Proximal Prostatic Stem Cells Are Programmed to Regenerate a Proximal-Distal Duc - 《stemcells》干细胞学杂志干细胞之家



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 ... 27 下一页

返回列表

查看: 405571\|回复: 263	go Proximal Prostatic Stem Cells Are Programmed to Regenerate a Proximal-Distal Duc [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 00:03 |只看该作者 |倒序浏览 |打印

作者：Ken Gotoa, Sarah N. Salma,b, Sandra Coetzeea, Xiaozhong Xionga, Patricia E. Burgerc, Ellen Shapirod, Herbert Lepord, David Moscatellia,e, E. Lynette Wilsona,c,d,e作者单位：aDepartment of Cell Biology, New York University School of Medicine, New York, New York, USA;bDepartment of Science, Borough of Manhattan Community College, New York, New York, USA;cDivision of Immunology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University

   ) R8 B8 ~+ a3 J
      6 Z$ X, G9 d( s/ y6 \
                     7 {' Y7 R. E1 p- o+ `# w
         0 e0 Q- ~5 d& K. Y/ m

            m( ?$ @% V) m3 ]" t, ]# A
         ' R$ @& c! |4 H# e. l4 f, [


      3 e3 I5 @) L+ H

      【摘要】
   Prostate carcinoma and benign prostatic hypertrophy may both originate in stem cells, highlighting the importance of the characterization of these cells. The prostate gland contains a network of ducts each of which consists of a proximal (adjacent to the urethra), an intermediate, and a distal region. Here, we report that two populations of cells capable of regenerating prostatic tissue in an in vivo prostate reconstitution assay are present in different regions of prostatic ducts. The first population (with considerable growth potential) resides in the proximal region of ducts and in the urethra, and the survival of these cells does not require the presence of androgens. The second population (with more limited growth potential) is found in the remaining ductal regions and requires androgen for survival. In addition, we find that primitive proximal prostate cells that are able to regenerate functional prostatic tissue in vivo are also programmed to re-establish a proximal-distal ductal axis. Similar to their localization in the intact prostate, cells with the highest regenerative capacity are found in the proximal region of prostatic ducts formed in an in vivo prostate reconstitution assay. The primitive proximal cells can be passaged through four generations of subrenal capsule grafts. Together, these novel findings illustrate features of primitive prostate cells that may have implications for the development of therapies for treating proliferative prostatic diseases. 2 a+ l9 X+ J8 j0 C" v  ^
      【关键词】 Proximal prostate stem cells Ductal axis Androgen sensitivity& x" @2 L+ p) Z1 s
   INTRODUCTION

The rodent prostate is an androgen-dependent organ with a ductal system that displays significant heterogeneity along the proximal-distal ductal axis. The different regions of prostatic ducts are heterogeneous in terms of morphology, telomerase expression, and levels of active transforming growth factor (TGF)-ß signaling . The evolution of androgen-independent prostate carcinoma may reflect the emergence of stem-like prostate tumor cells. The distribution of androgen-independent cells within the ductal regions of a normal prostate gland is currently unknown. The investigation of regional sensitivities to androgens may increase our understanding of both normal prostate physiology and the aberrant proliferation that occurs in prostatic diseases such as benign prostatic hypertrophy and prostate carcinoma.

We therefore isolated cells from different regions of prostatic ducts removed from donor androgen-replete or castrated animals and examined their ability to regenerate prostatic tissue in recipient androgen-replete as well as in androgen-ablated and reconstituted animals. This was done using an in vivo prostate reconstitution assay in which combinations of prostate cells and embryonic urogenital sinus mesenchyme (UGM) (inductive mesenchyme for prostatic tissue)  are inserted under the renal capsule (RC) of recipient animals, where they form prostatic tissue. We find that cells isolated from the intermediate and distal regions do not survive androgen ablation whereas those from the proximal region and the urethra are able to withstand prolonged androgen deprivation, indicating the presence of cells with stem cell properties in these areas. Additionally, we show for the first time that primitive proximal prostate cells, which regenerate functional prostatic tissue, are programmed to re-establish a proximal-distal ductal axis when inserted under the RC. Similar to the primitive cells in the intact prostate, the cells with the highest regenerative capacity are also found in the proximal region of ducts in the sub-RC prostatic tissue and can be passaged through four generations of sub-RC grafts.

MATERIALS AND METHODS. t0 }. \+ w! I1 q+ S* |! N

Animals
% i' Z1 g7 {" T4 t! K+ k! H
C57BL/6 mice (Taconic, Germantown, NY, http://www.taconic.com), athymic nude mice (National Institutes of Health, Bethesda, MD, http://www.nih.gov), CDIGS rats (Charles River Laboratories, Wilmington, MA, http://www.criver.com), and green fluorescent protein (GFP) transgenic mice (C57BL/6-TgN; The Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) were housed in a climate-controlled facility, and all animal care and procedures were performed in compliance with the New York University institutional review board requirements.$ l3 R! I2 c. l

Preparation of Dissociated Prostate and Urethra Cells' d* h$ [- [* i+ [4 o
" X- _% u3 d; {) J& _2 v, C7 A, X
Six-week-old C57BL/6 mice were sacrificed, and the urogenital tract was removed en bloc and transferred in Hanks' balanced salt solution (Mediatech, Inc., Herndon, VA, http://www.cellgro.com). The dorsal prostate (DP) was removed and dissected under a dissecting microscope in the presence of 0.5% collagenase (1.3 units/mg; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) . A portion of the urethra was removed (Fig. 1A) and was similarly digested. Cells were passed through a 40-µm nylon mesh (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), and viability was determined by trypan blue exclusion.

