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作者：Jennifer B. Gilnera, William G. Waltona, Kimberly Gushb, Suzanne L. Kirbya作者单位：Departments of aPathology and Laboratory Medicine andbPediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA , K4 e/ _' x l, s
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【摘要】
/ |" `+ R8 j( ^; c( V# X7 l8 I Hematopoietic stem cells (HSCs) have enormous potential for use in transplantation and gene therapy. However, the frequency of repopulating HSCs is often very low; thus, highly effective techniques for cell enrichment and maintenance are required to obtain sufficient cell numbers for therapeutic use and for studies of HSC physiology. Common methods of HSC enrichment use antibodies recognizing HSC surface marker antigens. Because antibodies are known to alter the physiology of other cell types, we investigated the effect of such enrichment strategies on the physiology and lineage commitment of HSCs. We sorted HSCs using a method that does not require antibodies: exclusion of Hoechst 33342 to isolate side population (SP) cells. To elucidate the effect of antibody binding on this HSC population, we compared untreated SP cells with SP cells treated with the Sca-1 c-Kit Lin¨C (SKL) antibody cocktail prior to SP sorting. Our findings revealed that HSCs incubated with the antibody cocktail had decreased expression of the stem cell-associated genes c-Kit, Cd34, Tal-1, and Slamf1 relative to untreated SP cells or to cells treated with polyclonal isotype control antibodies. Moreover, SKL antibodies induced cycling in SP cells and diminished their ability to confer long-term hematopoietic engraftment in lethally irradiated mice. Taken together, these data suggest that antibody-based stem cell isolation procedures can have negative effects on HSC physiology.
6 a" I1 C; ^9 z( [ 【关键词】 Hematopoietic stem cells Hematopoietic stem cell transplantation Side population cells Long-term engraftmentSca- c-Kit LinC' U3 T3 v2 W6 ~
INTRODUCTION
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Hematopoietic stem cells (HSCs) are defined by their ability to both self-renew and differentiate into all lineages of the hematolymphoid system. Efficient isolation and manipulation of this rare cell population has important implications in both basic research and clinical use. Isolating highly purified cells is important in studying the molecular mechanisms controlling HSC self-renewal and differentiation. Elucidating the mechanisms for maintaining stem cell properties may improve our ability to manipulate and potentially expand HSCs without losing their developmental potential. Furthermore, precise definition of the HSC phenotype will facilitate the most efficient possible purification of therapeutic cells for transplantation.
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) `7 c$ z6 H$ R8 Z/ S7 ^4 D3 JOver the past several decades, many groups have sought to define and purify long-term repopulating pluripotent hematopoietic stem cells. Protocols for the enrichment of HSCs are based on cell density .
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% S6 A. q% O* J3 }# m# f3 m; uTable 1. Methods used for isolation and enrichment of HSCs from WBM
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* l* {- A. ^, {) HThe dye exclusion-based approach is dependent on a functional property of HSCs, whereas most of the antigens used in antibody-based enrichment schemes have been shown to be nonessential to HSC function or may differ between species. Potential additional advantages to the dye exclusion-based enrichment are inclusion of facilitating cells for engraftment of HSCs; inclusion of progenitors for other cell types, such as endothelial cells; cost savings; and the avoidance of antibody-induced physiologic changes in the HSCs.
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It is well documented that monoclonal antibodies may be used to activate or otherwise alter the homeostasis of certain cell types . These data may be particularly pertinent to HSC isolation, as Sca-1 (Ly6a) and Thy1 are two GPI-anchored proteins that are frequently used to enrich long-term repopulating cells via antibody labeling and fluorescence-activated cell sorting (FACS). Thus, we hypothesized that the antibody cocktails commonly used in HSC purification might alter the normal physiology of stem cells.% H! K* a; O# W5 v/ j& A
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In this study, we investigated the effects of antibodies to stem cell surface marker antigens on HSCs by comparison of SP-sorted HSCs from untreated murine bone marrow cells and bone marrow cells from the same animals that had been incubated with the SKL antibody cocktail prior to SP sorting. We observed significant changes in the expression of stem cell-related genes with SKL antibody treatment that appeared to correlate with a loss of functional repopulating stem cells, possibly by inducing HSC cycling, as has been previously reported . Taken together, these results suggest that the antibodies used to label and sort HSCs may alter their physiology, thereby effectively reducing the yield of actual long-term repopulating HSCs.8 |& L- s1 z- n! M) R& X" l2 A# g
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MATERIALS AND METHODS
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Animals
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, T3 ^& K. z" T) y; W8 FC57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) were maintained in the animal facility at the University of North Carolina (Chapel Hill, NC) in accordance with Institutional Animal Care and Use Committee standards. All animals were housed in autoclaved microisolator cages and supplied with irradiated mouse chow and sterile water ad libitum. All mice used were between 7 and 16 weeks of age at the initiation of the experiments, and mice were age-matched within each experiment.
