enolase, e.g., were detected in monkey ES cell-derived, insulin-containing phenotypes, confirming the mixed nature of the population, about 50% of which were insulin C-peptide positive [21].


   GENETIC STRATEGIES IN ES CELL RESEARCH
TOP
 ABSTRACT
 INTRODUCTION
 ISOLATION AND CHARACTERIZATION
 CURRENT RESEARCH FOCUS
 GENETIC STRATEGIES IN ES...
SAFETY, EFFICACY, AND...
 REFERENCES
 
For ES cell-based transplantation, cell lines expressing a reporter gene for tracking cell fate are highly desirable. Transgenic cell lines that can be created using conventional transfection methods such as lipofectamine or adenoviral constructs so readily in the mouse are difficult to create in primates because of low transfection efficiency or transient gene expression [30]. However, lentiviral vectors have more recently allowed high-level sustained transgene expression in human and rhesus and cynomolgus monkey ES cells [17, 30–32].

Primate ES cell cultures are susceptible to contamination by feeder cells or spontaneously differentiated phenotypes. To improve the homogeneity of undifferentiated ES cell populations, genetic manipulation is possible based on the generation of a selectable phenotype by the expression of a marker under control of a specific promoter. For instance, the transcription factor Oct-4 is specifically expressed in totipotent cells of the mouse embryo and our studies indicate that Oct-4 is expressed only in the early, undifferentiated rhesus monkey stem cell [33]. A distal enhancer region of the gene is responsible for expression in the preimplantation embryo, the germ line, and ES cells [34]. This enhancer region coupled with a selectable marker, such as neomycin, or a reporter gene, such as eGFP, could be used in a gene construct that would allow selection of undifferentiated ES cells.

The ability to isolate homogenous populations of differentiated cells is particularly important in preclinical cell therapy trials. Contaminating cell populations may include undifferentiated ES cells that introduce safety risks because of their ability to support tumor formation [15], differentiated cells representing other lineages, and cells of the desired phenotype but at different stages of maturation or differentiation. Using transgenic approaches, ES cell populations enriched for a specific progenitor or lineage marker could be isolated. The enrichment of insulin containing pancreatic cell phenotypes under regulation by human insulin promoter serves as a paradigm. The human insulin promoter (HIP) gene fused to a marker protein such as eGFP would allow identification and selection of insulin-producing islet cell progenitors without prior cell fixation (Fig. 2). Coupling eGFP expression to the HIP would also provide a rapid screening method for monitoring differentiation and further enable the differentiation process by providing a small-scale, high-throughput screening method for the optimization of the differentiation protocol. Furthermore, the HIP-dependent expression of GFP could be used to simplify the tracking of ES cells following transplantation.



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  		FIG. 2. Schematic diagram of a HIP/GFP Vector. The human insulin promoter (HIP), from –100 to –400 base pairs, can be fused to an enhanced green fluorescent protein (eGFP) vector. Detection of GFP expression can be used as a surrogate for insulin gene expression, allowing isolation and purification of an insulin-producing, beta-like cell lineage

Knowledge concerning the specific gene expression patterns that arise as a consequence of cell developmental ontogeny can also be used to promote specific phenotype development. Expression of PDX1 as a pancreatic cell precursor gene introduced by homologous recombination into early ES cells could be used to promote differentiation of primate ES cells in vitro to pancreatic cell lineages. In addition to knocking in specific genes, gene silencing methods allow the knocking down of other genes using RNA interference technology, such as short interfering RNAs, or through transcription of short hairpin RNAs [35–37]. Continuing with the pancreatic islet development paradigm as an example, expression of Sonic hedgehog, a member of the hedgehog family of signaling, is important to the maintenance and progression of nonpancreatic endodermal tissue developmental [37]. Because endodermal lineages are thought to develop through a similar progenitor, the silencing or knocking down of sonic hedgehog should limit the progression of these progenitors down the exocrine pathway and subsequently increase the number of cells entering the endocrine differentiation pathway.


