|
  
- 积分
- 0
- 威望
- 0
- 包包
- 483
|
作者：Atsunori Tsuchiyaa,b, Toshio Heikea, Shiro Babaa, Hisanori Fujinoa, Katsutsugu Umedaa, Yasunobu Matsudab, Minoru Nomotob, Takafumi Ichidac, Yutaka Aoyagib, Tatsutoshi Nakahataa作者单位：aDepartment of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan;bDivision of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan;cDepartment of Gastroenterology, Juntendo University School of Medicine, Izunokuni, J ! |" Z9 z) n$ q! s$ X+ ^4 @
z# I# H G- P) [" [9 P- W
5 [ K8 @2 }4 [- |) O- J
( K! I" c w; w" h
5 z( F4 s* T. V: H0 S! c
1 h7 ?/ {5 x, k" g9 p
% X1 k# H! B6 v7 w# J
# Z" O6 s$ c! M0 o
8 D8 t3 I* ~7 L8 E' D3 V
: Z7 X, ]/ f, \ D$ Y: N( h
& N* H$ v; K( f, o7 M. U5 h
3 Y- j7 _) w7 ~+ C3 e
% _; \; B k1 @( r' Y& R 【摘要】0 m9 f& X- ?' a% }7 |8 H
Few studies on the long-term culture of postnatal mouse hepatic stem/progenitor cells have been reported. We successfully adapted a serum-free culture system that we employed previously to expand fetal mouse hepatic stem/progenitor cells and maintained them in culture over long periods. The expanded postnatal cells contained immature -fetoprotein-positive cells along with hepatocytic and cholangiocytic lineage-committed cells. These cells expressed CD49f but not CD45, CD34, Thy-1, c-kit, CD31, or flk-1, and oncostatin M induced their differentiation. This heterogeneous population contained side population (SP) cells, which express the ATP-binding cassette transporter ABCG2, and sca-1 cells. As mice aged, the frequency of SP and sca-1 cells decreased along with the ability of cultured cells to expand. Approximately 20%¨C40% of the SP cells expressed sca-1, but only a few sca-1 cells were also SP cells. Analysis of colonies derived from single SP or sca-1 cells revealed that, although both cells had dual differentiation potential and self-renewal ability, SP cells formed colonies more efficiently and gave rise to SP and sca-1 cells, whereas sca-1 cells generated only sca-1 progeny. Thus, SP cells are more characteristic of stem cells than are sca-1 cells. In regenerating livers, ABCG2 cells and sca-1 cells were detected around or in the portal area (the putative hepatic stem cell niche). The expanded cells share many features of fetal hepatic stem/progenitor cells or oval cells and may be useful in determining the mechanisms whereby hepatic stem cells self-renew and differentiate.9 s; k; M/ D" |
) g3 \1 C9 I |
Disclosure of potential conflicts of interest is found at the end of this article.
0 K, E3 o1 T' j9 D- y# p8 K5 T 【关键词】 Side population cells Sca- cells Serum-free medium Long-term culture Fluorescence-activated cell sorting analysis Hepatic stem cells: t( Z0 e; m9 P% }
INTRODUCTION
: w+ @8 S# `; {- `1 f
, T6 F" ], ?& ^3 f; rSerial cell transplantation studies . However, markers of all of these cells have not been comprehensively assessed, and thus their place in the stem cell developmental hierarchy remains unknown.% @- T, n, ?" \, j, I V
% L7 f3 Z2 p- n4 h1 K- U& [0 U/ W+ ~9 t
Recently, we established a long-term culture system that permits extensive expansion of normal mouse fetal hepatic stem/progenitor cells . This system employs serum-free medium containing B27, hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). This system generates large colonies more readily than previously reported systems. The method utilizes a replating procedure employing diluted trypsin with knockout serum replacement and CaCl2, thus maintaining cell-cell contact and viability. The resulting expanded fetal hepatic stem/progenitor cell populations are a heterogeneous mixture of cells including SP (5%¨C20%) and sca-1 (approximately 15%) cells. Using these two stem cell markers, we could further enrich the population of fetal hepatic stem/progenitor cells.- v$ s/ T0 h" g' Y1 g# E
; O/ B) J9 R3 v; t, rIn this study, we modified the culture system described above and succeeded in expanding postnatal hepatic stem/progenitor cells from undamaged postnatal livers. These cells could be maintained over the long periods by passage and are a heterogeneous mixture of cells that include SP and sca-1 cells. Our previous study suggested that these cells have stem cell characteristics , and, indeed, we show here that both types proliferate and have bipotential differentiation ability. We also analyzed the place in the hierarchy of stem cell development that these cells occupy by purifying single SP and sca-1 cells and evaluating their progeny. Finally, we examined the in vivo expression of the SP phenotype or sca-1 in normal and damaged livers. [/ h& y2 g" G# g- U: G0 `
7 a- D8 ?# E$ ?9 f
MATERIALS AND METHODS) ]- p* U7 A$ |4 [5 H1 H( |
: M& Z, t- ^5 \' pMice2 G) W1 a3 E+ `
, G6 u4 Q% [3 g; {" L$ _! I/ ?C57BL/6 mice (4-, 8-, 12-week-old; n = 5 per age group) were obtained from SLC (Hamamatsu, Japan, http://www.jslc.co.jp). Liver tissue served as the source of postnatal hepatic stem/progenitor cells. Regenerating livers were induced by injecting 4-week-old C57BL/6 mice intraperitoneally three times at 2-day intervals with anti-mouse Fas (0.3 mg/kg) (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) dissolved in phosphate-buffered saline (PBS). Five days after the last injection, regenerating livers were removed and analyzed. Mice were maintained according to Animal Protection Guidelines of Kyoto University (Kyoto, Japan).3 F6 J+ Q( E% e