Figure 1. The proximal region of mouse prostatic ducts and the urethra contain stem cells. (A): Schematic diagram of the prostate showing the protocol used to implant different regions of prostatic ducts under the RC. (B): Cells from the urethra (n = 15) or different regions of ducts (105) (all regions n = 14, proximal n = 37, intermediate n = 8, distal n = 26) were combined with UGM cells (2.5 x 105) and implanted under the RC. Grafts were harvested after 8 weeks, weighed, and used for immunocytochemical examination. Each bar represents the mean ¡À SD. (C): Prostatic tissue under the RC initiated with 105 proximal cells. Bar = 2 mm. (D): Prostatic tissue under the RC initiated with 105 distal cells. Bar = 2 mm. (E): A section of prostatic tissue arising from proximal cells showing basal cells (arrows) immunohistochemically stained using an antibody against K5 keratin. Bar = 40 µm. (F): A section of prostatic tissue arising from proximal cells showing luminal cells (arrows) immunohistochemically stained using an antibody against K8 keratin. Bar = 40 µm. (G): A section of prostatic tissue arising from proximal cells immunohistochemically stained with antibodies specific for prostatic secretory products (arrows). Bar = 40 µm. (H): A section of proximal prostatic tissue indicating no staining in tissues to which appropriate control antibodies were added, showing that staining is specific. Bar = 40 µm. Abbreviations: Lu, lumen of duct; RC, renal capsule; UGM, urogenital sinus mesenchyme.+ ?) c3 _# }+ [7 W5 y* ^
6 \- R: p/ r/ y
Preparation of UGM Cells  I9 V9 E+ D9 [6 f) l! y. O" \

UGM was isolated from the urogenital sinus of 18-day-old CDIGS rat embryos after digestion with trypsin (1%) at 4¡ãC for 90 minutes .

Implantation of Grafts Under the RC5 ?+ a- `$ P4 E+ I/ l3 C
8 d. Y5 Q( h2 t: A1 z6 Z
The grafts were implanted under the RC of intact or castrated athymic male mice  (tutorial for technique: http://mmmary.nih.gov/tools/Cunha001/index.html). Cells from the urethra or different regions of ducts (105 unless otherwise indicated) were combined with UGM cells (2.5 x 105) and resuspended in 15 µl of type 1 collagen (BD 354236; BD Biosciences, Bedford, MA, http://www.bdbiosciences.com). The collagen was allowed to gel at 37¡ãC for 15 minutes after which the grafts were inserted under the RC. Where indicated, androgens were administered by the subcutaneous implantation of testosterone pellets (5 mg; Innovative Research of America, Sarasota, FL, http://www.innovrsrch.com). Each experiment contained a set of grafts of UGM alone (3.5 x 105 cells) to ensure that tissue growth did not result from contaminating urogenital sinus epithelial cells. In addition, some experiments were done using prostate cells isolated from GFP transgenic mice (C57BL/6-TgN; The Jackson Laboratory) to ensure that tissue growth resulted from donor GFP-expressing cells and not contaminating epithelial cells in the UGM preparation (supplemental online Fig. 3). Grafts were harvested after 8 weeks of in vivo growth, weighed, and used for immunohistochemical examination.

Passage of Undissected Recombinant Tissue (All Regions) In Vivo1 X- a* h( {1 O/ A

The ability of proximal and distal regions of primary prostate cells to undergo multiple rounds of growth was assessed by serial in vivo passaging of recombinant tissue. Cells isolated from proximal and distal regions of prostatic ducts (1 x 105) were combined with UGM (2.5 x 105 cells) and implanted under the RC of intact 6-week-old male athymic nude mice. After 8 weeks, the recipient mice were sacrificed, and grafts from either proximal or distal cells (P1) (Fig. 2A) were retrieved and weighed. Grafts arising from either proximal or distal cells were minced finely, digested in collagenase (see above), and trypan blue-excluding cells were enumerated. These cells (1 x 105 cells) were combined with UGM (2.5 x 105 cells) and implanted into recipient mice to produce a "second passage (P2)" graft (Fig. 2A). This protocol was repeated until no tissue growth was noted.

Figure 2. Cells from the proximal region of primary prostate tissue can be serially passaged in vivo. (A): Schematic diagram showing the protocol used for passaging proximal and distal cells isolated from primary prostate tissue. Proximal (prox reg) and distal (dist reg) cells (105) were combined with UGM cells (2.5 x 105) and implanted under the RC of intact animals. Grafts arising from proximal and distal cells were harvested after 8 weeks, the entire graft (all reg) was digested with collagenase and trypsin, and cells (105) from each type of graft were again combined with UGM cells (2.5 x 105) and implanted under the RC for an additional 8 weeks. This process was repeated until no further tissue growth was noted. (B): The tissue arising from proximal and distal cells at each passage (P1¨CP4) was weighed. Proximal cells: P1 n = 37, P2 n = 8, P3 n = 8, P4 n = 7. Distal cells: P1 n = 26, P2 n = 7. P1, proximal versus distal: *, p & Z& A6 `* J$ `9 E& _/ d+ g; ~

