165 neural stem cells in neurospheres, embryoid bodies and the cns of advanced human embryos, in...

Neural Stem Cells in Neurospheres, Embryoid Bodies,and Central Nervous System of Human Embryos

A. Henry Sathananthan

Monash Immunology and Stem Cell Laboratories, Monash, Medical, Nursing and Health Sciences, Clayton, Vic. 3800, Australia

Abstract: The process of neurogenesis and formation of neural stem cells is reported in human neurospheres~NS! and embryoid bodies ~EB! derived from human embryonic stem cells, in vitro, and compared with neuraltissue formed in human ectopic embryos in week 4 ~stage 9!, developed in vivo. This morphological study wasdone using digital imaging by light microscopy and routine transmission electron microscopy. Both NS and EBform neural rosettes from the surface epithelium much like the process of neural tube formation from ectodermin the embryo. The rosette is the developmental signature of neuroprogenitors in cultures of differentiatingembryonic stem cells and is a radial arrangement of columnar cells that express many of the proteins expressedin neuroepithelial cells in the neural tube. The NS produce all of the major classes of progeny of the neural tube,some of which have been documented here. Specific neural markers expressed in the NS and the clinicalimplications of this study in cell therapy are also discussed.

Key words: neural stem cells, rosettes, neurospheres, embryoid bodies, embryos, TEM

INTRODUCTION

Embryonic stem cells ~ESC! are pluripotent and capable ofindefinite self-renewal in vitro ~Trounson, 2006; Sathanan-than, 2007a, 2007b!. They are very useful for deriving anycell type of the human body, including those of the nervoussystem, on prolonged culture. They can be induced sponta-neously or by directed differentiation to form neural ro-settes, embryoid bodies ~EB!, neurospheres ~NS! that arecomposed of neural stem cells ~NSC! and their progenitors,among other cell types ~Conley et al., 2004; Sathananthan &Trounson, 2005, 2007; Peh, 2007!. ESC usually form EBafter growth factor treatment and then develop into NS,when grown on a stromal cell monolayer ~Pankratz &Zhang, 2006; Schwartz et al., 2008; Koch et al., 2009!.Theneural rosette is the developmental signature of neuropro-genitors in cultures of differentiating ESC. Rosettes areradial arrangements of columnar cells that express many ofthe proteins expressed in neuroepithelial cells in the neuraltube. In addition to their similar morphology, neuroprogeni-tors within neural rosettes differentiate into the main classesof progeny of neuroepithelial cells as in vivo, namely neu-rons and their processes, astrocytes and oligodendrocytes~Wilson & Stice, 2006; Koch et al., 2009!.

The ectopic human embryo ~EE! is a useful model tostudy early development in vivo and will help us understandearly cell differentiation in ESC colonies, EB and NS. One ofthe first tissues to form in the embryo soon after theestablishment of the three germ layers ~ectoderm, endo-derm, and mesoderm in week 3! is neural progenitor tissuein the dorsal ectoderm, anterior to the primitive streak~Larsen, 1993!. This is believed to be induced by underlyingnotochordal tissue, an established concept in chordate em-

bryology. Neural development continues through weeks 4to 8 establishing the central nervous system ~CNS! and theperipheral nervous system of the embryo.

The aim of this article is to characterize the fine struc-ture of NSC in rosettes that differentiate spontaneouslywithin EB and NS and compare with the events that occurin vivo during early neurogenesis in EE. This study will alsohelp us recognize the microanatomy of cells studied byimmuno-labeling of NSC by fluorescent microscopy ~FM!in reports in the literature and also identify specific neuronsfor cell therapy to cure neural diseases.

MATERIALS AND METHODS

Seven NS, eight EB, and six EE ~weeks 3–4! were examinedby routine transmission electron microscopy ~TEM!. TheNS were derived spontaneously from three ESC lines ~hes 2,3, and 4! by mechanical isolation of rosettes and propaga-tion in a suspension culture ~Peh et al., 2003!. The rosette isan exquisite radial formation of cells and is easily identifiedby phase contrast in cell colonies. The EB were culturedfrom one cell line. The hes-2 was propagated in Dulbecco’smodification of Eagle’s medium ~DMEM! in the presence of20% fetal calf serum ~FCS! on a mouse embryonic fibro-blast feeder layer. Clumps of approximately 50–100 cellswere transferred to fresh DMEM and FCS and grown as EBin suspension culture ~Conley et al., 2004!. NS and EB werethen subjected to immunohistochemistry using specificmarkers and immunoreactivity analyzed by confocal micros-copy and on cryosections by these authors.

