标题: An Autogeneic Feeder Cell System That Efficiently Supports Growth of Undifferent [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:51 标题: An Autogeneic Feeder Cell System That Efficiently Supports Growth of Undifferent
a Centre for Stem Cell Biology and Developmental Genetics, University of Newcastle, Newcastle upon Tyne, UK; 8 r6 {7 D, \; J; a1 d; G; V7 P E; j9 R2 n
b School of Biological and Biomedical Sciences, University of Durham, Durham, UK;/ M3 K: L% F! y0 Y* D
/ {$ k2 c6 t. X. \: h( ic Newcastle Fertility Centre at Life, Newcastle Health Service, Newcastle upon Tyne, UK5 u2 s8 l4 m2 a7 B$ A. f; S
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Key Words. Human embryonic stem cells ? Pluripotency ? Differentiation ? Autogeneic feeder0 L: C; {& G4 e+ _) Q1 W+ D8 n
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Correspondence: M. Stojkovic, Ph.D. Centre for Stem Cell Biology and Developmental Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK. Telephone: 44-191-241-8638; Fax: 44-191-219-4747; e-mail: mio-drag.stojkovic@ncl.ac.uk ( D0 y6 {$ ]& F) l [- t0 W! L3 D* G8 X' K
ABSTRACT& q1 A; F% @' I5 u* U+ c4 R
4 D+ k1 K. U/ Y- z. o* P) y0 CHuman embryonic stem cells (hESCs) have the ability to differentiate into almost all adult cell types and hold great promise for regenerative medicine . Pluripotent hESC lines have now been derived from the inner cell mass (ICM) of surplus day 5 to day 8 human blastocysts , and continuous culture of isolated ICM cells and hESCs in an undifferentiated state requires the presence of feeder layers such as mouse embryonic fibroblast (MEF) cells , STO (SIM mouse embryo-derived thioquanine and ouabain resistant) cells , or a variety of human fetal, neonatal, and adult cells . Alternatively, hESCs may be cultured on dishes coated with animal-based ingredients with the addition of MEF cell-conditioned medium or in the presence of the specific pharmacological inhibitor of glycogen synthase kinase-3 .0 F+ a8 B# l. t$ D" [- h
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The use of feeder cells for the prolonged culture of undifferentiated hESCs, however, does limit their medical application: xenogeneic and allogeneic feeders bear the risk of transmitting pathogens and other unidentified risk factors , not all human feeders and cell-free matrices support the culture of undifferentiated hES cells equally well , and the availability of human cells from aborted fetuses or Fallopian tubes is relatively low. ) y" |& x0 `9 j+ O& a * O* e1 c! k1 S" t3 v8 YWe previously described the derivation of a new and fully characterized hESC line (hES-NCL1). After culture in a feeder-free system, the hES-NCL1 and commercially available hESC (H1 line) were found to spontaneously differentiate into cells with fibroblast-like morphology. We show in this article that the latter cells can be used as an autogeneic feeder system that efficiently supports the growth and maintenance of pluripotency of both autogeneic and allogeneic undifferentiated hESCs. ! ^/ z) V9 d: v* t0 D3 h # ^' w; L. i1 |! j! \MATERIALS AND METHODS . O S: l, o& ]. l( | ' B# f4 r- X+ [/ M6 OWhen colonies of both hESC lines were transferred and cultured in the absence of mouse embryonic fibroblasts, 86.8% (H1) and 90.0% (hES-NCL1) of the colonies attached showing the first signs of spontaneous differentiation. Of these, only colonies with fibroblast-like cells (Fig. 1A, B) which appeared as long, flat cells with an elongated, condensed nucleus (Fig. 1C) were further used for establishment of hES-dFs. Micro satellite analysis confirmed that the hES-NCL1 cells (Fig. 2A) and their autogeneic hES-dFs (Fig. 2B) have a common genetic origin. Flow cytometry (Fig. 3) revealed that very few cells showed expression of mesenchymal cell-specific markers CD106 (V-CAM1) and CD71 (transferrin receptor), and none expressed the endothelial-specific cell marker CD31 (PECAM-1). On the contrary, 94% and 82% of the hES-dF cells were stained with the CD44 and CD90 (THY-1) antibodies, respectively. Both antibodies were also presented in human foreskin fibroblasts (HFFs; Fig. 3).% H, w: b. n# j1 h1 |
