The Brainy Side of Human Embryonic Stem Cells

Transcription

1 Journal of Neuroscience Research 76: (2004) The Brainy Side of Human Embryonic Stem Cells Eran Hornstein and Nissim Benvenisty * Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel The recent isolation of human embryonic stem (ES) cells is evoking great hopes for their future utilization in cellreplacement therapies and human development research. The hallmarks of ES cells, pluripotency and selfrenewal capacity, suggest an infinite source for tissues of virtually all desired types. Specifically, human ES cells may potentially be the basis for effective treatments of a wide range of human neurodegenerative disorders. To enable the translation of this novel biomedical field into the clinic, mechanisms that control the differentiation of human embryonic stem cells into fully functional neuronal cells should be analyzed and controlled Wiley-Liss, Inc. Key words: human embryonic stem cells; neuron; differentiation; central nervous system Human embryonic stem cells (HESCs) are pluripotent cell lines derived from the inner cell mass of blastocyst stage embryos (Thomson et al., 1998; Reubinoff et al., 2000). HESCs keep the pluripotent characteristics of the inner cell mass and produce cells of various lineages. Under certain conditions, HESCs will spontaneously aggregate to form a spherical three-dimensional structure that is referred to commonly as an embryoid body (EB) (Itskovitz-Eldor et al., 2000). As its name suggests, the EB undergoes various processes that occur in early embryogenesis, generating tissues of the three embryonic germ layers. When transplanted into an immunodeficient host, HESCs differentiate to form teratomas. The pluripotency of HESCs is also exemplified in these teratomas, which are formed as a result of uncontrolled differentiation and are composed of multiple tissues (for recent reviews of HESCs biology refer to Pera et al., 2000; Eiges and Benvenisty, 2002). SPONTANEOUS DIFFERENTIATION OF HESCS INTO NEURONAL DERIVATIVES During normal development, neurulation is the first step in organogenesis. HESCs are therefore expected to differentiate spontaneously and directly into neural cells. Indeed, in vitro expression of neuronal markers can be detected readily during the differentiation process into EBs (Itskovitz-Eldor et al., 2000; Table I). The potential of HESCs to differentiate into neuronal tissue was attested further by use of markers such as neural adhesion molecules, neurotransmitters and neuroreceptors (Reubinoff et al., 2000; Table I). The neuronal cells, however, were part of a random mixture of many cell types, which is far from optimal in cases where isolation of neurons is necessary for applied purposes. Although manual selection of neurons by their morphology was described (Reubinoff et al., 2001), fluorescence activated cell sorting (FACS) is a more efficient strategy for isolating specific cell types. Human neuronal progenitors, derived from HESCs, were isolated using semiautomated sorting by antibodies for the PS- NCAM and A2B5 antigens, that are characteristic of neuronal progenitors (Carpenter et al., 2001). Even when sorting technology is applied, neuronal enrichment of cultures is essential for obtaining a sufficient amount of cells to establish novel therapeutic tools. Culture enrichment is needed especially for mass production of rare neuronal subtypes. The methodologies described below, for neuronal differentiation and enrichment, are also described schematically in Figure 1. INDUCED NEURONAL DIFFERENTIATION OF HESCS An attempt to restrict the phenotypic variability of spontaneously differentiating HESCs by the use of growth factors came on the heels of HESCs discovery (Schuldiner et al., 2000). It was shown that soluble ligands could control, to some extent, the fate of HESCs progeny. This was exemplified in BMP4, a known inhibitor of mouse neuronal tissue, which downregulated expression of neuronal markers. In contrast, nerve growth factor (NGF) and retinoic acid (RA) promoted neural cell fate (Schuldiner et al., 2000). Neural enrichment of up to 60% of cells in culture by NGF or RA was demonstrated by in situ hybridization with neurofilament light chain mrna (Schuldiner et al., 2001) and by immunostaining with an antibody against neurofilament heavy chain (Schuldiner et al., 2001; Fig. 2). Furthermore, RA induced the expression of neuron-specific genes such as dopamine (DA) receptor D1, serotonin receptor 2A and 5A, *Correspondence to: Dr. Nissim Benvenisty, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel. nissimb@mail.ls.huji.ac.il Received 8 July 2003; Revised 30 September 2003; Accepted 5 November 2003 Published online 8 March 2004 in Wiley InterScience (www. interscience.wiley.com). DOI: /jnr Wiley-Liss, Inc.