Ectopic gestational sacs were recovered intact by lapa-roscopic salpingectomy, fixed in glutaraldehyde, trans-ported, and dissected to recover the embryos for TEM.Though somewhat retarded in development, the embryospresent a normal structure ~Sathananthan & Selvaraj, 2007!.

Received March 24, 2010; accepted April 14, 2011*Corresponding author. E-mail: [email protected]

Microsc. Microanal. 17, 1–8, 2011doi:10.1017/S1431927611000584 MicroscopyAND

Microanalysis© MICROSCOPY SOCIETY OF AMERICA 2011

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The embryo sacs ~38–40 days old! were dissected, recover-ing embryos corresponding to stages 7–13 of the Carnegiecollection, post-fixed in osmium tetroxide, embedded inAraldite, and sectioned with diamond knives using anultramicrotome. Thick survey sections ~1 mm! and thinsections ~;70 nm! were used for LM and TEM. The thickswere stained with Toluidine blue and the thins with uranylacetate and lead citrate. Images were obtained by digitalmicroscopy using Leitz digital microscopes, JEOL and Phil-ips electron microscopes, and edited with Adobe PhotoshopCS2 and Paint Shop Pro 6. These procedures are describedin detail by Sathananthan and Nottola ~2007! and Sathanan-than and Trounson ~2007!. Thick sections ~LM!, in partic-ular, are very useful for comparison with frozen or paraffinsections, while TEM will help verify the identity of cellsultrastructurally.

RESULTS

Both NS and EB form spheroidal structures when fullydeveloped. They vary in their external form and internalorganization showing various levels of neural differentia-tion, among other cell types ~Sathananthan & Trounson,2007!. The mature NS form a compact mass of predomi-nantly neural progenitor stem cells associated with ESCencapsulated in a surface epithelium ~Figs. 1, 2!, equivalentto a week 3 to 4 human embryo and shows stages of earlyneural differentiation, mimicking differentiation in vivo~Larsen, 1993!. All stages of neurogenesis are evident exceptfor myelination of axons and formation of synaptic junc-tions within this time frame. The process seems to beaccelerated in NS, when compared to embryos in vivo.

The neural rosette is an exquisite aggregation of NSC,easily identifiable in sections ~Figs. 3–5!. Each rosette has acentral lumen with radiating cells. The lumen shows undif-ferentiated cells originating from the stroma, particularly inEB ~Figs. 1, 4!, resembling ESC documented previously~Sathananthan et al., 2002!. The rosette evidently arises byinvagination of the surface stratified epithelium ~Figs. 1, 2!,which resembles neuroectoderm of embryos ~Figs. 6, 7!.The epithelium has characteristic cell junctions like desmo-somes, apical centrioles, and mitotic cells ~Figs. 4, 5!. Thelumen is lined by a similar epithelium confirming its originsfrom the surface in both NS and EB. A neural tube-likeformation was occasionally seen associated with rosettes~Fig. 3!. Each epithelial cell is columnar with a nucleuscontaining a reticulated nucleolus, resembling NSC of em-bryos ~Fig. 7!. The cells radiating from rosettes also have asimilar configuration ~Fig. 4!. A few cells were degenerating.Progressive changes occur as the NSC radiate from therosette toward the periphery in mature NS ~Figs. 8, 9!. Thecells become more stellate developing branching processes,presumably dendrites and a few axons that make contactswith adjacent cells, best visualized by TEM. As the NSCapproach the periphery and matrix, there appear bundles ofelongated processes that give some NS a furry appearance.Apart from NSC progenitors, NS have undifferentiated ESC

showing the typical structure with large nuclei, scanty cyto-plasm, and ill-defined outlines ~Fig. 10!. The nonneuralcells detected in NS are probably astrocytes, which wereclosely associated with NSC and their processes ~Fig. 11!.Astrocytes are large cells and present prominent nuclei,small mitochondria, and lysosomes. Their structure con-forms with published images of astrocytes in atlases. Therewere no oligodendrocytes, Schwann cells, myelinated axons,and synaptic terminals. These will probably appear later onin the time scale of development.

The week 4 embryos ~24 h—stage 9! showed an openneural tube and three to four somites on either side of theCNS. The tube is lined by neuroectoderm showing a typicalstratified epithelium of NSC ~Figs. 6, 7!. The cells resembleNSC of neural rosettes. Underlying the neural groove is thenotochordal plate continuous with the endoderm on eachside. The notochordal cells represent the primitive embry-onic skeleton and has vacuolated cells, which are in closecontact with the overlying NSC ~induction!. Beneath neuro-ectoderm are the mesenchymal stem cells ~MSC! represent-ing mesoderm and the dorsal aorta containing haemopoieticstem cells ~HSC!. This stage is ideal to study all types ofstem cells in the embryo at the next level of cell differentia-tion after germ layer formation.