; F. b* M7 U7 r6 \% J7 F
Hepatic Cell Culture' t1 `8 v+ h+ i, A% S. Q
- ]9 r* s m! k n* y. B. L' KHepatic cells were dissociated using a 2-step collagenase method, collected by centrifugation at 500 rpm for 2 minutes, suspended in Dulbecco's modified Eagle's medium/F12 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) with B27 (Gibco, Grand Island, NY, http://www.invitrogen.com), ITS-X (Gibco), 10 mM HEPES (Nacalai Tesque Inc., Kyoto, Japan, http://www.nacalai.co.jp/en), antibiotics, 20 ng/ml EGF (Sigma-Aldrich), 20 ng/ml bFGF (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), and 10 ng/ml HGF (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), and finally plated onto 6-cm-diameter type 1 collagen-coated dishes (1 x 106 cells per dish). This standard medium was changed every 3 days. On day 14, cells were harvested using diluted trypsin and plated onto newly prepared collagen-coated dishes. On day 21, the colonies that contained more than 200 cells were counted and expanded.9 G8 U9 U- z$ t* Q1 H' P- O1 H
- }4 R' n) Q0 wReverse Transcription-Polymerase Chain Reaction Analysis) w# b" ~0 d2 g' R O9 W
2 X. j& R% h% t! p% H. N9 f5 P5 M, C; i
Total RNA was prepared by using TRIzol (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and reverse-transcribed using a SuperScript first-strand synthesis system (Invitrogen). cDNA was amplified by polymerase chain reaction (PCR) with the AmpliTaq Gold kit (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com), and PCR was performed with primers specific for transcripts encoding the following proteins: the immature cell marker -fetoprotein (AFP); hepatocyte-specific markers albumin (ALB), 1-antitrypsin (1-A), glucose-6 phosphatase (G-6-P) ; and internal control ß-actin (ß-act). The oligonucleotide primers were: AFP: 5'-ACT CAC CCC AAC CTT CCT GTC-3' 5'-CAG CAG TGG CTG ATA CCA GAG-3', ALB: 5'-CAT GAC ACC ATG CCT GCT GAT-3' 5'-CTC TGA TCT TCA GGA AGT GTA-3', 1-A: 5'-TCG ATC CTA AGC ACA CTG AGG-3' 5'-CGG CTT GTA AGA CTG TAG C-3', G-6-P: 5'-AAC CCA TTG TGA GGC CAG AGG-3' 5'-TAC TCA TTA CAC TAG TTG GTC-3', TAT: 5'-TCC AGG AGT TCT GTG AAC AGC-3' 5'-AGT ATA TGG TGC CTG CCT GC-3', TO: 5'-TGC GCA AGA ACT TCA GAG TGA-3' 5'-AGC AAC AGC TCA TTG TAG TCT-3', CK19: 5'-GTC CTA CAG ATT GAC AAT GC-3' 5'-CAC GCT CTG GAT CTG TGA CAG-3', BGP: 5'-GAA CTA GAC TCT GTC ACC CTG-3' 5'-GCC AGA CTT CCT GGA ATA GA-3', and ß-act: 5'-ATC CTG ACC CTG AAG TAC CCC ATT-3' 5'-CCA AGA AGG AAG GCT GGA AAA GAG-3'. The following conditions were employed for amplification: initial denaturation at 95¡ãC for 9 minutes followed by 28 (ALB, AFP, and CK19), 30 (ß-act), or 35 cycles (others) of 94¡ãC for 30 seconds, 56¡ãC for 30 seconds, 72¡ãC for 30 seconds, and a final extension step at 72¡ãC for 10 minutes. PCR products were then separated on 1.0% agarose gels.
. l5 G" z1 J$ a( H9 b2 l5 ~ R7 h& f- r2 z3 p. T' w, K
Flow Cytometry+ r2 n# F/ z4 a
7 R- X! \( ~( V6 n0 J2 Y' `
For fluorescence-activated cell sorting (FACS), we used the following markers: rat and human oval cell markers CD34 . After washing, the cells were analyzed or sorted by using FACSCalibur, BD LSR, or FACSVantage with the CellQuest program (Becton, Dickinson and Company). To analyze the relative developmental hierarchy of putative stem cells, expanded cells derived from 4-week-old mice were fractionated into four subpopulations using FACSVantage: SP sca-1¨C cells (group 1), SP sca-1 cells (group 2), main population (MP) sca-1 cells (group 3), and MP sca-1¨C cells (group 4). Our usual sorting procedure yielded purities of approximately 90%¨C95%. Single cells of each fraction were cultured in individual wells of 96-well type 1 collagen-coated dishes and confirmed microscopically. On day 14 after sorting, large colonies occupying more than 30% of the well surface were counted. Finally, five colonies representing SP sca-1¨C cells (group 1) and MP sca-1 cells (group 3) were randomly collected and expanded. On day 24 after sorting, the frequency of SP cells and sca-1 cells was determined and reverse transcription (RT)-PCR was performed with AFP, ALB, and CK19 primers." ~ _; _ }) m/ R/ R
1 d' F% y/ D/ \! j! J+ |4 w6 n! v7 \
Differentiation Culture Condition
" ?' i/ }! m# ?# X ~& L
+ |+ O2 H' L' k) v) fTo induce the differentiation of hepatocytic lineage cells, cells from 4-week-old mice that had been expanded for more than 6 weeks were employed. Expanded cells (5 x 105 cells per milliliter) were cultured with standard medium plus 10 ng/ml oncostatin M (OSM; R&D Systems) were performed. On day 9, the frequencies of SP cells, and sca-1 cells were determined using FACSCalibur, BD LSR, and FACSVantage.8 S7 A6 |8 F3 X
( q% y9 m' w/ |' `! C1 C+ T; f% s
Immunochemical Staining
5 S9 C6 p$ f8 m) `6 @: K4 C# z: S( q9 e5 ^# E
Immunocytochemistry was performed as described previously . Slides were then stained by using Histofine Simple Stain Max-PO (Nichirei, Tokyo, http://www.nichirei.co.jp/english/index.html) and DAB Substrate Kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) for sca-1, Vectastain ABC Kit and DAB Substrate Kit (Vector Laboratories) for anti-ABCG2 and anti-cow CK, and DAB Substrate Kit, M.O.M Kit, and Vectastain ABC Kit (Vector Laboratories) for anti-CK19. Nuclei were stained using Mayer's hematoxylin solution (Wako Chemical).
