Somatic Stem Cell Marker Prominin-1/CD133 Is Expressed in Embryonic Stem Cell–De - 《stemcells》2004-2005文献干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 25 下一页

返回列表

查看: 285740\|回复: 240	go Somatic Stem Cell Marker Prominin-1/CD133 Is Expressed in Embryonic Stem Cell–De [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 10:50 |只看该作者 |倒序浏览 |打印

a Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany;
5 N4 H0 f, ?: ^% u  \9 J8 `
b Medical Clinic and Polyclinic I, Carl Gustav Carus University, Dresden, Germany;0 K. Y* G7 b4 r* c6 H, R

c Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany;

d Laboratory of Cardiovascular Science, National Institute on Aging, NIH, Baltimore, Maryland, USA3 z$ F% b' Y& Q, H
  A) h  f/ U- l# p% u
Key Words. Mouse ? Embryonic stem cells ? Embryonic carcinoma cells ? Blastocysts ? Prominin-1 ? Nestin ? Nanog ? Neuronal ? Differentiation
4 e7 d% f* M3 Y4 z0 k
Correspondence: Anna M. Wobus, Ph.D., In Vitro Differentiation Group, Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany. Telephone: 49-39482-5256; Fax: 49-39482-5481; e-mail: wobusam@ipk-gatersleben.de

ABSTRACT
8 [5 p7 D) l, K8 o
Prominin-1 (CD133 or, for the human orthologue, AC133 antigen)  is a 115-/120-kDa cholesterol-binding glycoprotein  that belongs to a growing family of pentaspan membrane proteins expressed throughout the animal kingdom  (for review, see ). Prominin-1 is expressed on several primitive cells such as hematopoietic stem and progenitor cells derived from bone marrow, fetal liver, and peripheral blood , neural and endothelial stem cells , and developing epithelium  (for review, see ). The expression of prominin-1 is not limited to stem and progenitor cells but also occurs in adult epithelial cells, e.g., kidney proximal tubules , and in nonepithelial cells, notably rod photoreceptor cells .# \& E  g. L  a/ ^; X

Irrespective of cell type, prominin-1 is specifically associated with plasma membrane protrusions , and it binds to plasma membrane cholesterol . In epithelial cells, prominin-1 is selectively concentrated in microvilli present at the apical plasma membrane. The apical localization of prominin-1 is independent of the presence of functional tight junctions , which are known to prevent the lateral movement of transmembrane proteins and lipids of the extracellular membrane leaflet between the apical and basolateral plasma membrane . It was suggested that the specific retention of prominin-1 in microvilli may play an important role in the establishment and maintenance of apical-basal polarity of epithelial cells . Although the physiological function of prominin-1 has not yet been established, recent studies using biochemical, morphological, and genetic approaches suggest a role for this glycoprotein in the morphogenesis or physiology of plasma membrane protrusions .

Until now, prominin-1 was only described to be associated with somatic stem or progenitor cells. The in vitro models of undifferentiated embryonic stem (ES) and embryonic carcinoma (EC) cells allowed us to investigate prominin-1 expression in pluripotent ES cells and during in vitro differentiation. Mouse blastocysts were used to comparatively analyze the expression and localization of prominin-1 in early embryo-derived cells in vivo. The specific expression pattern suggested prominin-1 as a new marker to define ES-derived committed and early progenitor cells in vitro." d9 G. R! M% q8 i; T
( s! l! k- i. w, I* l" r" [1 g
MATERIALS AND METHODS

Transcript Abundance of Prominin-1, Nanog, and Nestin
- M$ s) q: y/ X( ^9 @' D
Prominin-1 transcripts were assayed as a function of the differentiation status in undifferentiated ES and EC cells, in cells committed to differentiation (48 hours), and after differentiation. Given the strong expression of prominin-1 in kidney , this tissue was used as positive control (i.e., 100%). Nanog was used as a marker for undifferentiated ES cells, whereas nestin was used for committed and neural progenitor cells.

In R1 ES cells cultured for 6 hours after passage (without FL), a low level of prominin-1 transcript was observed (~40% of the positive control), but the amount doubled when the cells were kept in an undifferentiated state for up to 48 hours (Fig. 1). The amount of prominin-1 transcript was also elevated in R1 colonies cultured for 7 days and in early EBs (5 days; 5 2 days), but it slightly decreased in EB outgrowths differentiated for 9 and 16 days and after neuronal differentiation (stages 4 8 days and 4 32 days). Interestingly, the cultivation of R1 cells for 7 and 21 days in the absence of LIF and spontaneous differentiation of EB outgrowths for 23 days reduced the prominin-1 transcript level twofold to threefold (Fig. 1). In P19 EC cells, the maximum level of prominin-1 transcript was found in cells cultured for 48 hours (Fig. 1). A downregulation of prominin-1 transcript was also observed in P19 cells cultured for a longer period, that is, 21 days (data not shown)., B! ^  A& e# g6 F: [

Figure 1. Prominin-1, nanog, and nestin transcript levels analyzed by semiquantitative reverse transcription–polymerase chain reaction. Nanog, prominin-1, and nestin transcripts were determined in adult kidney tissue. P19 (48 h) cells, undifferentiated R1 cells (6 h, 48 h), R1 cells cultivated as colonies for 7 days (7 d) in the presence of LIF, R1 cells further cultivated as monolayer in the absence of LIF for 7 and 21 days (w/o LIF; 7 d, 21 d), EBs cultivated in suspension for 5 days (5 d), EB outgrowths (5 2 d, 5 9 d, 5 16 d, 5 23 d), and ES-derived cells after neuronal differentiation at stages 4 8 d and 4 32 d were analyzed. The housekeeping gene ?-tubulin was used as internal standard. Each value (n = 3 experiments) represents mean ± SEM. Abbreviations: EB, embryoid body; ES, embryonic stem; LIF, leukemia inhibitory factor.

