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作者：Alexis J. Joannidesa, Christelle Fiore-Hricha, Alysia A. Battersbyb, Pandula Athauda-Arachchia, Isabelle A. Bouhona, Lydia Williamsb, Kristine Westmorea, Paul J. Kempb, Alastair Compstona, Nicholas D. Allenb, Siddharthan Chandrana作者单位：aCambridge Centre for Brain Repair, Department of Clinical Neurosciences, Cambridge, United Kingdom;bSchool of Biosciences, Cardiff University, Cardiff, United Kingdom 0 e$ Z/ V, o% K5 N% H9 {. ~
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7 n* o7 o; H( r! M" }9 B; B 【摘要】
. e+ j! P8 F: p3 V0 M9 O% y* g, Z The ability to differentiate human ESCs (hESCs) to defined lineages in a totally controlled manner is fundamental to developing cell-based therapies and studying human developmental mechanisms. We report a novel, scaleable, and widely applicable system for deriving and propagating neural stem cells from hESCs without the use of animal products, proprietary formulations, or genetic manipulation. This system provides a definitive platform for studying human neural development and has potential therapeutic implications. ) b! u; C) `7 r! }+ x
【关键词】 Human embryonic stem cells Neural stem cells Defined conditions Fibroblast growth factor
+ A' A$ o, z% d4 M1 k# b* g INTRODUCTION
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I: f7 k) b8 F7 u4 ^Realizing the promise of human embryonic stem cells as a source of defined cells for cell-based therapies or drug screening for neurological disorders requires totally defined and controlled differentiation conditions. In addition, the ability to control the conditions of neuralization will enable examination of the molecular mechanisms underlying human neural development. Although recent advances have been made in the derivation and propagation of undifferentiated human ESCs (hESCs) .
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& I! S; Y6 n: M4 \' {* e3 XInsights from mouse ESC (mESC) neuralization systems have informed and provided a platform for hESC studies. Several reports have demonstrated the in vitro capacity of mESCs to generate neurons , there is the fundamental difference of "timing." Human development is considerably longer than mouse development, and this is reflected in longer cell cycle times and greater numbers of divisions of human stem cells.
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The most widely applied hESC neuralizing systems use spontaneous differentiation, addition of retinoic acid to hESC aggregates, or coculture with stromal cells or conditioned media .9 ?: j n& U' T$ w0 ]
8 ^9 r& K8 K8 Q8 o; V& Y. t- yAlthough these findings are encouraging, a number of issues remain unresolved. Protocols that use only defined or human-derived products are imperative for generating clinical standard neural stem cells, since animal products raise the possibility of expression of nonhuman antigens and xenoinfections have relied on the use of commercial, proprietary formulations such as knockout serum replacement and B27 supplement, which are not fully humanized. In addition, the presence of retinoids in B27 has limited studies addressing physiological mechanisms of neural induction and patterning.
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0 @7 f% L4 o# Y6 vFurthermore, although hESC lines share fundamental properties, considerable differences exist with respect to growth rates, differentiation, and indeed methods used for maintenance and propagation and to show that efficient neuralization does not sacrifice scale.* }. C- F" q3 \6 i& V( x
6 X9 V4 ^' W G0 {& ~An ideal neural differentiation system should, therefore, contain only recombinant, chemically defined, or human-derived products and be applicable across hESC lines derived from different laboratories. In this study, we used the lines H9 and HUES9 to establish a simple, fully defined and controlled hESC neuralizing system.. p' [9 j' d: [9 u
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MATERIALS AND METHODS
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hESC Culture
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The hESC lines H9 (WiCell Research Institute, Madison, WI, http://www.wicell.org) and HUES9 (hES facility; Harvard University, Cambridge, MA, http://www.mcb.harvard.edu/melton/hues), derived in accordance with local and national guidelines, were used, between passages 21¨C60. Cells were cultured in knockout serum replacement (KSR) medium as previously described . KSR medium consisted of Knockout Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% Serum Replacement, 1% nonessential amino acids, 1 mM L-glutamine, 0.1 mM ¦Â-mercaptoethanol (all from Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and 4 and 10 ng/ml human fibroblast growth factor 2 (FGF2) (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) for H9 and HUES9, respectively.5 n) c& Y+ {5 ?. K! S1 u8 y+ R
7 g7 E& z$ ?' D9 p1 TNeural Stem Cell Generation and Propagation: q+ {" z" R. s1 n+ Q, S0 ~8 S
# d4 Z: z0 y$ z4 Q( a7 mTo generate NSC cultures, hESCs were washed in phosphate-buffered saline (PBS) and incubated in 1 mg/ml collagenase IV (Invitrogen) for approximately 90 minutes until colonies could be easily detached from the underlying feeder layer or Matrigel substrate by gentle pipetting. Detached colonies were placed in a 15-ml conical tube and allowed to settle by gravity for 5 minutes before being transferred with a sterile Pasteur pipette onto the lid of a 60-mm culture dish. Colonies were chopped at 150-µm intervals using a McIlwain tissue chopper (Mickle Engineering, Gomshall, U.K.) before being plated at a low density in neuralizing medium into bacteriological-grade 10-cm culture dishes on an orbital shaker to prevent sphere aggregation. Preliminary studies examined the ability of chemically defined medium (CDM) as described by Johansson and Wiles . SU5402 (R&D Systems) was used at a concentration of 10 µM. For terminal differentiation, NSCs were plated onto poly(D-lysine)/laminin-coated coverslips and cultured in DMEM/2% B27 or HNM without FGF2 and epidermal growth factor (EGF), for up to 35 days.
