标题: Derivation and Growing Human Embryonic Stem Cells on Feeders Derived from Themse [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:48 标题: Derivation and Growing Human Embryonic Stem Cells on Feeders Derived from Themse
a Center for Developmental Biology, Xinhua Hospital, Shanghai Second Medical University, Shanghai, P.R. China; - V, z: g! S }4 C, W9 g$ ]7 v; D) f j
b Laboratory of Stem Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, P.R. China;' B2 H6 x1 u5 `* {7 x
8 u( O- H- d3 v' m! Ic IVF Center of Women’s Hospital, School of Medicine, Zhejiang University, Zhejiang, P.R. China ( F* M3 `3 w8 V; v% E% V - L& n. H4 C4 O. Z5 n" sKey Words. Human embryonic stem cells ? Feeder, j! ^4 {. p+ y \1 @: C" C4 v. W
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Correspondence: Hui Z. Sheng, M.D., Ph.D., Center for Developmental Biology, 1665 Kong Jiang Road, Xinhua Hospital, Shanghai Second Medical University, Shanghai 200092, P.R. China. Telephone: 86-21-55570017; Fax: 86-21-55570017; e-mail: hzsheng@sh163.sta.net.cn " I0 f: S" G7 @# _! u6 I; W, a' ?/ d# I
ABSTRACT2 r. s" C9 l J
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Human embryonic stem cell (hESC) lines were first isolated by Thomson et al. in 1998. These cells have the potential to produce any type of cells of the body in an unlimited quantity and can be genetically altered . These characteristics make hESCs good candidates for cell-based therapies.0 C2 X% v6 u- R* p; `8 @6 s
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hESCs are in most cases cultured on mouse embryonic fibroblast (MEF). However, concerns arise that contaminations, such as rodent viruses or proteins introduced by MEF, may make hESCs unsuitable for therapeutic purposes. Alternative culture systems have therefore been invented to avoid the use of MEF. Some of these systems use human somatic cells or cell lines to substitute MEF. These include embryonic fibroblasts, adult fallopian tube epithelium , bone marrow stromal cells , foreskin fibroblasts , human cell lines (D551/CCL-10, CCL-2552), adult skin cells , and placenta cells . The other systems use a feeder-free environment that cultures hESCs in special media supplemented with Matrigel matrix plus MEF-conditioned medium , fibronectin plus transforming growth factor ?1 and basic fibroblast growth factor (bFGF) , or Matrigel in combination with activator of WNT pathway , respectively. 0 q' g+ K* z; h2 C0 [- g; S! I6 }, a! X$ N
Although these alternative systems provide solutions for minimizing pathogen contamination, each of them has its own limitations. For example, using human somatic cells as feeder, human materials will be needed frequently and tissues from different sources may bring variations to the culture. Using MEF-conditioned medium may still expose hESCs to animal pathogens. Feeder-free systems, using additional growth factors, will significantly increase the cost of the culture. In addition, until now it has not been reported that current feeder-free systems can be used to derive new hESC lines. Overall, each of these culture systems offers some advantages and disadvantages. Complementary to each other, they provide a variety of choices to meet the needs of different applications. " W$ Z. p4 u' K0 q4 k3 p6 M( ?8 y& G / d% b4 [3 f# [! B: P* } VIt was observed that in the feeder-free culture system, there were some stroma-like cells at the peripheral of the hESC colonies . It is possible that those differentiated cells may function as feeders to help maintain the rest of hESCs in an undifferentiated status. This observation has brought up the question of whether feeders can be derived from hESCs and used to support their own growth. This system would provide a growth environment for a broad range of hESC lines without increasing the possibility of heterogeneous contaminations. In this study, we show that hESCs can indeed produce feeder cells that are capable of supporting derivation and long-term growth of themselves. Potential applications of the method are discussed.; Y' y, } I* A
( O2 V/ s9 z: F6 `. f; N( C! V" MMATERIALS AND METHODS 4 |+ G4 s& a' [3 Q/ l. z 5 J4 ^& v6 N& w' kCharacteristics of EDFs2 A# O6 ^1 Q& w/ D4 H, E9 A9 r
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Early passages of differentiated hESCs were composed of various cells of different shapes as well as many cellular aggregates. As culture went on, cellular aggregates gradually disappeared and cells with a fibroblast-like morphology prevailed, which expressed at least some fibroblast cell markers, such as proline 4-hydroxylase and vimentin (Fig. 1) . These cells at the fifth or higher passages were used as feeders. EDF cells reached confluence and were split every 2–3 days in the early stages of culture. The interval between passages was gradually prolonged to 4–5 days. Using the current protocol, these cells can be passaged at least 15 times (approximately 20 PDs) and still maintain uniform fibroblast-like morphology. When EDF cells were transferred to the same culture medium supplemented with human serum instead of FBS, they maintained the same morphology and were passaged at the same interval (data not shown). " z+ D" {5 X4 Q D O# s7 G8 N* k0 c. `2 q8 b; }
Figure 1. Expression of fibroblast markers by human embryonic stem cell–derived feeder (passage 13) as detected by (A) immunofluorescent staining using a vimentin-specific antibody and (B) reverse transcription–polymerase chain reaction analysis. Lanes 1, 3, 5: expression of P4HB, Vimentin, and ?-actin by EDF cells, respectively. Lanes 2, 4, 6: negative controls of those genes (see Materials and Methods). M, 100-bp ladder. 5 ~$ j( L% v5 N ; r6 F* w# B& tThree hESC lines, H1, SH1, and SH2, have been used to derive EDFs. Eleven EDF subcultures were established from these lines, and all of them were able to support sustained growth of hESCs. We have also compared EDF from early passage (passage 5) to later passage (passage 15) for supporting the growth of hESCs. Both of them were effective. EDF cells can also be cryopreserved without losing their ability to proliferate and to support the growth of hESCs (data not shown). These results indicate that the growth-promoting property of EDFs is very stable and not influenced by either the number of passages or freeze-thaw cycles.& A* t; N- |3 J7 a. M( F