As expected, nanog transcripts were highly abundant in undifferentiated R1 cells cultured for 6 and 48 hours (the latter was set to 100%) and nearly absent in kidney (Fig. 1). Similar levels were measured in ES cells cultured in the presence of LIF for 7 days, in 5-day EBs, and in early EB outgrowths. Upon LIF removal or after spontaneous and neuronal differentiation, nanog was downregulated (Fig. 1). In parallel, we analyzed the level of nestin transcript, a neural stem cell–associated intermediate filament protein  but also a potential marker of multilineage progenitors . Nestin transcripts were found abundantly in undifferentiated R1 cells at 48 hours (set to 100%) and in R1 cells grown for 7 days in the presence of LIF. Undifferentiated R1 cells cultured for only 6 hours showed lower level of nestin transcript (~60%). A significant decrease of nestin transcript was observed in R1 cells differentiated for 7 days in the absence of LIF and at terminal differentiation stages (10%–20%; 21 days, 5 23 days). The nestin transcript level was at an intermediate level after neuronal differentiation at progenitor and terminal stages (30%–40%; 4 8 and 4 32 days; Fig. 1).

These data revealed that the transcript level of prominin-1 generally parallels that of nestin in ES cells, i.e., upregulation during the commitment to differentiation (R1 48 hours and 7 days with LIF) and downregulation after differentiation (R1 7 days and 21 days without LIF). Together with the observation that the level of nanog transcript is largely constant in undifferentiated R1 cells, irrespective of the commitment status (6 hours, 48 hours, and 7 days), these observations suggested that prominin-1 and nestin may play a role during commitment and early differentiation of ES cells in vitro; but to further characterize the cells, additional markers are necessary.3 _" X: a! j4 N5 l+ \
$ ?4 B6 Q- n9 Y) L+ x) h& \
Identification of Molecular Markers That Are Differentiation Responsive

To identify potential markers of early differentiation, we queried four SAGE library catalogs for potential early genes. Our rationale was that cytoskeletal-associated proteins are such markers, because they generally affect the distribution of membranous organelles and determine cell shape and cell polarity, which are hallmarks of differentiation. By identifying tags/transcripts that were absent or poorly expressed in undifferentiated ES and EC cells and that significantly (p ) Z" B, A) u* f$ h

A total of 169 cytoskeleton-associated tags could be specifically identified from the SAGE libraries. Several gene products (e.g., Tubulin 1a or 16 , Internexin, a neuronal intermediate filament protein, alpha ) showed significant decreases in tag abundance with differentiation (data not shown), but only five tags showed increased abundance with differentiation (Table 1). Three of these tags were associated with cytokeratin 18 (Krt1-18) and 19 (Krt1-19) or fibulin 1 (Fbln1) transcripts; however, each of these tags also matched one other sequence within the public domain (Table 1). It was therefore unclear if these tags actually corresponded to cytoskeletal-associated proteins. However, cytokeratins 8 and 18 are some of the earliest cytoskeletal components known to be coordinately expressed at four- to eight-cell-stage embryos , suggesting that these products represent authentic differentiation markers. Unlike Krt1-18, cytokeratin 8 (Krt2-8) did not show a significant differentiation response among the SAGE libraries: only four tags were present in one P19 SAGE catalog (3 0.5 days), and this tag sequence (AGCATTCATA) was absent in the three other catalogs. By Q-PCR, we also found that fibulin 1 is in fact differentiation-responsive (data not shown). Fibulin-1 is highly expressed in P19 cells at day 3 0.5, before decreasing by day 3 3 (Table 2), and it is prominent in fetal and adult hearts and lungs.

Table 1. Comparative SAGE analyses and targeted in silico analysis of ES (R1) and EC (P19) cells and differentiating EC-derived cells' B! s1 ~8 R& I5 S1 H  L9 Z

Table 2. BrdU labeling index of prominin-1–positive P19 (48-hour), R1 (48-hour), and differentiated R1 EB-derived (5 9-day) cells

One other potential differentiation-responsive tag corresponded to VCP, which has been shown to be important for membrane fusion, nuclear trafficking, and cell proliferation at the level of both cell division and apoptosis . Because this specific VCP tag increases with in vitro differentiation and is abundant in mouse forelimb SAGE libraries, we have included this product as a differentiation-responsive transcript.: W; ~& t; c7 |) |
+ t6 @7 l9 Y# k
Colocalization of Prominin-1 with Proteins Characteristic for Early Differentiating ES and EC Cells7 K3 Q* t9 \, I* f" W
  v" _  Q% Q7 Q8 ^/ {2 ~
Next we investigated the expression of prominin-1 protein and its distribution in undifferentiated and early differentiating R1- and P19-derived cells by indirect immunofluorescence using rat mAb 13A4 , which recognizes an epitope localized in the second extracellular loop of prominin-1 . Antibodies against proteins characteristic of undifferentiated cells, e.g., SSEA-1 , and of early differentiated cells, e.g., CK18, nestin, desmin, FRP-2, fibulin-1, and VCP, were also used.$ A+ o5 z- }  t

In undifferentiated R1 cells cultivated in the presence of LIF for 6 hours (or in the presence of mouse embryonic feeder layer cells, data not shown), fewer than 10% of prominin-1–positive cells were observed (Figs. 2A, 3A, 3B), although 28% of the cells were nestin-positive (Fig. 2A). However, in R1 cells that are committed to differentiation (48 hours), prominin-1 immunore-activity was easily detectable. Interestingly, prominin-1–positive cells mainly localized at the periphery of R1 cell colonies (Figs. 2B–2H). In contrast, undifferentiated cells expressing SSEA-1 were located in central areas of the aggregates (Fig. 2B). No coexpression of prominin-1 and SSEA-1 was observed (Fig. 2B, inset), except in singular cells at the clone periphery (Fig. 2B, see arrow), demonstrating that prominin-1 is not present in undifferentiated SSEA-1–positive ES cells and localizes to a distinct cell population at the periphery of ES cell colonies.

