- Email: [email protected]
Alterations in the bone marrow microenvironment may elicit defective hematopoiesis: a comparison of aplastic anemia, chronic myeloid leukemia, and normal bone marrow
Accepted Manuscript Alterations in bone marrow microenvironment may elicit defective hematopoiesis: a comparison of aplastic anemia, chronic myeloid leukemia and normal bone marrow Meerim Park, Chan-Jeoung Park, Young Wook Cho, Seongsoo Jang, Jung-Hee Lee, Je-Hwan Lee, Kyoo-Hyung Lee, Young Ho Lee PII:
S0301-472X(16)30621-X
DOI:
10.1016/j.exphem.2016.09.009
Reference:
EXPHEM 3466
To appear in:
Experimental Hematology
Received Date: 29 June 2016 Revised Date:
2 September 2016
Accepted Date: 19 September 2016
Please cite this article as: Park M, Park C-J, Cho YW, Jang S, Lee J-H, Lee J-H, Lee K-H, Lee YH, Alterations in bone marrow microenvironment may elicit defective hematopoiesis: a comparison of aplastic anemia, chronic myeloid leukemia and normal bone marrow, Experimental Hematology (2016), doi: 10.1016/j.exphem.2016.09.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Alterations in bone marrow microenvironment may elicit defective hematopoiesis: a comparison of aplastic anemia, chronic myeloid leukemia and normal bone marrow
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Running title: Alterations in BM microenvironment
Authors
Meerim Parka, Chan-Jeoung Parkb, Young Wook Chob, Seongsoo Jangb, Jung-Hee Leec, Je-Hwan Leec,
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Kyoo-Hyung Leec, and Young Ho Leed
a
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Affiliations
Department of Pediatrics, College of Medicine Chungbuk National University, Cheongju, Korea
b
Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine,
Seoul, Korea
Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine,
Seoul,Korea d
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c
Department of Pediatrics, Hanyang University Medical Center, Hanyang University College of
Correspondence
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Medicine, Seoul, Korea
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Chan-Jeoung Park, MD, PhD
Address: Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
Phone: 82-2-3010-4508 Fax: 82-2-478-0884
E-mail: [email protected]
Young Ho Lee, MD, PhD
ACCEPTED MANUSCRIPT Address: Department of Pediatrics, Hanyang University Medical Center, Hanyang University College of Medicine, Wangsimni-ro, 222, Seongdong-gu, Seoul, Korea Phone: 82-2-2290-8383
E-mail:[email protected]
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E-mail address for offprint request: [email protected]
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Fax: 82-2-2297-2380
ACCEPTED MANUSCRIPT Abstract Hematopoiesis involves complex interactions between hematopoietic cells and the bone marrow (BM) microenvironment; the specific causes and mechanisms underlying dysregulated hematopoiesis are unknown. Here, BM biopsy specimens from patients with aplastic anemia (AA), chronic myeloid
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leukemia (CML), and normal marrow were analyzed by semiquantitative immunohistochemistry to determine changes in the hematopoietic stem cell (HSC) compartment and BM microenvironment. HSC levels were lowest in AA and highest in CML. T- and B-lymphocytes were decreased in AA
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(p<0.01) and CML (p<0.01). Natural killer cells were observed in AA, but were absent in CML and normal controls (p<0.01). Macrophages and mast cells were absent in CML. There were significant
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differences between of AA and CML stromal cell components. No nestin+ cells were observed in CML and the mean number of SDF-1+ cells was lowest in CML. Osteopontin+ cells were higher in AA than in CML (p<0.01); osteonectin+ cells were higher in CML than in AA (p<0.01). There was no significant difference in the expression of osteocalcin between AA and CML. The number of endothelial cells was highest in CML and lowest in AA (p<0.01).Our findings suggest that changes in
CML.
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BM microenvironment components might be related to defective hematopoiesis leading to AA and/or
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Keywords: aplastic anemia, chronic myeloid leukemia, hematopoiesis, microenvironment
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ACCEPTED MANUSCRIPT Introduction Hematopoietic stem cells (HSCs) reside in specialized microenvironments (niches) in the bone marrow (BM) [1, 2]. The importance of the stem cell niche in regulating HSC function was first postulated in 1978, in a study demonstrating that the spleen is unable to support HSCs in the same
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way that BM can [3]. The stem cell niche is thought to provide signals that support key HSC properties, including self-renewal capacity and long-term multilineage repopulating ability. The marrow microenvironment is comprised of a cellular (stromal cells, osteoblasts, osteoclasts,
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endothelial cells, and immune cells) and a noncellular compartment including the extracellular matrix and the liquid milieu (cytokines, growth factors, and chemokines) [4]. Alterations of the BM
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microenvironment, of which mesenchymal stem cells (MSCs) are an important factor, can be a potential factor associated with hematopoietic impairment [5, 6]. Previous studies have suggested that various alterations occur in the MSCs of patients with different hematological diseases; however, how these alterations contribute to disease progression remains unclear [7-10]. In addition to MSCs, emerging data points to the role of osteolineage cells in the regulation of hematopoiesis; however, the
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results are heterogeneous [11-13].
