Acquisition of G0 state by CD34-positive cord blood cells after bone marrow transplantation

Experimental Hematology 2010;38:1231–1240

Acquisition of G0 state by CD34-positive cord blood cells after bone marrow transplantation Haruko Shimaa,b, Keiyo Takuboa, Naoko Tagoa, Hiroko Iwasakia, Fumio Araia, Takao Takahashib, and Toshio Sudaa a

Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine, Tokyo, Japan; b Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan (Received 10 March 2010; revised 4 August 2010; accepted 12 August 2010)

Objective. Hematopoietic stem cells are kept in a quiescent state in the hypoxic area of the bone marrow, which is essential for hematopoietic stem cell maintenance. However, when and how hematopoietic stem cells acquire their hypoxic state and maintain quiescence has not been fully elucidated. The aim of this study was to understand this process in human hematopoietic stem cells after bone marrow transplantation. Materials and Methods. Human CD34-positive cord blood cells were transplanted into nonobese diabetic/severe combined immunodeficient interleukin-2 receptor g chain knockout mice. Cell cycle and hypoxia assay analyses were performed, to identify changes in the characteristics of human hematopoietic stem cells following transplantation. Quantitative real-time reverse transcription polymerase chain reaction analysis was used to analyze the transcriptional changes accompanying this transition. Results. Engrafted primitive lineage-negative CD34-positive CD38-negative cells acquired hypoxic state and quiescence in the recipient bone marrow between 4 and 8 weeks, and between 8 and 12 weeks after transplantation, respectively. During 4 and 8 weeks after transplantation, changes in the transcription levels of G0 regulatory factors, such as CCNC and RBL1, and stem cell regulators, such as Flt3, were also seen, which may be related to the characteristic changes in the cell cycle or oxygenation state. Conclusions. Behavioral changes of hematopoietic stem cells in their cell cycle and oxygenation state during and after bone marrow engraftment may provide insights into hematopoietic stem cell regulation, mediating the improvement of clinical hematopoietic stem cell transplantation protocols and the eradication of leukemia stem cells. Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Quiescence acquisition in the bone marrow (BM) niche is crucial for the continued ability of hematopoietic stem cells (HSCs) to generate mature blood cells during hematopoiesis [1,2]. During engraftment, transplanted HSCs home to the specific BM niche of the recipient, where they initiate hematopoiesis and self-renewal. Most studies on engrafting cell tracking after transplantation have focused on this homing ability, providing some understanding of the molecular mechanisms underlying the HSC homing process Offprint requests to: Keiyo Takubo, M.D. or Toshio Suda, M.D., Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; E-mail: keiyot@ gmail.com or [email protected]

[3–6]. However, the behavior of engrafted HSCs after homing, such as how they lodge into the niche and acquire quiescence, remains unknown. In addition, HSCs localize in the endosteal zone, the most hypoxic area in the BM [7–9]. This hypoxic niche may have a low production level of reactive oxygen species, thereby protecting HSCs more efficiently [10,11]. However, when and how the engrafting HSCs become hypoxic have yet to be elucidated. Recent progress in the establishment of immunodeficient mice enabled the construction of a humanized mouse model [12,13]. Using nonobese diabetic/severe combined immunodeficient interleukin-2 receptor g chain knockout (NOG) mice, we previously successfully developed a mouse model of high human hematopoietic chimerism [8], which may provide insights into the previously mentioned

0301-472X/$ - see front matter. Copyright Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2010.08.004

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questions. In this study, cell cycle and hypoxia analyses of engrafted human cord blood (CB) cells in the recipient mouse BM were performed after transplantation to determine the process of quiescence and hypoxia acquisition. The relevant molecular factors underlying this process were also investigated.