Figure 2. Double immunofluorescence and confocal imaging analysis of prominin-1 (cell-surface labeling) and various intracellular marker proteins in R1 and P19 cells cultured for 6 hours (R1) and 48 hours (R1, P19). (A): Six-hour R1 cells showed no prominin-1–positive cells, whereas nestin-positive cells (green) were detected in approximately 28% of cells. (B): R1 (48-hour) cells showed almost no coexpression of prominin-1 (red) and SSEA-1 (green). SSEA-1–positive cells were localized in the center of ES cell aggregates, whereas prominin-1–positive cells were restricted to peripheral regions of the ES cell colonies. Prominin-1 was localized to the cell membrane (inset). (C–H): R1 (48-hour) cells showed partial coexpression of (C) prominin-1 (red) and nestin (green), (D) CK18 (green), and (E) desmin (green) and areas colabeled by (F) prominin-1 (green) and fibulin-1 (red), (G) VCP (red), and (H) FRP-2 (red), respectively, at the periphery of R1 cell aggregates. Hoechst 33342 (blue) was used to visualize cell nuclei. Insets show intracellular localization of proteins at higher magnification. As a control for the cell-surface labeling of (B–H) prominin-1, R1 (48-hour) cells after fixation with paraformaldehyde were saponin-permeabilized and incubated with prominin-1 antibody. Prominin-1 labeling was again detected at (I) the peripheral region of cell aggregates (red). (K–L): To control unspecific binding, R1 (48-hour) cells were labeled only with the secondary antibodies (Cy3 goat anti-rat antibody  , fluorescein isothiocyanate mouse anti-rabbit  ). (M–S): P19 cells cultured for 48 hours showed prominin-1 (red) expression in almost all cells and a partial coexpression with (M) SSEA-1 (green) and (N) nestin (green) but no colabeling with (O) desmin (green). P19 cells were colabeled by (P) prominin-1 (green) and fibulin-1 (red), (R) VCP (red), and (S) FRP-2 (red), respectively. Bars = 30 μm. Abbreviations: ES, embryonic stem; FRP, frizzled-related protein; SSEA, stage-specific embryonic antigen; VCP, valosin containing protein.3 F1 J/ {/ O& ~. ^" w
: f5 B, I/ V" Q- \" M
Figure 3. Semiquantitative immunofluorescence, FACS, and Western blot analysis of prominin-1 in P19 (48-hour) and R1 cells. (A): Semi-quantitative immunofluorescence analysis for prominin-1–positive Hoechst 33342–labeled P19 cells (48 hours); R1 cells cultured in the presence of LIF (6-hour, 48-hour, and 7-day colonies); and R1 cells differentiated for 2–28 days in the absence of LIF. For each value, 10 areas (~1,000 cells) of each coverslip (n = 3) of three independent experiments were analyzed. Values represent mean ± SD. (B–F): FACS analysis of prominin-1 immunoreactivity in (B) P19 cells (48 hours), (C) R1 6-hour cells, (D) R1 48-hour cells, (E) R1 7-day colonies, and (F) R1 cells after differentiation for 14 days (7 14 days) in the absence of LIF. The histograms represent the fluorescence of the prominin-1–immunolabeled cells (black) compared with corresponding control cells (gray; labeled with only secondary antibody) after gating the cells in a forward/side-scatter dot plot. (G): Western blotting analysis of prominin-1 (top panel, arrow) in undifferentiated R1 cells (48-hour and 7-day colonies) and R1 cells after further differentiation for 21 days (7 21 days). Adult kidney membrane and P19 (48-hour) cells were used as positive control. In each sample, the total amount of protein was visualized by Coomassie-blue staining (C.B.) (bottom panel). (H): Quantification of prominin-1 immunoreactivity during the differentiation of R1 cells. Prominin-1 immunoreactivity detected in (G) was quantified by densitometric scanning, normalized to the amount of protein detected by C.B. (reference band indicated by asterisks) (G), and plotted as percentage of prominin-1 immunoreactivity detected in undifferentiated R1 cells (7-day colonies). Each value represents the mean of two independent experiments. Abbreviations: FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; LIF, leukemia inhibitory factor.

