A variety of mammalian pluripotent stem cell lines have been artificially created and are used for different purposes. In addition to enabling studies on stem cell properties, such as stem cell renewal and differentiation, they have been applied in many other ways, notably as tools for studying mammalian gene function, modeling disease, and drug screening. There is also the potential for therapeutic applications (by generating appropriate cells to replace cells lost through injury or disease).
A major way to produce pluripotent stem cell lines involves obtaining suitable cells from the early mammalian embryo and manipulating them in vitro. As detailed in Section 4.1, certain cells have very high differentiation potential. The zygote and the blastomeres of cleavage embryos are totipotent, but are not numerous and not so amenable to culture. Later on, pluripotent cells are found in the undifferentiated inner cell mass (ICM) and in the epiblast of later-stage blastocysts (Figure 1B). These naturally pluripotent cells are not stem cells that renew themselves while producing more specialized cells: the ICM and epiblast are transient structures with short-lived founder cells that quickly give rise to differentiated tissues. However, the pluripotency of ICM/epiblast cells can be captured by culturing them under conditions that favor cell renewal and suppress differentiation. Success in this endeavour yields a pluripotent stem cell line that can undergo unlimited cell division.
Fig1. Early development of the mammalian embryo, from zygote to blastocyst. (A) Cleavage divisions. In mammals, the first cleavage is a normal meridional division (along the vertical axis), but in the second cleavage one of the two cells (blastomeres) divides meridionally while the other divides at right angles, equatorially (rotational cleavage). At the early eight-cell stage, the blastomeres are symmetrical and loosely packed with little contact between the cells. (B) From compacted embryo to blastocyst. Later, at about the eight-cell stage, the embryo undergoes compaction: the blastomeres flatten against each other to maximize cell cell contacts, and tight junctions begin to be formed between the cells (see Box 4.2). By the 16-cell morula stage there are two populations of cells. Outer cells are held together by tight junctions, sealing off the inside of the sphere, which has inner cells that have formed gap junctions, enabling small molecules and ions to pass between them. The morula does not have an internal cavity, but afterward fluid is secreted from the outer cells into the interior, producing a blastocyst—a hollow ball of cells with a fluid-filled internal cavity, the blastocoel. The blastocyst has an outer layer of cells, the trophoblast that will contribute to the chorion, plus an inner cell mass (ICM) located at one end of the embryo, the embryonic pole. Differentiation of ICM cells (buff-colored cells) continues until by the mid-blastocyst stage (64-cell stage) the ICM has fully differentiated into two cell populations that initially appear in a mosaic form (blue and yellow cells). By the later blastocyst stage (>100 cells) the two ICM cell populations have sorted out into two distinct layers: an outer epiblast and an inner hypoblast. The epiblast will give rise to all the cells of the organism plus components of the extra-embryonic membranes; the hypoblast gives rise to the yolk sac. For convenience, the zona pellucida surrounding the early embryo is not shown in part (A), and polar bodies are also omitted.
Germ cell progenitors are another special case. Although they express germ-line specific genes, primordial germ cells and other germ cell progenitors have genomes with very similar epigenetic settings to those of totipotent cells. They can readily be induced toward higher potency; when cultured under certain conditions they convert to pluripotency.
Many different types of pluripotent stem cell lines have been made by culturing cells from early mouse embryos, but according to the source of embryonic cells and the state of pluripotency, three major classes of pluripotent mouse cell line have been distinguished, as listed below.
• Embryonic stem cell (ESC). Formed by manipulating pre-implantation blastocyst cells in culture, the cells demonstrate a state of naive pluripotency resembling that of the early epiblast.
• Epiblast stem cell (EpiSC). Formed by manipulating later-stage, post-implantation epiblasts in culture, the cells are said to be in a primed pluripotency state: they express certain lineage-specific factors that make them more predisposed to differentiate than ESCs.
• Embryonic germ cell (EGC). Formed by culturing germ-line cells, such as primordial germ cells, that convert to pluripotency in vitro.
Origins of embryonic stem cells
ESCs have been particularly well studied and intensively used. They were first reported in 1981 after successful culturing of cells from the ICM of blastocysts from the 129 mouse strain. This mouse strain is unusual because males sporadically develop testicular teratocarcinomas, malignant germ cell tumors that can be maintained continuously by serial transplantation. Teratocarcinomas and related benign teratomas contain multiple tissue types that can represent the three germ layers, and fully differentiated structures can form, such as teeth and hair (Figure 2).