+ _/ ^8 J+ T; l& e# Q& C
$ k0 Y+ z, x' V; f4 x' zStatistical Analysis
) z3 \1 F4 c. A Q
5 I7 P1 o! l7 Y. R7 i: G+ E" q% l: gThe data are presented as mean ¡À SD. Student's t test was used to determine the statistical significance of observed differences.& X+ U: ]) S: j# _1 T- g
6 P+ L0 ^7 r7 H" A( J' @
RESULTS2 L1 K+ j- M1 P( m
/ ^2 R4 P2 k' Z' O+ C5 R$ MEffective Expansion of Postnatal Hepatic Stem/Progenitor Cells and Analysis of Their Characteristics
* @6 O5 f* D8 }- [4 J8 a- F) I8 X
$ M( n8 i1 e- U0 rWe previously showed that we could extensively expand fetal hepatic stem/progenitor cells using our standard medium . Therefore, we sought to expand postnatal hepatic stem/progenitor cells using the same medium. Hepatic cells originated from 4-, 8-, and 12-week-old male mice (n = 5, in each group). Initially, we directly cultured sorted cells obtained after liver dissociation; however, this procedure led to poor cell viability. Subsequently, we centrifuged a whole cell preparation at 500 rpm for 2 minutes, since our early studies showed that single fetal liver stem/progenitor cells that were expanded with our culture system could be collected by this centrifugation step. The resulting pellet consisted largely of mature hepatocytes, and the supernatant contained nonparenchymal cells. When the supernatant was cultured, the nonparenchymal cells, such as endothelial cells, hematopoietic cells, and stellate cells, showed rapid growth, but extensive expansion of hepatic stem/progenitor cells was not observed (data not shown). By contrast, when 1 x 106 pellet cells were plated on 6-cm type 1 collagen-coated dishes, rapid growth of endothelial cells, hematopoietic cells, and stellate cells was not detected, but rather expansion of hepatic stem/progenitor cells was observed. The plated hepatic cells first attached to dishes as a monolayer, and some formed floating and attaching spheroids that did not grow. By day 14, after repeated medium changes at 3-day intervals, most cells forming monolayer sheets or spheroids had died, leaving small polygonal living cells (Fig. 1A). On day 14, the cells were passaged for the first time using diluted trypsin, which maintains cell-cell contact. Harvested cells were replated on collagen-coated dishes using our standard medium. On day 21, we determined the frequency of the dishes containing large colonies (more than 200 cells) and found that the frequency of the dishes obtained from the 4-, 8-, and 12-week-old mice was 40%, 20%, and 0%, respectively. During subsequent culturing, these large colonies gradually expanded (Fig. 1B, 1C), and by day 45, stable expansion of cells was observed (Fig. 1D). These stable expanding cells formed heterogeneous populations containing immature AFP cells, hepatocytic lineage cells (ALB cells), and cholangiocytic lineage cells (CK7 cells) (Fig. 1E, 1F). The expanded cultures maintained these phenotypes after further passages. AFP cells were relatively smaller than the ALB cells and the CK7 cells (Fig. 1E, 1F). Notably, whereas cells from 4-week-old mice could be maintained for over 30 passages, cells from 8-week-old mice could only be maintained for approximately 20 passages and then gradually lost their ability to expand. Flow cytometric analysis of expanded heterogeneous day 45 cells revealed that they expressed CD49f ( low) but not CD45, CD34, Thy-1, c-kit, CD31, or flk-1 (Fig. 2A). This was true for both 4- and 8-week-old mouse-derived cultures. SP cells and/or sca-1 cells were also found in heterogeneous expanded cell populations; however, the frequencies of SP cells and sca-1 cells in the population varied depending on the age of the donor mouse. Thus, whereas 2%¨C4% and 15%¨C40% of the expanded cells from 4-week-old mice were SP and sca-1 , respectively, cell populations from 8-week-old mice showed fewer sca-1 cells (approximately 1%) and no SP cells (Fig. 2B, 2C). Thus, our culture system can successfully generate long-term postnatal hepatic cell cultures containing cells expressing immature markers.