In contrast to SSEA-1, nestin was highly coexpressed with prominin-1 at peripheral regions of ES cell clusters cultured for 48 hours (Fig. 2C). A partial coexpression of prominin-1 was also observed with fibulin-1 (Fig. 2F), VCP (Fig. 2G), and FRP-2 (Fig. 2H) and the intermediate filament proteins CK18 (Fig. 2D) and desmin (Fig. 2E). Like prominin-1, CK18, which is expressed during embryonic development in early endodermal and epithelial cells , was expressed by cells located mostly at the periphery of R1 cell aggregates (Fig. 2D). Control experiments with saponin permeabilization revealed that prominin-1 labeling was indeed located at the periphery of the ES cell aggregates (Fig. 2I), consistent with the results of prominin-1 cell-surface labeling without membrane permeabilization (Figs. 2B–2H). Control experiments showed no unspecific binding of the secondary antibodies in R1 (48-hour) cells (Figs. 2K, 2L). This coexpression pattern of prominin-1 with markers of early differentiation states suggests that prominin-1 may be involved in commitment and early differentiation of ES cells.
1 u5 I9 D7 W+ v0 f* g' V+ h" o
With regard to undifferentiated P19 EC cells, which do not grow as compacted cell colonies as R1 cells but rather grow in a homogeneous monolayer, prominin-1 immunoreactivity was detected in nearly all cells cultured for 48 hours (Figs. 2M–2S). The typical punctuate membrane staining observed suggests its association with microvilli, as previously reported .& M* }  A4 I" s# Y& y
# @0 b" F, ]& k3 e4 U- U6 W
Some prominin-1–positive P19 cells expressed SSEA-1 (Fig. 2M), which contrasts with the situation observed with R1 cells (Fig. 2B). Several prominin-1–positive P19 cells also expressed nestin (Fig.2N). No desmin was observed in undifferentiated P19 cells (Fig. 2O). Cytoplasmic proteins typical for ES cells committed to differentiation, such as fibulin-1, VCP, and FRP-2, were also detected in most prominin-1–positive P19 cells (Figs. 2P–2S, respectively).5 A, d# @+ m+ O) |
" `+ W, {' o5 E, c' a4 v
Proliferation Analysis of Prominin-1–Positive Cells5 g( g) ~2 O. k: p0 r9 N3 P
, V2 y1 e6 [( b) N& ]
To analyze the proliferation of prominin-1–positive cells, R1 and P19 cells were tested for BrdU incorporation. P19 cells cultured for 48 hours showed the highest BrdU labeling index (59.5%), followed by R1 cells (50.8%), whereas EB outgrowths after spontaneous differentiation showed a significantly lower value (34.4%) (Table 2). The maximal amount of BrdU-labeled/prominin-1–positive cells was found in P19 cells (58.1%), whereas significantly lower numbers of BrdU-labeled/prominin-1–positive cells were detected in undifferentiated R1 cells (31.3%) and in EB outgrowths after differentiation (16.4%) (Table 2).

These observations suggest that in undifferentiated P19 EC cells with a short cell cycle phase of 9–11 hours , prominin-1 is expressed in approximately 60% of the cells, whereas in R1 ES cells with a longer cell cycle phase (~12–14 hours), half of the cycling cell population expressed prominin-1. In differentiating ES cells (5 9 days), a lower percentage of prominin-1–positive cells is BrdU-labeled (Fig. 4A), suggesting that the proliferation efficiency of prominin-positive cells decreases when ES cells start to differentiate.$ g) ~3 f: ?- u% g

Figure 4. Semiquantitative imaging analysis of immunoreactivity in R1 cells at different stages of differentiation and in P19 cells. (A): Undifferentiated R1 cells (6 hours, 48 hours) and R1 cells cultivated as colonies for 7 days; differentiated R1 cells further cultured in the absence of LIF for 21 days; 5-day-old EBs; EB outgrowths at stages 5 2 d, 5 9 d, 5 16 d, and 5 23 d; R1 cells after neuronal differentiation at stages 4 8 d and 4 32 d; and 48-hour P19 cells were analyzed for immunofluorescence signals, and the percentages of immunopositive cells relative to the total number of Hoechst 33342–labeled cells were determined. For each value, 10 areas of each coverslip (n = 2 to 3) of three independent experiments were analyzed. Approximate percentage of immunolabeling was expressed as follows: (B): Coexpression profiles of cells positive for SSEA-1, nestin, cytokeratin 18, desmin, vimentin, E-cadherin, platelet-endothelial cell adhesion molecule-1, vascular endothelial–cadherin, ?III-tubulin, glial fibrillary acidic protein, and Oligodendrocyte (in %) in the prominin-1–labeled population (prominin-1 labeling = 100%). Specific proteins were investigated in the same cell populations and differentiation stages as described in (A). The approximate percentage of immuno-labeling was estimated according to (A). Abbreviations: EB, embryoid body; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; n.d, not determined; PECAM-1, platelet-endothelial cell adhesion molecule-1; SSEA, stage-specific embryonic antigen; VE, vascular endothelial.& ~5 ^/ m$ o0 T: u& T
! Q& I5 ]2 l/ m% h; f+ S
Prominin-1 Protein Abundance During In Vitro Differentiation; R3 L0 }/ Y5 @9 w/ E
8 y4 A2 Z& s; y* f
Our RT-PCR studies showed that (6-hour) R1 cells contained prominin-1 transcripts at 40% of the levels found in kidney tissue (Fig. 1); however, mRNA and protein levels were highly abundant in 48-hour R1 cells. Prominin-1 is therefore upregulated when undifferentiated ES cells become committed to differentiation. To confirm this finding, we performed semiquantitative immunofluorescence, Western blotting, and fluorescence-activated cell sorter (FACS) analysis using anti–prominin-1 mAb 13A4.7 |5 Q5 u& h9 w5 @7 E' {+ l2 q

Because all P19 cells were labeled by prominin-1, these cells were used as positive control (n=100%)in the semiquantitative immunolabeling assay (Fig. 3A). Prominin-1 staining was detected in approximately 60% of 48-hour R1 cells and 80% of 7-day colonies but was clearly reduced after differentiation in the absence of LIF (Fig. 3A).