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9 F5 P& P/ u, i" s S, r9 lTranscriptional Analysis+ ?: {7 |8 a1 Q i* J8 T
, ^4 r9 z, ? y' W @" {4 fTotal RNA was extracted from harvested NSCs using the RNeasy Mini Kit (Qiagen, Valencia, CA, http://www1.qiagen.com) according to the manufacturer's instructions. Samples were treated with RNAse-free DNase (Qiagen) to remove DNA contamination. cDNA was synthesized from 2.5 µg of RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen) and oligo(dT) primers according to the manufacturer's instructions. Polymerase chain reaction (PCR) was carried out using Taq polymerase (Invitrogen). Sequences and PCR conditions for each primer are shown in supplemental online Table 2. PCR products were separated on a 2% agarose gel and visualized with SYBR Green (Invitrogen). The expression of the housekeeping gene GAPDH was used to normalize PCRs.8 m1 B: ]" v9 j* y( G; c& H+ ?8 S
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Immunocytochemistry
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. f0 k9 n1 U! y: V& A$ IImmunocytochemistry was carried out using standard procedures. hESC colonies and coverslip plate-downs were fixed with 4% fresh paraformaldehyde (PFA) for 20 minutes at room temperature. NSC spheres were fixed with 4% fresh PFA for 30 minutes at 4¡ãC and cryoprotected with 30% sucrose before being embedded in OCT compound (BDH, Dorset, U.K., http://uk.vwr.com). Blocks were sectioned at 12-µm intervals using a Leica cryostat (Heerbrugg, Switzerland, http://www.leica.com). Fixed cells and sections were blocked for 1 hour at room temperature with PBS/5% goat serum/0.1% Triton X-100 and then incubated overnight with primary antibody at 4¡ãC. Secondary antibody (1:1000; Invitrogen) was applied for 1 hour at 37¡ãC in PBS/Hoechst (1:5000). Primary antibodies used included Oct4 (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), SSEA4 (1:50; Developmental Studies Hybridoma Bank ). Cells were viewed under a Leitz microscope with appropriate filters for cell identification and counting.! q1 f3 {" m7 Q' ^; O: r1 [
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Fluorescence-Activated Cell Sorting Analysis; j- J1 Q% ?6 W6 Z1 N
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Harvested NSCs were prepared for fluorescence-activated cell sorting (FACS) analysis as previously described ), and fixed in ice-cold 4% PFA for 30 minutes. Cells were incubated with primary antibodies (at concentrations similar to those for immunocytochemistry) in PFN with 0.14% saponin (Sigma-Aldrich) for 30 minutes at 4¡ãC. Secondary antibodies were applied for 30 minutes at 4¡ãC. Cells were then washed in PFN and analyzed using a CyAn flow cytometer (DakoCytomation). Data analysis was performed using Summit software, version 4.2 (DakoCytomation).