0 L& q3 H1 ^; }: n6 yFigure 2. Micro satellite comparison of (A) hES-NCL1 cells (passage 19) and (B) feeders (passage 5) derived from hES-NCL1 reveals identical profiles, indicative of a common genetic origin. * x2 v) ~) G4 O _( u, m; `0 A1 z9 r' f6 v
Figure 3. Flow cytometry analysis of fibroblast-like cells (passage 4) derived from hES-NCL1 (left panel) and human foreskin fibroblasts (HFFs) (passage 4; right panel) for the presence of CD31, CD44, CD71, CD90, and CD106. The red line represents the staining with the isotype control, and the green line shows staining with specific antibodies.% j: X; F! B# O" p( b
8 W& a. O: X5 a( m4 b: kThus far, the hES-NCL1 line has been cultured on autogeneic hES-dFs for over 44 passages and on feeder derived from H1 cells for over 22 passages. H1 cells were grown on autogeneic feeder for over 18 passages and on allogeneic feeder (derived from hES-NCL1) for 17 passages. Both the hES-NCL1 and H1 lines were grown on Matrigel in the presence of autogeneic hES-dF–conditioned medium for 14 and 12 passages, respectively. We found that both fresh and cryopreserved hESC colonies grown on fresh or cryopreserved hES-dFs (Fig. 4A, C, E) or on Matrigel (Fig. 4G) were dense and compact, and they exhibited the typical morphology of hESCs (Fig. 4B). The hES-NCL1 cells cultured on autogeneic hES-dFs or on Matrigel with the addition of autogeneic hES-dF–conditioned medium expressed markers typical of hESCs, such as the cell surface markers TRA-1-60 (Fig. 4D), SSEA-4 (Fig. 4F), and GTCM-2 (Fig. 4H). H1 cells cultured on autogeneic hES-dFs or on Matrigel with the addition of autogeneic hES-dF–conditioned medium expressed TRA-1-60 (Fig. 5D, H) and SSEA-4 (Fig. 5F). * [0 _: e3 M2 [ ' E$ Y5 C# u: \5 JFigure 4. Morphology and characterization of hES-NCL1 cells grown on (A–F) autogeneic hES-dF monolayers or (G, H) under feeder-free conditions with addition of autogeneic hES-dF–conditioned medium. (A): Five-day-old vitrified human embryonic stem cells (hESCs) cultured on frozen-thawed hES-dFs (passage 8). (B): Higher magnification of the same hESC colony. Note the typical morphology of human embryonic stem cells (hESCs): small cells with prominent nucleoli. (C, E): hESCs grown on hES-dFs stained with antibody recognizing the (D) TRA-1-60 and (F) SSEA-4 epitopes. (G): hESCs grown on Matrigel stained with antibody recognizing (H) the GTCM2 epitope . Bars: (A, E–H) 200 μm; (C, D) 100 μm; (B) 50 μm.) I1 s2 O8 ]" i, T* u6 i& V
9 [' d8 h- K6 L8 O( xFigure 5. Morphology and characterization of H1 cells grown on (A–F) autogeneic hES-dF monolayer or (G, H) under feeder-free conditions using hES-dF-conditioned medium. (A): Four-day-old human embryonic stem cell (hESC) colony (passage 11) cultured on hES-dF (passage 4). (B): Higher magnification of the same hESC colony shows typical morphology of hESCs. (C, E): H1 cells (passage 13) grown on autogeneic hES-dFs (passage 6) stained with antibody recognizing the TRA-1-60 (D) and SSEA-4 (F) epitopes. H1 cells (passage 10) grown on (G) Matrigel with addition of autogeneic hES-dF–conditioned medium