Examples of both dysregulated hematopoiesis extremes include BM failure in aplastic anemia (AA) and myeloproliferative diseases such as chronic myeloid leukemia (CML). AA is defined as BM
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hypocellularity and peripheral blood pancytopenia. In contrast, CML is a myeloproliferative neoplasm related to the presence of the BCR-ABL1 fusion gene. CML leukemia stem cells retain the ability to
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regenerate multilineage hematopoiesis and lead to the vast expansion of malignant myeloid cells, which retain differentiation capacity and displace residual normal hematopoiesis [14, 15]. The exact causes and mechanisms underlying dysregulated hematopoiesis in such diseases are not known and the role of the microenvironment itself in dysregulated hematopoiesis has not been well characterized. Previously, we reported on changes in the BM microenvironment of AA patients [16]. In order to gain a better understanding of the interaction between hematopoiesis and the BM microenvironment, we investigated changes in the HSC compartment and the BM microenvironment in patients with AA, CML in chronic phase (CML-CP), and normal marrow. 2
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Materials and methods
Patients
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Thirty newly-diagnosed patients (10 AA, 10 CML-CP, and 10 lymphoma patients without BM involvement [normal control]) were enrolled in this study. To approach a true BM microenvironment and reduce the risk of peripheral blood contamination, we examined BM biopsy specimens. BM
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specimens were obtained from the posterior iliac crest in all cases. In patients with AA, BM cellularity was less than 25% and at least two of the following criteria were fulfilled: an absolute neutrophil
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count less than 0.5 × 109/L, a platelet count less than 20 × 109/L, and a reticulocyte count less than 1%. The control subjects included age-matched patients whose BM was examined for staging work-up of non-Hodgkin’s lymphoma and proved to be normal without evidence of lymphoma involvement. The median age was 53.5 years (range: 25-74 years) for the AA patients, 51.0 years (range: 28-62 years) for the CML-CP patients, and 42.5 years (range: 26-62 years) for the control subjects. This study was
Medical Center.
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Immunohistochemistry
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approved by both the Institutional Review Board of Asan Medical center and Hanyang University
Semiquantitative immunohistochemical (IHC) staining was performed on the BM biopsy specimens
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for HSC antigens (CD34 and CD117), lymphocyte antigens (CD3, CD4, CD8, CD20, and CD56), a macrophage antigen (CD169), glycoproteins (osteopontin, osteonectin, and osteocalcin) synthesized by osteoblasts, nestin, and stromal cell-derived factor-1 (SDF-1). CD34+ cells were recognized into two cell types: small to medium sized mononuclear HSCs and elongated endothelial cells. CD117+ cells were differentiated into two cell types: small to medium sized mononuclear myeloid progenitor cells and oval shaped mast cells with condensed nuclei and abundant cytoplasmic granules. Details of the primary antibodies and staining procedures are provided in Table 1. IHC staining was performed using a Benchmark XT Autostainer (Ventana, Tucson, AZ, USA). All negative and positive controls 3
ACCEPTED MANUSCRIPT gave the expected results. Cells positive for all markers except osteocalcin were counted in 10 high-power fields (HPF,
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and the averages per HPF were calculated. Cells positive for osteocalcin were counted on the
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peritrabecular line on each slide and standardized by the mean length measured. Interpretation of the semiquantitative IHC results was performed independently by two observers.
Statistical analyses
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Analysis of variance (ANOVA) was used to test for differences between the three groups of bone marrow specimens. Pearson’s r correlation coefficient was calculated to identify the relationship
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between two variables. A value of P < 0.05 was used to define statistical significance. All analyses were conducted using SPSS version 18.0 software.
Results
± standard deviation.
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The results of the semiquantitative IHC staining are detailed in Table 2. Data are expressed as mean
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Hematopoietic stem/progenitor cells
Among the three groups, the number of CD34+ cells was the lowest in AA specimens. However,
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there was no significant difference in the number of CD34+ cells between CML-CP patients and control patients (Fig. 1A). The number of CD117+ cells was lowest in the AA specimens and highest in the CML specimens (Fig. 1B).
Lymphocytes, macrophages and mast cells T- (CD3, CD4, and CD8) and B-lymphocytes (CD20) were decreased in both the AA (p<0.01) and CML (p<0.01) specimens compared to the normal control specimens (Fig. 2A and B). Natural killer cells (NK cells, CD56) were occasionally observed in the AA specimens but were absent in the CML 4
ACCEPTED MANUSCRIPT and control specimens (p<0.01) (Fig. 2C). Macrophages (CD169) and mast cells were absent in the CML specimens (Fig. 2D and E). The number of mast cells was significantly higher in the AA group than in the other two groups (p<0.01)
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(Fig. 2E).
Stromal cell components
Nestin+ cells were not observed in the CML specimens and the number of nestin+ cells in the AA
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specimens was not significantly different from than in the control group (Fig. 3A).
The mean number of SDF-1+ cells was lowest in CML patients and there was no significant
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difference in the number of SDF-1+ cells between the AA and control specimens (Fig.3B). The number of osteopontin+ cells was higher in AA than in CML specimens (p<0.01), whereas the number of osteonectin+ cells was significantly higher in CML than in AA patients (p<0.01) (Fig. 3C and D). There was no significant difference in osteocalcin expression between the AA, CML, and control groups.
specimens (p<0.01).
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The number of endothelial cells was highest in CML specimens (p<0.01) and lowest in AA
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Interactions between each of the BM microenvironment components Macrophage levels showed a positive correlation with the number of nestin+ (r=0.68, p<0.01) and
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SDF-1+ cells (r=0.74, p<0.01; Fig. 4A). The number of mast cells also showed a positive correlation with the level of nestin+ (r=0.78, p<0.01) and SDF-1+ cells (r=0.73, p<0.01; Fig. 4B). Osteopontin (Fig. 4C) demonstrated a negative correlation with CD34+ (r=-0.40, p=0.03) and CD117+ cells (r=-0.62, p<0.01), whereas osteonectin (Fig. 4D) showed a positive correlation with CD34+ (r=0.39, p=0.03) and CD117+ cells (r=0.35, p=0.05). No association was found between osteocalcin+ cells and HSCs. The number of endothelial cells was positively correlated with the number of CD117+ cells (r=0.45, p=0.01; Fig. 4E). 5
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Discussion As shown in this study, the numbers of CD34+ and CD117+ cells were lowest in AA patients, implying they possessed the lowest level of HSCs. The incidence of CD34 positivity in CML patients
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has been reported in previous studies [17-19]. Banavali et al. [17] reported that CD34 positivity varied from 0-26% in patients with CML-CP, which is significantly lower than in patients with CML in accelerated phase or blast crisis. Other studies have reported that CD34 is non-reactive in patients
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with CML-CP [18, 19]. In the current study, there was no significant difference in the number of CD34+ cells between CML-CP and control patients. However, the number of cells positive for the
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CD117 antigen was highest in CML specimens. CD117 is expressed on early myeloid cells and its expression is independent of CD34 expression [20]. These findings indicate that patients with CMLCP still possess a higher level of immature myeloid cells compared to normal marrow. Decreased T- and B- lymphopoiesis was demonstrated in both AA and CML patients. The BM of CML-CP patients did not show any B-lymphocytes; Signer et al. [21] previously reported the
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occurrence of defective B-lymphopoiesis in CML. Interestingly, NK cells, macrophages, and mast cells were not identified in CML patients. In contrast, the BM of AA patients demonstrated increased
damage.