Passage of the Proximal Region of Recombinant Tissue In Vivo
* Y. F' b1 X' @3 {, X) i
Sub-RC grafts from cells isolated from the proximal region were digested with collagenase (see above), revealing a ductal network similar to that observed in a prostate removed from an animal (P1; Fig. 3B, 3C). To determine whether the sub-RC grafts maintained a proximal-distal axis and to ascertain whether cells within the proximal and distal regions of these grafts exhibited the differential growth capacity of proximal and distal cells isolated from a "primary" prostate (Fig. 3A), we dissected the recombinant tissue arising from proximal cells into proximal and distal regions. Single-cell suspensions of these regions were prepared (see above), and proximal and distal cells (1 x 105) were each combined with UGM (2.5 x 105 cells) and implanted into a second generation of recipient animals to produce a "P2" graft (Fig. 3A). The P2 graft arising from proximal cells was again dissected into proximal and distal regions and passaged as above into a third generation of recipient animals (P3, Fig. 3A). This protocol was repeated until no tissue growth was observed (Fig. 3A). After each tissue passage, animals were sacrificed after 8 weeks of in vivo growth and grafts were removed and weighed.% R/ e5 O: {( C5 k# V

Figure 3. Cells from the proximal region of recombinant tissue can be serially passaged in vivo. (A): Schematic diagram showing the protocol used for passaging proximal (prox reg) and distal (dist reg) cells isolated from recombinant tissue. Proximal and distal cells (105) were isolated after collagenase/trypsin digestion of successive passages of sub-RC tissue, combined with UGM cells (2.5 x 105) and implanted under the RC of intact animals until no further tissue growth was noted. Asterisk denotes minimal tissue growth. (B): The morphology of the prostatic ductal system of a collagenase-digested lobe of the dorsal prostate showing the prox and dist regions of ducts. Bar = 0.5 mm. (C): The morphology of the prostatic ductal system of collagenase-digested recombinant prostate tissue arising from proximal cells showing the prox and dist regions of ducts. This indicates that the recombinant tissue has the same morphology and proximal-distal ductal axis as a primary prostate. Bar = 0.5 mm. (D): The tissue arising from each passage of proximal and distal cells obtained from the recombinant tissue was weighed at each successive passage (P1¨CP4). Proximal cells: P1 n = 37, P2 n = 4, P3 n = 7, P4 n = 6. Distal cells: P1 n = 26, P2 n = 5, P3 n = 6, P4 n = 4. P1, proximal versus distal: *, p

Immunohistochemistry5 e+ L6 }4 a* M0 r8 w- k( Y

Grafts were fixed in 70% ethanol or 3% paraformaldehyde and embedded in paraffin, and sections were stained with hematoxylin and eosin. Immunohistochemistry was performed as described previously . Cytokeratin-5 was visualized using rabbit polyclonal antibodies (PRB-160P; Covance, Princeton, NJ, http://www.covance.com) and appropriate HRP-linked secondary antibodies (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). GFP was visualized using rabbit polyclonal antibodies (46¨C0092; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and the ABC staining kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Androgen receptors were visualized using rabbit polyclonal antibodies (sc-816; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com) and the ABC staining kit (Vector Laboratories). The specificity of staining was ascertained on sections using nonimmune serum or immunoglobulin G in place of primary antibodies. Sections were counterstained with hematoxylin.8 ]. H+ K% F. b0 x6 u
6 {# e! y3 e  m7 D" G
Isolation of 6 Integrin Expressing Cells# P0 x6 y1 }5 p
  f* f# V% z1 S& L- W6 o4 w
Samples were enriched for 6 integrin (CD49f)-expressing cells by immunomagnetic separation using antibodies to this antigen (BD 555734; BD Biosciences) and magnetically activated cell sorter (MACS) microbeads, magnetic columns, and the MiniMACS system (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com).

Cell Preparation and Fluorescence-Activated Cell Sorting Analysis

Prostatic cell digests obtained from either the proximal region or the remaining (intermediate and distal) regions were resuspended in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline containing bovine serum albumin . Cells were analyzed on a FACSCalibur flow cytometer (Becton, Dickinson and Company), using CellQuest software (Becton, Dickinson and Company).7 f) g' _4 e% V. m$ M+ L
' A- j! I) w: B# o# v7 p
Statistical Analysis1 x+ ]: `5 `+ ?9 U! d! I

The results are depicted as the means and SDs of each set of data. Comparisons between groups were made using the two-tailed, paired Student's t test, or the nonparametric two-tailed Mann-Whitney U test. A p value of & Y0 a  d* g5 M" U0 e
$ R9 c2 d8 e. R
RESULTS4 n# [5 q' g* H( C6 g+ W
& m6 g# `: Q) V3 z' x0 d5 V
Single Cell Populations of Proximal and Urethral Cells Form Large Amounts of Prostatic Tissue Under the RC