stained with antibody recognizing (H) the TRA-1-60 epitope. Bars: (A, C, D) 200 μm; (E–H) 100 μm; (B) 50 μm. & H' w3 o, _. X0 V! o1 {) k * v& g% q) b- j; J4 f- GTo test whether allogeneic hES-dFs support undifferentiated growth of hESCs, we cultured hES-NCL1 on hES-dFs derived from H1 and H1 cells on hES-dFs derived from hES-NCL1 cells. Indeed, the allogeneic feeder also supported undifferentiated growth of hESCs, as demonstrated by the presence of specific cell surface markers (Fig. 6A–F). In addition, hES-NCL1 cells cultured on Matrigel with allogeneic (H1) hES-dF–conditioned medium expressed cell surface marker TRA-1-60 (Fig. 6H). Flow cytometry analysis revealed that hES-NCL1 grown on either autogeneic or allogeneic feeders expressed TRA-1-60 antigen (passage 42, 66.3%; passage 20, 58.9%). When grown on autogeneic or allogeneic feeders, 70.8% and 68.6% of H1 cells expressed TRA-1-60 (passage 15 and 14, respectively).4 e7 n' l! Y) i# n, j1 r y8 C; R2 p
& ?: i- G, n# W% K3 l" U- l* fFigure 6. Morphology (A, C, E) and characterization (B, D, F) of hES-NCL1 and H1 cells grown on autogeneic or allogeneic hES-dFs. (A, C): hES-NCL1 cells (passage 18) grown on allogeneic (derived from H1 cells) feeder were stained with antibody recognizing the (B) TRA-1-60 and (D) SSEA-4 epitopes. (E): H1 cells (passage 11) grown on allogeneic (derived from hES-NCL1 cells) feeder stained with antibody recognizing (F) the TRA-1-60 epitope. H1 cells (passage 10) grown on (G) Matrigel with addition of allogeneic (hES-NCL1) hES-dF–conditioned medium stained with antibody recognizing (H) the TRA-1-60 epitope. Bars: (A, B, G, H) 200 μm; (C–F) 100 μm. 0 i' }5 F& f8 P1 L3 M/ Y6 o% _7 | k& k( ^3 q
RT-PCR analysis of both stem cell lines showed positive expression of OCT-4, NANOG, FOXD3, TERT, and REX-1 (Fig. 7A, B) when grown on their autogeneic feeders. The fibroblast-like cells derived from both hESC lines also expressed the telomerase reverse transcriptase (TERT) and REX1 in early passages, but none of the other markers characteristic of ESCs (see Fig. 7A, B).8 ^0 {8 p0 t0 Q( p! X# `
7 q8 D" K* @6 l. W; F; T! b4 mFigure 7. Characterization (A, B) and karyotyping (C, D) of hES-NCL1 and H1 cells grown on autogeneic hES-dF monolayer. Reverse transcription polymerase chain reaction (RT-PCR) analysis of (A) undifferentiated hES-NCL1 (passage 29) and (B) H1 (passage 14) cells grown on inactivated autogeneic hES-dF cells. The PCR products were obtained using primers specific for (lane 1) OCT-4, (lane 2) NANOG, (lane 3) FOXD3, (lane 4) TERT, (lane 5) REX1, and (lane 6) GAPDH. PCR was processed with ( ) or without (–) reverse transcriptase. (C): hES-NCL1 (passage 31) grown on hES-dF (passage 10) show a normal female karyotype (46, XX). (D): H1 (passage 16) grown on hES-dF (passage 3) show a normal male karyotype (46, XY). Abbreviations: NC, negative control. * g( B) h* {' j3 \! y5 ?$ g, i1 p. S* K3 k* D) q
Karyotyping of the hESCs showed that both hES-NCL1 and H1 lines grown on autogeneic feeders have normal female or male karyotypes (Fig. 7C, D).% O# L- } z; v5 D( y# z: k* B