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numbers of mast cells and NK cells, which could reflect cytotoxic and/or immune-mediated marrow
Whether a specific subpopulation of osteoblastic cells is interacting with HSCs is currently under
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investigation. Osteoblastic cells synthesize proteins such as SDF-1, osteopontin, osteocalcin, and osteonectin, which may constitute markers of osteoblast maturity or activation state. A number of recent studies have suggested that osteopontin is involved in CML pathogenesis [22, 23]. The level of osteopontin expression was shown to increase during CML progression; the highest levels were observed during the accelerated phase and blast crisis but decreased during hematological and cytogenetic remission [22]. In our study, the level of osteopontin was lowest in CML-CP patients. It seems that different phases of CML might reveal different levels of osteopontin with greatest changes during accelerated phase and blast crisis but declining during chronic phase. The analysis of BM from 6
ACCEPTED MANUSCRIPT patients with CML in accelerated phase and blast phase would give more information on the pathogenesis of CML. AA patients showed the lowest level of osteonectin expression; no significant difference in osteocalcin expression was observed between the three groups. These data suggest that
accordance with the dysregulated hematopoietic abnormalities.
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the subpopulations of osteoblastic cells comprising the osteoblastic niche differ significantly in
Recently, it was reported that SDF-1 promotes the growth and survival of BM stromal cells in an autocrine manner [24]. Reduction in BM SDF-1 levels is known as one of the mechanisms underlying
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impaired HSC homing and retention in CML BM [15]. In this study, the level of cells positive for SDF-1 was significantly lower in CML-CP patients than in the control subjects, indicating that
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recovery of SDF-1 expression remains incomplete. Interestingly, the number of SDF-1+ cells in AA specimens ranged broadly, thus we were unable to determine a statistically significant difference compared to the control subjects. The results obtained for the nestin+ cells were similar to the SDF-1 expression findings: no nestin+ cells were observed in CML specimens and the number of nestin+ cells in AA group ranged broadly, thus we were unable to determine a statistically significant difference
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compared with the control group. Nestin+ cells maintain HSC in BM and the depletion of nestin+ cells reduces HSC content in BM [25]. Such a wide range of nestin and SDF-1 level in the BM of AA patients may be partly due to the heterogeneous causes of AA. Our results suggest that the decreased
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levels of nestin+ cells and SDF-1+ cells are associated with CML pathogenesis; however, these cells are not significantly damaged in AA.
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This study identified multiple interactions between each of the BM microenvironment components, although further studies are needed to make firm conclusions. First, macrophages and mast cells showed a positive correlation with nestin+ cells and SDF-1+ cells, which were lowest in CML patients. This concept was supported by the study of Chow et al. [26]. They found that a reduction in BM mononuclear phagocytes led to a decrease in BM SDF-1 levels, the selective down-regulation of HSC retention genes in nestin+ niche cells, and egress of HSCs to the blood stream. Second, osteopontin showed a negative correlation with CD34+ cells and CD117+ cells, whereas osteonectin showed a positive correlation with CD34+ cells and CD117+ cells. Previously, Nilsson et al. [27] suggested 7
ACCEPTED MANUSCRIPT osteopontin as a physiologic-negative regulator of HSC proliferation showing a direct interaction between HSCs and osteopontin. It has been established that HSCs reside preferentially at the endosteal region within the BM where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fates [28-30]. Our findings indicate that osteoblast-derived
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extracellular proteins including SDF-1, osteopontin, and osteonectin seem to play important roles in HSC regulation and therefore hematopoiesis; however, osteopontin and osteonectin seem to play opposite roles in HSC regulation. Third, endothelial cells showed a positive correlation with CD117+
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cells. It has been suggested that the BM endothelium supports HSC proliferation by constitutive production of cytokines such as G-CSF, GM-CSF, and IL-6 [31]. BM endothelial cells express several
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genes implicated in HSC maintenance, including SDF-1, stem cell factor (SCF), and angiopoietin, and support the proliferation of HSCs in vitro [32]. Fernandez et al. [33] demonstrated that the hematopoiesis-enhancing activity of the BM endothelium is cell-to-cell contact dependent and largely mediated by Notch signaling. Taken together, our results suggest that alteration of the osteoblasts or sinusoidal endothelium can modify HSC levels and self-renewal function. Restoration of normal
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interactions between hematopoietic cells and the BM microenvironment could play a role in the recovery of BM failure or leukemia control.