The mouse prostate can be divided into ventral, dorsal, and lateral lobes, each of which contains an arborizing network of ducts that consists of a proximal (adjacent to the urethra), an intermediate, and a distal region . Androgen receptors were also evident in the sub-RC prostatic tissue (supplemental online Fig. 2). The histological appearance of tissue arising from cells isolated from different regions was similar.: w, h. r! C) L! h: ~0 H
" A& S& Y7 P$ Y
Each experiment contained grafts consisting of UGM alone to ensure that tissue growth did not result from contaminating urogenital sinus epithelial cells. The UGM grafts were weighed and examined microscopically to exclude urogenital sinus epithelial contamination. The average weight of UGM tissue alone was 8.3 ¡À 2.2 mg. In addition, some experiments were done using prostate cells isolated from GFP transgenic mice to ensure that tissue growth resulted from donor GFP-expressing cells and not contaminating epithelial cells in the UGM preparation (supplemental online Fig. 3)., o8 _7 O; r/ V" _

To determine the minimum number of cells capable of forming prostatic tissue, the sub-RC growth of varying numbers of proximal and distal cells (105 to 4 x 102 cells) was determined (supplemental online Fig. 4). The proximal region contained cells with 50-fold greater regenerative capacity than those from the distal region as 400 proximal cells formed prostatic tissue whereas 20,000 distal cells were required for tissue growth. A linear relationship between the tissue mass and inoculum dose was noted between 400 and 105 cells (supplemental online Fig. 4). The size of the UGM inoculum also affected tissue size with a linear relationship in its ability to support prostate tissue growth between 2.5 x 104 and 2.5 x 105 UGM cells (data not shown).3 ]( u1 f6 R) ?; C
' |9 ?/ |. N, x5 j& H5 ^8 A
Figure 4. Cells from the proximal region and the urethra survive prolonged androgen deprivation. (A): Schematic diagram showing the protocol used for examining the androgen sensitivity of prostate cells. (B): Cells from the urethra (n = 15) or different regions of ducts (105) (all regions n = 14, proximal n = 37, intermediate n = 8, distal n = 26) were combined with UGM cells (2.5 x 105) and implanted under the RC of intact animals (8w A ), castrated animals (16w A¨C) (all regions n = 3, proximal n = 3, intermediate n = 3, distal n = 5, urethra n = 2), or animals that had been castrated for 8 weeks followed by androgen supplementation for 8 weeks (8w A¨C/8w A ) (all regions n = 5, proximal n = 4, intermediate n = 6, distal n = 4, urethra n = 4). Grafts were harvested at the indicated times (A), weighed, and used for immunocytochemical examination. Each bar represents the mean ¡À SD. (C, E, G): Sections of prostate tissue from intact (C), castrated (E), and castrated and androgen-replenished (G) animals stained with hematoxylin and eosin. Bars = 50 µm. (D, F, H): Sections of prostate tissue from intact (D), castrated (F), and castrated and androgen-replenished (H) animals immunohistochemically stained using an antibody against -smooth muscle actin. Sections were counterstained with hematoxylin. Bars = 50 µm. Abbreviations: w, weeks; RC, renal capsule; UGM, urogenital sinus mesenchyme.5 t- G/ [, U6 g7 Z

These results indicate that isolated cells from the proximal region of ducts as well as urethral cells have considerably greater in vivo regenerative potential than do cells from other ductal regions." Y8 l* w0 Z  A' p6 M
/ h2 Y$ n4 A4 M/ r% ^3 A
Cells from the Proximal Region and the Urethra Survive Prolonged Androgen Deprivation: g& m" Q  {- E0 e  D
: @' |- P  b; E1 o: b6 j7 n
Because prostatic tissue can regenerate after androgen withdrawal and replenishment, we reasoned that cells from regions most enriched in androgen-independent cells having regenerative potential would survive prolonged androgen deprivation and would regenerate prostatic tissue to a greater extent than cells isolated from regions that consisted mainly of transit-amplifying cells. Transit-amplifying cells are considered to be progenitor cells that are capable of division and that represent a post-stem cell compartment.4 E+ a* x' r. r& w' _
; z) ]! m) F. ^/ e1 o$ i" o0 p
To determine the sensitivity to androgens, donor cells isolated from different regions of ducts of androgen-replete animals were implanted under the RC of (a) androgen-replete recipients and harvested after 8 weeks, (b) castrated recipients and harvested after 16 weeks, and (c) castrated recipients and harvested after 8 weeks of androgen deprivation followed by 8 weeks of androgen supplementation (Fig. 4A). Each group received donor cells isolated from one of the following: all regions of ducts, the proximal, intermediate, or distal region, or the urethra (Fig. 1A). Very little growth was noted when cells from any region were implanted in castrated recipients (Fig. 4B, center bar in each group). Cells isolated from both the proximal region and the urethra maintained full regenerative capacity through 8 weeks of androgen deprivation; the amount of prostatic tissue that was formed after subsequent exposure to androgens was comparable with that noted when cells from the proximal region or the urethra were implanted in androgen-replete animals (Fig. 4B, supplemental online Fig. 5). In contrast, the ability of cells isolated from the intermediate and distal regions to regenerate prostatic tissue was severely compromised by androgen deprivation (Fig. 4B, supplemental online Fig. 5), indicating that these regions contained cells that require androgen for survival. Intermediate or distal cells formed more tissue in intact animals (65 ¡À 26 mg or 25 ¡À 19 mg, respectively) compared with animals maintained in an androgen-deficient state for 8 weeks and subsequently exposed to androgens for an additional 8 weeks (5 ¡À 3 mg or 3 ¡À 1 mg respectively; p ' p+ W0 v0 k$ D  r% S7 X* V
) `; @, |6 ^* \4 E% q& ^
Figure 5. Castration enriches the primitive cells in the remaining regions of ducts. (A): Three-color FACS analysis was performed to determine the percentage of Sca-1 6 integrin Bcl-2  cells in the proximal (n = 3) and remaining (n = 3) regions of ducts in intact and castrated animals. *, p