7 L$ M% A6 G& U) _9 a
$ b5 m- t4 ?7 j6 CFigure 1. Culture and immunocytochemistry of expanded cells. (A¨CD): Time course of cell culture. These images were obtained from cultured cells from 4-week-old mice. (A): Cultures on day 14 just prior to the first passage: some small expanding cells can be seen. (B, C): Cultures on day 30: large colonies have formed. (D): Cultures on day 45: stably expanding cells can be detected. (E, F): Analysis of expanding cells shown in (D) with regard to -fetoprotein (AFP) expression (, x200).- l, u5 S5 l8 P4 ~4 K7 B. D
' B) \+ J5 y. T$ H+ F; J
Figure 2. Flow cytometric analysis of expanded cells. (A): Flow cytometric analysis of day 45 expanded cells showing that these heterogeneous cells are CD45¨C, CD34¨C, Thy-1¨C, c-kit¨C, CD31¨C, flk-1¨C, and CD49f low . Sca-1 cells are also detected. (B, C): Day 45 expanded cells originating from 4-week-old mice include side population cells (B), unlike cultures derived from 8-week-old mice (C). Abbreviations: 4W, 4-week-old mice; 8W, 8-week-old mice; APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.
: i5 R; f: {+ B3 J$ O! @2 J3 {! A( Y8 l3 B: Z5 n. L2 G
Differentiation Induced by Oncostatin M/ |8 y3 F: z. v8 I1 x9 b
8 d$ v7 s) `9 U. }8 O# v; _To determine whether expanded cells can differentiate into cells expressing hepatocytic lineage differentiation markers, we employed OSM treatment. Cultured cells from 4-week-old mice that had been expanded for over 6 weeks were differentiated in standard medium containing OSM. RT-PCR, PAS staining, and immunocytochemistry were performed 6 days later and flow cytometric analysis was performed on day 9. Before cells were treated with OSM, they expressed genes encoding AFP, ALB, 1-A, G-6-P, CK19, and BGP but did not express the hepatocytic differentiation markers TAT and TO (Fig. 3A). However, RT-PCR analysis revealed that TAT and TO mRNAs were induced by day 6 after OSM treatment (Fig. 3A). PAS staining and immunocytochemistry for CPSI also revealed relatively large PAS-positive (Fig. 3B, 3C) and CPSI-positive (Fig. 3D, 3E) cells on day 6. Thus, expanded cells can differentiate into mature hepatocytes. Flow cytometric analysis on day 9 revealed that OSM treatment reduced the growth rate by approximately 85% compared with untreated controls (data not shown). Interestingly, although 9 days of OSM treatment reduced the frequency of SP cells by approximately 45% (Fig. 3F, 3G), it increased the frequency of sca-1 cells by approximately 50% compared with the untreated controls (Fig. 3H).) J6 W* H9 ]7 w$ S* A- c- P5 o
5 R( x1 I, |8 {4 z2 I4 [: H# O
Figure 3. Induction of differentiation by oncostatin M (OSM) treatment. (A): Before adding OSM, expanded cells continued to express genes encoding AFP, ALB, 1-A, G-6-P, CK19, and BGP (lane 1). However, on day 6 after addition of OSM, hepatocytic differentiation markers TAT and TO are induced (lane 2). (B¨CE): Compared with untreated cells (B, D), OSM treatment generates cells strongly positive for periodic acid-Schiff (C) and carbamoyl phosphate synthetase I (, x200). Abbreviations: 1-A, 1-antitrypsin; ß-act, ß-actin; AFP, -fetoprotein; ALB, albumin; BGP, biliary glycoprotein; CK, cytokeratin; G-6-P, glucose-6 phosphatase; TAT, tyrosine amino transferase; TO, tryptophan-2,3-dioxygenase.3 O) A/ @( }0 ^/ Y1 s7 I
' ?3 U, ~: D% \8 G% x
SP Cells Are More Likely to Be Stem Cells Than Are Sca-1 Cells# v0 ]' a" _" Q& D( E8 `
, W- }8 r6 a/ l+ R
Next, we asked whether SP cells and/or sca-1 cells in our culture have stem cell characteristics and whether one of these two cell populations is derived from the other. Initially, we compared the size of these cells and the frequency of cells that showed both the SP attribute and expressed sca-1. Flow cytometric analysis revealed that SP cells were relatively smaller than sca-1 cells (Fig. 4A, 4B, 4D, 4E). Moreover, 20%¨C40% of the SP cells were also sca-1 , whereas less than 5% of the sca-1 cells were SP cells (Fig. 4A, 4C, 4D, 4F). Immunocytochemical analysis revealed that, although sca-1 cells were rarely also AFP and ALB cells, more than 20% of sca-1 cells were also CK7 (Fig. 4G¨C4I).5 I) O0 X7 Q; M. V' @
% w$ ?1 D. A9 c, U+ K5 m. a! ]7 K0 FFigure 4. Flow cytometry, immunocytochemistry, and RT-PCR of SP and sca-1 cells in the expanded populations derived from 4-week-old mice. (A¨CF): SP cells (A, B) are smaller than sca-1 cells (D, E), and 20%¨C40% of SP cells are sca-1 (A, C), whereas less than 5% of sca-1 cells exhibit the SP phenotype (D, F). Immunocytochemistry shows that, whereas sca-1 cells (, x100). Abbreviations: AFP, -fetoprotein; ALB, albumin; CK, cytokeratin; FSC, forward scatter; MP, main population; PI, propidium iodide; RT-PCR, reverse transcription-polymerase chain reaction; SP, side population; SSC, side scatter.8 i& {9 e5 h, ?& K/ ?0 Z