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; a- {9 \! R' x1 GFor transplantation experiments, SP cells were isolated from the bone marrow of B6 mice expressing the enhanced green fluorescent protein (eGFP) protein . Bone marrow from B6.SJL-Ptprca-Pepcb/BoyJ (CD45a) mice (Jackson Laboratory)¡ªwhich are congenic with B6 mice, carrying the SJL allele for CD45a¡ªwas used as a source of short-term engrafting cells., }3 a+ d- u" p% e
) K8 d; V, R T$ [% }8 m1 | \Preparation of Bone Marrow Cells
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Mice were euthanized, and femurs and tibias were removed. The marrow cavities were flushed with either sterile Hanks' balanced salt solution (HBSS) (HBSS with 2% fetal calf serum , 10 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin) or sterile Iscove's modified Dulbecco's medium (IMDM) supplemented with 5% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cell suspensions were filtered through a cell strainer to remove debris.
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: }8 x; ]' f3 h4 zHoechst 33342 Staining (With or Without Antibody Staining) for SP Isolation
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Bone marrow was harvested as described above in cold HBSS , and the resulting cell suspensions were centrifuged at 1,500 rpm (600g) for 12 minutes at 4¡ãC. Cell pellets were resuspended in prewarmed (37¡ãC) RPMI medium containing 2% FCS, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 µg/ml Hoechst 33342. Total nucleated cells (TNCs) were counted and brought to a final concentration of 1 x 106 TNCs per milliliter. Cell suspensions were incubated at 37¡ãC for 90 minutes and immediately centrifuged at 600g for 8 minutes at 4¡ãC. Cell pellets were resuspended in 1 ml of ice-cold flow suspension buffer (HBSS containing 2% FCS, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 µg/ml propidium iodide). For samples treated with SKL antibodies, cells were suspended in 1 ml of ice-cold HBSS and stained for 30 minutes on ice with the antibodies described below. Cells were then washed once with an excess of ice-cold HBSS and resuspended in 1 ml of ice-cold flow suspension buffer.9 d8 P/ ?' C5 b' ^- z$ v' G
6 y9 a0 T! r- g- Y2 J9 mAntibody Staining and FACS for SKL Isolation
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9 h# B# _1 I- ~) qBone marrow was harvested as described above and stained in a two-step procedure with fluorescein isothiocyanate-conjugated c-kit (clone 2B8; 3.75 µg per 107cells), biotinylated Sca-1 (clone E13-161.7; 3 µg per 107cells), and phycoerythrin-conjugated lineage marker antibodies (CD3:145-2C11, B220:RA3-6B2, TER119, Mac-1:M1/70, and Gr-1:RB6-8C5; 0.8 µg each per 107cells). The initial immunostaining was followed by incubation with a secondary antibody conjugated to streptavidin-PE-Cy5. (Antibodies were a gift from Dr. Lishan Su, University of North Carolina at Chapel Hill, or purchased from BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com/pharmingen). All incubations were done on ice, and washes were performed with ice-cold buffers. Aliquots of cells were stained individually for isotype controls.) Z0 W' S% ]& K5 R3 ]
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FACS6 }! |8 Y. L& |# s
8 I1 t6 H* J. D: H9 v0 r% C2 v9 i: tCell sorting was performed on a dual laser modular flow cytometer (MoFlo; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com). For SP sorting, an argon laser provided excitation of the Hoechst dye at 350 nm, and emission was monitored simultaneously at 405 nm (blue fluorescence) and 670 nm (red fluorescence) for analysis. SP cells were sorted based on the gate shown in Figure 1. A small sample of cells was run back through the cytometer following each sort to check the purity of the SP sorting (Fig. 1, right panel). For SKL sorting, excitation was provided by laser output at 488 nm, and fluorescence emission was monitored as appropriate for each fluorochrome. For reverse transcription-polymerase chain reaction (RT-PCR) analysis in SP compared with SKL studies, 100 cell samples were sorted into 0.2-ml microcentrifuge tubes containing 5 µl of cell lysis buffer (Cells-to-cDNA kit; Ambion, Austin, TX, http://www.ambion.com), moved immediately to dry ice, and then stored at ¨C70¡ãC. For RT-PCR studies comparing SP(plus antibody ) to SP(untreated), single cells or 20-cell pools were sorted (based on Hoechst dye exclusion; Fig. 1) into eight-well TurboCapture mRNA strips containing 15 µl of Buffer TCL (Qiagen, Valencia, CA, http://www.qiagen.com), moved immediately to dry ice, then stored at ¨C70¡ãC. For transplantation experiments, defined numbers of SP cells (enough for final doses of 10, 20, or 50 cells per animal) were sorted into sterile tubes containing sterile IMDM supplemented with 5% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 µg/ml gentamicin., n w8 |( c! S2 H
g; a, q5 L0 o% k$ r+ m% {' @8 {Figure 1. Sorting gate for SP cells based on Hoechst dye exclusion. Whole bone marrow cells were incubated with 5 µg/ml Hoechst 33342. Flow cytometric emission monitoring at both 405 nm and 670 nm allowed for selection of SP cells (gated, left panel), which exclude the Hoechst dye. The right panel demonstrates a postsort assessment of purity. Abbreviation: SP, side population.