FACS analysis confirmed the immunofluorescence data (Figs. 3B–3F). P19 cells cultured for 48 hours displayed prominin-1 immunoreactivity in approximately 93% of cells (Fig. 3B). R1 cells (6 hours) represented less than 10% of the undifferentiated cells (Fig. 3C), whereas an increase of prominin-1–positive cells was measured in R1 cells cultured for 48 hours (~50%; Fig. 3D) and after clonal growth in the presence of LIF in 7-day colonies (~55%; Fig. 3E). Therefore, FACS analysis clearly supported the immunofluorescence results, showing that undifferentiated ES cells grow as a heterogeneous population of cells. Spontaneous differentiation for 7 14 days resulted in a reduction of prominin-1 immunoreactivity by FACS analysis (~23%; Fig. 3F).
2 o3 o$ p4 x2 j/ y4 Y
Western blotting analysis confirmed the immunofluorescence and FACS data (Figs.3G, 3H). Significant protein levels were determined in P19 (48-hour) and R1 (48-hour, 7-day) cells, and a down-regulation of prominin-1 was obvious in spontaneously differentiated R1 cells (Fig. 3G) in agreement with immunofluorescence and PCR data. Prominin-1 immunoreactivity was quantified during the differentiation of R1 cells. The prominin-1 immunoreactivity of R1 cells (cultivated as colonies for 7 days) was set to 100%, whereas R1 cells cultivated for 48 hours and differentiated for 21 days showed approximately 40% prominin-1 immunoreactivity (Fig. 3H)./ r, D2 h1 n9 }1 P) _

Finally, to discriminate between various prominin-1 splice variants , we have performed Western blotting analysis using the I3 antiserum  instead of mAb 13A4. The I3 antiserum is directed against the cytoplasmic C-terminal domain of prominin-1.s1 variant  and does not detect any other known prominin-1 splice variant . The I3 antiserum, like the mAb 13A4 (Fig. 3G), recognized the prominin-1 band in P19 and R1 cells, indicating that this band was the s1 variant (data not shown).

Prominin-1 Is Expressed in ES-Derived Progenitors but Not in Differentiated Cells
. {$ ^8 S* \: w8 h7 O6 x) M0 t
Next, we analyzed the colocalization of prominin-1 with lineage-specific proteins in R1 cells during differentiation at early and terminal stages. Prominin-1–positive cells were detected at peripheral regions of early EB outgrowths without any colabeling with SSEA-1 2 days after plating of 5-day EBs (Fig. 5A). Central areas of EB outgrowths were prominin-1–negative (Fig. 5B, arrow); however, approximately 50%–75% of prominin-1–positive cells at the periphery of EB out-growths coexpressed nestin (Figs.4B,5B). CK 18 showed a high level of coexpression with prominin-1 (Fig. 5C), whereas desmin showed only a partial colocalization (Figs. 4B, 5D).2 ~* Z/ b* m3 f# D0 G" T8 q+ @

Figure 5. Double immunofluorescence and confocal imaging analysis of prominin-1 (cell-surface labeling) and various intracellular marker proteins in R1-derived EBs (5-day) differentiated for 2 (5 2 d, A–D) and 23 (5 23 d, E–N) days after EB plating, 32 days after neuronal differentiation (4 32 d, O–R), and Western blot analysis (S, T). (A–D): At 5 2 d, no SSEA-1 labeling (green) in prominin-1–positive cells (red) (A) was detected. Prominin-1–positive cells were not found in central areas of EB outgrowths (B, arrow) but localized at peripheral regions of EB outgrowths (B–D) and showed coexpression with nestin (green, B) and CK18 (green, C) and a partial coexpression with desmin (green, D). (E–N): In EB-derived cells differentiated for 23 days (5 23 d), coexpression of prominin-1 (red, E–L; green, M, N) with nestin (green, E), CK18 (green, F), and desmin (green, G) but no coexpression with vimentin (green, H), GFAP (green, I), ?III tubulin (green, K), platelet-endothelial cell adhesion molecule-1 (green, L), vascular endothelial–cadherin (green, M), and E-cadherin (green, N) was detected. (O-R): R1 cells after neuronal differentiation at terminal stage (4 32 d) showed coexpression of prominin-1 only in nestin-positive epithelial-like cells (green, O) but no coexpression in nestin-positive neuron-like cells (green, arrow, O) and in GFAP-labeled (green, P), ?III tubulin–labeled (green, Q), and oligodendrocyte-labeled protein (Olig) (green, R) cells. Hoechst 33342 (blue) was used to visualize cell nuclei. Bars = 30 μm. (S): Western blotting analysis of prominin-1 (top panel, arrow) in R1-derived EBs at stages 5 2 d, 5 16 d, and 5 23 d. Adult kidney membrane was used as positive control. In each sample, the total amount of protein was visualized by Coomassie-blue staining (C.B.) (bottom panel). (T): Quantification of prominin-1 immunoreactivity in R1-derived EBs at various stages. Prominin-1 immunoreactivity detected in (S) was quantified by densitometric scanning, normalized to the amount of protein detected by Coomassie-blue staining (reference band indicated by asterisks in ), and plotted as percentage of prominin-1 immunoreactivity detected in EBs at stage 5 16 d. Each value represents the mean of two independent experiments. Abbreviations: EB, embryoid body; GFAP, glial fibrillary acidic protein; PECAM-1, platelet-endothelial cell adhesion molecule-1; SSEA, stage-specific embryonic antigen.0 Q$ R5 Q9 B  D  W" D' x: Z

