Stem Cell Activity of Human Side Population and 6 Integrin-Bright Keratinocytes - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:57 |只看该作者 |倒序浏览 |打印

作者：Atsushi Terunumaa, Veena Kapoorb, Carole Yeea, William G. Telfordb, Mark C. Udeya, Jonathan C. Vogela 1 ^* G: [5 H# N% V7 q+ {

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      【摘要】
   The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP-KCs) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow-derived side population (SP) hematopoietic stem cells, have been identified in human skin, but their potential to function as keratinocyte stem cells (KSCs) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of ¦Â1 and 6 integrins and are distinct from SP-KCs, which express low levels of integrins, may be enriched for KSCs based on reported results of in vitro cell culture assays. When in vitro assays were used to measure total cell output of human SP-KCs and integrin-bright keratinocytes, we could not document their superior long-term proliferative activity versus unfractionated keratinocytes. To further assess the KSC characteristics in SP-KCs and integrin-bright keratinocytes, we used an in vivo competitive repopulation assay in which bioengineered human epidermis containing competing keratinocyte populations with different human major histocompatibility (MHC) class I antigens were grafted onto immunocompromised mice, and the intrinsic MHC class I antigens are used to quantify expansion of competing populations. In these in vivo studies, human SP-KCs showed little competitive expansion in vivo and were not enriched for KSCs. In contrast, keratinocytes expressing elevated levels of 6 integrin and low levels of CD71 (6-bright/CD71-dim) expanded over 200-fold during the 33-week in vivo study. These results definitively demonstrate that human 6-bright/CD71-dim keratinocytes are enriched with KSCs, whereas SP-KCs are not. 3 }$ S) M7 d4 H4 J  V
      【关键词】 Xenotransplantation Tissue engineering MHC class I genes Flow cytometry Nude mice
   INTRODUCTION

Human epithelia are maintained by tissue stem cells capable of self-renewal and extensive proliferation . In this article, we first used in vitro assays to measure the total cell output of SP-KCs and integrin-bright keratinocytes but found that these cells did not possess greater long-term proliferative capacity than total keratinocytes.5 H% w! {3 ?: i) @* [- I1 U: V5 u
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To assess the long-term repopulating ability of human KSC candidates such as SP-KCs and 6-bright/CD71-dim keratinocytes, in vivo assays are required. Several different in vivo approaches have been used to assess mouse or human KSC behavior such as rat trachea re-epithelization . In the in vivo competitive repopulation assay used in the present studies, bioengineered human epidermis was grafted onto immunocompromised mice, and intrinsic human major histocompatibility (MHC) class I antigens were used to distinguish between the competing keratinocyte populations and to quantify their relative self-renewal and long-term repopulating abilities. Grafts were serially transplanted every 8¨C16 weeks to better assess self-renewal and long-term repopulation.

MATERIALS AND METHODS
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Tissues and Cells: ^8 f3 m5 c3 Y( ^# k
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Human neonatal foreskin specimens were collected with informed consent of parents or guardians and institutional approval. Epidermal cells were prepared by stepwise processing with dispase (Discovery Labware, Bedford, MA, http://www.bdbiosciences.com) and trypsin (USB Corporation, Cleveland, http://www.usbweb.com/) .  m# j( f7 @9 G6 l* O

SP Cells- b6 Q8 c' i2 x! Z6 K

SP analysis/sorting was as described  with minor modifications. Briefly, epidermal cells were resuspended at 106 cells/ml in Hanks' balanced salt solution (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) containing 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2% fetal bovine serum (FBS), 25 U/ml penicillin G, and 25 µg/ml streptomycin (HHF buffer). Cells were preincubated at 37¡ãC for 15 minutes with or without 100 µM verapamil (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and incubated for 90 minutes at 37¡ãC with 10 µg/ml Hoechst 33342 (Sigma-Aldrich). Cells were subsequently stained with monoclonal antibody (mAb) described below, washed with HHF buffer containing Hoechst 33342 and verapamil if applicable, and stained with 5 µg/ml 7-aminoactinomycin D (Invitrogen). Expression profiles of cell surface molecules were not altered by the Hoechst 33342 incubation and staining. A FACSVantage DiVa (BD Biosciences¨CImmunocytometry Systems, Franklin Lakes, NJ, http://www.bdbiosciences.com) equipped with a 355-nm mode-locked solid-state laser and a 488-nm diode-pumped solid-state laser was used to detect and isolate SP-KCs and other populations of cells. In each experiment, all populations of cells (total, SP-KC, 6-bright/CD71-dim, or ¦Â1-bright) were isolated from the same pool of epidermal cells by staining the whole pool with both Hoechst 33342 and mAb.