Fig2. Teratomas, like teratocarcinomas, are tumors of germ cells converted to a pluripotent state that can differentiate into diverse somatic tissues. Teratomas and teratocarcinomas are, respectively, benign and malignant germ cell tumors. They have a disorganized collection of multiple different tissue types, and can have fully differentiated structures, such as teeth and hair, giving a bizarre appearance, as in this example of a teratoma. Both types of tumor arise from changes in germ cell progenitors. While expressing germ-line-specific genes, primordial and embryonic germ cells can be induced to a pluripotent state by genetic and epigenetic changes responsible for tumor formation and then differentiate to give diverse somatic tissues.
Teratocarcinomas are associated with the presence of embryonal carcinoma cells. These cells are not germ cells (which do not normally differentiate into other lineages); instead, they are proliferative pluripotent cells that closely resemble cells from the ICM in morphology, in ultrastructure, and in molecular markers. When injected into blastocysts, to test if they could behave like cells of the early embryo, cells from some embryonal carcinoma cell lines were able to colonize the host embryo and produce live-born chimeras (with cells originating from two zygotes). Even in the best cases, however, the efficiency was low. Embryonal carcinoma cells had another major drawback: they were genetically abnormal (having been derived from tumors).
Because of the difficulties with embryonal carcinoma cells, attention focused on deriving pluripotent stem cell cultures using blastocyst explants of the 129 mouse strain. Because pluripotent cells from the early embryo have a natural tendency to differentiate, the cell culture system was required to maintain the pluripotency of isolated ICM cells and suppress cell differentiation, while stimulating cell growth. That was possible by co-culturing with a layer of irradiated fibroblasts in the presence of medium containing fetal calf serum. The fibroblasts act as feeder cells: they are stimulated to produce matrix and growth factor support for the ESCs (but after having been irradiated they cannot divide). When grafted into adult mice, the cultured ESCs give rise to teratocarcinomas. The final proof of pluripotency was successful germ-line transmission following injection of ESCs into isolated blastocysts that were then implanted in a foster mother (Figure3).
Fig3. Development of an embryonic stem cell (ESC) line from the 129 mouse strain and how germ-line transmission can be demonstrated. (A) ESC isolation and chimera formation. To construct the ESC lines from the 129 mouse strain, blastocysts were excised from the oviducts of the ICM (inner cell mass) donor mouse and ICM cells were layered on top of a feeder cell layer of mouse embryonic fibroblasts in a culture dish with media supplemented by fetal calf serum. After various cell culture steps, a stable pluripotent ESC line was produced. To demonstrate germ-line transmission, ESCs can be injected into isolated blastocysts obtained from a mouse strain with a different coat color (such as C57B10/J, which has a black coat color that is recessive to the agouti color of the 129 strain). The resulting blastocysts can then be implanted into a pseudopregnant foster mother of the same strain as the donated blastocyst. Subsequent development of the introduced chimeric blastocyst results in chimeras with two populations of cells deriving from different zygotes (129 and C57B10/J in this case). The chimeras are readily identified because their coats have patches with different colors. (B) Germ-line transmission from chimeras. Breeding of male chimeras to C57B10/J mice can result in offspring with an agouti coat color, signifying a heterozygote: a sperm with a haploid strain 129 genome fertilized an egg with a haploid C57B10/J genome (the agouti coat color is dominant in the heterozygote).
Expanding the range of ESCs
The effectiveness of the cell culture system using fibroblast feeder cells and serum was heavily dependent on the genetic background of the inbred mouse strain; in practice, almost all stably pluripotent ESCs derived in this way are from the 129 mouse strain, or hybrids thereof. To isolate ESCs from other mouse strains and other mammals, there was a need to identify the extracellular signals (transmitted by feeder cells and serum) that promoted ESC self-renewal and suppressed differentiation. Leukemia inhibitory factor (LIF), which signals through the transcription factor STAT3, was quickly found to be an important factor, as was BMP4 (bone morphogenetic protein 4), which signals through SMAD transcription factors (Figure 4).