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+ l: W0 P; _5 Z* _1 ORT-PCR
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For SP Compared with SKL Studies. Samples were lysed by heating to 80¡ãC in a thermocycler block for 5 minutes and then cooled to 4¡ãC. DNase I (0.07 U/µl) treatment was performed to degrade genomic DNA in the samples. Samples were incubated at 37¡ãC for 30¨C45 minutes and then heated at 75¡ãC for 5 minutes to inactivate the DNase I. Reverse transcription was performed by the protocol provided with the Cells-to-cDNA kit (Ambion). The resulting cDNA was aliquoted into five individual 0.2-ml PCR tubes and stored at ¨C20¡ãC until PCR was performed. Amplification of specific genes was performed using enzymes and reagents supplied with the HotStarTaq Polymerase system (Qiagen, Hilden, Germany, http://www1.qiagen.com). Supplemental online Table 1 lists all primers, PCR cycling conditions, and references used. cDNA from murine bone marrow nucleated cells was used as a positive control for expression of all genes except the endothelial growth factor receptors Kdr (Flk-1) and Tek (Tie-2); for these genes, cDNA from the endothelial cell line PY4.1 was used as the positive control .. ] J) E% S o+ {
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For SP( Ab) Compared with SP(Untreated) Studies. Cell lysates were allowed to hybridize to TurboCapture mRNA tubes for 90 minutes at room temperature and then washed three times with wash buffer. Based on the solid-phase synthesis protocol provided in the TurboCapture manual, cDNA was generated using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), dNTPs (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com), and the immobilized poly(dT) on the hybridization tubes as primers. Since the synthesized cDNA was covalently linked to the TurboCapture mRNA tubes, several rounds of PCR could be run on the same samples. Following cDNA synthesis, each sample was tested for the presence of template using hypoxanthine phosphoribosyltransferase (HPRT) as an internal control. Samples that tested negative for the HPRT message were discarded, whereas samples that tested positive for HPRT were then assayed for other genes of interest. Supplemental online Figure 1 shows an example of results for the HPRT RT-PCR. Using these criteria, the number of samples analyzed was as follows: 20 SP without antibody (NoAb), 40 samples (total of 254 PCRs); 20 SP with antibody ( Ab), 40 samples (total of 254 reactions); single SP NoAb, 55 samples (total of 272 reactions); single SP( Ab), 49 samples (total of 243 reactions). Samples were assayed for gene expression using a two-step nested PCR procedure. Round one of PCR was conducted in 50 µl using PCR Master Mix (Promega, Madison, WI, http://www.promega.com) and gene-specific primers A and B listed in supplemental online Table 2. Then, 2 µl of the PCR product from round 1 was used as a template for a second round of PCR using gene-specific primers C and D listed in supplemental online Table 2. PCR conditions were as follows: 40 cycles of 94¡ãC for 45 seconds, annealing temperature (supplemental online Table 2) for 45 seconds, and 72¡ãC for 45 seconds. The PCR product from round 2 was then run on a gel containing ethidium bromide and scored as positive or negative for gene expression.' \* T; C3 _" N& @8 ]1 f
! R: M1 O' L. S' w7 x& SReal-Time Quantitative RT-PCR3 R+ m; K8 {4 v9 G& r
6 f' x7 t+ r+ N9 hPools of 200¨C500 SP cells were sorted (based on Hoechst dye exclusion) into eight-well TurboCapture mRNA strips containing 15 µl of Buffer TCL. Cell lysates were allowed to hybridize to TurboCapture tubes for 90 minutes at room temperature and then washed three times with wash buffer. Based on the solution-phase synthesis protocol provided in the TurboCapture manual, cDNA was generated using SuperScript II reverse transcriptase (Invitrogen), dNTPs (Amersham Biosciences), and Random Hexamer Primers (Invitrogen). The resultant cDNA was then subjected to quantitative real-time PCR analysis using the ABI Prism 7000 sequence detection system (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com). We used commercial TaqMan gene expression assays (Applied BioSystems) as the gene-specific probe and primer sets for analysis of all genes studied. Real-time PCRs were set up using TaqMan Universal PCR Master Mix (Applied BioSystems).