Materials and methods Mice and CB cell transplantation Six-week-old female NOG mice were purchased from the Central Institute for Experimental Animals (Chiba, Japan) and bred in a pathogen-free environment according to the guidelines of Keio University School of Medicine. Purchased CD34þ CB cells (Sanko Junyaku, Tokyo, Japan) were transplanted into sublethally irradiated (2.4 Gy) NOG mice via the tail vein (5  104 cells per mouse). These recipient mice were sacrificed at 4, 8, 12, or 16 weeks after transplantation to harvest their BM mononuclear cells (MNCs). The collected BM MNCs were counted with a Coulter counter (Beckman Coulter, Fullerton, CA, USA) and prepared for further analyses. Antibodies and flow cytometric analysis The collected BM MNCs were evaluated for human chimerism and repopulation of the human stem/progenitor population with mouse anti-human CD34 (clone 581), CD38 (HIT2), CD45 (HI30), and rat anti-mouse CD45 (30-F11) monoclonal antibodies (BD Biosciences, San Diego, CA, USA). Human lineage cells were stained with a biotinylated antibody cocktail for lineage markers (Miltenyi Biotec, Bergisch Gladbach, Germany) and subsequently with perinidin chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5)conjugated or phycoerythrin-Cy7 (PE-Cy7) conjugated streptavidin (BD Biosciences). Flow cytometric analyses were performed with a FACSVantage SE (BD Biosciences). BrdU labeling assay The transplanted NOG mice were intraperitoneally injected with 1 mg 5-bromodeoxy-uridine (BrdU; Sigma-Aldrich, St Louis, MO) every 8 hours starting 24 hours before sacrifice. Eight hours after the last injection, BM MNCs were harvested and stained with antibodies (human CD34, CD38, and lineage markers) to distinguish the human stem/progenitor cell fraction. Cells were then fixed, permeabilized, and stained with a mouse anti-BrdU antibody (BD Biosciences) according to manufacturer’s protocol. The population of BrdU-labeled cells was quantified by flow cytometry. Immunohistochemistry Sixteen weeks after transplantation, recipient NOG mice were injected with BrdU as described here and sacrificed 24 hours after the first injection. Femurs were treated for frozen BM sections as described previously [14], immunostained with biotylated mouse anti-BrdU antibody (Calbiochem, Darmstadt, Germany), and imaged using Alexa Fluor 488conjugated streptavidin (Invitrogen, Carlsbad, CA, USA). 40 ,6-Diamidine-20 -phenylindole dihydrochloride (Invitrogen) was used to identify cell nuclei. Fluorescent images were captured and analyzed with laserscanning confocal microscopy (FV-1000; Olympus, Tokyo, Japan).

Cell cycle analysis and hypoxia assay Cell cycle analysis was performed with pyronin Y (PY) staining as follows: collected BM MNCs were stained with 2.5 mg/mL Hoechst 33342 (Invitrogen) at 37 C for 45 minutes, followed by another 45-minute incubation at 37 C with 1 ug/mL PY. Cells were further stained with other surface antibodies (human CD34, CD38, and lineage markers) to distinguish the human stem/progenitor cell fraction. For cell cycle analysis using flow cytometry with Ki67 assay system, BM MNCs were stained for surface antibodies (human CD34, CD38, and lineage markers), fixed and permeabilized using Cytofix/Cytoperm Kit (BD Bioscience) according to manufacturer’s protocol, and stained with fluorescein isothiocyanateconjugated mouse anti-Ki67 antibody (BD Bioscience) and Hoechst 33342. The cells were then analyzed by flow cytometry as described earlier. To determine the oxygen state of engrafted human cells, transplanted recipient NOG mice were intraperitoneally injected with 60 mg/kg of the molecular cellular hypoxia probe pimonidazole (Chemicon, Temecula, CA, USA), and sacrificed 90 minutes after injection. Harvested BM MNCs were treated for hypoxia analysis as described previously [7,8]. Fluorescein isothiocyanateconjugated Hypoxyprobe-1-Mab1 (Chemicon) was used at 0.75 mg/mL for the flow cytometric detection of pimonidazolepositive cells. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from 2  104 engrafted human lineage (Lin)CD34þCD38 cells or LinCD34þCD38þ cells in recipient BM 4 or 8 weeks after transplantation. The complementary DNA was reverse-transcribed using Superscript VILO (Invitrogen). Expression levels of genes related to cell cycle, hypoxia, and stem cell regulation, as well as chemokines, niche factors, and apoptotic factors, were detected using the ABI 7500 Fast Real-Time PCR System with TaqMan Gene Expression Assay Mixes (Applied Biosystems, Foster City, CA, USA), except for CDKN1A and CDKN1C. For CDKN1A and CDKN1C, primer sets purchased from TaKaRa Bio (Shiga, Japan) were used. b-actin was used as an internal control. Statistical analysis Statistical significance was determined by Tukey’s multiple comparison test. To compare two groups in the pimonidazole, qRT-PCR, and immunocytochemical assays, two-tailed Student’s t test was used.