Our study has several limitations to consider, including the small patient population, which hinders
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the ability to draw firm conclusions. Secondly, we did not use an isotype control for each antibody as a test control. Considering this is a study of semiquantative IHC staining to identify changes in the
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HSC compartment and the BM microenvironment in AA and CML, it would have no significant problem with yielding results. Thirdly, the antibody used in this study detects full length osteopontin, but it is recently suggested that thrombin-cleaved osteoponin is the dominant form present in the BM [34]. Currently, investigators use either osteopontin antibody or anti- thrombin-cleaved osteopontin antibody to analyze BM specimen. For better understanding of BM microenvironment, thrombincleaved osteopontin should be also analyzed in future studies. Whether malignant hematopoietic cells disrupt the normal hematopoietic microenvironment or the emergence of a malignant environment better suited to support malignant cells was not addressed in 8
ACCEPTED MANUSCRIPT this study. Similarly, changes in the BM microenvironment of AA patients could either contribute to the pathogenesis of AA, or be caused by impaired hematopoiesis. A better understanding of the interaction between each of the BM microenvironment components may provide potential therapeutic targets for myeloproliferative disease as well as BM failure.
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In conclusion, our findings suggest that changes in the components of the BM microenvironment including lymphocytes, macrophages, mast cells, and stromal cells, might be related to defective
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hematopoiesis that could lead to AA and/or myeloproliferative disease.
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Authorship statement
M. Park performed the study, analyzed the data, and wrote the manuscript; C.J. Park and Y.H. Lee designed the study, performed the study, analyzed the research data, and reviewed the manuscript; Y.W. Cho and S. Jang contributed to the analysis of data; and K. Hwang, J.H. Lee, J.H. Lee and K.H.
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Conflicts of interest: none
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Lee contributed to data collection.
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ACCEPTED MANUSCRIPT Figure 1. Comparative analyses of (A) CD34+ cells and (B) CD117+ cells in aplastic anemia, chronic myeloid leukemia, and normal control; *p<0.05.
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(A) CD34+ cells
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(B) CD117+ cells
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ACCEPTED MANUSCRIPT Figure 2. Comparative analyses of (A) CD3+ T cells, (B) CD20+ B cells, (C) CD56+ natural killer cells, (D) macrophages (CD169) and (E) mast cells in aplastic anemia, chronic myeloid leukemia, and normal control; *p<0.05.
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(A) CD3+ T cells
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(B) CD20+ B cells
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(C) CD56+ natural killer cells
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(D) Macrophage
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(E) Mast cells
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ACCEPTED MANUSCRIPT Figure 3. Comparative analyses of cells positive for (A) nestin, (B) SDF-1, (C) osteopontin, and (D) osteonectin in aplastic anemia, chronic myeloid leukemia, and normal control; *p<0.05.
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(A) Nestin
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(B) SDF-1
(C) Osteopontin
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(D) Osteonectin
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ACCEPTED MANUSCRIPT Figure 4. Correlations between the number of (A) macrophages and SDF-1+ or nestin+ cells, (B) mast cells and SDF-1+ or nestin+ cells, (C) osteopontin+ cells and CD34+ or CD117+ cells, (D) osteonectin+ cells and CD34+ or CD117+ cells, and (E) endothelial cells and CD117+ cells.
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(A) macrophages and SDF-1+ or nestin+ cells
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(B) mast cells and SDF-1+ or nestin+ cells
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(C) osteopontin+ cells and CD34+ or CD117+ cells
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(D) osteonectin+ cells and CD34+ or CD117+ cells
(E) endothelial cells and CD117+ cells
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Table 1 Primary antibodies, clones, reaction temperatures, dilution factors, and incubation time used for immunohistochemistry Clone
Species
Clonality
Manufacturer
Reaction temperature
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Primary antibodies
QBEND/10
Mouse
Monoclonal
Leica Biosystems Newcastle, Ltd (Newcastle, UK)
CD117
YR145
Rabbit
Monoclonal
CD3
UCHT1
Rabbit
CD4
SP35
CD8
Incubation time (min)
Room temperature
1:50
60
Cell Marque, Inc (Rocklin, CA, USA)
42 ˚C
1:100
32
Polyclonal
Dako (Glostrup, Denmark)
42 ˚C
1:100
32
Rabbit
Monoclonal
Cell Marque, Inc
42 ˚C
1:100
32
C8/144B
Mouse
Monoclonal
Dako
42 ˚C
1:100
32
CD20
L26
Mouse
Monoclonal
Leica Biosystems Newcastle, Ltd
42 ˚C
1:250
20
CD56
123C3
Mouse
Monoclonal
Invitrogen Corporation (Camarillo, CA, USA)
42 ˚C
1:100
32
CD169
HSn 7D2
Mouse
Monoclonal
Abcam Inc. (Cambridge, MA, USA)
Room temperature
1:25
60
Osteopontin
OP3N
Mouse
Monoclonal
Leica Biosystems Newcastle, Ltd
42 ˚C
1:50
40
Osteocalcin
OC4-30
Mouse
Monoclonal
Abcam Inc
37 ˚C
1:200
32
Osteonectin
15G12
Mouse
Monoclonal
Leica Biosystems Newcastle, Ltd
Room temperature
1:100
60
SDF-1
MM0211-9N26
Mouse
Monoclonal
Abcam Inc
Room temperature
1:40
60
Nestin
196908
Mouse
Monoclonal
R&D System (Boston, MA, USA)
42 ˚C
1:4000
32
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SDF-1 = stromal cell-derived factor-1
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CD34
Dilution
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ACCEPTED MANUSCRIPT Table 2 Analysis of semiquantitative immunohistochemical staining of bone marrow cells from patients with aplastic anemia (AA), chronic myeloid leukemia (CML), and control subjects AA
CML
Control
CD34
1.36 ± 1.22
14.31 ± 15.44
CD117
2.84 ± 2.06
34.00 ± 21.83
Hematopoietic stem cells
9.49 ± 3.02
0.013
11.17 ± 4.25
<0.01
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Lymphocytes, macrophages, and mast cells
P value
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Antigen
18.67 ± 12.88
26.28 ± 10.44
70.83 ± 30.70
<0.01
CD4
1.56 ± 1.25
0.74 ± 1.14
9.87 ± 3.65
<0.01
CD8
12.41 ± 9.79
16.96 ± 6.27
41.17 ± 14.11
<0.01
CD20
1.01 ± 1.07
0.00 ± 0.00
7.37 ± 4.98
<0.01
CD56
0.11 ± 0.12
0.00 ± 0.00
0.00 ± 0.00
<0.01
CD169
1.53 ± 0.96
0.00 ± 0.00
1.05 ± 1.07
<0.01
Mast cells
2.34 ± 1.85
0.00 ± 0.00
0.49 ± 0.41
<0.01
5.79 ± 2.08
0.10 ± 0.32
4.45 ± 1.11
<0.01
113.40 ± 88.54
261.70 ± 323.93
145.20 ± 149.28
0.275
0.40 ± 0.17
2.13 ± 0.73
2.60 ± 1.10
<0.01
2.96 ± 2.76
0.43 ± 0.49
1.94 ± 0.47
<0.01
8.04 ± 8.41
0.00 ± 0.00
3.62 ± 1.21
<0.01
39.20 ± 21.46
186.90 ± 140.95
18.00 ± 7.64
<0.01
Osteocalcin Osteonectin SDF-1
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Nestin
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Osteopontin
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Stromal cell components
Endothelial cells
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AA = aplastic anemia; CML = chronic myeloid leukemia; SDF-1 = stromal cell-derived factor-1 Results are represented as mean ± standard deviation of positive cell number/high power field (×400). Osteocalcin is expressed as mean ± standard deviation of positive peritrabecular line length (µm). Analysis of variance (ANOVA) was used to test for differences between the three types of bone marrow specimens.