Antibodies5 }, Y* ]3 K9 L: l

Anti-human cytokeratin 14 mAb (clone LL002; Biomeda, Foster City, CA, http://biomeda.com/) and Alexa Fluor 647 goat anti-mouse IgG3 secondary antibody (Invitrogen) were used to detect keratin 14 in cells permeabilized by Cytofix/Cytoperm (BD Biosciences¨CPharmingen, San Diego, http://www.bdbiosciences.com). Anti-HLA-A2 (clone BB7.2), anti-CD29 (¦Â1 integrin; clone MAR4), anti-CD49f (6 integrin; clone GoH3), and CD71 (clone M-A714) mAbs (all from BD Biosciences¨CPharmingen) were used for flow cytometry. For adherence assays, anti-HLA-A,B,C antibody (clone V1029; Biomeda) was used after conjugation using a Zenon Alexa Fluor 488 mouse IgG2a labeling kit (Invitrogen).1 Y+ T4 B  k$ V* `( g5 A. X

Cell Cycle Analysis

Cells were fixed in 70% ethanol for 20 minutes on ice, incubated with 40 µg/ml RNase for 20 minutes at 37¡ãC, and resuspended in phosphate-buffered saline (Invitrogen) containing 2% FBS and 3 µg/ml propidium iodide (Sigma-Aldrich). Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences¨CImmunocytometry Systems) in linear mode.7 Z! v4 x' c. W+ Y4 _# M  G1 i
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Adherence Assay. H0 n  f  M& K4 w! z- c+ N
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Multiwell 96-well culture plates (Discovery Labware) were seeded with mitomycin C-treated 3T3 cells, human fibroblasts (passage 3), or collagen gels . Collagen IV-coated plates were purchased from Discovery Labware. Flow-purified keratinocytes were stained with anti-HLA-A,B,C antibody and 4 µM ethidium homodimer-1 (Invitrogen) and seeded onto the plates in triplicate. Living keratinocytes (HLA-A,B,C-positive and ethidium homodimer-1-negative) adhering in 60 minutes were counted and are shown as percentages relative to the number of total living keratinocytes initially seeded.
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In Vitro Assays

Colony-forming efficiency was measured as described .- f5 @7 d  q! K5 Y9 h: j

Human Skin Equivalents- v" r0 f/ ?  H$ j; A' O  r

Procedures to construct and graft human skin equivalents (HSEs) were based on established methods , before being grafted as HSEs onto the backs of 6- to 8-week-old male NIH Swiss nu/nu mice (NCI-Frederick Animal Production Area, Frederick, MD, http://web.ncifcrf.gov/researchresources/apa/), which were housed and used in accordance with institutional guidelines.

For serial transplantation experiments, individual grafts of HSE were excised and epidermal cell suspensions were prepared. Heavy pigmentation of grafts allowed the clear distinction between human skin (HSE) and surrounding mouse skin (Fig. 3A) so that 99% of the epidermal cells prepared from grafts were HLA-A,B,C( ) human cells (supplemental online Fig. 1). Each graft yielded 2 to 3 million epidermal cells, and one third of the epidermal cells were used for flow cytometry analysis to determine the fractions of human cells that were HLA-A2( ) and HLA-A2(¨C). The remaining epidermal cells were combined with those from other grafts in the same group without further purification and seeded onto collagen gels to generate new sets of HSE. At the second transplantation (day 158), HLA-A2( ) cells from the grafts were combined with equal numbers of HLA-A2(¨C) cultured human keratinocytes (passage 3) to ensure good graft take.
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Immunostaining

To observe the distribution of HLA-A2( ) keratinocytes within grafts, 6-µm-thick frozen sections were fixed with acetone, incubated with primary mAb against HLA-A2 (1:50, clone BB7.2; BD Biosciences¨CPharmingen) or mouse IgG2a isotype control (1:50, clone G155-178; BD Biosciences¨CPharmingen) for 60 minutes at room temperature, and then incubated with goat anti-mouse Ig FITC-conjugated F(ab')2 fragments (Invitrogen, BioSource, Camarillo, CA, http://www.biosource.com/) for 45 minutes at room temperature.# }# ^$ ^$ I6 E# {/ I  w