# p1 v! I i9 k3 iNext, to determine whether sca-1 cells are derived from SP cells or vice versa, expanded cells were fractionated into four groups, namely SP sca-1¨C (group 1), SP sca-1 (group 2), MP sca-1 (group 3), and MP sca-1¨C (group 4), and subjected to single cell culture analysis. Fourteen days after sorting, large colonies occupying more than 30% of the well surface were counted. The frequencies of large colonies per 300 fractionated cells were 42.6 ¡À 10.5 (group 1), 37.6 ¡À 2.3 (group 2), 20.3 ¡À 8.6 (group 3), and 8.6 ¡À 1.2 (group 4) (Fig. 4J). Thus, SP cells formed colonies more readily than did sca-1 cells. To analyze further the relationship between SP and sca-1 cells, five large colonies from group 1 (SP sca-1¨C) and group 3 (MP sca-1 ) were randomly picked and cultured. On day 24, after being sorted, cells were subjected to flow cytometry to determine the frequencies of SP and sca-1 cells and analyzed for expression of AFP, ALB, and CK19. Flow cytometric analysis revealed that, whereas the SP sca-1¨C cells gave rise to both SP cells and sca-1 cells, MP sca-1 cells produced mainly sca-1 cells and rarely produced SP cells (Fig. 4K). RT-PCR also revealed that, whereas the progeny of either SP sca-1¨C or MP sca-1 cells expressed genes encoding ALB and CK19, cells derived from SP sca-1¨C cells expressed higher levels of AFP than those derived from MP sca-1 cells (Fig. 4K). These results indicate that, although SP cells and sca-1 cells both have self-renewal ability and dual differentiation potential, SP cells are more immature than sca-1 cells and give rise to sca-1 cells.; @: r, o4 C) z" h
& U& Z$ T. g5 b* X5 i; x2 X5 x
SP Cells and Sca-1 Cells Reside in the Periportal Area of Regenerating Livers6 U( \& R+ B: a/ k6 k
$ @9 H; `% z0 T! l6 }' C/ U! dWe next determined whether cells expressing SP and sca-1 stem cell markers are found in normal or regenerating livers by searching for ABCG2-expressing and sca-1-expressing cells. SP phenotype (the ability to exclude dyes) arises from the expression of the ATP-binding cassette transporter ABCG2/BCRP1 , and thus SP cells in the liver were identified on the basis of their ABCG2 expression. To induce immature hepatic cells, 4-week-old mice were injected three times at 2-day intervals with anti-mouse Fas (0.3 mg/kg) dissolved in PBS. Five days after the last injection, regenerating livers were removed and analyzed. Many ductular proliferating cells that were positive for anti-cow CK and exhibiting oval cell morphology were apparent (Fig. 5A¨C5D). The luminal surface of the ductular proliferating cells in the portal area strongly expressed ABCG2 (Fig. 5F). By contrast, in normal liver, only the canalicular membrane of hepatocytes weakly expressed ABCG2 (Fig. 5E). With regard to sca-1, sca-1 positivity in liver was not restricted to hepatic stem/progenitor cells but was also seen in endothelial cells, hematopoietic cells, and other cells including hepatic stem/progenitor cells (data not shown). Endothelial cells around the portal area in the regenerating liver strongly expressed sca-1 (Fig. 5H), whereas the same cells in the normal liver showed much weaker expression (Fig. 5G). In the regenerating liver, although sca-1-expressing cells were mainly endothelial cells, some terminal bile duct cells and interlobular bile duct cells also expressed sca-1 (Fig. 5H). Ductular proliferating cells expressing ABCG2 rarely expressed sca-1 (data not shown). These observations indicate that the portal area is a likely hepatic stem cells niche.; g0 g# Z) M8 U) V! P0 j# i# Z
) I4 T. j0 T- z5 i
Figure 5. Immunohistochemical analysis of cytokeratin (CK)19, ABCG2, and sca-1 expression in normal and regenerating livers. (A, B): Histological analysis of normal liver (A) and regenerating liver damaged by anti-mouse Fas (B). Many ductular proliferating cells resembling oval cells are detected in damaged but not normal livers (B). (C, D): Immunohistochemical analysis of damaged liver indicates CK19 bile duct cells (C) and ductular proliferating cells positive for anti-cow CK, which detects bile ducts and oval cells (, x400).- V4 ?: _3 c( M* t6 w# p) O
! H7 T: m2 a+ K8 c. C' VDISCUSSION R; f2 P/ G! H# C
9 k" M2 f/ H0 s" ]
Our Culture System Enables Expansion of Postnatal Hepatic Stem/Progenitor Cells In Vitro
" Z& `5 l3 @6 g5 T6 I) k+ |7 P
3 v/ E1 ^+ A" ITo date, many reports describe expansion of fetal hepatic stem/progenitor cells but few address expansion of postnatal hepatic stem/progenitor cells. Moreover, long-term expansion with passage of normal postnatal mouse hepatic stem/progenitor cells has not been achieved. In addition, phenotype of postnatal hepatic stem/progenitor cells and how these cells are related developmentally to other stem cells have not been thoroughly analyzed. Recently, we established a culture system that permits the extensive expansion of mouse fetal hepatic stem/progenitor cells. This culture system allows large colonies to be subcultured in a way that maintains cell-cell contact. Subculturing of postnatal hepatic stem/progenitor cells is a major impediment to their long-term expansion. Here, when we used our culture system with postnatal hepatic stem/progenitor cells, we could expand and maintain them over multiple passages. Notably, whereas fetal hepatic stem/progenitor cells easily form expanding spheroids, which then form large monolayer colonies after being plated on collagen, postnatal hepatic cells did not form expanding spheroids and generated monolayer colonies at an extremely low rate (Table 1). Such rare large colonies became increasingly rarer as the age of donor mice increased (Table 1). Nevertheless, our ability to expand postnatal hepatic cells with this culture system indicates that the postnatal liver contains cells with extensive growth ability.. L# |' d1 J4 H