2 170 Hornstein and Benvenisty TABLE I. Properties of Neuronal Derivatives From Human Embryonic Stem Cells Methodology Cell type Marker RT-PCR Neurons Nestin a,b Pax-6 a,b NF-H (neurofilament heavy chain) c Dopamine hydroxylase c NSE (neuron specific enolase) b NF-M b Glutamate decarboxylase a Dopamine receptor D1 (DRD1) d Serotonin receptor 2A (5HT2A) d Serotonin receptor 5A (5HT5A) d Dopa decarboxylase (DDC) d Astrocytes/oligodendrocytes GFAP (glial fibrillary acidic protein) b Phosphoprotein enriched in astrocytes (PEA-15) c GABA A 2 receptor a Myelin basic protein (MBP) b Plp, a myelin proteolipid protein b Immunostaining Neurons A2B5 b,e,f Astrocytes/oligodendrocytes N-CAM (neural cell adhesion molecule) a,b,e g Musashi-1 g 70 kda neurofilament protein a,b,g III tubulin a,b,e,g NSE b Nestin (intermediate filament protein) a,b,e,g MAP 2 (microtubule-associated protein 2) a,b,e,g Synaptophysin a,b,e Tyrosine hydroxylase b,e,g Vimentin a,b 160 kda neurofilament protein a,b 200 kda neurofilament protein a,d,g Glycine e Glutamate a,b,e,g Serotonin b GABA b,e,g Glutamate decarboxylase (GABA synthesis) a,b GD3 and GD2 gangliosides f GFAP (Glial fibrillary acidic protein) b,e g O4 oligodendrocyte-specific glycolipids b,g Hyaluronate receptor (astrocytes) e NG-2 (oligodendrocyte progenitors) b 2,3 -cyclic nucleotide 3 -phosphodiesterase (oligodendrocytes) b RNA in situ hybridization Neurons NF-L (neurofilament light chain) d,h Fura-2 imaging Neurons Voltage-gated ionic currents e Whole-cell patch clamp a Reubinoff et al., b Reubinoff et al., c Schuldiner et al., d Schuldiner et al., e Carpenter et al., f Draper et al., g Zhang et al., h Itskovitz-Eldor et al., Neurons Response to neurotransmitters: GABA, glutamate, glycine, dopamine, acetylcholine, ATP e and of dopa decarboxylase, a key enzyme in the synthesis of both serotonin and dopamine (Schuldiner et al., 2001; Table I). In a separate study, induced differentiation of human ES cells into neuroectodermal tissue was manifested in the expression of several ganglioside markers (Draper et al., 2002; Table I). The functional activity of human neurons generated in a similar manner, was assessed in vitro by Carpenter et al. (2001), who revealed that neurons can synthesize and respond to neurotransmitters and are electrically active. An empirical approach to researching neuronal enrichment could prove limited when applied to achieve specific neuronal cell types. Great efforts are being taken to

3 Neuronal Derivatives From Human ES Cells 171 Fig. 1. Scheme for neural differentiation and enrichment of human embryonic stem cells. Fig. 2. Immunostaining for the neurofilament heavy chain (NF-H) of a 20-day-old EB. Neuronal differentiation of HESCs is marked by morphology of axon-like processes. Figure can be viewed in color online via replace current protocols with methods that will accurately drive cells into a precise subtype fate. In this context, insights into normal pathways of neurogenesis can be applied in a rational manner to direct mouse ES cells into specific CNS neuronal subtypes at high efficiency. For example, Lee et al. (2000) used understanding of embryonic differentiation mechanisms (Ye et al., 1998) to generate midbrain dopaminergic neurons from mouse ES cells in vitro. Similarly, utilizing knowledge gained from embryonic development, Wichterle et al. (2002) manufactured spinal motor neurons in culture system. The recapitulation of embryonic neurogenesis in vitro, as exemplified in the mouse, is an attractive option. This is especially true for HESCs, because this method may test knowledge gathered from neurodevelopment in rodents that will prove useful in the study of human neurogenesis. GENETIC MANIPULATION OF HESCS The obvious difficulty of producing homogenous populations of neuronal cells may be overcome by genetically manipulating cells for lineage selection. This can be carried out using ES cell lines expressing fluorescent marker genes (Eiges et al., 2001) or dominant selectionencoding gene (Zwaka and Thomson, 2003), driven by a cell-specific promoter. Although some concerns exist about the introduction of transgenes into the human genome, this technology can be accessed efficiently and used to produce pure cell populations. Ying et al. (2003) recently reported mouse ES cell lines in which a green fluorescent protein (GFP) coding sequence was knocked-in to replace the open reading frame of Sox1, the earliest marker of neuroectoderm, or of Tau, a marker of mature neurons. Indeed, these precise lineage tracers facilitated the detection