Results Reconstitution of human hematopoiesis in NOG mouse BM To characterize human HSCs after transplantation and during reconstitution of the hematopoietic system, a hematopoietically humanized NOG mouse model was used. Each recipient NOG mouse was transplanted with 5  104 CD34þ CB cells. At 4, 8, 12, and 16 weeks after transplantation, BM MNCs were harvested for analysis. The average human chimerism was 91.1% 6 4.7% (n 5 6) at 4 weeks after transplantation (Fig. 1A, B). The total number of human MNCs plateaued between 4 and 8 weeks after transplantation, and the numbers of human LinCD34þCD38 HSC-containing

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Figure 1. Hematopoietic reconstitution of CD34-positive CB cells in NOG mice. A total of 5  104 human CD34þ CB cells were transplanted to a NOG mouse. The BM MNCs were harvested at 4, 8, 12, and 16 weeks (W) after transplantation for analysis (n 5 6 for each time point). (A) Surface markers of engrafted human cells. BM MNCs were analyzed for expression of hCD45, mCD45, hLineage, hCD34, and hCD38 by flow cytometry. Representative fluorescence-activated cell sorting profiles are shown. (B) Human chimerism in recipient mouse BM. Each dot represents an individual mouse. Average levels of O90% of human chimerism were achieved 4 weeks after transplantation and maintained thereafter. (C) The number of total MNCs in the BM of femurs and tibias were counted. The number of cells significantly increased 8 weeks after transplantation (mean 6 standard deviation [SD]; *p ! 0.01, n 5 6). (D) The number of human LinCD34þCD38 cells was counted. The number of human LinCD34þCD38 cells significantly increased 16 weeks after transplantation (mean 6 SD, *p ! 0.05, n 5 6).

fraction were comparable until 12 weeks and were increased 16 weeks after transplantation (Fig. 1C, D). The average percentage of viable human LinCD34þCD38 fraction after transplantation was 0.63% (4 weeks), 0.39% (8 weeks), 0.56% (12 weeks), and 0.82% (16 weeks). These data suggest that engrafted human LinCD34þCD38 cells resumed in vivo reconstitution activity for human hematopoiesis between 12 and 16 weeks after transplantation. Cell cycle state of engrafted human HSCs during reconstitution process after transplantation Next, the cell cycle state of human primitive cells after engraftment was examined. A short-term BrdU labeling assay was used to detect proliferating cells. NOG mice transplanted with CB cells were administered BrdU three times at 8-hour intervals, and sacrificed 8 hours after the last injection (Fig. 2A). A micrograph of immunostained recipient BM 16 weeks after transplantation is shown in Figure 2B. BrdU-positive cells labeled in green, of which DNA was synthesized at least once during 24 hours before

sacrifice, were unlikely to localize in endosteal zone where osteoblastic niche is located [9]. Nonhematopoietic cells in the recipient BM were far less than the amount of BrdUnegative cells shown in the micrograph (approximately 2% of total MNCs), suggesting that noncycling hematopoietic cells were likely to localize in the osteoblastic niche. Flow cytometric analysis was used to determine changes in the proportion of BrdU-positive cells following engraftment (Fig. 2C). The proportion of all MNCs that were BrdUpositive decreased with time after transplantation. In both LinCD34þCD38þ and LinCD34þCD38 populations, most cells were BrdU-positive until 8 weeks, and this positive population decreased 12 weeks after transplantation (Fig. 2D). These data suggest that transplanted CB stem and progenitor cells become less proliferative between 8 and 12 weeks after transplantation. The cell cycle state was further evaluated by PY expression and DNA content analyses. The proportion of PYnegative G0 cells in the human LinCD34þCD38 fraction was !5% (n 5 3) at 4 and 8 weeks after transplantation,