# T* L, `+ B+ b6 JThe hESC cells grafted into SCID mice consistently developed into teratomas, demonstrating the pluripotency of hES-NCL1 cells grown on autogeneic hES-dFs. Teratomas were primarily restricted to the site of injection, and their histological examination revealed advanced differentiation of structures representative of all three embryonic germ layers, including cartilage, skin, muscle, primitive neuroectoderm, neural ganglia, secretory epithelia, and connective tissues (Fig. 8).( d& Q8 D" h# o
' a9 B( \( @$ D: _& j! t" p0 UFigure 8. Histological analysis of teratomas formed from grafted colonies of hES-NCL1 (passage 23) grown on inactivated autogeneic hES-dF in testis (A–C) and kidney (D–F) of severe combined immunodeficient (SCID) mice. (A): Neural epithelium (ne). (B): Aggregation of glandular cells with characteristic appearance of secretory acini (sa). (C): Cartilage (cart). (D): Wall of respiratory passage showing epithelium (ep), submucosa (sm), and submucosal glands (sg). The epithelium contains occasional ciliated cells and numerous goblet cells secreting mucin (m). (E): Two types of epithelia: respiratory (top), keratinised skin (bottom). Submucosal glands (sg) located beneath pseudostratified ciliated (in parts) epithelium (ep). Structures of the skin include epidermis (ed), dermis (dm), and cornified layer (c). Note that the stratum granulosum (arrow) is characterized by intracellular granules, which contribute to the process of keratinization. Occasional mitotic indices (m) are seen in the basal layer. (F): High magnification image of skin, showing greater detail of dermis (dm), epidermis (ed), and cornified layer (c). Again, the stratum granulosum is visible (arrow). Scale bars: (A, B, C) 100 μm; (D, E) 25 μm; (F) 17.5 μm. * `! N L, g$ m1 J& P8 c 5 m+ `3 A7 j! a% X) G! R4 VWhen hES-NCL1 and H1 cells grown on hES-dFs were cultured in the absence of hES-dFs, spontaneous differentiation into neuronal precursor, beating cardiomyocytes, and endodermal cells was observed (Fig. 9). This demonstrates that both hESC lines grown on autogeneic feeders have the ability to differentiate into cells of all three germ layers also under in vitro conditions.4 |- W3 H& j' p- a3 a, [ r
+ @. L. z- j6 A: fFigure 9. Spontaneous differentiation of hES-NCL1 (A–C) and H1 (D–E) cells grown on autogeneic feeders and then in feeder-free culture conditions. Human embryonic stem cells (hESCs) from both lines differentiate into (A, D) neuronal precursor cells, (B,E) beating cardiomyocytes, and (C,F) endodermal cells. Specific staining the green color〞represents cells stained with (A, D) nestin, (B, E) -actinin (sarcomeric), and (C, F) -fetoprotein antibodies. Red and blue colors represent cell-nuclei stained with (A, D) propidium iodide or (F) Hoechst 33342, respectively. In (B, E, C), the bright field and fluorescent images were recorded at the same time. Scale bars: (A–C) 100 μm; (F) 50 μm. , H+ j: G4 _. E1 ^' ^: I8 [5 {6 x2 q4 g- E
DISCUSSION H8 C( ^+ t6 ~$ t0 @" {
5 n5 i9 z+ D4 n% yWe thank M. Choudary and A. Elliott for the technical support; Complement Genomics, Sunderland for sample analysis; and Dr. M. Pera for the donation of specific antibody. This work was supported by Newcastle University Hospitals Special Trustees, One Northeast Regional Development Agency, Newcastle Health Charity, the Department of Health and MRC (UK) grant no. G0301182.. F" ]) o- R9 s3 S
9 u% |0 l0 x& T$ E; SREFERENCES # }! {8 x) P* u% }* x3 }3 a9 }% N: B5 M! i6 T: C
Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cells. STEM CELLS 2001;19:193–204. 1 w8 z6 B a& h& s0 m7 Z( o ; e$ A* l; z# A# _Schuldiner M, Eiges R, Eden A et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res 2001;913:201–205.6 c; \. h. s) T- m$ q1 \0 y
4 @3 c8 r8 f$ ~3 K
Zhang S-C, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neuronal precursors from human embryonic stem cell. Nat Biotechnol 2001; 19:1129–1133. 7 R# o7 M! W) N0 |: @' ^! v& j/ ~2 b6 } i5 k$ m+ }
He J-Q, Ma Y, Lee Y et al. Human embryonic stem cells develop into multiple types of cardiac myocytes. Circ Res 2003;93:32–39. $ G v: L4 S9 ^2 W% p ; ?5 f& B8 Y+ \1 LThomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell line from human blastocysts. Science 1998;282:1145–1147.( t8 T% b' v2 l" d1 k; X$ [, P