of precursors and mature neurons (Ying et al., 2003). In HESCs, progenitors for neurons can be identified similarly by transfecting nestin-gfp expressing construct (Drukker and Benvenisty, 2003). Alternatively, ES cells may be manipulated genetically to express a transcription factor that forces commitment to a specific lineage. For instance, exogenous expression of Nurr1, a pivotal transcription factor in specification of dopaminergic neurons, facilitated differentiation of mouse ES cells into the midbrain dopaminergic neuronal lineage in vitro (Chung et al., 2002; Kim et al., 2002). Efficient generation of midbrain precursors and dopamine neurons was assayed by selective upregulation of midbrain DA-specific markers (e.g., tyrosine hydroxylase, homeobox protein Ptx3) (Chung et al., 2002; Kim et al., 2002), and was followed by functional analysis that demonstrated their survival and function after grafting into lesioned rodent striatum (Kim et al., 2002). IS THERE AN OPTIMAL PATH FOR THE GENERATION OF DESIRED NEURON SUBTYPES? Combination of Shh and FGF8 promote ventral midbrain fate in neural plate explants (Ye et al., 1998) and efficiently promote dopaminergic neuronal fate of mouse ES cells (Lee et al., 2000). A similar neuronal population was obtained by the use of a PA6 coculture system (Kawasaki et al., 2000), which directs cells into neuronal fate by upregulation of Oct3/4 (Shimozaki et al., 2003). Although it may be possible to generate a specific cellular subtype by following the embryonic cues (Wichterle et al., 2002), there could be multiple, independent ways of generating ES-derived neurons in vitro that do not occur in vivo. One area of difficulty is the classification of neuron subtypes, as this is based on expression of markers and can be difficult to interpret. Cells derived through different protocols can express similar markers without necessarily

4 172 Hornstein and Benvenisty demonstrating equal functionality. For instance, undifferentiated HESCs showed positive immunoreactivity for the neural marker -III tubulin, and a subpopulation of -aminobutyric acid (GABA)-positive cells did not coexpress any other neuronal markers (Carpenter et al., 2001). In one more example, the motor neurons of the HB9 / mouse are indistinguishable from normal counterparts in vitro; however, in the context of the central nervous system (CNS), defects are revealed in cell body migration and axon pathfinding (Thaler et al., 1999). Phenotypic markers could therefore prove unreliable. It is plausible that only a few differentiation protocols will produce neurons that are functional in vivo, whereas other methods of obtaining neurons from HESCs could lead to an artifactual expression of markers. Only a definitive in vivo assay that explores neuronal phenotype and function after implantation into the CNS of laboratory animals can provide satisfactory quality control for neurons generated in vitro. IN VIVO DIFFERENTIATION OF HESCS In vitro-generated mouse neural precursors, which were transplanted into rodent CNS, responded to host environmental signals. Local cues, rather than neuronautonomous mechanisms, guided both differentiation into neuronal subtypes, as well as their migration in defined CNS streams (Brustle et al., 1997). Information within the CNS instructs implanted cells to acquire proper phenotypes (Brustle et al., 1997, 1998; Gage, 2000). In this regard, it has been shown recently that undifferentiated HESCs, which were implanted in direct contact with the axial structures of chick embryos, acquired a neuronal fate, probably by confined in vivo cues (Goldstein et al., 2002). Taken together, the role of the local environment in controlling the fate of undifferentiated cells might be more significant than we expect. It is thus possible that less-thanperfect in vitro control of phenotypes may be sufficient for practical therapeutic purposes. Two reports describe the implantation of HESCderived neurons into mouse CNS. Both groups demonstrated incorporation of neuronal derivatives to the host tissue after transplantation into the ventricles of newborn mice. In addition, migration of the human progenitors followed established brain streams and resulted in a widespread distribution within the host brain. These studies also demonstrated differentiation in vivo into neural lineages by immunochemistry (Reubinoff et al., 2001; Zhang et al., 2001; Table I). In the past, when human neuronal stem cells were implanted into the CNS of rodents, histological analysis showed that they expressed phenotypic