* [' F! i) `" V& E- l) W
Table 1. Comparison of expanded cells derived from postnatal livers with those derived from fetal livers
: K$ ?4 t" R9 J; i8 a* Z. |, Y( s3 e# Z" ]! B& e. ~
Expanded Cells Contain SP and Sca-1 Cells in a Developmental Hierarchy
7 Z4 ?$ z f1 @1 y) s
/ S+ X1 ?, j- j3 M1 y$ D8 MThe expanded postnatal liver cells were heterogeneous populations containing immature AFP cells, hepatocytic lineage cells, and cholangiocytic lineage cells. Flow cytometric analysis of these heterogeneous cells revealed that they expressed CD49f ( low ) but not CD45, CD34, Thy-1, c-kit, CD31, or flk-1. Such marker expression indicates that this cell population is similar to previously reported hepatic stem cells .3 F' Y: Q" P0 H/ Q$ [
% B$ g9 y- S4 v8 O' c% bHepatic Stem Cells with Highly Regenerative Potential Decrease with Age& e5 {+ M; [; ]9 R$ d: l
+ M7 k6 c, e) c" Y' xIn comparing the number of SP and sca-1 cells, we found that there were fewer SP cells in the expanded postnatal hepatic stem/progenitor cell population (2%¨C4%) than in expanded fetal hepatic stem/progenitor cell populations (more than 5%). This observation suggests that the number of stem cells decreases with age. This idea is supported by observations that (a) SP cells could be expanded from livers of 4-week-old but not 8- or 12-week-old mice, (b) the expanded population from the livers of 8-week-old mice contained fewer sca-1 cells (approximately 1%) than the 4-week-old liver derived population (15%¨C40%), and (c) although cells originating from 4-week-old mice could be maintained for over 30 passages, cells from 8-week-old mice could only be maintained for about 20 passages and gradually lost their ability to expand. Thus, we estimate that the number of stem cells with the greatest regenerative ability decreases with age as does liver regenerative potential.
$ w9 h- f; q( D" s2 U" @- D/ H" N c' E4 n/ ~0 M' P9 ^, D g4 Q: M
The Periportal Area Is the Niche of Hepatic Stem/Progenitor Cells, I; S. M( @% f% t
( I6 h5 w. T% g1 \) ]We next analyzed which cells in the normal or regenerative livers of 4-week-old mice express the SP cell marker ABCG2 and which express sca-1. In the normal liver, some mature hepatocytes express ABCG2, although the main expression of sca-1 was by the endothelial cells around the portal area. In contrast, in regenerating livers, ductular proliferating cells expressed ABCG2 and some bile duct cells expressed sca-1. These results reveal that ABCG2 and sca-1 cells exist in the portal area (the putative hepatic stem cell niche). However, the distribution of ABCG2 and sca-1 cells differed. In particular, ABCG2 cells were found in the vicinity of the canal of Hering, supporting a previous report suggesting that hepatic stem cells are located near this location. Notably, our immunocytochemical analysis revealed that sca-1 cells also tended to express CK7 more often than AFP and ALB, suggesting that sca-1 cells are more likely to be of the cholangiocytic than the hepatocytic lineage.
- y7 ~& H; `% o; u5 B1 z. F/ N- }+ h
Y. _: s# K8 `9 f0 \9 a6 r J0 Q2 YUtility of Our Expanded Cell System( A, R Z( |) b" @5 ]) K- }9 w$ {/ g
* ^" v7 w' A3 m8 W: {Our expanded cell populations may be useful in analysis of self-renewal and differentiation of hepatic stem cells. Recent studies showed that, although embryonic stem cells are attractive hepatic stem cell sources for such study, this cell source is problematic because it is difficult to differentiate highly enriched populations of hepatic cells. By contrast, our culture system expands only hepatic cells, making it ideal for characterizing various hepatic stem/progenitor cells in the liver and for assessing their relative position in a developmental hierarchy.! |1 c- z* `2 H: `
x9 u. ~- X5 ^DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST& O* Z/ J, a3 J/ R
/ a; _1 W8 ~% E/ s3 ` ]3 u( S: c
The authors indicate no potential conflicts of interest.
, S) T1 ?: T9 k/ h3 H5 {# E& }
8 z$ W' ?& C" y. z9 N EACKNOWLEDGMENTS
8 r7 S* G8 ~+ y0 h/ X3 t7 g9 n/ G* o
This work was supported by a Grant-in-Aid for Creative Scientific Research (13GS0009) and a Grant-in-Aid for Scientific Research (B) (15039219) from the Ministry of Education, Science, Technology, Sports and Culture of Japan.
