The fine structure of human embryonic stem cells

Articles

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IntroductionHuman embryonic stem (ES) cells are derived from the innercell mass (ICM) of the developing blastocyst in vitro. The ICMcells are isolated by immunosurgery and cultured on murine orhuman embryonic fibroblast monolayers (Thomson et al.,1998; Reubinoff et al., 2000). The ICM grows as a compactcolony on a murine embryonic fibroblast feeder layer,producing large numbers of tightly adherent cells with a largenuclear:cytoplasmic ratio (see Figure 1C in Reubinoff et al.,2000). Continued overgrowing culture of ES cell coloniesresults in differentiation of cells, particularly those at theperimeter. To maintain pluripotentiality, ES cell colonies arebroken up into smaller colonies for passage and can becryopreserved by simple vitrification techniques for transport

and re-establishment (Reubinoff et al., 2001). Human ES cellswill spontaneously differentiate into a wide range of cell andtissue types in vitro and in vivo and can be directed intoparticular lineages, such as neural stem cell types (Reubinoff etal., 2000).

Bongso et al. (1994) cultured human blastocysts beyond thenormal 6–7 day preimplantation period and allowed the cells toattach and outgrow on plastic dishes. Further development ofisolated human ICM and their co-culture with a variety offeeder cells was carried out by Bongso and colleagues(Trounson and Pera, 2001). Thomson et al. (1998) andReubinoff et al. (2000) derived ES cell lines frommicrosurgically isolated human ICM cells and characterizedtheir surface antigenic properties using various markers. The

The fine structure of human embryonic stem cellsHenry Sathananthan has retired from teaching microanatomy at La Trobe University,Melbourne and is now involved in full-time research as Hon. Associate Professor at theMonash Institute of Reproduction & Development (MIRD), Monash University. His currentinterests are in centrosomal dynamics in development, evaluation of human blastocysts andembryonic stem cells. Henry has launched his own visual website:www.sathembryoart.com, which is also linked to MIRD, and his aim is to publicise hisimages of embryo microstructure on the web, with bimonthly updates.

Henry Sathananthan1, Martin Pera, Alan TrounsonMonash Institute of Reproduction and Development, Monash University, Melbourne, Australia1Correspondence: Monash Institute of Reproduction and Development, 27–31 Wright St, Clayton Victoria 3168,Australia; e-mail: [email protected]

Professor Henry Sathananthan

Keywords: culture, differentiation, embryonic stem cells, human, ultrastructure

RBMOnline - Vol 4. No 1. 56–61 Reproductive BioMedicine Online; www.rbmonline.com/Article/392 on web 26 November 2001

AbstractThe fine structure of human embryonic stem (ES) cell colonies was analysed by transmission electron microscopy (TEM)after 35 passages of in-vitro culture. Most cells formed compact, saucer-shaped colonies with epithelioid cells on theperiphery and polygonal cells within the colony. Three morphological types of cells were identified based on their finestructure: undifferentiated cells resembling inner cell mass (ICM) cells of blastocysts; protein-synthesizing cells at the onsetof cellular differentiation; and compact masses of secretory cells resembling unicellular goblet cells of the intestine. Thepredominant cell type was the undifferentiated ES cells resembling ICM cells of blastocysts. These cells had large nucleicontaining reticulated nucleoli, well-developed rough endoplasmic reticulum (RER), Golgi complexes, elongated tubularmitochondria, lysosomes and typical centrosomes with centrioles associated with microtubules and microfilaments,organizing the cytoskeleton. Some ES cells have very large nuclei and scanty cytoplasms with fewer organelles.The isolatedor attached protein-synthesizing cells at the onset of differentiation had extensive RER and large Golgi complexes. Themorphologically differentiated cells formed compact colonies and resembled goblet-like cells in microstructure. They hadRER and large Golgi complexes associated with secretory vesicles. The epithelioid cells at the periphery were columnar andlargely polarized by centrosomes associated with Golgi complexes. Epithelioid cells in all three categories had specializedcell junctions (desmosomes), anchored by tonofilaments, and surface blebs. Isolated cells were seen on the surface, towardsthe centre of the colony, and their free surfaces had microvilli and larger blebs. Approximately 3–5% of all cells were mitotic,with typical bipolar spindles organized by centrosomes, pivotally located at the poles, and appeared to resemble typicalsomatic cells.