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ACCEPTED MANUSCRIPT Decreased T- and B- lymphopoiesis was demonstrated in both AA and CML.
Natural killer cells were observed in AA, but were absent in CML and control.
There were significant differences between of AA and CML stromal cell components.
Alteration of the osteoblasts or sinusoidal endothelium could modify HSC levels.
Changes in the BM microenvironment could lead to AA and/or CML.
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S0301-472X(16)30621-X
DOI:
10.1016/j.exphem.2016.09.009
Reference:
EXPHEM 3466
To appear in:
Experimental Hematology
Received Date: 29 June 2016 Revised Date:
2 September 2016
Accepted Date: 19 September 2016
Please cite this article as: Park M, Park C-J, Cho YW, Jang S, Lee J-H, Lee J-H, Lee K-H, Lee YH, Alterations in bone marrow microenvironment may elicit defective hematopoiesis: a comparison of aplastic anemia, chronic myeloid leukemia and normal bone marrow, Experimental Hematology (2016), doi: 10.1016/j.exphem.2016.09.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Alterations in bone marrow microenvironment may elicit defective hematopoiesis: a comparison of aplastic anemia, chronic myeloid leukemia and normal bone marrow
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Running title: Alterations in BM microenvironment
Authors
Meerim Parka, Chan-Jeoung Parkb, Young Wook Chob, Seongsoo Jangb, Jung-Hee Leec, Je-Hwan Leec,
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Kyoo-Hyung Leec, and Young Ho Leed
a
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Affiliations
Department of Pediatrics, College of Medicine Chungbuk National University, Cheongju, Korea
b
Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine,
Seoul, Korea
Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine,
Seoul,Korea d
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c
Department of Pediatrics, Hanyang University Medical Center, Hanyang University College of
Correspondence
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Medicine, Seoul, Korea
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Chan-Jeoung Park, MD, PhD
Address: Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
Phone: 82-2-3010-4508 Fax: 82-2-478-0884
E-mail: [email protected]
Young Ho Lee, MD, PhD
ACCEPTED MANUSCRIPT Address: Department of Pediatrics, Hanyang University Medical Center, Hanyang University College of Medicine, Wangsimni-ro, 222, Seongdong-gu, Seoul, Korea Phone: 82-2-2290-8383
E-mail:[email protected]
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E-mail address for offprint request: [email protected]
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Fax: 82-2-2297-2380
ACCEPTED MANUSCRIPT Abstract Hematopoiesis involves complex interactions between hematopoietic cells and the bone marrow (BM) microenvironment; the specific causes and mechanisms underlying dysregulated hematopoiesis are unknown. Here, BM biopsy specimens from patients with aplastic anemia (AA), chronic myeloid
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leukemia (CML), and normal marrow were analyzed by semiquantitative immunohistochemistry to determine changes in the hematopoietic stem cell (HSC) compartment and BM microenvironment. HSC levels were lowest in AA and highest in CML. T- and B-lymphocytes were decreased in AA
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(p<0.01) and CML (p<0.01). Natural killer cells were observed in AA, but were absent in CML and normal controls (p<0.01). Macrophages and mast cells were absent in CML. There were significant
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differences between of AA and CML stromal cell components. No nestin+ cells were observed in CML and the mean number of SDF-1+ cells was lowest in CML. Osteopontin+ cells were higher in AA than in CML (p<0.01); osteonectin+ cells were higher in CML than in AA (p<0.01). There was no significant difference in the expression of osteocalcin between AA and CML. The number of endothelial cells was highest in CML and lowest in AA (p<0.01).Our findings suggest that changes in
CML.
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BM microenvironment components might be related to defective hematopoiesis leading to AA and/or
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Keywords: aplastic anemia, chronic myeloid leukemia, hematopoiesis, microenvironment
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ACCEPTED MANUSCRIPT Introduction Hematopoietic stem cells (HSCs) reside in specialized microenvironments (niches) in the bone marrow (BM) [1, 2]. The importance of the stem cell niche in regulating HSC function was first postulated in 1978, in a study demonstrating that the spleen is unable to support HSCs in the same
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way that BM can [3]. The stem cell niche is thought to provide signals that support key HSC properties, including self-renewal capacity and long-term multilineage repopulating ability. The marrow microenvironment is comprised of a cellular (stromal cells, osteoblasts, osteoclasts,
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endothelial cells, and immune cells) and a noncellular compartment including the extracellular matrix and the liquid milieu (cytokines, growth factors, and chemokines) [4]. Alterations of the BM
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microenvironment, of which mesenchymal stem cells (MSCs) are an important factor, can be a potential factor associated with hematopoietic impairment [5, 6]. Previous studies have suggested that various alterations occur in the MSCs of patients with different hematological diseases; however, how these alterations contribute to disease progression remains unclear [7-10]. In addition to MSCs, emerging data points to the role of osteolineage cells in the regulation of hematopoiesis; however, the
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results are heterogeneous [11-13].