markers relevant to their anatomic location (Brustle et al., 1998; Fricker et al., 1999; Wu et al., 2002). Such experiments have not yet been carried out on HESCs. In addition, long-term survival and correction of pathologic phenotypes should be investigated in the future. Similarly, the fact that teratomas or teratocarcinomas were not detected cannot be taken as an unequivocal demonstration of cell safety, because the follow-up period was relatively short and the number of animals experimented upon was limited. In summary, HESCs can be induced to differentiate into neural derivatives that express lineage-specific markers under appropriate differentiation conditions. In-depth biological understanding and improved biotechnological protocols will eventually allow us to use HESCs to bring long-awaited healing tools to patients with neurodegenerative diseases and enable studies of human neurogenesis in vitro. REFERENCES Brustle O, Choudhary K, Karram K, Huttner A, Murray K, Dubois-Dalcq M, McKay RD Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat Biotechnol 16: Brustle O, Spiro AC, Karram K, Choudhary K, Okabe S, McKay RD In vitro-generated neural precursors participate in mammalian brain development. Proc Natl Acad Sci USA 94: Carpenter MK, Inokuma MS, Denham J, Mujtaba T, Chiu CP, Rao MS Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol 172: Chung S, Sonntag KC, Andersson T, Bjorklund LM, Park JJ, Kim DW, Kang UJ, Isacson O, Kim KS Genetic engineering of mouse embryonic stem cells by nurr1 enhances differentiation and maturation into dopaminergic neurons. Eur J Neurosci 16: Draper JS, Pigott C, Thomson JA, Andrews PW Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat 200: Drukker M, Benvenisty N Genetic manipulation of human embryonic stem cells. In: Chiu A, Rao MS, editors. Human embryonic stem cells. Totowa: Humana Press. p Eiges R, Benvenisty N A molecular view on pluripotent stem cells. FEBS Lett 529: Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty N Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol 11: Fricker RA, Carpenter MK, Winkler C, Greco C, Gates MA, Bjorklund A Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain. J Neurosci 19: Gage FH Mammalian neural stem cells. Science 287: Goldstein RS, Drukker M, Reubinoff BE, Benvenisty N Integration and differentiation of human embryonic stem cells transplanted to the chick embryo. Dev Dyn 225: Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 6: Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa SI, Sasai Y Induction of midbrain dopaminergic neurons from es cells by stromal cell-derived inducing activity. Neuron 28: Kim JH, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, Lee SH, Nguyen J, Sanchez-Pernaute R, Bankiewicz K, McKay R Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson s disease. Nature 418: Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 18: Pera MF, Reubinoff B, Trounson A Human embryonic stem cells. J Cell Sci 113:5 10.

5 Neuronal Derivatives From Human ES Cells 173 Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T Neural progenitors from human embryonic stem cells. Nat Biotechnol 19: Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nat Biotechnol 18: Schuldiner M, Eiges R, Eden A, Yanuka O, Itskovitz-Eldor J, Goldstein RS, Benvenisty N Induced neuronal differentiation of human embryonic stem cells. Brain Res 913: Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 97: Shimozaki K, Nakashima K, Niwa H, Taga T Involvement of oct3/4 in the enhancement of neuronal differentiation of es cells in neurogenesis-inducing cultures. Development 130: Thaler J, Harrison K, Sharma K, Lettieri K, Kehrl J, Pfaff SL Active suppression of interneuron programs within developing motor neurons revealed by analysis of homeodomain factor hb9. Neuron 23: Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM Embryonic stem cell lines derived from human blastocysts. Science 282: Wichterle H, Lieberam I, Porter JA, Jessell TM Directed differentiation of embryonic stem cells into motor neurons. Cell 110: Wu P, Tarasenko YI, Gu Y, Huang LY, Coggeshall RE, Yu Y Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat. Nat Neurosci 5: Ye W, Shimamura K, Rubenstein JL, Hynes MA, Rosenthal A FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 93: Ying QL, Stavridis M, Griffiths D, Li M, Smith A Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 21: Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19: Zwaka TP, Thomson JA Homologous recombination in human embryonic stem cells. Nat Biotechnol 21: