Embryonic stem (ES) cells were first derived from the inner cell mass (ICM) of inbred mouse embryos in 1981 by Martin, Evans and Kaufman. Recently, ES cells were successfully derived from non-human primate and human embryos. The National Institutes of Health listed 64 human ES cell lines available for research in 2001; however, only a few had been characterized and studied. Similarly, less than 7 of the more than 20 monkey ES cell lines have been well characterized apart from establishing pluripotency and genetic stability. Even in the mouse, most ES cell studies have been performed with a single inbred mouse cell line. The extent of diversity among primate ES cell lines is currently unknown.
Primate and mouse ES cells are similar in their ability to self-renew and differentiate into cells representing all three embryonic germ layers. They are different in cell/colony morphology, growth requirements and molecular signatures defining various developmental stages. For example, leukemia inhibiting factor (LIF) is able to sustain undifferentiated murine, but not primate ES cell growth. Thus, primate ES cells are routinely cultured on a mouse feeder layer, i.e., mitotically suppressed mouse embryonic fibroblasts. 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.
Safety is one of the most important issues for ES cell-based therapy. In addition to mouse cell contamination, tumor formation or other somatic cell transformations from grafted ES cell progeny must be considered. The likelihood of transplanting pluripotent ES cells as a therapeutic approach is low because of this problem. In contrast, their differentiated progeny, which also survive and integrate well in host tissues, are unlikely to form tumor cells. Although differentiated ES cell progeny can be produced in large quantity, enrichment and purification of the desired phenotypes for transplantation must be ensured. Clearly, the efficacy of ES cell-based transplantation must be tested rigorously with each and every phenotype, preferably in non-human primate models.

Derivation of monkey ES cell lines

Different laboratory groups have not directly compared existing primate ES cell lines. A comparative undertaking is important for several reasons. First, it helps to standardize a protocol with simplified growth requirements for primate ES cell lines, or to derive a primate ES cell line that replicates faster, is more amenable to subcloning and can be maintained in the absence of feeder layers or conditioned medium. Second, it characterizes cell line-specific developmental potential for studies of primate development or in drug discovery. Finally, it is prudent for investigators to characterize several lines before transplantation efforts begin. The quality of ES cells among different cell lines, especially the ability to self-renew and differentiate into a desired lineage, must be ascertained, at least by in vitro assessment, before selection for transplantation studies.

Characterization of undifferentiated primate ES cells

Primate ES cells are routinely cultured on mouse feeder cells because the molecular pathway and the key molecules required to maintain pluripotency are unknown. A manual method to select undifferentiated monkey ES cells for propagation and differentiation studies is adopted. The procedure can enrich a monkey ES cell population to near homogeneity as indicated by the high percentage of cells expressing several pluripotent ES cell markers. This unique repertoire of markers includes the stage-specific embryonic antigens (SSEA) 3 and 4, glycoproteins TRA-1-60 and TRA-1-81, specific enzymes such as alkaline phosphatase and telomerase, and transcriptional factors Oct4, Rex1 and GDF-3 that are rapidly down regulated upon differentiation. The expression of markers such as SSEA-1 or Sox-1 in primate ES cell populations indicates that some of cells may have undergone differentiation. While none of these markers alone carries absolute predictive value for pluripotency, their presence as a group is highly informative.
The ultimate identification of, and hence ability to compare, pluripotent ES cell lines might be based on genome wide expression analysis. By applying DNA microarray technology, a set of "stemness" genes has been tentatively identified in different murine stem cell populations. A similar approach has been initiated in parthenogenetic monkey ES cells. Extending the analysis to genes that are up- or down-regulated upon differentiation allows identification of putative genes associated with pluripotency that, in turn, could lead to the isolation of factors critical to the propagation of undifferentiated ES cells in a defined medium without the confounding contamination of serum or feeder cells.

Differentiation of monkey ES cells

Primate ES cells spontaneously differentiate when cultured in serum-containing medium but in the absence of feeder cell support. This process is hastened by aggregating ES cells in hanging drops, in suspension or in overgrowth cultures. In all cases, ES cells retain the ability to differentiate into phenotypes representing the three embryonic germ layers. In general, the hanging drop or suspension approach is appropriate when chemicals are used to induce lineage-specific differentiation. This is primarily because both approaches result in the formation of pluripotent embryoid bodies (EBs), spherical ES cell aggregates (Figure 1). EBs form within days, allowing chemical induction to occur early in differentiation without the interference of serum or feeder cells. In contrast, the overgrowth approach usually takes weeks, enabling the selection of lineage-specific cells by morphological assessment. For example, one interesting possibility based on studies conducted in mice is the derivation of gametes from ES cells. ES cell-derived gonadal germ cells were obtained either by prolonged culture of overcrowded ES cells forming primitive oocytes or by EB formation and induction forming spermatogonial cells.
It is unknown whether the gender orientation of ES cell differentiation is genetically determined and/or protocol-dependent. Certainly, epigenetic influences, including cell-to-cell and cell-to-molecule interactions at critical times, can bias ES cell differentiation toward a single lineage. It has been postulated that ES cells in culture will differentiate toward the neural lineage by default, i.e., without genetic or epigenetic influences. In practice, spontaneous differentiation of ES cells rarely produces cells of a single lineage, although the proportion of neural cells may be greater than other lineage-specific cells.
Monkey ES cell-derived EBs have been induced and differentiated into glial (Figure 2) and neuronal phenotypes (ectodermal lineage) (Figure 1) as well as insulin producing, pancreatic beta cell-like phenotypes (endodermal lineage). Cardiomyocytes (mesodermal lineage) after spontaneous differentiation of a monkey ES cell line (ORMES-2) were also identified

Fig. 1. Neuronal differentiation of monkey ES cells into embryoid bodies (EBs) (left); neuronal differentiation of monkey ES cells under the influence of monkey brain tissue crude extracts (right).