Examples of both dysregulated hematopoiesis extremes include BM failure in aplastic anemia (AA) and myeloproliferative diseases such as chronic myeloid leukemia (CML). AA is defined as BM
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hypocellularity and peripheral blood pancytopenia. In contrast, CML is a myeloproliferative neoplasm related to the presence of the BCR-ABL1 fusion gene. CML leukemia stem cells retain the ability to
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regenerate multilineage hematopoiesis and lead to the vast expansion of malignant myeloid cells, which retain differentiation capacity and displace residual normal hematopoiesis [14, 15]. The exact causes and mechanisms underlying dysregulated hematopoiesis in such diseases are not known and the role of the microenvironment itself in dysregulated hematopoiesis has not been well characterized. Previously, we reported on changes in the BM microenvironment of AA patients [16]. In order to gain a better understanding of the interaction between hematopoiesis and the BM microenvironment, we investigated changes in the HSC compartment and the BM microenvironment in patients with AA, CML in chronic phase (CML-CP), and normal marrow. 2
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Materials and methods
Patients
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Thirty newly-diagnosed patients (10 AA, 10 CML-CP, and 10 lymphoma patients without BM involvement [normal control]) were enrolled in this study. To approach a true BM microenvironment and reduce the risk of peripheral blood contamination, we examined BM biopsy specimens. BM
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specimens were obtained from the posterior iliac crest in all cases. In patients with AA, BM cellularity was less than 25% and at least two of the following criteria were fulfilled: an absolute neutrophil
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count less than 0.5 × 109/L, a platelet count less than 20 × 109/L, and a reticulocyte count less than 1%. The control subjects included age-matched patients whose BM was examined for staging work-up of non-Hodgkin’s lymphoma and proved to be normal without evidence of lymphoma involvement. The median age was 53.5 years (range: 25-74 years) for the AA patients, 51.0 years (range: 28-62 years) for the CML-CP patients, and 42.5 years (range: 26-62 years) for the control subjects. This study was
Medical Center.
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Immunohistochemistry
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approved by both the Institutional Review Board of Asan Medical center and Hanyang University
Semiquantitative immunohistochemical (IHC) staining was performed on the BM biopsy specimens
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for HSC antigens (CD34 and CD117), lymphocyte antigens (CD3, CD4, CD8, CD20, and CD56), a macrophage antigen (CD169), glycoproteins (osteopontin, osteonectin, and osteocalcin) synthesized by osteoblasts, nestin, and stromal cell-derived factor-1 (SDF-1). CD34+ cells were recognized into two cell types: small to medium sized mononuclear HSCs and elongated endothelial cells. CD117+ cells were differentiated into two cell types: small to medium sized mononuclear myeloid progenitor cells and oval shaped mast cells with condensed nuclei and abundant cytoplasmic granules. Details of the primary antibodies and staining procedures are provided in Table 1. IHC staining was performed using a Benchmark XT Autostainer (Ventana, Tucson, AZ, USA). All negative and positive controls 3
ACCEPTED MANUSCRIPT gave the expected results. Cells positive for all markers except osteocalcin were counted in 10 high-power fields (HPF,
400),
and the averages per HPF were calculated. Cells positive for osteocalcin were counted on the
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peritrabecular line on each slide and standardized by the mean length measured. Interpretation of the semiquantitative IHC results was performed independently by two observers.
Statistical analyses
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Analysis of variance (ANOVA) was used to test for differences between the three groups of bone marrow specimens. Pearson’s r correlation coefficient was calculated to identify the relationship
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between two variables. A value of P < 0.05 was used to define statistical significance. All analyses were conducted using SPSS version 18.0 software.
Results
± standard deviation.
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The results of the semiquantitative IHC staining are detailed in Table 2. Data are expressed as mean
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Hematopoietic stem/progenitor cells
Among the three groups, the number of CD34+ cells was the lowest in AA specimens. However,
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there was no significant difference in the number of CD34+ cells between CML-CP patients and control patients (Fig. 1A). The number of CD117+ cells was lowest in the AA specimens and highest in the CML specimens (Fig. 1B).
Lymphocytes, macrophages and mast cells T- (CD3, CD4, and CD8) and B-lymphocytes (CD20) were decreased in both the AA (p<0.01) and CML (p<0.01) specimens compared to the normal control specimens (Fig. 2A and B). Natural killer cells (NK cells, CD56) were occasionally observed in the AA specimens but were absent in the CML 4
ACCEPTED MANUSCRIPT and control specimens (p<0.01) (Fig. 2C). Macrophages (CD169) and mast cells were absent in the CML specimens (Fig. 2D and E). The number of mast cells was significantly higher in the AA group than in the other two groups (p<0.01)
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(Fig. 2E).
Stromal cell components
Nestin+ cells were not observed in the CML specimens and the number of nestin+ cells in the AA
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specimens was not significantly different from than in the control group (Fig. 3A).
The mean number of SDF-1+ cells was lowest in CML patients and there was no significant
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difference in the number of SDF-1+ cells between the AA and control specimens (Fig.3B). The number of osteopontin+ cells was higher in AA than in CML specimens (p<0.01), whereas the number of osteonectin+ cells was significantly higher in CML than in AA patients (p<0.01) (Fig. 3C and D). There was no significant difference in osteocalcin expression between the AA, CML, and control groups.
specimens (p<0.01).