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Articles - Fine structure of human embryonic stem cells - H Sathananthan et al.

techniques included light microscopy (LM) usinghistochemical, molecular and antigenic markers of cells grownin colonies, as well as karyotype analysis. The fine structure ofnon-human primate ES cells was reported by Thomson andMarshall (1998) and of mouse ES cells in embryoid bodies byDesbaillets et al. (2000). Both transmission electronmicroscopy and scanning electron microscopy (TEM andSEM) were used in the mouse study. However, neither themonkey nor the mouse studies showed details of fine structure(TEM). This report elucidates the ultrastructure of human EScells after approximately 150 population doublings in vitro andcompares them with the ICM cells from which they originate.The fine structure of ICM cells in human blastocysts has beenreported previously by Sathananthan et al. (1990, 1993,1999a,b).

Materials and methodsES cell cultureHuman ES cell line HES-2 was grown in monolayer culture ona feeder cell layer of mouse embryonic fibroblasts, as describedpreviously (Reubinoff et al., 2000). Growing colonies sevendays old were dissected using drawn-out glass capillaries andharvested using dispase in the 35th passage of culture. Thefragments and some colonies were rinsed in phosphate-buffered saline (PBS) and fixed for TEM as described below.Approximately 70% of cells in colonies at this stage of growthwere positive for the stem cell marker TRA-1-60, a surfacecarbohydrate epitope (Reubinoff et al., 2000).

The ES cells and colonies were routinely fixed inglutaraldehyde/osmium tetroxide, dehydrated and processedfor TEM, as described for human embryos (Sathananthan,1993). Survey sections (1 µm) and thin sections (70 nm) werestained with toluidine blue and uranyl acetate/Reynold’s leadcitrate, respectively, and examined by LM and TEM.

ResultsUndifferentiated human ES cells (group 1)The undifferentiated human ES cells grew in saucer-shapedcolonies, thickened at the rim and thinning out towards thecentre. Isolated ES cells could be found on their free surfacesor forming compact groups within the colony (Figure 1). Thecells at the periphery were usually epithelioid and polarized,developing specialized cell junctions consisting of desmosomesanchored by bundles of tonofilaments (Figures 2 and 3). Thepolarity of these cells appeared to be determined bycentrosomes with typical centrioles that organize the spatialarrangement of cellular organelles. The cells in groupsresembled ICM cells, were polygonal and had large nuclei(Figure 4) and sometimes indistinct cell membranes. Thenuclear:cytoplasmic ratio was high in these undifferentiatedcells. Desmosomes were less evident between cells, and gapjunctions were not found, in contrast to the ICM cells ofblastocysts. All cells had nuclei with one to three reticulatednucleoli, associated with centrosomes, each consisting of twocentrioles with pericentriolar material nucleating microtubulesand microfilaments, as seen in cells of blastocysts (typicalsomatic centrosomes). Some centrioles showed satellites ofpericentriolar material nucleating microtubules. The usualcellular organelles, such as elongated mitochondria, rough

Figure 2. Epithelioid human ES cells at the surface of thecolony (human embryonic stem (ES) cells cultured in vitro after35 passages). Transmission electron microscopy (TEM)original magnification ×3500; scale 2 µm = 7 mm.

Figure 1.Colony of undifferentiated human embryonic stem(ES) cells cultured in vitro after 35 passages. Light microscopy(LM) original magnification ×200.

Figure 3. Cell junction between two surface cells (humanembryonic stem (ES) cells cultured in vitro after 35 passages).TEM original magnification ×35 000; scale 200 nm = 7 mm.

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endoplasmic reticulum (RER), Golgi complexes, lysosomesand phagosomes were present, as reported in ICM cells (Mohrand Trounson, 1982; Sathananthan et al., 1990, 1993, 1999a,b).However, no lipid was found. These cellular organelles wereparticularly evident in isolated ES cells and in those at the onsetof cellular differentiation. Occasionally, cells with clear (lesselectron-dense) cytoplasm were found, similar to those withinthe ICM of blastocysts.

Cells at the onset of cellular differentiation(group 2)The cells at the onset of cellular differentiation displayed amicrostructure conforming to typical protein-synthesizing cells.Mitochondria were oval to tubular, with dense matrices anddistinct cristae. RER was often extensive and tubular and

studded with ribosomes (Figure 5). Free ribosomes andpolyribosomes were also evident. Golgi complexes wereextensive, consisting of circular juxta-nuclear profiles (Figure6) often associated with centrioles, and polarized in peripheral,epithelioid cells. Lysosomal activity was also evident, whilesome cells had autophagic vacuoles resembling primitiveendoderm cells of blastocysts (Figure 4).

Differentiated cells (group 3)The differentiated cells were clearly undergoing cellularspecialization in compact colonies (Figures 7 and 8). Theyresembled goblet cells of the intestinal epithelium in manyrespects—therefore, they were probably endodermal in origin.They presented features of secretory cells. In addition to theusual organelles, there were Golgi complexes associated withlarge secretory vesicles, which were more translucent than thesurrounding cytoplasm. The majority of cells showed thesesecretory characteristics (Figure 8). Those on the periphery,however, were distinctly epithelioid with specialized celljunctions—desmosomes anchored by bundles oftonofilaments forming a terminal web. The cells werepolarized by apical centrosomes associated with Golgicomplexes and microtubules with nuclei towards their bases,as in goblet cells.

Mitotic metaphases were rare (3–5% of cells) in all cells.These had typical bipolar spindles with polar, doublecentrioles (Figures 9 and 10), similar to those of interphasecells. Perhaps the cells undergo synchronized cycles ofinterphase and mitosis, a feature also common to ICM cells ofblastocysts.

Figure 4. Undifferentiated ES cells within the colony (humanembryonic stem (ES) cells cultured in vitro after 35 passages).TEM original magnification ×4375; scale 2 µm = 8 mm.

Figure 5. Isolated cell at the onset of differentiation – roughendoplasmic reticulum (RER) (human embryonic stem (ES)cells cultured in vitro after 35 passages). TEM originalmagnification ×7000; scale 2 µm = 13 mm.

Figure 6. Isolated cell at the onset of differentiation – Golgi(human embryonic stem (ES) cells cultured in vitro after 35passages). TEM original magnification ×8750; scale 1 µm = 8mm.

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Dense, inactivated mouse fibroblasts were seen both within andoutside the colonies, while the free surfaces of ES cells showedboth microvilli and larger blebs of cytoplasm. A few ES cellswere degenerating, with pyknotic nuclei and dense chromatinor wrinkled nuclear envelopes. These were probablyundergoing apoptosis (programmed cell death), also evident inblastocyst ICM.

DiscussionUndifferentiated ES cells very similar toICM cells of blastocystsThese cells have similar cell components to those of ICM cells– reticulated nucleoli in nuclei, RER, Golgi complexes,lysosomes, elongated mitochondria and typical centrosomes –but do not have lipid globules. The cells lining the periphery ofthe colony tend to be epithelioid, resembling trophoblast cellsof blastocysts. Those towards the centre of the colony are morepolygonal in shape, with close cell contacts, while isolated

rounded cells rarely have specialized cell junctions. These EScells show the lowest degree of cellular differentiation,compared to the other two groups of cells, conforming in manyrespects to the ICM cells of blastocysts (Mohr and Trounson,1982; Sathananthan et al., 1990, 1993, 1999a,b).

Establishment of ES cells in embryosCultures of ES cells at early stages of differentiation bear someresemblance to post-blastula stage embryos. A recent study ofan abnormal human embryo grown in culture for nine daysshowed a compact mass of undifferentiated cells that resembledboth ICM cells and ES cells (CY Fong, AH Sathananthan, ABongso, unpublished). This embryo had a solid core of cellsclosely resembling ICM cells and a surface epitheliumresembling trophoblast cells. In addition, there were superficialclumps of syncytiotrophoblast cells, and a primitive amnionwas developing on one side of the embryo, enclosing anamniotic cavity. As expected, this post-implantation embryoshowed abnormalities, because normal development would be

Figure 7. Differentiated colony with goblet-like cells (humanembryonic stem (ES) cells cultured in vitro after 35 passages).LM original magnification ×200.

Figure 8. Differentiated colony showing goblet-like cells(human embryonic stem (ES) cells cultured in vitro after 35passages). TEM original magnification ×3500; scale 2 µm = 7 mm.

Figure 10. Centriole in mitotic cell (Figure 9) (humanembryonic stem (ES) cells cultured in vitro after 35 passages).PCM = pericentriolar material. TEM original magnification×87 500; scale 100 nm = 8 mm.