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Figure 2. Decreased frequency of BrdU-positive cell population 12 weeks (W) after transplantation in engrafted human CD34þ cell subsets. (A) Protocol of short-term BrdU labeling at 4, 8, 12, and 16 weeks after transplantation before analysis. (B) Immunohistochemical detection of BrdU in the BM section of recipient femur prepared 16 weeks after transplantation. Sections were stained with 40 ,6-diamidine-20 -phenylindole dihydrochloride (DAPI) (gray) and BrdU (green). Dotted line indicates the bone edge. Most of the BrdU-positive cells were localized apart from the endosteal area. (C) Representative fluorescenceactivated cell sorting profiles of BrdU-labeled cells in BM. Engrafted human LinCD34þCD38þ cells and LinCD34þCD38 cells were analyzed for BrdU expression. (D) Frequencies of BrdU-positive cells 4, 8, 12, and 16 weeks after transplantation. The BrdU-positive cell population significantly decreased 12 weeks after transplantation in both LinCD34þCD38þ cells and LinCD34þCD38 cells (mean 6 standard deviation; *p ! 0.05, n 5 3).

which increased to 29.0% 6 6.6% (n 5 3) at 12 weeks (Fig. 3A). This tendency was also confirmed with the cell cycle analysis by Ki67 staining (Fig. 3B). In contrast, the proportion of PY-positive G1 cells decreased from 92.3% 6 1.0% (n 5 3) at 8 weeks to 58.2% 6 5.3% (n 5 3) at 12 weeks (Fig. 3A). The S/G2/M fraction was constantly low during the experimental period but showed a slight increase at 12 weeks after transplantation (Fig. 3A). These data suggest that most of LinCD34þCD38 cells were in a rapid-cycling state until 8 weeks, but a portion of these cells entered into a quiescent state between 8 and 12 weeks after transplantation. Oxygen state of engrafted human HSCs during the reconstitution process after transplantation We previously demonstrated that engrafted human HSCs are hypoxic in the mouse BM [8]. To investigate how HSCs acquire their hypoxic state during hematopoietic reconstitu-

tion after transplantation, we performed a pimonidazolelabeling assay. The intracellular retention level of pimonidazole can be used to describe the relative hypoxic status of the cell. CB cell-transplanted mice were administered pimonidazole 90 minutes before sacrifice, and then BM cells were prepared for flow cytometric analysis. At 4 weeks after transplantation, the pimonidazole retention level was similar among the human Lin, LinCD34þCD38þ, and LinCD34þCD38 fractions. However, at 8 weeks after transplantation, the rate of hypoxic cells changed with each cell fraction (Fig. 4A). The relative median fluorescence intensity of intracellular pimonidazole in LinCD34þCD38 fraction against that of LinCD34þCD38þ fraction increased significantly at 8 weeks after transplantation and remained high at 12 weeks (Fig. 4B). These results indicate that engrafted LinCD34þCD38 cells were hypoxic soon after transplantation and maintained the hypoxic state thereafter,

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Figure 3. Increased frequency of quiescent cells in engrafted human HSCs 8 weeks (W) after transplantation. (A) Engrafted human LinCD34þCD38þ cells or LinCD34þCD38 cells were stained with PY and analyzed with flow cytometry for cell cycle status. The frequency of the quiescent PY-negative cell population was significantly increased 12 weeks after transplantation, compared with 4 and 8 weeks (mean 6 standard deviation [SD]; *p ! 0.05, **p ! 0.01, n 5 3). (B) Engrafted human LinCD34þCD38þ cells or LinCD34þCD38 cells were stained with Ki67 and fluorescence-activated cell sorting was used for cell cycle analysis. The Ki67-negative cell population tended to increase in size 12 weeks after transplantation in human LinCD34þCD38 cells (mean 6 SD; *p ! 0.05, n 5 3).