Because of the colocalization of prominin-1 with proteins characteristic for neural (nestin), epithelial (CK18), and (partially) mesodermal (desmin) cell types in early differentiating ES cells, we analyzed the lineage-specific expression of prominin-1 after continued differentiation of R1 cells. Lineage-specific markers of glial (GFAP), neuronal (?III-tubulin), epithelial (E-cadherin), endothelial (VE-cadherin, PECAM-I), and meso-dermal (vimentin) cells were analyzed at a terminal stage (5 23 days; Figs. 5E–5N).! _" m2 ~5 N. j9 m% W) u
' @  d  N3 Z% n* X( z
At the terminal differentiation stage, 5%–25% of R1-derived cells were labeled by prominin-1 (Fig. 4A), and a partial coexpression of prominin-1 in nestin-positive (Fig. 5E), CK18-positive (Fig. 5F), and desmin-positive (Fig. 5G) cells was found. However, significant differences in the morphology of cells labeled by prominin-1 and nestin (Fig. 5E), respectively, were observed. Whereas most cells that coexpressed prominin-1 and nestin showed an epithelial morphology, most cells labeled only by nestin (prominin-1–negative) revealed the typical morphology of neuron-like cells. Several cells at this terminal differentiation stage were also labeled by vimentin (Fig. 5H), GFAP (Fig. 5I), ?III-tubulin (Fig. 5K), PECAM-I (Fig. 5L), VE-cadherin (Fig. 5M), and E-cadherin (Fig. 5N); however, no coexpression of prominin-1 was observed with any of these proteins (Fig. 4B). Western blotting confirmed the upregulation of prominin-1 in EB outgrowths at stage 5 16 days but showed a downregulation at stage 5 23 days (Fig. 5S). The quantification of prominin-1 immunoreactivity (maximum value at stage 5 16 days was set to 100%) revealed approximately 50% prominin-1 immunoreactivity at stages 5 2 days and 5 23 days (Fig. 5T).

Because of the relatively high level of coexpression of prominin-1 with nestin (known as neural stem cell marker) in R1-derived cells (Figs. 2C, 4B, 5B), we analyzed prominin-1 expression specifically during neuronal differentiation . R1-derived nestin-positive progenitor cells at early stage of neural differentiation (4 8 days) showed high coexpression with prominin-1 (Fig. 4B), whereas after induction of neuronal differentiation, glial (GFAP), neuronal (?III-tubulin), and oligodendrocytic (oligodendrocyte-specific protein) cells were all prominin-1–negative (Figs. 5O–5R). Prominin-1 and nestin were coexpressed in some epithelial-like clusters (Fig. 5O), but prominin-1 was never found in nestin-positive cells of neuronal morphology (Fig. 5O, see arrow).
7 }7 N2 V3 x4 _) z
We also tested the colocalization of prominin-1 with endodermal (hepatic and pancreatic) lineage markers, including -fetoprotein, albumin and cytokeratin 19, Islet-1, and insulin, respectively, but did not see prominin-1 labeling in any of these cells (data not shown).
( @8 b7 l4 `- i, @* S: q4 Q
In conclusion, we found that prominin-1 expression is maintained when ES cells undergo commitment to differentiation into neural, epithelial, and (partially) mesodermal cells, suggesting a role of prominin-1 for the specification of these precursor cells. At terminal stages of differentiation, however, prominin-1 is never coexpressed with proteins that are typically found in specialized cell populations with the exception of prominin-1 expression in nestin-positive cell types, which define potential neural precursor cells. This might suggest a specific role of prominin-1 expression at least in neural precursor cells.

Prominin-1 Is Expressed in Trophectoderm Cells of Mouse Blastocysts& |. o* v5 X% c3 _" [3 K8 r
  e- U- A$ C6 [- O5 I! _$ @0 a
Given that prominin-1 is expressed in early progenitor cells, we have investigated the presence of this marker in early embryos in vivo. RT-PCR analysis showed the absence of prominin-1 transcript in 3.5-day p.c. blastocysts (Fig. 6A). Under the same condition, nestin was barely detectable, whereas nanog attained maximal levels (Fig. 6A). Remarkably, after plating of blastocysts followed by 4 days of culture, the level of prominin-1 as well as nestin transcript showed maximal levels (Fig. 6A), whereas the amount of nanog transcript was decreased (Fig. 6A).' ^" c1 @# v0 S

Figure 6. Prominin-1 expression in mouse blastocysts. (A): The abundance of prominin-1, nanog, and nestin transcripts in blastocysts isolated at day 3.5 postconceptionem (3.5 d) and in blastocysts cultivated for an additional 4 days (3.5 4 d) was assayed by reverse transcription–polymerase chain reaction. The mRNA level of prominin-1 and nestin determined in cultivated blastocysts (3.5 4 d) and of nanog in isolated blastocysts (3.5 d) was set to 100%, and the respective other value was expressed relative to this. The housekeeping gene ?-tubulin was used as internal standard. Each value (n = 3 experiments with 15 to 20 blastocysts) represents mean ± SEM. (B–G): Double immunofluorescence analysis of prominin-1 (cell-surface labeling) and various proteins in blastocysts cultivated for 4 days (3.5 4 d) (n = 15). Prominin-1 was localized at the apical domain of trophoblast cells (B, red overlay with DIC). Cells were double-labeled for (C) prominin-1/stage-specific embryonic antigen-1 (SSEA-1), (D) prominin-1/nestin, (E) prominin-1/CK18, and (F) prominin-1/desmin and analyzed by confocal microscopy. Partial coexpression of prominin-1 with (E) CK18 and (F) desmin was found in the trophoblast cells (see arrows). Corresponding phase micrograph of (G) a blastocyst cultivated for 4 days is shown, in which Hoechst 33342 (blue) was used to visualize cell nuclei. Bar = 30 μm (B, D–G) and 10 μm (B). Abbreviation: DIC, differential interference contrast.. J+ q6 h: F6 X. w) V

To verify prominin-1 abundance in cells of the inner cell mass (ICM), the founder cells of ES cells, we investigated prominin-1 expression in plated blastocysts by indirect immunofluorescence (Fig. 6). Using either cell-surface immunolabeling (Figs. 6B–6F) or immunolabeling after PFA fixation and saponin permeabilization (data not shown), prominin-1 (red) was detected in trophoblast cells but not in ICM cells (Figs. 6B–6F). Prominin-1 was localized at the apical domain of trophoblast cells (Fig. 6B), and no coexpression with SSEA-1 (green, localized in ICM cells) was found (Fig. 6C). Comparable results are obtained upon saponin permeabilization (not shown). Nestin was not detected in ICM and trophoblast cells (Fig. 6D), but a partial colocalization of prominin-1 (red) with CK18 (green; Fig. 6E) and with desmin (green; Fig. 6F) was found in trophoblast cells (see arrows).8 e$ d/ |* ~" O# o7 n  s; N
' F# i) V0 V6 v
DISCUSSION. [. x+ e- S/ s7 ]4 F5 q