Histological examination of the sub-RC tissue removed from intact animals showed prostatic ducts containing basal and luminal cells (Figs. 1E, 1F, 4C). The ducts were enveloped by a thin band of smooth muscle (Fig. 4D) as is noted in normal prostate . When implants of either intermediate or distal cells were placed in androgen-deprived animals, no evidence of epithelial cells or ducts was noted (data not shown). However, when implants originating from proximal or urethral cells were examined, small rudimentary ducts were noted 16 weeks after androgen deprivation (Fig. 4E, 4F). This indicates that some epithelial cells survived and formed small ductal structures in the absence of androgen. These ducts are the likely source of the primitive cells from which the tissue regenerated after androgen administration. Histological examination of the tissue arising from proximal or urethral cells after androgen deprivation and subsequent regeneration indicated an extensive ductal network surrounded by significantly more smooth muscle tissue (Fig. 4H) than was noted in intact animals (Fig. 4D). These data show that two populations of cells capable of regenerating prostatic tissue in androgen-replete animals are present in the prostate. The first population resides in the proximal region and the urethra, and the survival of these cells does not require the presence of androgens. The second population (with more limited growth potential) is found in the remaining ductal regions and requires androgen for survival., o0 L+ Y5 B2 l1 _) }4 E2 D5 C3 M
" D+ ]+ m% s& W; w, `  I
Castration results in the involution of the gland and the loss of significant numbers of cells primarily from distal regions, with the proximal region being least affected . We therefore compared the phenotype and growth potential of cells isolated from the proximal and remaining regions of ducts from androgen-replete and castrated animals to determine whether cells with primitive regenerative features were enriched in the remaining regions of ducts after involution.! U* f! \0 U! q2 t; x( a
0 ?3 R3 S$ s, }+ |
We have previously shown that cells coexpressing the antigens Sca-1, 6 integrin, and Bcl-2 are concentrated in the proximal region of ducts . We therefore determined the coexpression of these antigens on cells in different ductal regions in androgen-replete and castrated animals to ascertain whether their incidence was altered by involution of the gland (Fig. 5A). Castration resulted in a 3.4-fold increase (p 2 T5 z, M! ^: E% e
! ]$ d8 t- k% Q5 ]% e
We next compared the proliferative potential of cells isolated from different regions of ducts from both androgen-replete and castrated animals. Cells isolated from the remaining regions of ducts of castrated animals formed 5.3-fold more sub-RC tissue than did cells isolated from the remaining regions of ducts of intact animals (p # c  d( F; L3 H5 U+ u
; v4 }! O) ^" d1 [- A2 S
Cells from the Proximal Region Can Be Serially Passaged In Vivo9 H) S+ T% `2 j- H7 {& P8 I$ ]% |8 }
: z& K7 w/ H3 y( x
Passage of cells isolated from undissected recombinant tissue (i.e., all regions). Because stem cells have high regenerative potential, we determined whether the RC tissue obtained after implantation of proximal cells could be serially passaged in vivo more frequently than tissue arising from distal cells. Cells (105) were isolated from the proximal and distal regions of primary prostates and implanted under the RC (Fig. 2A). Prostatic tissue obtained from proximal and distal cell sub-RC implants was weighed and digested. Cells (105) from each of these digests were combined with UGM and re-implanted under the RC of a second animal (P2). This process was repeated until no prostatic tissue growth was noted (Fig. 2A, 2B). Cells from the proximal region can be serially passaged four times, whereas distal cells can be passaged twice. In addition, as noted previously in primary implants (Fig. 1), cells isolated from the proximal region formed larger amounts of prostatic tissue at each consecutive passage than did cells isolated from the distal region, indicating that proximal cells were capable of more extensive division than distal cells.3 l' b) {# b0 a$ f8 F" Z  i3 R

Passage of cells isolated from the proximal region of recombinant tissue. We wished to determine whether the sub-RC prostatic tissue formed an "organ" that maintained a physiologically distinct proximal-distal ductal axis similar to that observed in the prostate and whether cells isolated from different regions of the sub-RC graft displayed similar disparate growth properties to that noted in primary prostatic ducts. Cells were therefore isolated from proximal and distal ductal regions of the primary prostate (Fig. 3A, 3B) and implanted under the RC. Microdissection of the sub-RC tissue mass obtained from proximal cells (Fig. 3A, 3C) revealed an interconnected series of ducts consisting of proximal and distal regions very similar to that obtained from the primary prostate (Fig. 3B). Quite remarkably, when these ducts were dissected into proximal and distal regions and isolated cells were reimplanted under the RC, the cells from the proximal region once again formed large amounts of prostatic tissue (228 ¡À 83 mg) compared with cells obtained from the distal region (5 ¡À 4 mg; p
! M# h" S0 X% {! z  o* P4 B7 D- A
These data indicate that primitive proximal cells are programmed to re-establish a proximal-distal ductal axis under the RC (Fig. 3C) and have extensive regenerative capacity, as they can be passaged through four generations of sub-RC grafts. Cells isolated from the proximal region of successive grafts form considerably more sub-RC tissue (Fig. 3; 228 mg P2, 76 mg P3) than those isolated from similar passages from the entire graft (Fig. 2; all regions, 78 mg P2, 2 mg P3). This confirms that the proximal region of sub-RC tissue is similar to the proximal region of a primary prostate gland because it is also enriched in primitive cells with high regenerative capacity.