1 X" ~9 o7 X4 Z) k% O7 ~& vDerivation and Growth of hESC Lines on EDF9 p3 P! T9 ?/ ^% u2 J+ ?
- v4 I: E: ~: x# s& n$ I$ C/ u% b$ zTo examine whether new hESC lines could be derived on EDF, inner cell mass of the blastocyst was dissected and plated on EDFs. Cells with morphology typical of hESCs grew out of the cell cluster on the second week after plating. Once dissociated and passaged to new EDFs, these cells continued to proliferate to form a new hESC line, SH7. The SH7 cells appeared as round, monolayer colonies with clearly defined edges. Under high magnification, individual ESCs showed a high nucleus-to-cytoplasm ratio and prominent nucleoli (Fig. 2). They are positive for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-4, as shown by immunofluorescent analysis (Fig. 3). At present, they have been passaged 30 times (>180 days).' @8 T& B: B m4 k5 L- u
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Figure 2. Morphology of human embryonic stem cell (SH7) colonies, which were derived on human embryonic stem cell–derived feeder. (A): Outgrowth of inner cell mass 9 days after plating. SH7 cell line at passage 5 (B), passage 24 (C), and passage 32 (D). Pictures are taken in different magnifications (see bars) to show various aspects of the colonies. Scale bar = 100 μm." S/ ^- q! j4 h5 M( Z) v
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Figure 3. Fluorescent immunocytochemistry of H1 cells on mouse embryonic fibroblast (MEF) (passage 67), H1 cells on human embryonic stem cell–derived feeder (EDF) (passage 30), and SH7 cells on EDF (passage 28). Lane 1, OCT-4; lane 2, SSEA-3; lane 3, SSEA-4; lane 4, TRA-1-60; lane 5, TRA-1-81. Scale bar = 100 μm. , c$ c6 h# }2 P9 ^1 _* [2 c8 r% w5 u" C& M `! K
hESCs previously grown on MEF can be easily adapted to EDFs. Usually at the first one or two passages, some colonies tended to differentiate, but the percentage of differentiated colonies never exceeded 20% of all the colonies in the culture. Most colonies maintained an undifferentiated morphology after transferring from MEF to EDF. Similarly, when they were first transferred from EDFs to MEFs, a certain percentage of colonies also differentiated spontaneously. Such spontaneous differentiation seen during transition soon reduced to a normal range in subsequent passages using the same type of feeder, either MEFs or EDFs. hESCs on EDF were usually round, flat colonies with a large surface area, maintained the same morphology as they grow on MEFs (Fig. 4). Under high magnification, individual ESCs on EDFs remained round and small, with a high nucleus:cytoplasm ratio and prominent nucleoli (Fig. 4E). The H1, SH1, and SH2 lines were plated on either EDF or MEF, and their growth rates were compared. The three lines grown on EDF had a PD time of approximately 38, 35, and 37 hours, respectively, and on MEF, of approximately 37, 33, and 38 hours, respectively. At the time of writing this article, the H1 line had been maintained on EDF for 52 passages (>300 days), the SH1 line for 31 passages (>150 days), and the SH2 line for 34 passages (>200 days).2 \3 c* X. R, I
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Figure 4. Phase-contrast micrographs of feeders, human embryonic stem cells (hESCs), and embryoid bodies (EBs). (A): Mouse embryonic fibroblast (MEF) feeder cells. (B): Colony of H1 ESCs on MEF layers at passage 50. (C): Human embryonic stem cell–derived feeder (EDF) cells. (D): Colony of H1 ESCs on EDF layers at passage 24. (E): Colonies of H1 ESCs on EDF at high magnification. (F): Day-3 EBs derived from H1 cells, which were cultured on EDF for 20 passages. Scale bar = 100 μm.3 h! j) n( H4 O* b' T
( R& T) a' `' F) M5 KMarker Expression of hESCs Grown on EDFs , n* |$ C4 t: w" X; u: x: }9 B* Z3 j3 `1 y