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Quantification and Statistical Analysis) Y/ A- d E. a+ B* L
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All experiments were repeated at least three times unless stated otherwise. Cells were viewed using a Leitz microscope at high magnification (x40) for determination of hESC colony fragment sizes after seeding in HNM and NSC sphere diameter in subsequent culture using a calibrated eyepiece. A minimum of 20 random fields were counted for each experiment. Neuralization experiments were performed a minimum of six times per line, with quantitative immunocytochemical analysis undertaken twice for each line. The proportion of positive cells was expressed as a mean ¡À SE. A paired t test was used to determine any significant difference in mean cell counts between lines for the markers studied. p values for statistical significance in all other two-sample comparisons were calculated using Student's unpaired t test. Statistical analysis was carried out using GraphPad Prism 3.03 (GraphPad Software, Inc., San Diego, http://www.graphpad.com).. } [5 d- E3 A a2 L R% B
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Karyotypic Analysis: q7 ~4 z# H4 n1 {, ]
) d& O9 K* S4 s5 q/ F7 |Chromosome number and size was scored using G-banding by the Department of Hematology, Cambridge University Hospitals NHS Trust.
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+ A$ m) e. T3 uRESULTS. G, o7 b3 U% n3 B
. t5 p2 M: Q; |% J& aNeuroectodermal Specification of hESCs Under Defined Conditions( G+ X v( [" _' I! u
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Human embryonic stem cell lines H9 and HUES9 were maintained on irradiated mouse fibroblast feeder layers or under feeder-free conditions. These lines are from distinct laboratories and are maintained by different methods. Propagation of H9 relies on collagenase or mechanical passaging, thus maintaining cell-cell contact. In contrast, HUES9 is propagated as a single-cell suspension following trypsin treatment. Recent studies have used defined conditions to neuralize mESCs . Against this background, initial studies led us to develop an optimized HNM for hESCs (described in Materials and Methods). HNM has a fully humanized, defined formulation, devoid of serum, exogenous mitogens, retinoids, and other known neural inducers (supplemental online Table 1), thus giving control over the extrinsic signaling environment.* m& ]& z0 V3 t$ a. I0 a
5 O' Z1 x* q. G: TTo initiate neuroectodermal differentiation, hESC colonies were selectively detached, seeded in HNM and cultured as free-floating embryoid bodies (EBs) in suspension culture. Preliminary studies showed that neural specification occurred over approximately 16 days. However, EBs were heterogeneous in nature. Analysis revealed a correlation between EB size and degree of neurogenesis; small aggregates had a uniform, compact structure and were highly neurogenic, whereas larger aggregates had a nonuniform internal organization, with limited neurogenic regions dispersed as rosette islands (Fig. 1). This finding is consistent with previous mESC observations showing that low cell plating density in suspension culture is critical for efficient neurogenesis , was present throughout. Importantly, negligible expression of pluripotent, endodermal (HNF-3¦Â) and mesodermal (T, MYOD, SMA) markers was evident by day 16 (D16) (Fig. 2C).
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Figure 1. Efficiency of neural specification in human neuralizing medium is dependent on fragment size. Representative phase contrast and immune micrographs of day 16 cultures showing large EBs with a cystic structure, limited neuralization (Pax6 ) in isolated rosette islands, and maintenance of Oct4 expression (A), and smaller spheres with a compact, uniform structure, high neurogenicity, and absence of Oct4 expression (B). Scale bars = 100 µm.& E5 _6 P6 o. c" m- L
; S( H: v Y5 X) d s6 I8 p3 W$ _Figure 2. Predictable neuralization of small hESC colony fragments in human neuralizing medium (HNM). (A): Scatter representation of hESC colony size before and after mechanical chopping (bars represent mean values). (B): Representative phase micrographs of detached hESC colonies (i), chopped hESC colony fragments (ii), and homogeneous sphere formation and growth (iii¨Cvi) over 16 days in HNM. (C): Representative reverse transcription-polymerase chain reaction temporal expression analysis of chopped hESC colonies in HNM, showing loss of pluripotent (POU5F1 and NANOG) and mesendodermal markers (T, MyoD, SMA, and HNF-3¦Â) and acquisition of neuroectodermal markers (NCAM, PAX6, and SOX1). Expression of the pluripotent and neuroectodermal marker SOX2 was maintained throughout the time course. Scale bars = 300 µm. Abbreviations: D, day; ES, embryonic stem; hESC, human ESC.# ^# u' N, c4 n. j4 n