Fig4. Extrinsic signaling pathways that feed into reinforcing or antagonizing naive pluripotency. Simplified schematic of various signaling cascades that affect self-renewal. Blue arrows indicate activation of indicated target, whereas red T-bars show inhibition or blockade. Solid lines indicate a direct or known downstream target; dashed lines indicate indirect/inferred effects. Small, filled red circles indicate small-molecule inhibitors. BMP4 is present in serum and functions via SMADs to activate Id genes that repress differentiation-promoting transcription factors. Leukemia inhibitory factor (LIF) signaling affects many pathways but primarily acts via JAK-mediated phosphorylation of STAT3, which activates Tcfp2l1 and Klf4. Canonical Wnt signaling blocks GSK3 (glycogen synthase kinase-3) activity leading to stabilization of β-catenin, which in turn abrogates TCF3-mediated repression of pluripotency genes including Esrrb. CHIR009021 closely mimics Wnt signaling by inhibiting GSK3. FGF signaling activates the MAPK pathway leading to phosphorylation of MEK kinases, which in turn phosphorylate and activate ERK. Activated ERK promotes transition to a ‘‘primed’’ state of pluripotency that is blocked by the MEK inhibitor PD0325901. TF, transcription factor; -R, receptor. (Adapted from Hackett JA & Surani MA [2014] Cell Stem Cell 15:416–430; PMID 25280218. With permission from Elsevier.)
Subsequently, activation of the Wnt/β-catenin signaling pathway and inhibition of the FGF (fibroblast growth factor)/MAPK (mitogen-activated protein kinase) pathway were also found to be very important in maintaining renewal of ESCs while suppressing differentiation. A screen then identified chemical ways of manipulating these two path ways using two small-molecule inhibitors: PD0325901 inhibits MEK1/MEK2 (kinases that phosphorylate MAPK) and as a result inhibits FGF/MAPK signaling; CHIR009021 inhibits glycogen synthase kinase-3 (GSK3) to promote Wnt signaling through β-catenin (see Figure 4). The use of these two small-molecule inhibitors in culture conditions (known as 2i culture) does away with the need for feeder cells and serum by increasing the efficiency of keeping ESCs in a “ground state” of naive pluripotency. By using these conditions (sometimes with the addition of LIF), ESCs could be isolated from diverse mouse strains, and for the first time it was possible to develop rat ESCs, which were first reported in 2008, a full 27 years after the first mouse ESC lines.
Mouse EpiSC and EGC lines
Mouse epiblast stem cell (EpiSC) lines were established by culturing later-stage egg cylinder epiblasts. The culture conditions do not use LIF or 2i but instead use the fibroblast growth factor FGF2 and activin-A. Compared to ESCs, the cells are more heterogeneous and are associated with a state of pluripotency known as primed pluripotency that has certain disadvantages (Table 1).
Table1. PROPERTIES OF TWO MAJOR PLURIPOTENCY STATES IN MAMMALIAN PLURIPOTENT STEM CELL LINES
Primordial germ cells that will normally develop into mature gametes can be isolated from the gonadal ridge of E7.5 mouse embryos and cultured in vitro, leading to pluripotent embryonic germ cell (EGC) lines that are virtually identical to ESCs.
Human pluripotent stem cell lines
Human EGCs, derived from primordial germ cells of embryos and fetuses from 5 to 10 weeks old, were first cultured in the late 1990s. To isolate human embryonic stem cell lines, surplus embryos arising from in vitro fertilization (which were donated with con sent) were cultured to the blastocyst stage and the ICM cells were cultured.
The first human pluripotent stem cell lines derived from ICM cells of the blastocyst were reported in 1998, but as well as being difficult to manipulate, they had some very different properties from mouse ESCs. Now, it is widely accepted that they are the human counterparts of mouse EpiSCs (which were not reported until 2007). Like the mouse EpiSCs, the human “embryonic stem cell” lines exhibit primed pluripotency and associated features (see Table 1). Presumably, during the explant procedure, developmental progression continued further than anticipated.
In order to isolate genuine human counterparts of mouse ESCs, attempts were made to convert the human “embryonic stem cells” from primed pluripotency to naive pluripotency. However, culturing them in 2i medium alone (using inhibitors of MEK and GSK3; see Figure 4) causes the cells to differentiate into cells resembling neural stem cells. Additional chemical compounds and/or growth factors were needed to supplement the medium (see Theunissen TW et al. [2014]; PMID 25090446, for an example).