6 l( ^& a3 M0 \4 x+ v/ P 【参考文献】
$ @: M" W0 a' K4 x+ r* v# p 5 Y$ i+ b# o4 D
- q4 O7 W3 ]6 b q1 Z g0 R
Rajvanshi P, Kerr A, Bhargava KK et al. Efficacy and safety of repeated hepatocyte transplantation for significant liver repopulation in rodents. Gastroenterology 1996;111:1092¨C1102.- `! @. _3 ~4 ?# F: X3 |
! s; c# W+ t+ x
Overturf K, al-Dhalimy M, Ou CN et al. Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol 1997;151:1273¨C1280. e. K3 \/ W! R% V6 F" L- |5 T0 ?
- M1 L6 M3 l8 T. Y3 R
Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229¨C1234.
+ D: W" @$ f1 l/ x& C$ x5 N( H$ u' c( T
Ruhnke M, Ungefroren H, Nussler A et al. Differentiation of in vitro-modified human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells. Gastroenterology 2005;128:1774¨C1786.
2 n8 \' N3 o" I# C2 b% h* \% `& O! R# Y4 v9 }+ N1 I
Omori N, Omori M, Evarts RP et al. Partial cloning of rat CD34 cDNA and expression during stem cell-dependent liver regeneration in the adult rat. Hepatology 1997;26:720¨C727.3 v1 z4 ~1 P$ n+ {" ~5 Z
7 H' C( R& z# h' A" S/ r# J! `
Crosby HA, Kelly DA, Strain AJ. Human hepatic stem-like cells isolated using c-kit or CD34 can differentiate into biliary epithelium. Gastroenterology 2001;120:534¨C544.
9 T& H+ W6 T5 E/ S. P4 ]
$ P# W# z9 Z6 s2 rMatsusaka S, Tsujimura T, Toyosaka A et al. Role of c-kit receptor tyrosine kinase in development of oval cells in the rat 2-acetylaminofluorene/partial hepatectomy model. Hepatology 1999;29:670¨C676.
" [# ~; y! a1 w. Y3 N
1 X6 j, i7 U; ^2 M0 T/ {Fujio K, Evarts RP, Hu Z et al. Expression of stem cell factor and its receptor, c-kit, during liver regeneration from putative stem cells in adult rat. Lab Invest 1994;70:511¨C516.* F* n% x2 F8 i3 s1 y; a3 i$ E# m9 F7 R
6 ~8 P. \# D: m% E4 O/ g
Blakolmer K, Jaskiewicz K, Dunsford HA et al. Hematopoietic stem cell markers are expressed by ductal plate and bile duct cells in developing human liver. Hepatology 1995;21:1510¨C1516.) E6 y( N" Y, d6 Z
/ H. @& F |: F2 FPetersen BE, Goff JP, Greenberger JS et al. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 1998;27:433¨C445.$ [) O' ~0 I8 n; C
9 ~7 n/ t5 V0 `' c- r
Petersen BE, Grossbard B, Hatch H et al. Mouse A6-positive hepatic oval cells also express several hematopoietic stem cell markers. Hepatology 2003;37:632¨C640.9 ] G& Z2 L2 h) B3 r0 t+ p' a
1 B M; I3 L3 h! h
Tsuchiya A, Heike T, Fujino H et al. Long-term extensive expansion of mouse hepatic stem/progenitor cells in a novel serum-free culture system. Gastroenterology 2005;128:2089¨C2104.; z$ K. G3 Q3 A
) x2 q8 l3 w3 g
Shimano K, Satake M, Okaya A et al. Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1. Am J Pathol 2003;163:3¨C9.
" R8 `2 t* v! M4 g6 x
) u4 [8 {3 d9 a, N( Y+ XBordoni V, Alonzi T, Agrati C et al. Murine hepatocyte cell lines promote expansion and differentiation of NK cells from stem cell precursors. Hepatology 2004;39:1508¨C1516.
# T5 J% p1 ` o, D" S& u& y: b( l8 ~
Spagnoli FM, Amicone L, Tripodi M et al. Identification of a bipotential precursor cell in hepatic cell lines derived from transgenic mice expressing cyto-Met in the liver. J Cell Biol 1998;143:1101¨C1112.& L( h3 p) X+ K- Z
, E4 \) S+ p. J8 d! m$ G0 {1 _Dumble ML, Croager EJ, Yeoh GC et al. Generation and characterization of p53 null transformed hepatic progenitor cells: Oval cells give rise to hepatocellular carcinoma. Carcinogenesis 2002;23:435¨C445.
& x+ o0 h$ V3 K" l3 y% m6 e" K4 ^1 J6 E3 |0 X
Azuma H, Hirose T, Fujii H et al. Enrichment of hepatic progenitor cells from adult mouse liver. Hepatology 2003;37:1385¨C1394.& O$ N/ }/ H+ U! ~ b; {' q8 n
3 z+ c5 y) M$ IBlock GD, Locker J, Bowen WC et al. Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium. J Cell Biol 1996;132:1133¨C1149.
1 ?' J0 U7 W3 N3 a0 A' T5 P- K5 m- Z- S6 I
Parent R, Marion MJ, Furio L et al. Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology 2004;126:1147¨C1156.; V6 [; T2 L8 f
1 |& [, c- U8 G" s
Tsao MS, Smith JD, Nelson KG et al. A diploid epithelial cell line from normal adult rat liver with phenotypic properties of ¡®oval' cells. Exp Cell Res 1984;154:38¨C52.6 [) `* e [, C7 D( f: |* w8 s% \
8 P5 l; U8 t1 \, XSanchez A, Factor VM, Espinoza LA et al. In vitro differentiation of rat liver derived stem cells results in sensitization to TNFalpha-mediated apoptosis. Hepatology 2004;40:590¨C599.