This work was supported by the Deutsche Forschungsgemein-schaft (WO 503/3-2) and Fonds der Chemischen Industrie, Germany (to A.M.W.). We are grateful to S. Sommerfeld, K. Meier, O. Weiss, D. Schrader, and B. Claus for excellent technical assistance, Drs. J. Czyz, A. Rolletschek, and C. Wiese (IPK Gatersleben) for helpful discussions, and Dr. A. Navarrete-Santos (Department of Anatomy and Cell Biology, University of Halle) for the preparation of 5-day EBs.
7 N& s; k0 |/ l  d$ S" w
REFERENCES

Fargeas CA, Corbeil D, Huttner WB. AC133 antigen, CD133, prominin-1, prominin-2, etc.: prominin family gene products in need of a rational nomenclature. STEM CELLS 2003;21:506–508.- T) j$ ]) l, I/ }$ c; G% e

Weigmann A, Corbeil D, Hellwig A et al. Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells. Proc Natl Acad Sci U S A 1997;94:12425–12430.1 o1 S; P; {* d
6 P$ o2 A* p  F6 ~5 y% a
Roper K, Corbeil D, Huttner WB. Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane. Nat Cell Biol 2000;2:582–592.

Fargeas CA, Florek M, Huttner WB et al. Characterization of prominin-2, a new member of the prominin family of pentaspan membrane glycoproteins. J Biol Chem 2003;278:8586–8596.- w( W5 {# I9 r+ P+ J) a) R
$ d6 U. t5 N; v. C" f! q' ?) ]8 Y
Corbeil D, Roper K, Fargeas CA et al. Prominin: a story of cholesterol, plasma membrane protrusions and human pathology. Traffic 2001;2:82–91.% K* k& X& X- W$ g1 `) N" C

Yin AH, Miraglia S, Zanjani ED et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 1997;90:5002–5012.
1 R8 Y+ L3 w9 h. F2 g
Corbeil D, Roper K, Hellwig A et al. The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. J Biol Chem 2000;275:5512–5520.
4 \' K( }) _8 z9 l# g! @
Uchida N, Buck DW, He D et al. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 2000;97:14720–14725.
* v, g1 [+ P# F8 R  N
Peichev M, Naiyer AJ, Pereira D et al. Expression of VEGFR-2 and AC133 by circulating human CD34  cells identifies a population of functional endothelial precursors. Blood 2000;95:952–958.) m+ T/ Y0 p  k7 @4 @6 L5 g: f0 M

Richardson GD, Robson CN, Lang SH et al. CD133, a novel marker for human prostatic epithelial stem cells. J Cell Sci 2004;117:3539–3545.) \- v9 c+ k$ e$ _( W

Bhatia M. AC133 expression in human stem cells. Leukemia 2001;15:1685–1688.
3 e" c& Z3 ]5 }, I4 K" b7 @/ s
Maw MA, Corbeil D, Koch J et al. A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration. Hum Mol Genet 2000;9:27–34.
8 a6 ~8 H+ w" g9 ?- o* e1 ?/ E& e
Corbeil D, Roper K, Hannah MJ et al. Selective localization of the poly-topic membrane protein prominin in microvilli of epithelial cells: a combination of apical sorting and retention in plasma membrane protrusions. J Cell Sci 1999;112:1023–1033.! [1 \3 ?% g6 C3 J6 i1 N
; _- c7 b. k& i( Q0 g1 A
Giebel B, Corbeil D, Beckmann J et al. Segregation of lipid raft markers including CD133 in polarized human hematopoietic stem and progenitor cells. Blood 2004;104:2332–2338.
3 F+ R5 k9 \$ ~2 S
Dragsten PR, Blumenthal R, Handler JS. Membrane asymmetry in epithelia: is the tight junction a barrier to diffusion in the plasma membrane? Nature 1981;294:718–722.4 ^. P% w7 y' `# f, p' D
' B3 x$ }" M$ _9 O7 ?
van Meer G, Gumbiner B, Simons K. The tight junction does not allow lipid molecules to diffuse from one epithelial cell to the next. Nature 1986;322:639–641.
/ y3 w( G0 w2 T: j5 z+ h. g
Kosodo Y, Roper K, Haubensak W et al. Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells. EMBO J 2004;23:2314–2324.

Nagy A, Rossant J, Nagy R et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A 1993;90:8424–8428.
! O) ^3 x9 {% b, K' T( Y3 @$ b) z' H
Wobus AM, Guan K, Yang HT et al. Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation. Methods Mol Biol 2002;185:127–156.! U+ j6 p! [  R1 r* |

McBurney MW, Rogers BJ. Isolation of male embryonal carcinoma cells and their chromosome replication patterns. Dev Biol 1982;89:503–508.
5 o0 y3 n# N5 n9 ]; Z
Okabe S, Forsberg-Nilsson K, Spiro AC et al. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech Dev 1996;59:89–102.

Rolletschek A, Chang H, Guan KM et al. Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors. Mech Dev 2001;105:93–104.
: [+ w" {6 G- d: _$ A3 k
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–159.
# v' I( K4 K3 a# o
Anisimov SV, Tarasov KV, Riordon D et al. SAGE identification of differentiation responsive genes in P19 embryonic cells induced to form cardiomyocytes in vitro. Mech Dev 2002;117:25–74.