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" T7 v5 o4 U2 s/ O! E0 A# dData were analyzed using the 2¨CCT method as previously described . Briefly, data are presented as the fold change in gene expression normalized to an internal control gene (Actb) and relative to a calibrator sample (untreated SP cells).
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- u# O+ k; m" j% B( pTransplantation. Z+ z* ^. ^# M6 \# C: Z
9 H* z1 |# H. X; L2 z) x! ~Wild-type B6 female mice (CD45b) were lethally irradiated (9.5 Gy of total body irradiation from a 137Cs source) and anesthetized with tribromoethanol (Avertin; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for retro-orbital injection of the indicated dose of green fluorescent protein-positive (GFP ) SP cells plus 2 x 104 whole bone marrow cells from B6-CD45a mice. Since the frequency of HSCs in whole bone marrow is 1 in 105 cells, the B6-CD45a whole bone marrow cells are considered to be short-term repopulating cells. The transplanted animals were maintained in microisolator cages with autoclaved food and acidified (pH 2.0) sterile water supplemented with neomycin sulfate for the first 2 weeks after transplant.; B! C) H, {- q4 K
" h W0 R4 Q' \3 L& {9 \Analysis for Donor Cell Repopulation
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At various days following transplantation, mice were anesthetized using tribromoethanol, and approximately 100 µl of peripheral blood was collected from the retro-orbital sinus into 1.5-ml Eppendorf tubes containing 10 µl of EDTA (25 mg/ml). Red blood cells were lysed using ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2 EDTA, pH 7.4). The remaining cells were washed in phosphate-buffered saline with 2% FCS and then stained with PE-anti-CD45a (A20; BD Pharmingen). Dual-laser FACS analysis was performed with a FACScan machine (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Samples were gated based on high forward scatter (to avoid residual debris from ACK lysis step), and SP-derived cells were identified based on fluorescence intensity of eGFP, whereas B6-CD45a-derived cells were identified based on fluorescence intensity of PE.
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% N, }6 z+ f, k/ VCell Cycle Analysis
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) ^+ p# e+ n' j* n& w7 y CSP cells were sorted based on Hoechst dye efflux and then resuspended in 250 µl of HBSS buffer with 0.1% sodium citrate and 50 µg/ml propidium iodide. Cells were incubated on ice for 10 minutes then analyzed using flow cytometry (488 nm excitation) and Modfit software (Verity Software House Inc., Topsham, ME, http://www.vsh.com) to determine the percentage of cells in each phase of the cell cycle.
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7 t7 u. K8 J e& D7 PStatistical Analysis
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; ^0 z2 t0 T% hTwo-tailed Student's t tests were performed for comparisons of quantitative RT-PCR results. SigmaStat software (San Jose, CA, http://www.systat.com) was used for statistical calculations./ T7 O/ o+ X+ N
5 b% V2 x( [6 @+ s B) T& k @9 n$ TRESULTS& ~7 }3 K4 l2 O2 r" ? y
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Stem Cell-Related Gene Expression Is Altered in SP Cells Compared with SKL Cells/ A/ G6 w4 I" G" k: |1 ]( W- Z1 {+ R
% q: i; G' k$ I8 n1 \! FTo compare gene expression in two defined cell populations that are highly enriched for hematopoietic stem cell activity, RT-PCR analyses were performed with sorted samples of SP and SKL cells. Because of the rarity of these cell populations in bone marrow, we chose to examine the expression of select genes individually rather than in an array format. Considerable amplification of transcripts would be required to generate enough starting material for analysis by array, which can introduce significant bias because of variable amplification efficiency. We thus studied the expression of 18 separate genes. Detection of a housekeeping gene, Hprt1, was used as a positive control for each sample, so samples scoring negative for Hprt1 were not included in the analysis. We analyzed a total of 231 PCR results from 48 SKL sorted samples and 224 PCR results from 46 SP sorted samples (each sample represented a pool of 20 cells). Each sample was scored as positive or negative for gene expression, and overall gene expression frequency was defined as the percentage of positive samples in all samples analyzed.