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The number of endothelial cells was highest in CML specimens (p<0.01) and lowest in AA
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Interactions between each of the BM microenvironment components Macrophage levels showed a positive correlation with the number of nestin+ (r=0.68, p<0.01) and
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SDF-1+ cells (r=0.74, p<0.01; Fig. 4A). The number of mast cells also showed a positive correlation with the level of nestin+ (r=0.78, p<0.01) and SDF-1+ cells (r=0.73, p<0.01; Fig. 4B). Osteopontin (Fig. 4C) demonstrated a negative correlation with CD34+ (r=-0.40, p=0.03) and CD117+ cells (r=-0.62, p<0.01), whereas osteonectin (Fig. 4D) showed a positive correlation with CD34+ (r=0.39, p=0.03) and CD117+ cells (r=0.35, p=0.05). No association was found between osteocalcin+ cells and HSCs. The number of endothelial cells was positively correlated with the number of CD117+ cells (r=0.45, p=0.01; Fig. 4E). 5
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Discussion As shown in this study, the numbers of CD34+ and CD117+ cells were lowest in AA patients, implying they possessed the lowest level of HSCs. The incidence of CD34 positivity in CML patients
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has been reported in previous studies [17-19]. Banavali et al. [17] reported that CD34 positivity varied from 0-26% in patients with CML-CP, which is significantly lower than in patients with CML in accelerated phase or blast crisis. Other studies have reported that CD34 is non-reactive in patients
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with CML-CP [18, 19]. In the current study, there was no significant difference in the number of CD34+ cells between CML-CP and control patients. However, the number of cells positive for the
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CD117 antigen was highest in CML specimens. CD117 is expressed on early myeloid cells and its expression is independent of CD34 expression [20]. These findings indicate that patients with CMLCP still possess a higher level of immature myeloid cells compared to normal marrow. Decreased T- and B- lymphopoiesis was demonstrated in both AA and CML patients. The BM of CML-CP patients did not show any B-lymphocytes; Signer et al. [21] previously reported the
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occurrence of defective B-lymphopoiesis in CML. Interestingly, NK cells, macrophages, and mast cells were not identified in CML patients. In contrast, the BM of AA patients demonstrated increased
damage.
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numbers of mast cells and NK cells, which could reflect cytotoxic and/or immune-mediated marrow
Whether a specific subpopulation of osteoblastic cells is interacting with HSCs is currently under
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investigation. Osteoblastic cells synthesize proteins such as SDF-1, osteopontin, osteocalcin, and osteonectin, which may constitute markers of osteoblast maturity or activation state. A number of recent studies have suggested that osteopontin is involved in CML pathogenesis [22, 23]. The level of osteopontin expression was shown to increase during CML progression; the highest levels were observed during the accelerated phase and blast crisis but decreased during hematological and cytogenetic remission [22]. In our study, the level of osteopontin was lowest in CML-CP patients. It seems that different phases of CML might reveal different levels of osteopontin with greatest changes during accelerated phase and blast crisis but declining during chronic phase. The analysis of BM from 6
ACCEPTED MANUSCRIPT patients with CML in accelerated phase and blast phase would give more information on the pathogenesis of CML. AA patients showed the lowest level of osteonectin expression; no significant difference in osteocalcin expression was observed between the three groups. These data suggest that
accordance with the dysregulated hematopoietic abnormalities.
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the subpopulations of osteoblastic cells comprising the osteoblastic niche differ significantly in
Recently, it was reported that SDF-1 promotes the growth and survival of BM stromal cells in an autocrine manner [24]. Reduction in BM SDF-1 levels is known as one of the mechanisms underlying
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impaired HSC homing and retention in CML BM [15]. In this study, the level of cells positive for SDF-1 was significantly lower in CML-CP patients than in the control subjects, indicating that
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recovery of SDF-1 expression remains incomplete. Interestingly, the number of SDF-1+ cells in AA specimens ranged broadly, thus we were unable to determine a statistically significant difference compared to the control subjects. The results obtained for the nestin+ cells were similar to the SDF-1 expression findings: no nestin+ cells were observed in CML specimens and the number of nestin+ cells in AA group ranged broadly, thus we were unable to determine a statistically significant difference
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compared with the control group. Nestin+ cells maintain HSC in BM and the depletion of nestin+ cells reduces HSC content in BM [25]. Such a wide range of nestin and SDF-1 level in the BM of AA patients may be partly due to the heterogeneous causes of AA. Our results suggest that the decreased
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levels of nestin+ cells and SDF-1+ cells are associated with CML pathogenesis; however, these cells are not significantly damaged in AA.
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This study identified multiple interactions between each of the BM microenvironment components, although further studies are needed to make firm conclusions. First, macrophages and mast cells showed a positive correlation with nestin+ cells and SDF-1+ cells, which were lowest in CML patients. This concept was supported by the study of Chow et al. [26]. They found that a reduction in BM mononuclear phagocytes led to a decrease in BM SDF-1 levels, the selective down-regulation of HSC retention genes in nestin+ niche cells, and egress of HSCs to the blood stream. Second, osteopontin showed a negative correlation with CD34+ cells and CD117+ cells, whereas osteonectin showed a positive correlation with CD34+ cells and CD117+ cells. Previously, Nilsson et al. [27] suggested 7
ACCEPTED MANUSCRIPT osteopontin as a physiologic-negative regulator of HSC proliferation showing a direct interaction between HSCs and osteopontin. It has been established that HSCs reside preferentially at the endosteal region within the BM where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fates [28-30]. Our findings indicate that osteoblast-derived
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extracellular proteins including SDF-1, osteopontin, and osteonectin seem to play important roles in HSC regulation and therefore hematopoiesis; however, osteopontin and osteonectin seem to play opposite roles in HSC regulation. Third, endothelial cells showed a positive correlation with CD117+
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cells. It has been suggested that the BM endothelium supports HSC proliferation by constitutive production of cytokines such as G-CSF, GM-CSF, and IL-6 [31]. BM endothelial cells express several
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genes implicated in HSC maintenance, including SDF-1, stem cell factor (SCF), and angiopoietin, and support the proliferation of HSCs in vitro [32]. Fernandez et al. [33] demonstrated that the hematopoiesis-enhancing activity of the BM endothelium is cell-to-cell contact dependent and largely mediated by Notch signaling. Taken together, our results suggest that alteration of the osteoblasts or sinusoidal endothelium can modify HSC levels and self-renewal function. Restoration of normal
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interactions between hematopoietic cells and the BM microenvironment could play a role in the recovery of BM failure or leukemia control.