Anisimov SV, Tarasov KV, Tweedie D et al. SAGE identification of gene transcripts with profiles unique to pluripotent mouse R1 embryonic stem cells. Genomics 2002;79:169–176.! Q& h/ Z  ~7 H5 c
' o0 O1 T4 {: I3 q2 T: C9 h
Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990;60:585–595.' ~$ |4 Z- l! \" T

Wiese C, Rolletschek A, Kania G et al. Nestin expression: a property of multi-lineage progenitor cells? Cell Mol Life Sci 2004;61:2510–2522.- t; G# o% d+ G% f* L8 k. g1 E

Coonen E, Dumoulin JC, Ramaekers FC. Intermediate filament protein expression in early developmental stages of the mouse: a confocal scanning laser microscopy study of in vitro fertilized and in vitro cultured pre-implantation mouse embryos. Histochemistry 1993;99:141–149.
0 f# B/ v9 x0 y8 Y, m
Dai RM, Li CC. Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nat Cell Biol 2001;3:740–744.
* x+ y, B. l. r! x1 G6 k
Solter D, Knowles BB. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci U S A 1978;75:5565–5569.- w4 p! D! x# v# M+ A# X! A
+ i/ g8 }( n" o) y- J. a4 X1 u
Kemler R, Morello D, Babinet C et al. Surface antigens in early embryonic development. Results Probl Cell Differ 1980;11:107–111.

Rohwedel J, Sehlmeyer U, Shan J et al. Primordial germ cell-derived mouse embryonic germ (EG) cells in vitro resemble undifferentiated stem cells with respect to differentiation capacity and cell cycle distribution. Cell Biol Int 1996;20:579–587.) u  {! P/ O: Z

Fargeas CA, Joester A, Missol-Kolka E et al. Identification of novel Prominin-1/CD133 splice variants with alternative C-termini and their expression in epididymis and testis. J Cell Sci 2004;117:4301–4311.
% I# P  }* x; r7 `/ q
Chambers I, Colby D, Robertson M et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643–655.
, f5 b, `6 D# X  r, Z  J
Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;113:631–642.7 N# p) a& R$ s9 M7 W  V7 n

Hesse M, Franz T, Tamai Y et al. Targeted deletion of keratins 18 and 19 leads to trophoblast fragility and early embryonic lethality. EMBO J 2000;19:5060–5070., d7 \1 @2 i, n! \% ?

Pan TC, Sasaki T, Zhang RZ et al. Structure and expression of Fibulin-2, a novel extracellular-matrix protein with multiple Egf-like repeats and consensus motifs for calcium-binding. J Cell Biol 1993;123:1269–1277.
( }/ s2 X) M$ B! V( q
Spence SG, Argraves WS, Walters L et al. Fibulin is localized at sites of epithelial mesenchymal transitions in the early avian embryo. Dev Biol 1992;151:473–484.; o0 j+ D! H: v. I- L2 H
0 C* O* T( C! J7 R, ]+ J! A
Zhang HY, Kluge M, Timpl R et al. The extracellular matrix glycoproteins BM-90 and tenascin are expressed in the mesenchyme at sites of endothelial-mesenchymal conversion in the embryonic mouse heart. Differentiation 1993;52:211–220.

Castoldi M, Chu ML. Structural and functional characterization of the human and mouse fibulin-1 gene promoters: role of Sp1 and Sp3. Biochem J 2002;362:41–50.$ S& c& X& ?# p2 s  P

Roger P, Pujol P, Lucas A et al. Increased immunostaining of fibulin-1, an estrogen-regulated protein in the stroma of human ovarian epithelial tumors. Am J Pathol 1998;153:1579–1588.) A) \2 z* a, x6 e+ E: ^
3 h( w9 [# l8 d1 V- k
Singh SK, Hawkins C, Clarke ID et al. Identification of human brain tumour initiating cells. Nature 2004;432:396–401.. }/ e3 @- L+ ?' R

Asai T, Tomita Y, Nakatsuka S et al. VCP (p97) regulates NF kappa B signaling pathway, which IS important for metastasis of osteosarcoma cell line. Jpn J Cancer Res 2002;93:296–304., r' p) r; ?! R

Cattaneo E, McKay R. Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor. Nature 1990;347:762–765.& W! Y1 z% L, J/ {# G& F  t" y
! d8 c# c/ S$ F) N1 l
Wang Y, Macke JP, Abella BS et al. A large family of putative transmembrane receptors homologous to the product of the Drosophila tissue polarity gene frizzled. J Biol Chem 1996;271:4468–4476.+ G6 P0 _0 s+ Z3 `$ e5 F6 Z/ ]
8 M1 p) [% C! m. r
Kim AS, Anderson SA, Rubenstein JL et al. Pax-6 regulates expression of SFRP-2 and Wnt-7b in the developing CNS. J Neurosci 2001;21:RC132.' a) g& w; N: P: F
( V" X! M$ X/ ?5 C2 l) A; Q' H
Aubert J, Dunstan H, Chambers I et al. Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat Biotechnol 2002;20:1240–1245./ F) W' |  H- |/ ~& m# N  ~
" B, C0 n$ F* j5 v4 U
Leimeister C, Bach A, Gessler M. Developmental expression patterns of mouse sFRP genes encoding members of the secreted frizzled related protein family. Mech Dev 1998;75:29–42.0 u4 `) Z! G0 d8 ~; A' Y3 C
: k9 K& }) O' Y, r
Potgens AJ, Bolte M, Huppertz B et al. Human trophoblast contains an intracellular protein reactive with an antibody against CD133: a novel marker for trophoblast. Placenta 2001;22:639–645.(Gabriela Kaniaa, Denis Co)

收藏0 分享0 顶0 踩0