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$ @" l' g% q; g, u( A* aFor this analysis, we focused on genes relevant to hematopoietic cell physiology, which can be organized into the following basic categories: transcription factors (Tal1 and Gata1) . In contrast, our data demonstrate a higher frequency of expression of the macrophage inflammatory protein-1 (Ccl3) receptors Ccr1, Ccr4, and Ccr5 in SP cells. From these results, we made the general observation that the SP and the SKL population clearly have different gene expression profiles with respect to genes important to hematopoietic stem cell physiology, but we could not conclude that there is a causal effect of antibody binding in inducing such gene expression differences.2 i2 ~ P5 a- c. M6 Z" P: b
0 D' H; d0 y0 c4 J" c2 R7 CTable 2. Differences in frequency of gene expression in SKL cells compared with SP cells3 r/ D2 p3 N, |9 h1 [2 h' @
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The SKL Antibody Cocktail Reduces the Frequency of Expression of Stem Cell-Related Genes in SP Cells
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The SP and SKL isolation protocols differ fundamentally in that the SKL protocol involves the use of antibodies that bind to cell surface molecules. We therefore hypothesized that the presence of these antibodies may actually give rise to some of the gene expression differences observed between the two HSC populations. However, the use of antibodies is not the only difference between SP and SKL isolation protocols, so any phenotypic differences seen by direct comparison of the two populations may not be caused solely by the presence of the SKL antibody cocktail. Thus, in all subsequent studies, we compared untreated cells sorted on the basis of Hoechst dye exclusion (SP untreated) with the same population of cells that were incubated with the SKL antibody cocktail prior to sorting on the basis of Hoechst dye exclusion (SP SKL Ab). Figure 1 illustrates the sorting gate used to isolate SP cells from whole bone marrow, and the right panel shows a representative check of postsort purity.) U6 r4 y4 U5 s0 [5 Q
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To determine whether the presence of the SKL antibody cocktail alters gene expression within the SP, we conducted RT-PCR analyses for hematopoietic cell-related genes on untreated SP cells and SKL antibody-treated SP cells. We studied the expression of 19 different genes, which can be organized into the following general categories: transcription factors (Tal1, Gata1, Runx1, and Tgif) . Detection of a housekeeping gene, Hprt1, was used as a positive control for each sample, so samples scoring negative for Hprt1 were not included in the analysis. Samples were scored as positive or negative for gene expression and then expressed as the percentage of positive samples among all samples analyzed (frequency). At least 8 and as many as 22 RT-PCR samples were analyzed for each gene. We first analyzed the expression of these genes in cDNA samples collected from 20-cell pools of sorted cells. Interestingly, we saw large differences in the frequency of expression of many of the genes studied between untreated SP cells and SP cells that had been treated with the SKL antibody cocktail (Fig. 2A). The antibody-treated samples were generally less likely to express key HSC-related transcription factors Tal1, Gata1, and Runx1 as compared with untreated samples. At the same time, antibody-treated samples showed altered expression frequencies of Epor, Csf1r, Mpo, and Pecam1, which are markers of differentiated endothelial or hematopoietic cells. Moreover, the MIP-1 (Ccl3) receptor Ccr1, which may be important in maintaining quiescence in HSCs, was less frequently expressed in the antibody-treated SP samples. In contrast, expression of the integrin Itga2b and the SDF-1 (Cxcl12) receptor Cxcr4, which are both key molecules in HSC homing, was more frequent in the antibody-treated samples than in untreated samples.2 U7 J0 @$ h J% }2 y
' f0 }' |* X3 |Figure 2. Frequency of gene expression in untreated and Sca-1 c-Kit Lin¨C Ab-treated SP cells. Reverse transcription-polymerase chain reaction analysis of cDNA samples from 20-cell pools (A) or single cells (B). Each sample was scored as positive or negative for expression of the indicated genes. Results are displayed as the percentage of positive results in all samples analyzed. The total number of samples analyzed for each gene and condition ranged from 8 to 22. The presence of the Ab cocktail resulted in distinct heterogeneity in expression of many hematopoietic stem cell-relevant genes within the SP. Abbreviations: Ab, antibody; n/d, analysis not done; noAb, no antibody; SP, side population.