3 |* g* o! [+ r/ T+ I5 w7 E a' ^8 ^6 R, \4 Z" n r
Nagai H, Terada K, Watanabe G et al. Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells. Biochem Biophys Res Commun 2002;293:1420¨C1425.
/ K# y7 b w* M7 ^; u* m
" B" V/ f% B$ SOh BK, Lee CH, Park C et al. Telomerase regulation and progressive telomere shortening of rat hepatic stem-like epithelial cells during in vitro aging. Exp Cell Res 2004;298:445¨C454.! C' c3 t4 r. S0 {; }
1 `# ~0 W' ~* \0 Z2 Z. ]; ^Eichhorst ST, Krueger A, Muerkoster S et al. Suramin inhibits death receptor-induced apoptosis in vitro and fulminant apoptotic liver damage in mice. Nat Med 2004;10:602¨C609.+ P9 O4 i8 M0 b* i
+ Z3 B4 |' U+ B; j6 K6 S$ @# ]# ^Suzuki A, Zheng Y, Kondo R et al. Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. Hepatology 2000;32:1230¨C1239.
& R6 _9 p0 n* `
( _9 M- g- t5 `- C' [3 |Hoppo T, Fujii H, Hirose T et al. Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver. Hepatology 2004;39:1362¨C1370.; Y [2 `1 I+ h$ o+ U
0 x1 W* ?' b; }7 Y! V8 h* p& F: h5 ]1 FUmeda K, Heike T, Yoshimoto M et al. Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro. Development 2004;131:1869¨C1879." |4 o6 `6 o9 D& A. T; ]" L) c2 }1 {
; M6 x& C% G) S) z! T/ AMedina J, Arroyo AG, Sanchez-Madrid F et al. Angiogenesis in chronic inflammatory liver disease. Hepatology 2004;39:1185¨C1195.$ T' h1 z( C7 z9 |; c# h
' p4 D9 l; i* w
Iida M, Heike T, Yoshimoto M et al. Identification of cardiac stem cells with FLK1, CD31, and VE-cadherin expression during embryonic stem cell differentiation. Faseb J 2005;19:371¨C378.( E6 k5 w; s3 D1 p7 M
( q a+ G4 w, ^/ f- |Alison MR. Tissue-based stem cells: ABC transporter proteins take centre stage. J Pathol 2003;200:547¨C550.) @4 Y" T0 e$ \7 D
0 V) T0 C0 g% J: E( ^( P8 Y
Kamiya A, Kinoshita T, Ito Y et al. Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer. Embo J 1999;18:2127¨C2136.
# z# Y) ?3 p6 k1 K1 z% ?$ M9 ?' P& k
Miyajima A, Kinoshita T, Tanaka M et al. Role of Oncostatin M in hematopoiesis and liver development. Cytokine Growth Factor Rev 2000;11:177¨C183.! b: T2 I7 _% Y# ~
- }9 c Z: B% D: c
Strick-Marchand H, Morosan S, Charneau P et al. Bipotential mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes. Proc Natl Acad Sci U S A 2004;101:8360¨C8365.! D6 H+ T' R1 e6 n# v: @
7 A5 U: q/ K# d% H* X/ |& v
Minguet S, Cortegano I, Gonzalo P et al. A population of c-Kit(low)(CD45/TER119)- hepatic cell progenitors of 11-day postcoitus mouse embryo liver reconstitutes cell-depleted liver organoids. J Clin Invest 2003;112:1152¨C1163.$ S% A! `/ T' a
+ X' Z/ v, g8 S/ y Q
Kofman AV, Morgan G, Kirschenbaum A et al. Dose- and time-dependent oval cell reaction in acetaminophen-induced murine liver injury. Hepatology 2005;41:1252¨C1261.& Y9 _' y. P: @
' n; ~4 g' _8 @9 R' _' [Zhou S, Schuetz JD, Bunting KD et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028¨C1034.
5 Q4 G+ y) }( A6 k! W5 B u9 h1 d5 A* f9 m6 B6 Q0 F
Malhi H, Irani AN, Gagandeep S et al. Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. J Cell Sci 2002;115:2679¨C2688.7 q' r% ^) U! v5 V* b
3 Y' a& W [/ u! w/ q4 |Strick-Marchand H, Weiss MC. Inducible differentiation and morphogenesis of bipotential liver cell lines from wild-type mouse embryos. Hepatology 2002;36:794¨C804.; x5 X; t/ T( z) Z
" H" ^& r4 k. y1 l
Lazaro CA, Croager EJ, Mitchell C et al. Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes. Hepatology 2003;38:1095¨C1106.! [: j. F3 |6 \& B. q4 t9 b9 |
* n9 c2 l, _# l ^& e
Kamiya A, Gonzalez FJ, Nakauchi H. Identification and differentiation of hepatic stem cells during liver development. Front Biosci 2006;11:1302¨C1310.
) x3 I; P, C5 m3 X* r9 w* |# Q3 r* f# ]2 s0 `
Teratani T, Yamamoto H, Aoyagi K et al. Direct hepatic fate specification from mouse embryonic stem cells. Hepatology 2005;41:836¨C846.8 o9 W4 Y; T& E
5 y, P. q9 _7 MYamamoto H, Quinn G, Asari A et al. Differentiation of embryonic stem cells into hepatocytes: Biological functions and therapeutic application. Hepatology 2003;37:983¨C993. |
|
发表于 2009-3-5 00:56
发表于 2009-5-6 23:40