Fig. 2. Differentiation of monkey ES cells into glial phenotypes; morphology in culture.

Perspectives and limitations of ES cell research and applications

Since the isolation of human ES cells, an explosion in ES cell research is based on the expectation that these cells will become a valuable source for replacement therapy to cure cellular degenerative diseases. At the same time, adult stem cells isolated from various tissues carry similar expectations for regenerative medicine, given that de-differentiation (from tissue-specific stem cells to pluripotent stem cells) and trans-differentiation (between tissue-specific stem cells) become possible.
ES cell-based therapy is a viable approach to cure degenerative conditions such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, spinal cord injury, diabetes and cardiac dystrophy, to name a few. The road to achieve these goals will be long, and the challenge ahead is daunting. Some of the immediate limitations in primate ES cell research and applications include the following:
(1) The optimization of undifferentiated ES cell culture is key to standardize successful culture of undifferentiated primate ES cells and to scale-up the production of differentiated progeny for transplantation. The solution may rely on the discovery of "stemness" genes and their signaling pathways. Using mouse feeder cell-conditioned medium or human cell co-cultures cannot circumvent this requirement, but these alternatives are helpful in animal research. A feeder layer- and serum-free culture system for human ES cells employing a combination of serum replacement and growth factors has been reported. Validation of this method in different primate ES cell lines will be valuable. Methods to quantitatively identify and isolate undifferentiated ES cells from spontaneously differentiated phenotypes during culture must be established. Moreover, the quality of pluripotent ES cells over multiple passages, such as chromosomal normalcy and differentiation potential, must be maintained.
(2) A characterization and comparison among primate ES cell lines is essential to define line specificity. The full potential of ES cell applications can not be realized by a few cell lines studied by a small number of laboratories. A concerted effort to create an ES cell bank with well-characterized cell lines and standardized culture procedures for distribution to scientists will greatly facilitate primate ES cell research. Creating new monkey ES cell lines for unbiased characterization should provide invaluable information about ES cell line diversity that is unable to be performed at the present time in the United States with federal funding on human ES cell lines.
(3) Phenotype-specific differentiation and enrichment protocols are critical to the application of ES cells for therapy. Differentiation protocols derived from mouse ES cell research must be applied to primate ES cells as soon as feasible, as it is most certain that modifications will have to be made to overcome species differences. Enrichment of the desired phenotype for transplantation will probably be essential, with the quality of primate ES cells or progeny ensured after enrichment.
(4) Genetic manipulations such as functional gene knockins, including disease-specific genes, differentiation-promoting genes and immunocompatible genes are highly desirable. Recent success in gene transfer by homologous recombination and lentiviral vectors is encouraging, but these procedures need to be applied to other cell lines, by other laboratories, and improved for better efficiency. The quality and potential of genetically modified primate ES cells should be tested rigorously in animal models.
(5) Development of animal models for transplantation studies is a crucial step toward successful clinical applications. In many cases, results using rodent models cannot be translated to human patients. Currently there are no discriminative non-human primate models of cellular degenerative diseases because gene-knockout and cloning procedures in the monkey are both time consuming and technically challenging. A few experimental models, such as MPTP-induced hemiparkinsonian monkeys, are therefore extremely valuable for ES cell-based therapeutic studies. The development of additional disease models with nonhuman primates is urgent.

At least 15 rhesus monkey ES cells lines are available at the Oregon National Primate Research Center. These cell lines have been characterized by their chromosomal normalcy, growth patterns and differentiation potential. A panel of molecular signatures that include embryonic surface antigens, enzymes and transcriptional factors, defines the pluripotency of undifferentiated monkey ES cells. Highly purified nestin+/Musashi1+ progenitor cells can be produced by a neural selection protocol. These progenitor cells are multipotent, if not pluripotent, capable of differentiating into insulin-producing endodermal cells, various neuronal phenotypes and glial cells expressing Schwann cell markers and myelinating proteins. Differentiation into certain populations, such as the serotonergic phenotypes, is induced by the selection protocol. Differentiation into other lineages, such as the dopaminergic phenotypes, is either inhibited or requires additional epigenetic influences such as those present in extracts of the dopaminergic striatum. However, enrichment of dopaminergic phenotypes by directional differentiation of mouse ES cells followed by appropriate growth factor induction has been successful. The potential exists for the differentiation and enrichment of monkey ES cells into dopaminergic neurons or myelinating glial phenotypes that serve as suitable cell sources for transplantation studies in animal models of Parkinson Disease, multiple sclerosis and spinal cord injury. Such translational studies, studies, particularly in nonhuman primate models, are critical steps to understand the safety, feasibility and efficacy of ES cell-based therapy for the treatment of neural degenerative diseases.