while other more mature cells became less hypoxic between 4 and 8 weeks after transplantation. Regulation of engrafted human HSCs during reconstitution process after transplantation To understand the molecular regulatory mechanism in human LinCD34þCD38 cells accounting for cell cycle and hypoxic status changes during hematopoietic reconstitution, a qRT-PCR analysis was used against a subset of genes, including known key regulators for cell cycle, hypoxia, and stem cell maintenance as well as chemokines and apoptotic factors. Samples were prepared from the recipient BM at 4 or 8 weeks after transplantation, when engrafted human HSC-containing fraction showed initial characteristic changes apart from more differentiated cells (Table 1). Figure 5 shows the gene expression levels that changed by more than twofold between 4 and 8 weeks after transplantation. Among the G0/G1 regulators, cyclin C (CCNC), E2F transcription factor 4 (E2F4), and retinoblastoma-like 1 (p107) (RBL1) messenger RNA (mRNA) expression levels

were significantly decreased at 8 weeks compared with 4 weeks after transplantation. Expression levels of cyclindependend kinase (CDK) inhibitors showed unremarkable changes, except for CDK inhibitor 1C (p57) (CDKN1C). The expression levels of hypoxia-related genes were also examined, revealing a significant decrease in solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) expression. Among the stem-cell regulating factors, fms-related tyrosine kinase 3 (FLT3) was significantly upregulated between 4 and 8 weeks after transplantation, which was the most striking change in this period. The upregulation of FLT3 may contribute to the hypoxic state and subsequent acquisition of quiescence in engrafted human LinCD34þCD38 cells.

Discussion Long-term HSCs are quiescent in the BM niche and give rise to all types of mature hematopoietic cells throughout life [15]. Transplanted HSCs may also acquire a quiescent

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Figure 4. Engrafted human HSCs acquire relatively hypoxic state 8 weeks (W) after transplantation. (A) Representative histogram of engrafted human cells analyzed for pimonidazole at 4 and 8 weeks after transplantation. (B) Relative median fluorescence intensity (MFI) of pimonidazole in human LinCD34þCD38þ cells or LinCD34þCD38 cells at 4, 8, and 12 weeks after transplantation. The relative hypoxic state, as indicated by pimonidazole-positive staining, was acquired in LinCD34þCD38 cells 8 weeks after transplantation compared with LinCD34þCD38þ cells (mean 6 standard deviation; *p ! 0.0.05, **p ! 0.01, n 5 3).

state at some point in the recipient BM niche. Previous reports have suggested that the homing of transplanted hematopoietic cells into recipient BM is not specific, but that subsequent lodgment of engrafted cells in the recipient niche may be selective [3]. Based on this concept, the performance of hematopoietic cells during BM reconstitution after transplantation was analyzed, specifically with respect to determining the factors related to HSC maintenance. Our humanized NOG mouse model, representing high human hematopoietic chimerism as early as 4 weeks after transplantation, allowed us to investigate the characteristics of engrafting human HSCs in the BM. Most of the engrafted primitive cells were in a rapid-cycling state at early stage after transplantation, but acquired a quiescent state in the recipient BM between 8 and 12 weeks after transplantation. The BM niche is proposed to be hypoxic [7,8,15,16]. Because reactive oxygen species regulation is crucial for HSC maintenance, hypoxia is presumed to be one factor related to HSC regulation or maintenance [8,17]. Preconditional irradiation exposure leads to destruction of the BM vasculature and stroma, as well as the apoptosis of cycling hematopoietic cells. Hematopoietic regeneration requires vasculature and stromal reconstruction, which may be important to restore the oxygen gradient in the BM [18–21]. Previous data suggested that after 650950 Gy irradiation,

normal blood vessels were hardly observed at day 10 after irradiation [21]. There is a possibility that BM blood vessel injury by irradiation may have resulted in hypoxia of all the cells at 4 weeks. However, as the blood vessels recover, oxygen gradient may be restored in the BM and LinCD34þCD38 were likely to localize in the hypoxic area, while others did not. Primitive CB cells reportedly exhibit increased self-renewal activity when cultured in a hypoxic state, acquiring stem cell characteristics [22]. Moreover, hypoxia-inducible factor1alpha (HIF-1alpha) is reported to negatively regulate cell proliferation. For example, HIF-1a induces cell cycle arrest by directly displacing Myc-binding from the promoter of cdkn1a [23,24]. Thus, with the progress of BM reconstruction after irradiation and transplantation, engrafted HSCs may have adapted to the hypoxic area [22], resulting in subsequent acquisition of quiescence. From this perspective, the expression levels of mRNAs related to the cell cycle and hypoxic metabolism were examined at 4 and 8 weeks after transplantation (Table 1). Cell cycle regulators, cyclins, and G0/G1 regulators were first examined. Because murine long-term HSCs express the highest mRNA levels of most CDK inhibitors and the lowest of most cyclins in multipotent BM cells [25], we hypothesized that the expression levels of these genes may change along with the transition of cell cycle state. In LinCD34þCD38 fraction, CCD2, CCNG1, and CCNB2

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Table 1. Relative expression ratio of subset of genes after transplantation (8 wk/4 wk) Gene symbols G1 cyclins Cyclin D1 Cyclin D2 Cyclin D3 Cyclin E1 Cyclin E2 S-G2/M cyclins Cyclin A2 Cyclin G1 Cyclin G2 Cyclin B1 Cyclin B2 Cyclin F G0/G1 regulators Cyclin C E2F transcription factor 4 E2F transcription factor 6 Retinoblastoma 1 Retinoblastoma-like 1 (p107) Retinoblastoma-like 2 (p130) CDK inhibitors Cyclin-dependent kinase inhibitor 1A (p21, Cip1) Cyclin-dependent kinase inhibitor 1B (p27, Kip1) Cyclin-dependent kinase inhibitor 1C (p57, Kip2) Hypoxia-associating factors Hypoxia-inducible factor 1, alpha subunit (HIF-1a) Hypoxia-inducible factor 2, alpha subunit (HIF-2a) Solute carrier family 2 (facilitated glucose transporter), member 1 (Glut-1) Solute carrier family 2 (facilitated glucose transporter), member 3 (Glut-3) Chemokines and differentiation marker and niche factors CD44 molecule v-myc myelocytomatosis viral oncogene homolog Chemokine (C-X-C motif) receptor 4 v-myc myelocytomatosis viral related oncogene, neuroblastoma derived TEK tyrosine kinase, endothelial Cadherin 11, type 2, OB-cadherin Cadherin 2, type 1, N-cadherin Cadherin 5, type 2 Integrin, alpha 4 Integrin, alpha 5 Integrin, beta 1 Integrin, beta 2 Stem cell regulating factors fms-related tyrosine kinase 3 v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog BMI1 polycomb ring finger oncogene Catenin (cadherin-associated protein), beta 1 Myeloproliferative leukemia virus oncogene Notch homolog 1, translocation-associated Apoptotic factors BCL2-associated X protein B-cell CLL/lymphoma 2 Myeloid cell leukemia sequence 1 Tumor protein p53

Ratio (8 wk/4 wk)

p Value

CCND1 CCND2 CCND3 CCNE1 CCNE2

0.67 1.26 1.06 1.2 1.18

0.22 0.03 0.77 0.2 0.5

CCNA2 CCNG1 CCNG2 CCNB1 CCNB2 CCNF

1.23 0.55 0.71 0.71 0.63 2.36

0.23 0.002 0.29 0.11 0.03 0.27

CCNC E2F4 E2F6 RB1 RBL1 RBL2

0.3 0.23 0.96 1.02 0.26 0.84

0.04 0.005 0.8 0.94 0.01 0.36

CDKN1A CDKN1B CDKN1C

1 0.85 0.3

0.98 0.05 0.007

HIF1A HIF2A SLC2A1 SLC2A3

0.21 UD* 0.15 0.62

0.09

CD44 MYC CXCR4 MYCN TEK CDH11 CDH2 CDH5 ITGA4 ITGA5 ITGB1 ITGB2

0.86 0.75 1.29 0.88 UD 0.59 UD* at 4W UD* 0.65 1 0.91 1.38

0.26 0.14 0.047 0.71

FLT3 KIT BMI1 CTNNB1 MPL NOTCH1

2.69 0.77 0.69 0.98 1.52 1.44

0.004 0.008 0.24 0.93 0.2 0.11

BAX BCL-2 MCL1 TP53

0.85 0.86 0.5 1.09

0.53 0.63 0.23 0.71

0.01 0.04

0.31

0.006 0.94 0.31 0.05

More than twofold difference with significance (p ! 0.05) indicated with bold. UD 5 undetectable.

showed a significant change of less than twofold. For the CDK inhibitors, CDKN1C expression levels decreased significantly, by more than twofold. Long term-HSCs were

reported to show the highest level of CDKN1C in murine studies [25,26]. As LinCD34þCD38 fraction is not fully purified with long-term HSCs, further study on the function

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Figure 5. Quantitative RT-PCR analysis of gene expression related to the regulation of cell cycle, hypoxia, and stemness in human HSCs after engraftment in NOG mice. Engrafted human Lin-CD34þCD38þ cells or LinCD34þCD38 cells were collected at 4 and 8 weeks after transplantation, and the relative expression of various genes were analyzed (tested genes are listed in Table 1). Genes that specifically changed by more than twofold between 4 and 8 weeks after transplantation in LinCD34þCD38 cells are shown, along with their relative expression levels. Each value was normalized to b-actin expression (mean 6 standard deviation; *p ! 0.05, **p ! 0.01, n 5 3).

of CDKN1C in human HSCs is needed. Despite that marked increase of PY-negative or BrdU-negative population has not yet occurred at 8 weeks after transplantation, CCNC showed a significantly decreased expression level at this time point (Table 1, Fig. 5). Cyclin C is upregulated in G0 cells, forming a complex with Cdk3 and phosphorylated Rb to promote exiting from a transient cell cycle arrest [27]. A recent study on human HSCs demonstrated that CCNC knockdown enhances the quiescence and engraftment potential of hematopoietic stem/progenitor cells [28]. Therefore, acquisition of quiescence in LinCD34þCD38 cells during 8 and 12 weeks after transplantation may have resulted from CCNC transcriptional downregulation already observed at 8 weeks after transplantation. Rb and two related proteins, p107 and p130, are known to regulate E2F activity in G0/G1 and contribute to G0 maintenance [29]. Consistent with a previous report that RBL1 is the least expressed in long-term HSCs [25], the RBL1 expression level was significantly decreased at 8 weeks compared with 4 weeks after transplantation. In contrast, E2F4, which reportedly displays a stable protein level throughout the cell cycle [30], also displayed decreased expression. These data suggest that changes in the expression levels of G0/G1 cell cycle regulators may promote engrafting HSCs to acquire quiescence in the recipient BM niche. Hypoxia-related factors were also examined. In general, HIF-1a mRNAs are constitutively and ubiquitously