To test whether EDFs were able to support proliferation of hESCs in an undifferentiated status, we examined expression of undifferentiated markers of the hESC lines: the SH7 line, which was derived and maintained on EDF (passage 28), and the H1 line grown on either MEF (passage 67) or EDF (passage 30). All three lines were positive for OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (Fig. 3) and negative for SSEA-1 (data not shown). These cells also displayed strong alkaline phosphatase activity (data not shown). To ensure undifferentiated status of hESCs maintained on EDF, we also examined differentiated markers of the H1 cell line grown on either MEF or EDF. Although EBs formed from H1 cells (grown on EDF for 20 passages) expressed markers representative of all three germ layers, the H1 cell line grown on either MEF or EDF expressed OCT-4 and NANOG but none of the differentiated markers (Fig. 5). These data indicated that hESCs remained undifferentiated after long-term culture on EDFs. : F% `& N% f2 R1 N9 P 0 [5 k9 s) i0 ]# P, f$ u: } j, qFigure 5. Reverse transcription–polymerase chain reaction analysis of gene expression of H1 cells cultured on human embryonic stem cell–derived feeder (EDF) (passage 20) (lane 2) and on mouse embryonic fibroblast (passage 60) (lane 3). Human embryonic stem cells cultured on both feeders expressed OCT4 and Nanog but not differentiated markers. After embryoid body formation, H1 cells grown on EDF expressed differentiated markers representative of the three germ layers (lane 1).7 U4 ], h6 @- o
" h5 I( a; [3 i" ePluripotency of hESCs on EDF % m K- K. `, \6 ~ ! O! j$ V' t% A, ~* s9 Z* F% WhESCs that had been cultured on EDF long term were tested for their potential to form EBs and teratomas. These cells formed EBs when cultured insuspension (Fig.4F). After 1 month, the EBs were examined for their gene expression using an RT-PCR assay. Those EBs expressed genes representative of all three primary germ layers, including nestin, neurofilament L (ectoderm), alpha-fetoprotein, albumin (endoderm), and BMP4 (mesoderm) (Fig. 5). 2 d8 I3 t* t/ a1 u" C6 a! v; { U% Y
hESCs grown on EDF for 11 passages formed teratomas when injected into NOD/LtSz-scid mice. The teratomas contained a variety of tissue types, including epithelial cells containing melanin (ectoderm, Fig. 6A), muscle and cartilage (mesoderm, Figs. 6A, 6D), and columnar epithelium with goblet cells (endoderm, Figs. 6B, 6C). Therefore, hESCs grown on EDFs retained pluripotency, as demonstrated by both in vivo and in vitro assays. ' ^- U9 ^- V7 T8 }0 {. X S# C9 h: ]4 k
Figure 6. Histology of differentiated elements in teratomas formed by H1 embryonic stem cells after 11 passages on human embryonic stem cell–derived feeder. (A): Epithelium with cells containing melanin and striated muscle. (B): Gut-like epithelium with mucous-containing cells and glandular epithelium. (C): Columnar epithelium with goblet cells. (D): Cartilage. Scale bar = 100 μm. % V8 E. _+ g5 Q. A; ^ ! P, d; y! L$ J, L8 A, T7 |Karyotype of hESCs on EDF' [7 x! O$ r/ s8 U0 X
& H5 s/ P" w' Y* f. VWe counted 30 karyotypes for the H1 cells after 18 passages on EDF. Each of them contained 46 chromosomes. Of the six samples on which we performed G-band analysis, all of them showed a normal 46,XY karyotype (Fig. 7). 0 ?$ w) H# j: M* s Y4 l @ ' H8 @. d$ g% A9 ]4 UFigure 7. Normal 46,XY karyotype of H1 cells cultured on human embryonic stem cell–derived feeder for 18 passages. . m) _8 N- j, \: h& d8 S0 q$ w: A- R
DISCUSSION& M' [6 f' U& @0 @: Z" ?
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Q.W., Z.F.F., and F.J. contributed equally to this article. The authors would like to thank Jianxin Chu for analysis of the teratomas; Tianlong Gao for assistance with the confocal microscopy, and Youming Zhu and Ailian Liu for technical assistance. We thank Drs. Michal Amit and J. Itskovitz-Eldor for sharing their protocol for hESC karyotyping. The authors would also like to thank Drs. Ray and Gayla Sessions for valuable suggestions on this manuscript. This work was funded by the National Basic Research Program (001CB5099), Projects of Shanghai Science, and Technology Development Foundation (03DJ14017). * O$ ?( _( W4 V" x; C* K2 A, Z/ ~1 T, i6 `8 d/ O b. K) A8 m
DISCLOSURES 8 @: i% q4 E6 J/ B5 C5 b& [8 v$ C! l; i4 g: c+ s
The authors indicate no potential conflicts of interest.2 W+ @! e3 j' \0 E4 \9 B
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