" W0 R6 r) v4 n' f4 e7 m4 l( ?Reubinoff BE, Pera MF, Fong C-Y et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.0 n `0 ^ T6 l. f; D# O+ ^
, E' k) o7 @( [, m" Z+ ~7 ]Richards M, Fong C-Y, Chan W-K et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cell lines. Nat Biotechnol 2002;20:933–936.( Q3 ~7 N5 A; D
0 Y$ |9 n' S6 B$ Q" q3 I
Hovatta O, Mikkola M, Gertow K et al. A culture system using foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003; 18:1404–1409.; O1 t9 G+ _# U' F' ~8 y! W! u7 H
# T- U7 ]+ W+ [9 lMitalipova M, Calhoun J, Shin S etal. Humanembryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526. 2 ?9 x: E0 h: w4 g3 y' J% J p N! }* Z
Pickering SJ, Braude PR, Patel M et al. Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. RBM Online 2003;7:353–364. % E: p1 _! `5 b: | s5 Z - V% t; T' F! w1 C! q6 iStojkovic M, Lako M, Stojkovic P et al. Derivation of human embryonic stem cells from day 8 blastocysts recovered after three-step in vitro culture. STEM CELLS 2004;22:790–797. , b$ f/ \# j) w, x K2 S3 o6 { . T& h& w8 ^9 i$ `. yPark JH, Kim SJ, Oh EJ et al. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 2003;69:2007–2014.& X6 D" `! d2 F& h: y2 t2 t
$ u' W- ]; q# V) }% w0 xRichards M, Tan S, Fong C-Y et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. STEM CELLS 2003;21: 546–556.& }- N5 v8 A5 P: i5 l
6 n: t) f4 ~% KAmit M, Margulets V, Segev H et al. Human feeder layers for human embryonic stem cells. Biol Reprod 2003;68:2150–2156. * k9 c3 N9 w9 X1 c+ ]5 A' |0 W. L" e
Cheng L, Hammond H, Zhaohui Y et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.3 n0 m, Q1 J2 x) e& B/ a, `
! T A) c9 K s9 Y" jXu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.$ a2 G5 ?( G" Q U- M" h6 D8 Q
g* g/ p* }, L
Rosler ES, Fisk GJ, Ares X et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 2004;229:259–274.& n/ Q3 N. x V6 p+ B2 M H; b. [$ A
& _* b$ p b- {0 T
Sato N, Meijer L, Skaltsounis L et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63. 5 V' f! M% j- e: x $ Q8 R1 B/ _5 r4 o3 Y' ]Reubinoff BE, Pera MF, Vajta G et al. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 2001;10:2187–2194.3 ]" P' e3 Z; T3 \6 y* m" N4 K' _: Y
; g. e. `1 M8 V4 v4 N1 Z$ R, ^Lim JW, Bodnar A. Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics 2002;2:1187–1203./ R( A' p o+ ~# @4 u1 N5 {) S
% } a- R3 q3 ^" B) S. y
Carpenter MK, Rosler ES, Fisk GJ et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 2004;229:243–258.' c* T, A- k, k8 S" j0 L! m
1 T( l# R( @8 C- m A$ ?1 Q) ~; XAmit M, Shariki C, Margulets V et al. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–845.; H' K* |6 u1 k2 H. w( R& f
. C( b6 i/ ~: y# M5 u
Gearhart J. New human embryonic stem cell lines: more is better. N Engl J Med 2004;350:13./ r2 k! p6 C: ?2 [
6 P3 i! b" P. ^Linge C, Green MR, Brooks RF. A method for removal of fibroblasts from human tissue culture systems. Exp Cell Res 1989;185:519–528. + I6 j% }3 h- r/ W @* J * {& K0 @. C" V. Q1 vFromigue O, Louis K, Dayem M et al. Gene expression profiling of normal human pulmonary fibroblasts following coculture with non-small-cell lung cancer cells reveals alterations related to matrix degradation, angiogenesis, cell growth and survival. Oncogene 2003;22:8487–8497.2 s- |$ B! q# Z7 f$ e" l" v
& U" U! L' u$ xHenderson JK, Draper JS, Baillie HS et al. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. STEM CELLS 2002;20:329–337.(Petra Stojkovica, Majlind)作者: 石头111 时间: 2015-6-7 15:59