Statistical Analyses
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Results were expressed as mean ¡À range. Data were analyzed by Mann-Whitney U test. P values less than .05 were considered to be significant.) d/ z  O& J) [* l6 E
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RESULTS4 R5 ]: F1 D$ C
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We have determined that SP-KCs in human epidermis represent a small population (0.3%) of epidermal cells (Fig. 1A) that express the basal cell marker keratin 14 (Fig. 1B) and are quiescent compared with total keratinocytes (Fig. 1C)  (Fig. 1D). To resolve these apparently contradictory results, we hypothesized that SP-KCs might represent primitive KSCs that do not yet express high levels of 6 and ¦Â1 integrin, analogous to the primitive status attributed to SP hematopoietic stem cells. In addition, we sought to determine whether the 6-bright population is enriched for keratinocytes that exhibit stem cell behavior in vivo. To test these hypotheses, we used both in vitro and in vivo assays to compare the long-term repopulating stem cell behaviors of SP-KCs and keratinocytes expressing high levels of integrins.. k# T, q3 g! R0 Y; A5 h6 r
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Figure 1. Human epidermal side population keratinocytes (SP-KCs). (A): SP-KCs (left, gated) are sensitive to verapamil (right). (B): SP-KCs are mostly positive for a basal cell marker keratin 14. (C): SP-KCs are quiescent having 4% in S and G2/M phases of the cell cycles (right), whereas the total population has 12% in S and G2/M (left). (D): SP-KCs express low levels of 6 and ¦Â1 integrins. Abbreviations: FSC, forward scatter; SP, side population.+ s( ~  T) G: a+ l8 F; q
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Because SP-KCs do not express high levels of 6 or ¦Â1 integrin, we first assessed their ability to adhere to extracellular matrices used for in vitro assays. SP-KCs, as well as total keratinocytes and ¦Â1-bright cells, were purified ( 90%) using flow cytometry, seeded onto extracellular matrices or feeder layers, and the percentage of adherent cells was assessed after 1 hour. SP-KC and ¦Â1-bright cells bound to extracellular matrices or feeder layers to similar extents (Fig. 2A). Next, we assessed the in vitro colony-forming efficiency of SP-KCs on irradiated 3T3 feeder cells. Whereas ¦Â1-bright cells and 6-bright/CD71-dim cells, both of which are believed to be enriched for KSCs based on in vitro assay results . Consistent with results of the colony formation assays (Fig. 2B), SP-KCs showed much lower cell output compared with the other populations of keratinocytes during the initial 2 weeks (Fig. 2C, bottom panel). However, after the first passage at week 2, all three populations had equivalent growth rates (Fig. 2C, top panel) and all populations senesced by 3 months, demonstrating comparable life spans (Fig. 2C, top panels). Interestingly, all three populations in the first experiment (Fig. 2C, left) developed similar 6 and ¦Â1 integrin expression profiles by day 26 (Fig. 2D). Thus, in vitro assays did not reveal superior longevity or long-term repopulating ability in SP-KCs, ¦Â1-bright cells, or 6-bright/CD71-dim cells.

Figure 2. In vitro growth potential of keratinocytes. (A): SP and ¦Â1 integrin-bright keratinocytes adhere to each extracellular matrix equivalently well. (B): Colony formation assay for total, SP, ¦Â1 integrin-bright, and 6 integrin-bright/CD71-dim keratinocytes. The photo is from one of three independent sets of experiments, whereas the graph represents the average of all three sets. Values in the graphs are of the summed colony areas (top) or the numbers of colonies (bottom) relative to those of total cell control. Data shown in the graph are means ¡À S.E.M. of three independent sets of experiments. *, p ! P+ m" r6 H# y7 C" @# L8 b

To better assess the stem cell characteristics of SP-KCs and 6-bright/CD71-dim keratinocytes, we employed an in vivo competitive repopulation assay that we developed to quantify the self-renewal and long-term repopulating abilities of human KSC candidates . The relative percentage of competing HLA-A2( ) or HLA-A2(¨C) keratinocytes in grafted HSEs, after many epidermal turnover or replacement cycles in vivo and after serial transplantation, should reflect the relative numbers of KSCs in the initial competing HLA-A2( ) and HLA-A2(¨C) populations. A population that is enriched for KSCs will thus yield greater percentages of keratinocytes within grafts in long-term competitive studies.4 Q! ~$ K" b* H
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Figure 3. In vivo assessment of long-term repopulating ability. (A, B): Human skin equivalents (HSEs) generated from the mixtures of HLA-A2( ) primary and HLA-A2(¨C) cultured keratinocytes are grafted repeatedly onto the backs of nude mice. At every transplantation, a portion of keratinocytes are analyzed by flow cytometry to measure the percentage of HLA-A2( ) cells. (C): Hematoxylin and eosin staining of an HSE graft shows multilayered epidermis resembling the human tissue. Original magnification, x150. (D): HLA-A2( ) epidermal cells in a 21-week graft form a column-like structure (arrows), suggesting their clonal repopulation. White dots denote the border between epidermis and dermis. Dermis (on the bottom right corner) shows high background signals. Original magnification, x640. (E): Side population keratinocytes (SP-KCs) express HLA-A2 and HLA-A,B,C. (F): Percentages of HLA-A2( ) flow-sorted primary keratinocytes seeded onto artificial dermis to construct HSEs. (G): Percentages of HLA-A2( ) keratinocytes at the time of grafting (after a 4-day in vitro organ culture). Because bright background signals from HLA-A2(¨C) cultured keratinocytes are commonly observed after the 4-day organ culture, measurements may not be as accurate as those of primary cells (F) or cells from grafts (H). (H): Percentages of HLA-A2( ) cells at 22 weeks after grafting (day 158). (I): Total and 6-bright/CD71-dim HLA-A2( ) primary keratinocytes, but not SP-KCs, overwhelm HLA-A2(¨C) cultured keratinocytes (left panel). Data shown represent the average ¡À S.E.M. of four replicated samples. An independent experiment with SP-KCs and 6-bright/CD71-dim keratinocytes (right panel; n = 1 for each) shows consistent results. *, p

We first confirmed that the SP-KCs to be introduced into HSEs expressed HLA-A2 molecules at levels comparable with those of other keratinocytes (Fig. 3E). Three distinct populations of HLA-A2( ) keratinocytes were purified by flow-sorting for comparison in the in vivo assay: total keratinocytes, SP-KCs, and 6-bright/CD71-dim keratinocytes. Each population of HLA-A2( ) keratinocytes was immediately mixed with HLA-A2(¨C) cultured keratinocytes (passage 3) and seeded on collagen gels to generate four pieces of HSE per keratinocyte population. Because only a small number of SP-KCs can be collected from primary epidermal tissue samples by flow-sorting, and a minimum of 500,000 keratinocytes are required for successful HSE grafting onto immunocompromised mice, we seeded a mixture of 5,000 of the HLA-A2( ) flow-purified primary keratinocyte populations to be evaluated and 1,000,000 of HLA-A2(¨C) competing total keratinocytes (passage 3) onto each collagen gel resulting in an initial percentage of HLA-A2( ) keratinocytes of 0.5% (Fig. 3F). At the time of grafting (after a 4-day in vitro organ culture), the HSEs for each population contained comparable percentages (0.18%¨C0.25%) of HLA-A2( ) cells (Fig. 3G). The decline in the percentages of HLA-A2( ) keratinocytes during the 4-day culture period may reflect a time lag in initiating cell division in primary keratinocytes in contrast to the competing HLA-A2(¨C) passage-3 cultured keratinocytes that are already actively cycling and may expand more efficiently during the short in vitro culture period.

All 12 HSE grafts (four for each of three HLA-A2( ) populations) were successfully grafted and maintained for 8¨C16 weeks to achieve multiple epidermal turnover cycles (five or more cycles) , who did not observe a growth advantage of 6-bright/CD71-dim cells during a 3-month observation period in vivo. An independent set of in vivo competitive repopulation experiments demonstrated consistent results for SP-KCs and 6-bright/CD71-dim cells (Fig. 3I, right). Based on the results of these in vivo competitive repopulation assays, we concluded that the 6-bright/CD71-dim keratinocyte population is enriched for KSCs, whereas SP-KCs do not demonstrate the KSC behavior of long-term extensive proliferation.

DISCUSSION
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Although it has been hypothesized that the SP phenotype may be a global marker of stem cells in various tissues, reliable in vivo assays are required to demonstrate stem cell behavior, especially for epithelial tissues where the biological function of SP cells remains controversial .

Another advantage of our approach is that minimal in vitro cellular manipulation, and thus unwanted selection, occurs before the grafting procedure. Because not all 6-bright/CD71-dim keratinocytes are KSCs, this in vivo assay can now be used in conjunction with different panels of cell surface markers to more precisely define the KSC subpopulations that exist within the 6-bright/CD71-dim population. This assay can also serve as a prototype for developing an in vivo assay for human squamous cell carcinoma stem cells. Ultimately, the ability of an in vivo assay to recapitulate human squamous cell carcinomas will be necessary to assess the stem cell behavior and cancer-causing abilities of putative cancer stem cell candidates. Finally, this approach should be adaptable to assess the self-renewal and long-term repopulating abilities of stem cell candidates from other human epithelial tissues.+ K2 H: ?' Q' R

DISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS* M( w6 B! T  @% W

We thank Melissa B. Shaya, Priya Batra, and Michelle J. Dyck for assistance in surgical procedures. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.7 {/ \- o+ s6 a# \8 I
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