$ x; W C# R) |) x$ uFGF2 Supports Propagation of hESC-Derived NSCs1 O& G0 g( ?- s0 i& u5 `
7 S+ J! r& K! c" u' YAlthough HNM efficiently supported the neuralization of hESCs, culture in HNM alone did not support longer-term propagation of the hESC-derived NSCs much beyond D16. Therefore, to improve cell yield, we determined the effect of human recombinant FGF2, an established mitogen for human fetal-derived NSCs , we first determined the optimal time point for adding FGF2 to HNM to support NSC proliferation. Addition of FGF2 at D0 increased total cell yield but, as expected, maintained a significant persistence of Oct4 expression (Fig. 3A). However, studies using an FGF receptor inhibitor (SU5402) suggested that endogenous FGF activity was also necessary for neuralization. Treatment of cultures from D0 with SU5402 resulted in a significant reduction of expansion and nestin expression by D4 (Fig. 3A) and 1 [+ A! _6 E, g9 u" r4 I2 x" y% r
, E8 P/ ~- ]/ \+ ~2 i6 t. s! O" [Figure 3. Role of FGF signaling in HNM cultures. (A): Micrographs and table showing the effect of addition of FGF2 or the FGF receptor pharmacological inhibitor SU5402 on human ESCs from day 0 (D0) to D4 in HNM. (B): Graphs demonstrating sphere growth (i), cell yield (ii), and persistence of Oct4 (iii) at D16, following addition of FGF2 at D4, D8, or D12. *, p 3 R1 Z0 V% v) m! |+ c, W
- p5 ]% Z4 n; S) C# r. gSupplementing cultures with FGF2 from D8 onwards was found to be optimal for maximizing NSC yield without "contaminant" undifferentiated hESCs: cell expansion and continued sphere growth after D8 was greatly augmented, with a significant increase in cell yield (p - Y. m+ a$ J) i% J8 E
- G$ E2 |$ p; B* a$ OThe combination of mechanical chopping of hESC colonies at D0, neuralization in HNM, orbital shaking, and addition of FGF2 at D8 resulted in cell acquisition of nestin and Pax6 protein expression by D16 with loss of Oct4 and SSEA4 expression (Fig. 4). Together, these findings are consistent with the generation of a highly enriched hESC-derived NSC population. There was no significant difference between H9 and HUES9 in the mean proportion of cells expressing these markers at the time points studied (p = .439).; m* _5 I' H$ y3 v3 l1 m& K
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Figure 4. Neuralization of H9 and HUES9 under defined conditions. (A): Schematic of culture system with corresponding representative phase micrographs. (B): Representative immune micrographs and graphs showing quantification of the temporal profile of Oct4, SSEA4, Nestin, and Pax6 in H9 and HUES9 cultured in HNM at D0, D8, and D16 demonstrating efficient neuralization under defined conditions. Scale bars = 100 µm. Abbreviations: D, day; ES, embryonic stem; FGF, fibroblast growth factor; hESC, human ESC; HNM, human neuralizing medium.
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$ K: {! G' Y8 {8 k1 uLong-Term Propagation of hESC-Derived NSCs- ~' {# t" M7 \3 F/ t) w+ ]* \
+ Z! f9 f7 w7 F% n6 A$ I/ p7 xFollowing neuralization, NSCs at D16 were dissociated and subsequently propagated in HNM under conditions similar to those used for human fetal-derived NSCs ; automated mechanical passaging and addition of FGF2 together with EGF resulted in long-term NSC propagation with complete loss of Oct4/SSEA4 at all subsequent time points analyzed (data not shown). Using this system, NSCs had an overall expansion in excess of 250,000-fold by D100 (Fig. 5A) and maintained a stable karyotype (supplemental online Fig. 1). The behavior of hESC-derived NSCs was comparable across H9 and HUES9. The use of the orbital shaker allowed up to 3 x 107 cells to be cultured in a single 10-cm plate, thus making large-scale expansion practical. Propagated NSCs maintained high expression of NSC markers Nestin and Musashi1 at D50 and later time points analyzed (Fig. 5B). To date, we have established 12 NSC lines using this method, which have been propagated for up to 180 days. NSCs could also be cryopreserved, allowing efficient banking and recovery of expanded neuralized cell stocks. Critically, NSCs were multipotent and could be differentiated into ¦Â-III-tubulin neurons, GFAP astrocytes, and O4 oligodendrocytes (Fig. 5C). Differentiated neurons expressed multiple markers of maturation, including the cytoskeletal protein MAP2ab and SynapsinI (Fig. 5D), and were electrically active (data not shown). Neuronal subtype analysis revealed GABAergic and glutaminergic differentiation (Fig. 5D).$ s5 G- \* k7 O/ Q! s