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There were several genes in the study that were expressed with very high frequency in both treated and untreated 20-cell pooled samples; therefore, we analyzed gene expression at the single-cell level to further elucidate any additional differences in expression frequency between untreated and antibody-treated SP cells (Fig. 2B). In these single-cell samples, we noted a considerable decrease in the frequency of expression of the HSC markers Kit and Ly6a (Sca-1) in the antibody-treated samples. Single-cell analysis also revealed an increase in Abcg2 expression frequency in antibody-treated SP cells. Overall, these findings indicate that the presence of the SKL antibody cocktail results in distinct heterogeneity in expression of many genes important to hematopoietic stem cells and their progeny in the SP. However, the net result of this gene expression heterogeneity is inconclusive, as it is based on expression frequency alone. Thus, we used these results to direct both quantitative gene expression analyses and in vivo analysis of mice transplanted with antibody-treated versus untreated SP cells.9 |1 E5 R- w, t' p6 k
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Quantitative Studies Confirm That Reduced Gene Expression Is Specific to SKL Antibody Cocktail1 e9 M! D' k2 o" }( ^) R& g
. \7 T/ G, R9 Y: @' W" n- Y, BTo further confirm and quantitate the gene expression differences observed, we next conducted quantitative RT-PCR analyses on some of the key genes that were altered upon antibody treatment of the SP cells. As an additional control, we tested samples that were treated with the isotype controls of the SKL antibody cocktail to determine whether any observed changes were specific to the SKL antibodies or simply a nonspecific consequence of having antibodies in solution with the cells. It is notable that in these studies, the approximate time from exposure to the antibodies until analysis of gene expression was a minimum of 4 hours, thereby allowing significant time for some alteration in gene expression if induced by antibody exposure. We found significant reductions in mRNA levels of Kit, Cd34, Tal1, and Slamf1 from untreated SP samples to SKL antibody-treated SP samples (p
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Figure 3. Quantitative gene expression in SP cells with or without Ab treatment. Quantitative PCR analyses from pools of 200¨C500 cells are shown as the level of gene expression in the Ab-treated samples relative to the level of expression in the untreated samples. The numbers in parentheses indicate the numbers of individual cell sorts analyzed. The SKL Ab cocktail caused marked downregulation of several genes known to be important to hematopoietic stem cell physiology; this downregulation did not occur in isotype Ab-treated samples. Abbreviations: Ab, antibody; SKL, Sca-1 c-Kit Lin¨C; SP, side population.
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3 \: c% R c, x3 O- |# u* S) lSKL Antibody-Treated SP Cells Show Reduced Engraftment of Lethally Irradiated Recipients Compared with Untreated SP Cells1 ?4 r4 C, q* U, b( j
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Given the nature of the genes whose expression was altered in the SKL antibody cocktail-treated SP cells, we hypothesized that the antibody-treated cells may have altered functional ability to repopulate the hematopoietic system in lethally irradiated recipients as compared with untreated SP cells. We sorted untreated or SKL antibody-treated SP cells from the bone marrow (BM) of eGFP-transgenic mice so that we could follow engraftment of the SP cells using GFP fluorescence. Transplant recipients (CD45b genotype) were lethally irradiated and then given a mixture of BM cells containing 10, 20, or 50 SP cells (CD45b genotype, GFP ) and 2 x 104 whole bone marrow cells (CD45a genotype) via retro-orbital injection (Fig. 4A). Following transplantation, peripheral blood was collected from recipient animals and analyzed by flow cytometry approximately once per month to monitor the percentage of eGFP (SP-derived) cells as a measure of engraftment and hematopoietic repopulation by the transplanted cells (Fig. 4B). In mice receiving 20 SP cells, we noted over a period of several months that the percentage of SP (eGFP ) engraftment was significantly lower in the animals that received SKL antibody-treated SP cells than in the animals that received untreated SP cells (Fig. 5A). In particular, many mice receiving antibody-treated cells died during the 4th month post-transplant, which suggests that a number of SP-derived cells in those mice functioned as short-term progenitors and that there were not enough long-term stem cells to keep the mice alive. Furthermore, the mice that survived past the 4th month showed very few SP-derived cells in their peripheral blood, suggesting either deficient engraftment or a possible loss of long-term repopulating potential after SP cell exposure to the SKL antibodies. Figure 5B summarizes the engraftment of SP-derived cells in mice receiving all doses of SP cells. Once again, many of the mice died at approximately 4 months post-transplant, when the short-term progenitor cells were exhausted, and the SP cell engraftment was significantly lower in surviving mice that received SKL antibody-treated SP cells. These findings indicate that a functional consequence of SKL antibody binding to SP cells is to significantly reduce their ability to stably engraft lethally irradiated mice, particularly when transplanting small numbers of cells.4 w. h- R6 Q/ \9 N