6 Integrin-Expressing Proximal Cells Have High In Vivo Proliferative Potential4 `- H& X. t, ]. Z
& H. Q- k; |! ?" N0 Y7 \
6 integrin is present on primitive cells from a number of origins . To determine whether cells expressing this antigen were enriched after castration and to ascertain whether 6 integrin-expressing cells had greater regenerative capacity than cells not expressing this antigen, we performed two sets of experiments. We first determined expression of this antigen on proximal and remaining cells from androgen-replete and castrated animals. The proximal region of intact animals contained 2.8-fold (p

Figure 6. 6 Integrin-expressing cells are enriched in the proximal region of ducts and form more prostatic tissue under the RC than those depleted of this antigen. (A): The expression of 6 integrin in the proximal and remaining regions of intact (n = 6) and castrated (n = 4) animals. *, p

DISCUSSION% o8 f% }- M& z# f
" W" H) E3 E$ w6 V" u
We show that ductal regions of androgen-replete animals vary markedly in their ability to survive androgen ablation. Two distinct populations of cells with different androgen sensitivities are capable of sub-RC organ reconstitution. The proximal region and the urethra contain cells that regenerate prostatic tissue far more robustly than cells isolated from the intermediate or distal regions of ducts. These cells survive androgen ablation and regenerate prostatic tissue fully once androgens are re-administered. Both these attributes would be expected in a stem cell population. A second population, residing in the intermediate and distal regions of ducts, is capable of a more limited organ reconstitution and is androgen-sensitive in terms of their survival. Cells in these regions are unable to regenerate prostatic tissue after androgen deprivation. These may be the cells that are referred to as transit-amplifying cells and that are considered to be a post-stem cell population. Because all of the regions of the ducts express equivalent levels of androgen receptors and 5--reductase , the differences in sensitivity to androgens are not due to regional variations in expression of these molecules.5 U9 M( }2 I6 j2 I3 b
" s6 U3 B8 B  I* C; \
Although we and others  used fragments of epithelium of embryonic origin.2 P" v% f% r( Q
" Z- ?$ y! o% `( f' y& E
We also show that, in the remaining regions of ducts, castration results in the enrichment of primitive cells that are capable of forming prostatic tissue. Cells removed from the remaining regions of ducts of an involuted prostate formed more tissue under the RC than did cells isolated from these regions of an androgen-replete prostate. Castration results in the loss of large numbers of epithelial cells from the distal regions of ducts . The remaining regions of ducts of intact animals have very few of these primitive cells. These results support the idea that the cells lost as a result of castration are differentiated nonregenerative cells. It is interesting that isolated cells removed from remaining regions of ducts and placed in castrated recipients for 2 months followed by androgen administration could not reconstitute prostatic tissue under the RC whereas cells isolated from the remaining ductal regions of an involuted prostate removed from an androgen-deprived animal can regenerate prostatic tissue. This discrepancy may be due to the enrichment of primitive cells in the remaining ductal regions that follows castration and involution as this process results in the loss of differentiated cells. Thus, a larger number of primitive cells would be present in the remaining regions of castrated prostates than in the remaining regions of androgen-replete prostates.% p. `" i* ?$ a, ]* H& T3 W* }

We also show that primitive cells in the proximal region are programmed to regenerate a proximal-distal ductal axis through consecutive passages in a prostate reconstitution assay. The most definitive test of stem cell function is their ability to reconstitute an organ. Serially transplanted bone marrow can reconstitute lethally irradiated mice .4 f$ k, h3 Y  N3 W& [8 S$ M, |

The appearance of the dissected sub-RC tissue was remarkably similar to that noted in an intact prostate gland (Fig. 3B, 3C), and primitive cells were concentrated in the proximal regions of both the intact gland and the sub-RC tissue. This indicates that primitive prostate cells are programmed to regenerate an organ with regional differences in regenerative capacity. Isolated cells from the proximal regions of sequential grafts could be passaged four times before senescence and formed large amounts of prostatic tissue (228 ¡À 83 mg) that greatly exceeded the size of a primary prostate gland (11 ¡À 1 mg). This indicates that primitive cells in the proximal region of the sub-RC grafts have considerable regenerative capacity. It is not surprising that primitive proximal cells cannot be passaged indefinitely; experimental evidence indicates that hematopoietic stem cells show signs of aging and have a limited functional lifespan . This is likely to be an important discriminator between healthy stem and tumor stem cells.
5 A9 D: @7 N9 g
6 Integrin is the only common protein expressed by stem cells of a number of origins and is present on the primitive cells of different tissues .