7 D( a0 L, @3 `, W/ w" `8 wFigure 5. Scaleable propagation and characterization of human ESC (hESC)-derived NSCs. (A): Graph showing overall expansion hESC-derived NSCs over 100 days from one representative hESC plate. (B): Fluorescence-activated cell sorting characterization and quantification of day 50 NSCs for the NSC markers Nestin and Musashi1. (C): Phase and immune micrographs demonstrating multipotent differentiation of hESC-derived NSCs into neurons (¦Â-tubulin ), astrocytes (GFAP ) and oligodendrocytes (O4 ). (D): Immune micrographs demonstrating maturation (MAP2ab , SynapsinI ), and GABAergic and glutaminergic subtype differentiation of NSC-derived neurons after 14 days of terminal differentiation. Scale bars = 50 µm. Abbreviations: GFAP, glial fibrillary acidic protein; HNM, human neuralizing medium.5 |. I7 c9 q2 M7 Z0 O+ d
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DISCUSSION
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The generation of large numbers of enriched human-derived neurons has significant therapeutic implications. Our findings establish a simple, efficient, and scaleable system to generate almost limitless numbers of neural stem cells from human embryonic stem cells without genetic modification or the use of serum or other animal-derived products.1 B" k9 z+ s8 n; {
: E6 r' w# f I8 s& t8 lThe considerable therapeutic promise of NSCs for regenerative neurology is largely focused on cell replacement strategies, targeted at site-specific repair of focal central nervous system disorders, such as Parkinson . Furthermore, the availability of defined human neurons to enable high-throughput screening would greatly promote novel drug development and testing.5 L4 ]+ C D, B# G L! ^
' X% `) k' c4 ]) n$ a4 e$ eThere is thus a great need for the establishment, to a clinical and pharmaceutical standard, of systems for efficient and bulk generation of enriched NSCs, a process that must run in tandem with comparable efforts for derivation of hESC lines. Insights from several reports of neural derivation from hESCs are valuable in demonstrating their utility in generating neural and defined cell types . However, the reported methods are not readily scaleable and critically use serum and/or other nonhuman products. This study is notable for the use of automation, thus promoting speed, uniformity, and scale. In addition, the reported system removes the need for multiple steps that involve replating-based selection./ C& A3 q" \ c# I3 z; ~, [1 v
1 P$ X+ z. _1 Y) |- `, u: uOur findings suggest a requirement for autocrine FGF signaling in neuralization of hESCs, an observation in line with mESC reports . The defined conditions described in this report will thus enable future studies exploring the molecular mechanism(s) underlying the differential effects of FGF signaling on hESC self-renewal and neuroectodermal differentiation.
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There are two established methods for the propagation of NSCs: substrate-free neurosphere and adherent monolayer cultures. The majority of studies have used substrate-free conditions to propagate human NSCs. In addition, recent studies have extended earlier reports using substrate conditions to propagate human NSCs .
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. {& a, |/ e4 P* J+ RCONCLUSION: Y. x- \5 T& V
% e* R3 S9 b3 V1 UIn summary, we report a simple, cost-effective, and fully defined system for deriving and propagating neural stem cells from human embryonic stem cells. The culture process is also highly amenable to automation and expansion to bulk culture. The fully humanized and generic culture conditions enable this system to be used to generate clinical-grade neural stem cells for therapeutic applications. Furthermore, the scaleable nature of this system as a source of human neurons permits high-throughput drug screening. Finally, the use of totally defined and minimal growth factor conditions allows the analysis of molecular pathways involved in human embryonic stem cell neural specification and terminal differentiation. We have confirmed the reproducibility of our findings over two human embryonic stem cell lines derived from independent laboratories. It would be of great interest to examine the reported system with lines derived under defined conditions, thus generating definitive clinical-grade neural stem cells.* B4 D, N) p% V: v f7 _
, Z; I! N$ I4 N$ M: [5 m4 f* ^5 G4 UDISCLOSURES
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$ o& N+ e( K* ~. gThe authors indicate no potential conflicts of interest.2 L9 y Z! E# g( n! ~
2 O; L/ D+ O Z; [ACKNOWLEDGMENTS
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# ^+ W! v9 N/ g3 P% {% v1 P! B5 jIsabelle Bouhon passed away on October 6, 2005. We thank the Developmental Studies Hybridoma Bank for providing antibodies. Michelle Jackson, Nigel Miller, Pam Tyers, and Craig Secker provided valuable technical assistance. This work was supported by a Medical Research Council Stem Cell Strategic Grant (N.D.A., S.C.). A.J.J. is supported by a Merck Sharpe and Dohme Neuroscience Studentship, the Cunning Fund, and the University of Cambridge M.B./Ph.D. program. P.A. is supported by the Gates-Cambridge Scholarship Fund.
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