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Figure 4. Transplantation and engraftment analysis for untreated versus Sca-1 c-Kit Lin¨C antibody-treated SP cells. (A): Lethally irradiated recipient animals (CD45b genotype) received small numbers of untreated or antibody-treated GFP SP cells and 2 x 104 CD45a unfractionated bone marrow cells. (B): Each month post-transplant, peripheral blood cells were harvested and analyzed by flow cytometry to determine their origin (SP donor, STR donor, or endogenous). Abbreviations: GFP, green fluorescent protein; PE, phycoerythrin; SP, side population; STR, short-term repopulating.4 E" t) T) n+ R0 r! C
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Figure 5. Engraftment of transplanted SP cells. Flow cytometric analysis determined the percentage of recipient PB derived from the transplanted SP cells (GFP ). (A): Values shown are the mean ¡À SEM of the percentage of GFP cells in all living mice at each time point (indicated by numbers in parentheses). The graph represents the average of two separate transplants in which the mice received a dose of 20 SP cells (with or without Sca-1 c-Kit Lin¨C Ab). (B): Summary of all transplants performed, using multiple different doses of SP cells. Values shown are the mean ¡À SEM of the percentage of GFP cells as a function of the starting dose of SP cells. Beginning approximately 4 months post-transplant, the mice receiving SKL Ab-treated SP cells showed significantly reduced levels of SP-derived cells in their PB compared with the mice receiving untreated SP cells. Abbreviations: Ab, antibody; GFP, green fluorescent protein; noAb, no antibody; PB, peripheral blood; sp, side population; SP, side population.$ \: T, l- b/ |/ G1 T0 b
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The SKL Antibody Cocktail Markedly Increased the Numbers of Cycling SP Cells
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4 j) K, c8 J- i6 ^, NTo explore a possible mechanism for the observed effects of the SKL antibody cocktail on the phenotype of SP cells, we conducted cell cycle analysis of the SP with or without SKL antibody treatment. Untreated SP cells are usually a quiescent population, with more than 90% of cells in G0/G1, approximately 8% of cells in S phase, and 2% of cells in G2/M (Fig. 6A). When these cells were exposed to the SKL antibody cocktail prior to Hoechst sorting, the percentage of cells in S phase more than doubled to almost 20%, with a marked reduction of cells in G0/G1 (Fig. 6B). Importantly, when the cells were treated with isotype control antibodies prior to sorting, the cell cycle profile of isotype-treated cells was very similar to that of untreated cells (2% increase in S-G2/M). These findings suggest that the SKL antibody cocktail induces cells within the SP to enter the cell cycle, which may in part explain the changes in gene expression profile and stem cell functionality seen between untreated SP cells and SKL antibody-treated SP cells.* t8 F$ j6 W6 j2 z) e, [) E
/ |, e: b7 b- YFigure 6. Cell cycle analysis of SP cells with or without Ab treatment. (A): Representative flow cytometric histograms showing the DNA content of SP-sorted cells with or without prior Ab treatment. Percentages indicate the portion of the cell population in the designated phase of the cell cycle as determined by Modfit software. (B): Summary of cell cycle phase distribution of SP cells following the indicated Ab treatments. Note the large increase in S-phase cells caused by treatment with the SKL Ab cocktail. Graphs represent the average of three separate experiments. Abbreviations: Ab, antibody; SKL, Sca-1 c-Kit Lin¨C; SP, side population.
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( l6 d6 z8 V* V' E# `9 [- [DISCUSSION
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( F' K3 I, k$ t* TThe two major determinants of successful hematopoietic stem cell transplantation are (a) repopulation of all components of the hematolymphoid system via multipotent differentiation of the stem cells, and (b) long-term maintenance of all cell types via self-renewal of the stem cells. Many different protocols have been established to isolate or enrich the stem cells for transplants, and in all cases, the desired cell population makes up a small fraction of the original cell source. In addition, available stem cell numbers may be reduced depending on the source of the cells (e.g., umbilical cord blood) or by manipulation prior to transplantation (e.g., gene therapy). Therefore, it is important to understand the impact that a chosen enrichment protocol will have on HSC function to maximize transplant efficiency.