DISCUSSION

Neural RosetteThe NS serves as a model for neurogenesis in vivo in severalrespects ~Wilson & Stice, 2006!. Evidently multipotentialneuroprogenitors in NS express many of the same genes asneuroepithelial cells in the neural tube. The rosette arrange-ment itself is noteworthy, and the molecular mechanism ofits formation could reveal if and how formation of NSreflects specification of neuro-ectoderm in vivo. The originsof the rosette have been clearly traced to a neuro-epithelium-like organization in both NS and EB, and there seems littledoubt that their homologies are similar, if not identical.Formation of NS is also sensitive to bone morphogenicprotein ~BMP! signaling, as is formation of the neural tube.Significantly, NS produce all of the major classes of progenyof the neural tube, including brain and motor neurons.

NeurospheresTEM reveals that NS present an orderly spectrum of neuraldifferentiation mimicking that seen in the human embryoduring weeks 3 to 4 of neurogenesis. In NS, we have acompact, homogenous, self-renewing mass of NSC encapsu-lated in an epithelium, which resembles neuroectoderm ofembryos, differentiating spontaneously in vitro from ESC. Asignificant difference is that NSC differentiation occurs in acompact structure in space, and the events of neurogenesisseem to be accelerated in time. Thus it is an excellent modelto unravel the process of neural differentiation, includingdendrites, axons, and nonneural glial cells such as astro-cytes. We have, however, not seen myelination of axons northe formation of synaptic junctions, perhaps due to the

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early stages of differentiation recapitulated in NS. Myelina-tion begins in the fourth month of development ~Larsen,1993!. Hopefully, this could be induced by directed neuraldifferentiation using specific growth factors. Dottori andPera ~2008! have established an in vitro model of human

neural differentiation derived from ESCs with noggin, aBMP antagonist. Treatment with noggin for 14 days resultsin the efficient induction of NSC.

The NS assay ~NSA! has proved to be a powerful toolfor the detection and expansion of neural stem and progen-

Figure 1. Origin of rosettes from the neurosphere surface epithelium: the stratified epihelia ~E! resemble neuroecto-derm in embryos. The epithelium invaginates, breaks away from the surface, and forms rosettes that resemble neuraltube-like formations ~NT!. Note undifferentiated cells ~C! in lumen. Magnifications: �200 and �1,000.

Figure 2. Embryoid body ~left! showing origins of rosettes: the epithelial invagination ~Inv! is possibly the origins of arosette simulating a neural groove. MSC � mesenchymal stem cells. Note other tissues. Magnifications: �40 and�1,000.


Neurogenesis in Neurospheres and Embryoid Bodies 3

itor cells from the developing and adult CNS. The NSA candemonstrate the presence of NSC that can differentiate intothe three main phenotypes found in the CNS: neurons,astrocytes, and oligodendrocytes, if the NSA cells are main-tained in an undifferentiated proliferative state by culturingthem in a serum-free medium supplemented with a mito-

gen such as epidermal growth factor ~Wilson & Stice, 2006;Peh, 2007; Koch et al., 2009!. NSC begin to proliferate afterabout 24 h in culture and form small clusters of cells~rosettes! by 2–3 days. By day 5–7 the clusters group intoNS, which measure 100–200 mm in diameter, composed ofapproximately 10,000 cells. The NS can then be dissociated,

Figure 3. Neural rosette and tube-like formation in embryoid body: classical image of a neural rosette ~R! showingradiating neural cells. Magnification �1,000. Right: Neural tube-like formation associated with ESC. Note stratifiedepithelium. Magnification �400.

Figure 4. Neural rosette with radiating neurons: note stratified epithelium with dividing cell ~M! and surface special-izations. Undifferentiated cells ~C! are found in the lumen above. Right: Elongated neurons radiating below this rosette.Note nuclear structure and degenerated cell ~D!. Magnification �3,500.

Figure 5. Surface epithelia showing centrioles, desmosomes of neurospheres, and a rosette tip. Surface epithelia withdesmosomes ~D! and centrioles ~C!. Rosette tip epithelium ~right!. Note cilium arising from centriole ~star!. Magnifica-tions: �5,250, �35,000, and �13,125.



plated, and differentiated into nerve cells in 1 to 2 weeks ofculture.