expressed regardless of the level of oxygen tension, and HIF-1a regulation occurs through the proteasomal degradation system according to the oxygen level [31,32]. The hypoxic induction of HIF-1a reportedly occurs without an accompanying change in mRNA abundance. This may occur because the HIF-1a mRNA level is regulated by autonegative feedback by HIF-1a, or because the accumulation of the aHIF transcript for the 30 -untranslated region of HIF1-a may increase HIF-1a mRNA instability [33]. Consistent with this, no significant change in the HIF1A expression level was observed. Meanwhile, the levels of SLC2A1 and SLC2A3 were significantly decreased. Although glucose transporters are positively regulated through the coordination of glucose utilization and uptake by HIF-1a in hypoxia [34], an increased proportion of quiescent cells may cause decreased glucose transporter transcription, despite acquisition of the hypoxic state. The most striking change observed was that of the FLT3 expression level, which increased more than twofold between 4 and 8 weeks after transplantation. Flt3 is an HSC differentiation marker, and Flt3 expression in murine HSCs correlates with a loss of stemness [35]. However, the expression pattern of Flt3 supposedly differs in humans; cells capable of longterm reconstitution are contained in the Flt3þ population in human CD34þ CB or BM cells [36–38]. Flt3 ligand reportedly stimulates primitive and more committed CD34þ progenitor cells in vitro and in vivo, even during the postirradiation

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recovery phase, while FLT3 inhibition impairs progenitor cell growth and increases the rate in apoptosis [39–43]. Because it was noted that Flt3 signaling might support cell survival through MCL1 upregulation, which inhibits apoptosis [39,44], some apoptotic factors were also examined. However, no such relationship was observed in the xenotransplantation model described in this study (Table 1). Collectively, FLT3 upregulation may have contributed to the acquisition of quiescence in LinCD34þCD38 cells, supporting the previous finding that Flt3 may help promote BM recovery after transplantation or after myeloablative chemotherapy [40]. It is our future interest to demonstrate the functional role of FLT3 to the cell cycle or oxygenation state in human HSCs. Although quiescence is proposed to be crucial for the maintenance of long-term HSCs, engrafted primitive CB cells were able to maintain their stemness during nonquiescent period at early stage after transplantation. For this, we could think of two possibilities. Firstly, because long term-HSCs, which might have been always quiescent after transplantation, are only a minority in the Lin CD34þCD38 cell fraction and thus the results in the experiments in this study did not reflect the characteristics of those cells. Secondly, as shown previously that HSCs regularly enter and exit cell cycle and reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress [45], CB stem cells may be cycling soon after transplantation, but acquire dormancy and maintain stemness a certain period after transplantation. Several studies on identification of human HSCs by evaluating the capability of serial repopulation in immunodeficient mice demonstrated that human HSCs reside in the Lin CD34þCD38 fraction [46,47]. Because LinCD34þCD38þ fraction contained cells with short- but not long-term repopulating ability, we used the surface marker combinations of LinCD34þCD38 and LinCD34þCD38þ for HSCcontaining fraction and more differentiated progenitor fraction, respectively [48]. Although limiting dilution analysis showed that severe combined immunodeficientrepopulating cells were enriched by 1500-fold in LinCD34þCD38 fraction compared with unfractionated MNCs, the frequency of severe combined immunodeficientrepopulating cells in LinCD34þCD38 fraction was 1 in 617 cells [48]. Thus, we have to be aware that long-term HSCs are only a minority in LinCD34þCD38 cell fraction. The differences in post-transplantation behaviors such as cell cycle status and hypoxic conditions between LinCD34þCD38 and Lin CD34þCD38þ populations may reflect the characteristic differences between HSCs and progenitors in the engraftment process. In order to further assess the characteristics change of engrafting long-term HSCs following transplantation HSC purification with additional surface markers may be required. In conclusion, using an NOG mouse model, we clarified the acquisition processes of quiescence and the hypoxic state by engrafted human HSCs in the BM. It is important for oncologists to have an idea that most of the primitive

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HSCs and progenitors are still in the cell-cycling state even after the confirmation of stable engraftment for as long as 2 months after HSC transplantation. Understanding the characteristic changes of engrafting human HSCs after transplantation may allow determination of the molecular mechanism regulating HSCs, allowing the detection of new targets for optimizing transplantation therapies and therapeutic strategies for leukemia.

Acknowledgments We thank Ms. Ayako Kumakubo for technical support. This work was supported by Grants-in-Aid for Young Scientists (B) (H.S. and K.T.) and a Grant-in-Aid for Scientific Research on Priority Areas (K.T.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Conflict of Interest Disclosure No financial interest/relationships with financial interest relating to the topic of this article have been declared.

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