Figure 9. Differentiated cell in mitosis (human embryonic stem(ES) cells cultured in vitro after 35 passages). TEM originalmagnification ×8750; scale 1 µm = 8 mm.

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ensured only after implantation in the endometrium. Evidently,there has been an extensive proliferation of ICM cells withinthe embryo. The inner mass of cells resembles undifferentiatedES cells (group 1) in many respects, and it is possible that EScells are established during this period of development in weektwo, before the three primary germ layers are established inweek three. A report of a day 9 human embryo in vitro waspublished by Edwards and Surani (1978), but no TEMexamination was done. Nikas (1999) reported the surfacestructure of a day 8 old blastocyst using SEM, which portraysonly surface images of trophoblast cells. Lindenberg et al.(1986) have documented the implantation of a humanblastocyst on endometrial cells in vitro.

Cells at the onset of cell differentiation(protein-synthesizing cells)These cells are isolated or attached and have reticulatednucleoli within nuclei, highly developed RER and extensiveGolgi complexes associated with small secretory vesicles,characteristic of protein synthesizing cells in somatic tissues(Fawcett, 1981; Sathananthan, 1996). These cells seem to be inan intermediate state of differentiation between group 1 andgroup 2 cells.

Differentiated cells resembling gobletcells These cells present features of secretory cells, resemblinggoblet cells of the intestinal epithelium. The majority of cellsshowed these secretory characteristics, whilst those towards thecentre of the colony resemble undifferentiated group 1 cells.Peripheral cells were distinctly epithelioid, with specialized celljunctions, and were polarized by apical centrosomes associatedwith Golgi complexes with nuclei towards their bases, as ingoblet cells. The centrosome is known to define polarity in cells(Edwards and Beard, 1997) and is defined as the cell centre(Boveri, 1901; Sathananthan et al., 1991, 1996); it is usuallylocated close to the nucleus in somatic cells and organizes thewhole cytoskeleton of the cell. Goblet-like cells were thecommonest specialization of ES cells seen in some of thecolonies after 35 passages.

Cell specializationOne of the problems associated with ES cell culture is cellspecialization. The general aim is to produce a culture ofundifferentiated, pluripotent cells for future directedspecialization to particular tissues of the human body.Following culture in the absence of mouse embryonicfibroblasts, different types of cells spontaneously appear(Thomson et al., 1998; Reubinoff et al., 2000). These includetrophoblast cells and derivatives of all three germ layers,including: gut epithelium (endoderm); cartilage, bone andmuscle (mesoderm); and neural and squamous epithelium(Thomson et al., 1998). In the present study, the predominantcell type appearing in the differentiating cultures was thegoblet-like gland cell, presumably endodermal in origin. Theroof of the yolk sac will later become the embryonic gut, whilstthe yolk sac per se will line the inside of the trophectoderm inthe hatching blastocyst (days 7–9). These glandular cells areunlike the squamous epithelial cells that line the primitive yolksac or Heuser’s membrane alongside the trophectoderm. The

latter, which originate from the hypoblast in a day 7 blastocystafter delamination of cells from the ICM, also havecharacteristic phagocytic vesicles (Sathananthan et al., 1990,1993, 1999a,b).

More systematic work needs to be done by TEM to identifyspecific cell types in ES cell cultures. This, in conjunction withother techniques already used – molecular, histochemical andantigenic probes – should be most valuable in isolating celltypes in ES cell cultures. TEM permits precise demonstrationof the fine structure of different cell types (Fawcett, 1981;Sathananthan, 1996) and should be an invaluable tool in ES cellresearch. It will be interesting to see the evolution of finestructure in ES cells, particularly in those that would eventuallydifferentiate into nerve tissue (neuroblasts) that have beenproduced in our laboratories (Reubinoff et al., 2000). This hasnot been possible in vivo after implantation. Combined withhistochemical or molecular techniques, this would be excitingat the level of the TEM. Programmed differentiation of cellsfrom primitive ES cells will eventually become possible, so thata population of nerve, muscle, fibroblast or even endodermalcells could be generated without contamination of other celltypes. The ES cells provide the ability to study these cellularprocesses in vitro for the first time in humans. It is likely thatmost of these cell types are differentiating in ES cell cultures,and it might be possible to identify different cell types ifspecializations have progressed considerably. Such cells wouldserve as excellent models to unravel early embryonic celldifferentiation in the laboratory.

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The fine structure of human embryonic stem cells