Embryoid BodiesAlthough EB show neural rosettes and their surface origins,they present almost every other type of tissue progenitorsdifferentiating from ESC, documented by TEM ~Sathanan-than & Trounson, 2005, 2007!. Hence the isolation of pureNSC from other cell types present will be a problem. Thereare neural progenitors in ESC colonies, as well, demon-strated by fluorescent markers, after 7 days in culture ~Peh,2007!. Neural rosette clusters will be easier to isolate incolonies after 16 days in culture ~Pankratz & Zhang, 2006!.Hence NSC are differentiated in early passages of ESC,

mimicking a week 3 human embryo, when neural differen-tiation begins during embryogenesis.

Neural MarkersIdeally, these morphological studies should have gone hand-in-hand with FM studies using specific markers. The NSCin rosettes and NS are reactive to neuronal markers such asNestin, Sox2, Sox1, Musashi-1, Pax-6, Ssea1, NeuN, beta IIItubulin, and the stem cell marker Oct-4 ~Peh, 2007; Kochet al., 2009!. Beta III tubulin stains the neural processes,which contain microtubules, while Oct-4 labels the ESC, asexpected. Prolonged differentiation over 2 weeks show astro-cytes, reactive to GFAP, also demonstrable by TEM. Otherneural markers specific to various types of neurons stain

Figure 6. Neural groove epithelium of a day 24 ectopic human embryo: the neural ectoderm is composed of stratifiedcolumnar cells. Note neural induction by underlying notochordal cells. DA � dorsal aorta with HSC. MSC �mesenchymal stem cells. Magnifications: �200 and �1,000.

Figure 7. Neural epithelium of 24 D human embryo. Note stratified epithelium with surface microvilli ~MV! and amitotic cell ~M!. Primordial NSCs have nuclei with reticulated nucleoli. Magnifications: �1,000 and �3,300.



later, on prolonged culture and passaging of NSC ~Kochet al., 2009!. Thus we have confirmation that NSC derivedfrom ESC react positively to various neural markers ~Peh,2007!. The ultimate confirmation, of course, will be to

combine immunofluorescence with TEM by labeling anti-bodies with gold or heavy metals that can be detected in thecells and processes and their organelles. Unlike FM, LM andTEM of thin sections can show specific localization of

Figure 8. Neurons and processes radiating from rosette to periphery LM and TEM: neurons are nucleated, somevacuolated ~above!. The peripheral processes arise from neurons ~below!. Magnifications: �1,000 and �8,750.

Figure 9. Neurons and processes—central region to periphery of NS: neurons form processes ~above! and elongatedprocesses ~below! toward periphery. Magnifications: �3,500, �7,000, and �26,250.



antibodies within each cell type. This is the gold standardwe need to achieve, combining FM with TEM ~Sathanan-than & Trounson, 2007!.

Clinical ApplicationsThere are obvious applications in cell therapy, particularlyusing NSC in the treatment of neurodegenerative diseases,such as Parkinsons ~Pruzak & Isacson, 2007; Sathananthan,2007b!. This work is directed toward generating dopaminer-gic ~DA! neurons. The objective is to produce a homog-enous population of neurons in humans. Here too rosettesare formed in 14–21 days, which can be mechanically iso-lated and propagated into DA neurons. This approach hasbeen used to treat Parkinsonian rats with some success~Ben-Hur et al., 2004!. The directed differentiation of ESCinto spinal motor neurons is another important therapeuticmilestone ~Hu & Zhang, 2007!. This protocol again usesneural tube-like rosettes in EB, which form in about 10–14 days. Fortunately, neural rosettes in culture are easy toidentify by phase contrast due to their exquisite configura-tion, and the TEM has clearly demonstrated they are com-posed of cells, closely akin to the neuro-epithelium of

human embryos. Unlike ESC colonies that grow on plates,NS and EB are more compact aggregates of cells and needto be cryosectioned for immunofluorescence ~FM! or thinsectioned for TEM. Niclis et al. ~2009! have recently devel-oped an ESC model of Huntington disease ~HD!, anotherdegenerative disorder in the brain. They also generated NSfrom HD-ESC that differentiated into neurons and astro-cytes. So there is plenty of scope and potential for researchin the treatment of neurodegenerative conditions.

ACKNOWLEDGMENTS

This project was approved by the Hospital InstitutionalHuman Ethics Committee, Monash Medical Centre, Mo-nash University, Melbourne, Australia. Figures 1–11 arereproduced from the Sathananthan ~2007a! DVD.

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Figure 10. Undifferentiated ESC ~potential NSC! in neurospheres: the ESC have large nuclei with scanty cytoplasm andill-defined outlines. �1,000 and �3,500.

Figure 11. Nonneural cells—astrocytes: astrocytes have large nu-clei, small mitochondria and lysosomes. Note process between thecells ~P! and a centriole ~C!. Magnifications: �4,375 and �13,125.



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165 neural stem cells in neurospheres, embryoid bodies and the cns of advanced human embryos, in...

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