   SAFETY, EFFICACY, AND FEASIBILITY ISSUES
TOP
 ABSTRACT
 INTRODUCTION
 ISOLATION AND CHARACTERIZATION
 CURRENT RESEARCH FOCUS
 GENETIC STRATEGIES IN ES...
 SAFETY, EFFICACY, AND...
REFERENCES
 
The transplantation of human ES cell-derived populations into patients is impacted by ethical and safety concerns and the NHP should, therefore, serve as a valuable, clinically relevant model. With the established ability to drive relatively large quantities of GFP-expressing ES cells into a desired phenotype by a reproducible in vitro differentiation protocol, a new set of challenges arises in assessing their potential use in cell-based therapies. First, and perhaps foremost, is the issue of homogeneity of the cell population to be grafted, a subject that has already been discussed. Additional safety issues have recently been reviewed [38], including the possibility of infectious disease transmission from donor to recipient. While there are feeder cell lines under development that minimize this risk (primate foreskin or placental cell feeders), most existing human ES cell lines have been grown on mouse embryonic fibroblasts and their use in clinical therapy is, therefore, unacceptable.

With ES-derived cell populations, graft rejection is also a potential problem; immunosuppressive therapy, while possible, is undesirable because of the need for its prolonged use and the potential for serious side effects. Alternatives in this case include deriving your own ES cell line by therapeutic cloning [39], genetically modifying cells to make them transparent to the immune system, or creating ES cell banks that support HLA matching with the recipient. All of these options will clearly require further investigation, much of which could be conducted in the NHP. At the genetic level, while the risks are impossible to anticipate at this juncture, epigenetic regulation of imprinted gene expression is an area of concern; namely, do ES cells retain the allele-specific gene expression characteristics of the embryo from which they were derived and, if not, does it matter to differentiated cell fate and functions.

In considering efficacy of the transplant, it is, of course, critical that the physiological function of the phenotypes available for transplant be well characterized (in the case of pancreatic islet phenotypes, spontaneous insulin production and induced release in response to a glucose challenge). First approximations in this regard are now available in rodent systems, e.g., the ability to abrogate streptozotocin-induced diabetes in mice with transplanted ES cell-derived, insulin-containing phenotypes [40]. When grafted, do the cells retain function for prolonged periods of time and are they regulated normally, as hypo- or hyperactivity may be less desirable than no activity? The three-dimensional relationship between cells in vivo is also undoubtedly critical to these questions and we may be asking the impossible by transplanting isolated populations of a single phenotype. Finally, will it be desirable to transplant a mixture of cells from the same lineage including both precursor as well as terminally differentiated functional cells to prolong function?

Feasibility concerns associated with transplantation include the availability of safe and convenient delivery systems. Obviously, there is quite a difference between portal vein injection of pancreatic islet phenotypes (convenient and medically simple) versus midbrain placement of dopaminergic neuronal phenotypes in the treatment of Parkinsonian patients. Will repeated grafting be required? Can adequate cell numbers be achieved given that massive cell losses may occur before colonization at the transplant site? Precise tissue targeting, grafted cell fate, and host-transplant integration are issues that also must be considered. Again, the mixture of cells to transplant is germane, as terminally differentiated populations don't replenish. Meaningful answers to all these questions could and perhaps should derive from translational studies in NHPs.

In conclusion, there are clearly a number of significant hurdles yet to surmount in this field of research; however, the potential for effective, cell-based, disease treatment remains promising, the emphasis for stem cell research from the biomedical research community is clear, and the push is on to further evaluate the potential of these fascinating cells.


   FOOTNOTES
 
1 Supported by National Institutes of Health grants RR15199, to D.P.W., HD18185 to Richard L. Stouffer (H-C.K), RR00163 to Peter Kohler (L.L.), and NS41601 to K.-Y.F.P. Back

2 Correspondence: Don P. Wolf, Oregon National Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006. FAX: 503 533 2494; wolfd{at}ohsu.edu Back

Received: 10 March 2004.

First decision: 9 April 2004.

Accepted: 21 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 ISOLATION AND CHARACTERIZATION
 CURRENT RESEARCH FOCUS
 GENETIC STRATEGIES IN ES...
 SAFETY, EFFICACY, AND...
 REFERENCES
 

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