Stem cells generally reside in specialized niches that form a microenvironment that maintains their primitive phenotype. The proximal region of ducts provides a protective niche for prostatic stem cells which may permit them to survive in the absence of androgen. This region is least affected by castration in terms of apoptosis and cell loss .

SUMMARY
. j. y2 t+ y: t) B1 e9 U
The data in this manuscript show that two distinct cell populations with different androgen sensitivities are located in different regions of prostatic ducts. The proximal region contains primitive cells with attributes of stem cells. They survive in the absence of androgen and are able to regenerate large amounts of prostatic tissue after androgen replenishment. The remaining regions of ducts contain cells with limited regenerative capacity that are unable to withstand androgen withdrawal. Interestingly, primitive proximal cells are programmed to maintain a functional proximal-distal ductal axis through successive passages in a sub-RC prostate reconstitution assay. The prostate is an androgen-sensitive organ that is the site of considerable pathology as both prostate cancer and benign prostatic hyperplasia are common diseases. Because prostate carcinoma evolves into an androgen-independent disease that may reflect the emergence of cells with stem-like properties, the identification of cells within regions of prostatic ducts capable of withstanding androgen ablation may lead to advances in elucidating the biology of proliferative prostatic diseases. Because the prostate is a nonessential organ, it may be possible to develop a therapy that targets and ablates the stem cell compartment prior to the development of proliferative abnormalities in this gland.2 l& Y& [" e$ ?$ A, _: _) v/ T

DISCLOSURES
) A2 J1 D' f$ k) r; B
The authors indicate no potential conflicts of interest.
6 O& k. s! M8 \: _5 \2 L
ACKNOWLEDGMENTS

This work was supported by National Institutes of Health Grant DK52634, Department of Defense Grant W81XWH-04-1-0255, the University of Cape Town Faculty Research Fund, and the South Africa Medical Research Council. We thank Dr. Susan Logan, Department of Urology, New York University School of Medicine, for her assistance with the staining for androgen receptors.( ~% F' I% r& u( C! S- K: W
      【参考文献】
( ?2 b3 s( ?2 R5 c$ I+ Y) Y9 p6 X

Sugimura Y, Cunha GR, Donjacour AA. Morphological and histological study of castration-induced degeneration and androgen-induced regeneration in the mouse prostate. Biol Reprod 1986;34:973¨C983.+ y0 s% U. S9 O& e
3 {6 c( i* b5 J; u; s6 g
Rouleau M, Leger J, Tenniswood M. Ductal heterogeneity of cytokeratins, gene expression, and cell death in the rat ventral prostate. Mol Endocrinol 1990;4:2003¨C2013.& `# P0 Y: ?: A2 a) U/ E9 ?" `/ _

Salm SN, Burger PE, Coetzee S et al. TGF-&szlig; maintains dormancy of prostatic stem cells in the proximal region of ducts. J Cell Biol 2005;170:81¨C90.

Tsujimura A, Koikawa Y, Salm S et al. Proximal location of mouse prostate epithelial stem cells: A model of prostatic homeostasis. J Cell Biol 2002;157:1257¨C1265.  w* B1 }7 i# y

Burger PE, Xiong X, Coetzee S et al. Sca-1 expression identifies stem cells in the proximal region of prostatic ducts with high capacity to reconstitute prostatic tissue. Proc Natl Acad Sci U S A 2005;102:7180¨C7185.% h# p) R" E, H8 @- z. c( c
& S4 E: G0 X0 v' ?5 G$ ~0 Y0 ^: k. F
Xin L, Lawson DA, Witte ON. The Sca-1 cell surface marker enriches for a prostate-regenerating cell subpopulation that can initiate prostate tumorigenesis. Proc Natl Acad Sci U S A 2005;102:6942¨C6947.0 G6 g7 g% U) }7 }6 u  q& H2 y" |
( d9 `" [: w) [7 e9 }) w8 w) [
Reya T, Morrison SJ, Clarke MF et al. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105¨C111./ y* d* L$ a- e
9 o/ V" ~- P( B3 J6 {+ @& A
Passegue E, Jamieson CH, Ailles LE et al. Normal and leukemic hematopoiesis: Are leukemias a stem cell disorder or a reacquisition of stem cell characteristics? Proc Natl Acad Sci U S A 2003;100 (suppl 1):11842¨C11849.

Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730¨C737.

Al-Hajj M, Wicha MS, Benito-Hernandez A et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100:3983¨C3988.
; D' o% o9 ]4 i
Al-Hajj M, Clarke MF. Self-renewal and solid tumor stem cells. Oncogene 2004;23:7274¨C7282.

Kim CF, Jackson EL, Woolfenden AE et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005;121:823¨C835.# y8 G! F" L' t0 e; X. Y

Cunha GR, Donjacour A. Mesenchymal-epithelial interactions: Technical considerations. Prog Clin Biol Res 1987;239:273¨C282.; {  Y1 Q8 ^  Y# H
) m  G3 f1 B. N* r7 H
Norman JT, Cunha GR, Sugimura Y. The induction of new ductal growth in adult prostatic epithelium in response to an embryonic prostatic inductor. Prostate 1986;8:209¨C220.