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We describe here the negative impact of certain antibodies to cell surface proteins on the long-term repopulating ability of HSCs from the SP fraction of murine whole bone marrow. Our initial gene expression profile analysis comparing SKL-sorted cells to SP-sorted cells revealed many differences in the frequency of expression (percentage of samples positive for expression) of many genes related to HSC activation, homing, cycling, growth factor responsiveness, differentiation, and gene transcription. Overall, the SP was less transcriptionally active with respect to HSC-related genes and showed less evidence of differentiated cells. The only genes studied that showed a significantly higher frequency of expression in SP cells were the chemokine receptors Ccr1, Ccr4, and Ccr5, which bind to the common ligand MIP-1 (Ccl3). In several reports, MIP-1 has been shown to be a negative regulator of proliferation of hematopoietic stem cells . Thus, the higher frequency of expression of proliferation- and migration-inhibitory chemokine receptors, coupled with lower frequency of expression of growth factor receptors and markers of differentiation, suggests a more quiescent phenotype of SP cells compared with SKL cells." V: g+ ]( @) L! ~( v5 ?; w
4 L# ~1 k" H# D" ZThe fundamental difference between SKL sorting and SP sorting of HSCs is the use of antibodies to cell surface proteins. We hypothesized that antibodies within the SKL sorting cocktail are a cause of transcriptional changes in HSCs that may lead to altered function. To directly test this hypothesis, we compared the effects of SKL antibody exposure versus no antibody exposure on a single HSC population (SP cells). We again found significant differences in the expression of a number of stem cell-relevant genes that was not seen after exposure to irrelevant isotype control antibodies, suggesting some loss of the stem cell phenotype. Furthermore, through in vivo transplantation studies using small numbers of stem cells, we showed that exposure to the SKL antibody cocktail resulted in a significant loss of long-term repopulating potential of SP HSCs.
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Two general mechanisms that could explain the defect we saw in SKL antibody-treated SP cell long-term repopulating capability are as follows: (a) a localization defect (including homing, migration, and engraftment in the niche), and (b) an alteration in intrinsic stem cell properties (i.e., multipotentiality and self-renewal). Our quantitative PCR results demonstrate that, at least at the transcriptional level, the SKL antibodies do not alter the expression of any known molecules important to HSC homing or migration. Therefore, a localization defect is unlikely to explain the reduced long-term repopulation shown by SKL-treated SP cells.8 H- X" M* g1 y
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Our results support the idea of an intrinsic loss of stem cell properties caused by antibody exposure as demonstrated by significant changes in cell surface marker phenotype and cell cycle profile. Expression levels of Kit, Slamf1, and Cd34 were significantly downregulated in response to SKL antibody exposure, suggesting a possible loss of both long-term and short-term repopulating HSCs .
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5 G2 X4 k/ H" _Quantitative RT-PCR analysis revealed a significant upregulation of p21 (Cdkn1a) expression upon SKL antibody exposure. Although p21 in HSCs is most often described by its role in maintaining quiescence by the inhibition of cell cycling . These studies are in accordance with our findings showing increased p21 gene expression and increased cell cycling by exposing SP cells to the SKL antibody cocktail.1 p0 q4 z, H" ~8 p
( e3 T& T2 E1 j0 }8 yThe potential effect of our findings on the possible detrimental effects of antibody-based enrichment strategies may be relevant to stem cell isolation in nonhematopoietic tissue types as well. Cell surface antigens useful for stem or progenitor cell isolation have been identified in several other tissue types, including neural cells in brain, skeletal muscle, and liver, our results suggest that SP-based stem cell isolation may also have similar advantages over antibody-based enrichment in tissues other than bone marrow.
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DISCLOSURES. C% v! O- r3 u& d* y) M
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The authors indicate no potential conflicts of interest.& ?& i! H9 ]$ _# b* d
) } D1 ~$ y, a! P# kACKNOWLEDGMENTS+ ?! i- E, v: T3 W
+ q2 V& U% k5 v1 F. g( W+ SWe thank Drs. Nobuyo Maeda, Mark Majesky, Karen McKinnon, and Donald Cook for critical reading of the manuscript. We also thank Larry Arnold, Megan Gottlieb, and Nancy Martin of the University of North Carolina Flow Cytometry Facility for assistance and advice with cell sorting and FACS analysis. This work was supported by NIH Grants K18HL072848 (to S.L.K.) and T32HL069768 (to Nobuyo Maeda) and by a Burroughs Wellcome Fund Career Development Award (to S.L.K.). J.B.G. is a trainee on NIH Grant T32HL069768.
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