Chung LW, Cunha GR. Stromal-epithelial interactions: II. Regulation of prostatic growth by embryonic urogenital sinus mesenchyme. Prostate 1983;4:503¨C511., d7 z% F: X0 |% u

Sugimura Y, Cunha GR, Donjacour AA. Morphogenesis of ductal networks in the mouse prostate. Biol Reprod 1986;34:961¨C971.
8 \1 ?: t7 M+ V/ w2 c) c
Salm SN, Takao T, Tsujimura A et al. Differentiation and stromal-induced growth promotion of murine prostatic tumors. Prostate 2002;51:175¨C188.
. V% X/ r9 A2 ?- u
Takao T, Tsujimura A, Coetzee S et al. Stromal/epithelial interactions of murine prostatic cell lines in vivo: A model for benign prostatic hyperplasia and the effect of doxazosin on tissue size. Prostate 2003;54:17¨C24.0 U% G& W& g8 A% x3 A8 j* B- N9 u

Kim MJ, Cardiff RD, Desai N et al. Cooperativity of Nkx3.1 and Pten loss of function in a mouse model of prostate carcinogenesis. Proc Natl Acad Sci U S A 2002;99:2884¨C2889.

Kinbara H, Cunha GR. Ductal heterogeneity in rat dorsal-lateral prostate. Prostate 1996;28:58¨C64.
+ j4 R; r& u- ?# {$ X
Donjacour AA, Cunha GR. Assessment of prostatic protein secretion in tissue recombinants made of urogenital sinus mesenchyme and urothelium from normal or androgen-insensitive mice. Endocrinology 1993;132:2342¨C2350.
: @; K$ D+ a! p
He WW, Sciavolino PJ, Wing J et al. A novel human prostate-specific, androgen-regulated homeobox gene (NKX3.1) that maps to 8p21, a region frequently deleted in prostate cancer. Genomics 1997;43:69¨C77.
) G- }0 E! b4 `
Nemeth JA, Lee C. Prostatic ductal system in rats: Regional variation in stromal organization. Prostate 1996;28:124¨C128.
4 P1 D. S7 Z. l, p8 J4 P/ N) o
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.

Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci U S A 2000;97:10960¨C10965.! F. n* j3 D9 J9 R. _' f

Shinohara T, Avarbock MR, Brinster RL. beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999;96:5504¨C5509.4 a9 M4 H2 Q. L
  J1 n4 u, a2 f/ H( j9 m% `( P* j
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.
. y# @& x+ n; n
Jackson KA, Majka SM, Wulf GG et al. Stem cells: A minireview. J Cell Biochem 2002;38 (Suppl):1¨C6.% i) v: B1 c/ B% k' p! {

Tumbar T, Guasch G, Greco V et al. Defining the epithelial stem cell niche in skin. Science 2004;303:359¨C363.
9 U7 h/ N; `7 w& J
Fortunel NO, Otu HH, Ng HH et al. Comment on " ¡®Stemness¡¯: Transcriptional profiling of embryonic and adult stem cells" and "a stem cell molecular signature". Science 2003;302:393 author reply 393.
- p$ e5 G/ H, O& [
Prins GS, Cooke PS, Birch L et al. Androgen receptor expression and 5 alpha-reductase activity along the proximal-distal axis of the rat prostatic duct. Endocrinology 1992;130:3066¨C3073.
% V$ S6 E/ g9 f/ G
Xin L, Ide H, Kim Y et al. In vivo regeneration of murine prostate from dissociated cell populations of postnatal epithelia and urogenital sinus mesenchyme. Proc Natl Acad Sci U S A 2003;100 (suppl 1):11896¨C11903.
7 Q' T: Y& A7 ]
Chen J, Astle CM, Harrison DE. Genetic regulation of primitive hematopoietic stem cell senescence. Exp Hematol 2000;28:442¨C450.% p: M% @/ M* X, S, A$ a
0 s8 \/ H" p+ b1 }+ Y% c( g- C
Maggio-Price L, Wolf NS, Priestley GV et al. Evaluation of stem cell reserve using serial bone marrow transplantation and competitive repopulation in a murine model of chronic hemolytic anemia. Exp Hematol 1988;16:653¨C659.

Jones RJ, Celano P, Sharkis SJ et al. Two phases of engraftment established by serial bone marrow transplantation in mice. Blood 1989;73:397¨C401.& n' {+ Y  o# H
/ k% e$ }. |3 G# p% H
Geiger H, Van Zant G. The aging of lympho-hematopoietic stem cells. Nat Immunol 2002;3:329¨C333.
5 f/ {  X  F- ^& ?9 Q) i
Collins AT, Habib FK, Maitland NJ et al. Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(1)-integrin expression. J Cell Sci 2001;114Pt 21:3865¨C3872.: E5 j7 _3 n, U

Banerjee PP, Banerjee S, Zirkin BR et al. Telomerase activity in normal adult Brown Norway rat seminal vesicle: Regional distribution and age-dependent changes. Endocrinology 1998;139:1075¨C1081.

Caporaso GL, Lim DA, Alvarez-Buylla A et al. Telomerase activity in the subventricular zone of adult mice. Mol Cell Neurosci 2003;23:693¨C702.  d) u2 ]) J, v. V- l  x1 f. I

Wright WE, Piatyszek MA, Rainey WE et al. Telomerase activity in human germline and embryonic tissues and cells. Dev Genet 1996;18:173¨C179.

收藏0 分享0 顶0 踩0