Our study has several limitations to consider, including the small patient population, which hinders
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the ability to draw firm conclusions. Secondly, we did not use an isotype control for each antibody as a test control. Considering this is a study of semiquantative IHC staining to identify changes in the
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HSC compartment and the BM microenvironment in AA and CML, it would have no significant problem with yielding results. Thirdly, the antibody used in this study detects full length osteopontin, but it is recently suggested that thrombin-cleaved osteoponin is the dominant form present in the BM [34]. Currently, investigators use either osteopontin antibody or anti- thrombin-cleaved osteopontin antibody to analyze BM specimen. For better understanding of BM microenvironment, thrombincleaved osteopontin should be also analyzed in future studies. Whether malignant hematopoietic cells disrupt the normal hematopoietic microenvironment or the emergence of a malignant environment better suited to support malignant cells was not addressed in 8
ACCEPTED MANUSCRIPT this study. Similarly, changes in the BM microenvironment of AA patients could either contribute to the pathogenesis of AA, or be caused by impaired hematopoiesis. A better understanding of the interaction between each of the BM microenvironment components may provide potential therapeutic targets for myeloproliferative disease as well as BM failure.
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In conclusion, our findings suggest that changes in the components of the BM microenvironment including lymphocytes, macrophages, mast cells, and stromal cells, might be related to defective
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hematopoiesis that could lead to AA and/or myeloproliferative disease.
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Authorship statement
M. Park performed the study, analyzed the data, and wrote the manuscript; C.J. Park and Y.H. Lee designed the study, performed the study, analyzed the research data, and reviewed the manuscript; Y.W. Cho and S. Jang contributed to the analysis of data; and K. Hwang, J.H. Lee, J.H. Lee and K.H.
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Conflicts of interest: none
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Lee contributed to data collection.
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progenitors. Blood. 1995;86:3353-3363. Chute JP, Muramoto GG, Dressman HK, et al. Molecular profile and partial functional analysis of novel endothelial cell-derived growth factors that regulate hematopoiesis. Stem Cells. 2006;24:1315-1327.
33.
Fernandez L, Rodriguez S, Huang H, et al. Tumor necrosis factor-alpha and endothelial cells modulate Notch signaling in the bone marrow microenvironment during inflammation. Exp Hematol. 2008;36:545-558.
34.
Grassinger J, Haylock DN, Storan MJ, et al. Thrombin-cleaved osteopontin regulates 12
ACCEPTED MANUSCRIPT hemopoietic stem and progenitor cell functions through interactions with alpha9beta1 and
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alpha4beta1 integrins. Blood. 2009;114:49-59
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ACCEPTED MANUSCRIPT Figure 1. Comparative analyses of (A) CD34+ cells and (B) CD117+ cells in aplastic anemia, chronic myeloid leukemia, and normal control; *p<0.05.
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(A) CD34+ cells
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(B) CD117+ cells
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ACCEPTED MANUSCRIPT Figure 2. Comparative analyses of (A) CD3+ T cells, (B) CD20+ B cells, (C) CD56+ natural killer cells, (D) macrophages (CD169) and (E) mast cells in aplastic anemia, chronic myeloid leukemia, and normal control; *p<0.05.
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(A) CD3+ T cells
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(B) CD20+ B cells
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(C) CD56+ natural killer cells
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(D) Macrophage
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(E) Mast cells
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ACCEPTED MANUSCRIPT Figure 3. Comparative analyses of cells positive for (A) nestin, (B) SDF-1, (C) osteopontin, and (D) osteonectin in aplastic anemia, chronic myeloid leukemia, and normal control; *p<0.05.
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(A) Nestin
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(B) SDF-1
(C) Osteopontin
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(D) Osteonectin
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ACCEPTED MANUSCRIPT Figure 4. Correlations between the number of (A) macrophages and SDF-1+ or nestin+ cells, (B) mast cells and SDF-1+ or nestin+ cells, (C) osteopontin+ cells and CD34+ or CD117+ cells, (D) osteonectin+ cells and CD34+ or CD117+ cells, and (E) endothelial cells and CD117+ cells.
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(A) macrophages and SDF-1+ or nestin+ cells
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(B) mast cells and SDF-1+ or nestin+ cells
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(C) osteopontin+ cells and CD34+ or CD117+ cells
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(D) osteonectin+ cells and CD34+ or CD117+ cells
(E) endothelial cells and CD117+ cells
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Table 1 Primary antibodies, clones, reaction temperatures, dilution factors, and incubation time used for immunohistochemistry Clone
Species
Clonality
Manufacturer
Reaction temperature
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Primary antibodies
QBEND/10
Mouse
Monoclonal
Leica Biosystems Newcastle, Ltd (Newcastle, UK)
CD117
YR145
Rabbit
Monoclonal
CD3
UCHT1
Rabbit
CD4
SP35
CD8
Incubation time (min)
Room temperature
1:50
60
Cell Marque, Inc (Rocklin, CA, USA)
42 ˚C
1:100
32
Polyclonal
Dako (Glostrup, Denmark)
42 ˚C
1:100
32
Rabbit
Monoclonal
Cell Marque, Inc
42 ˚C
1:100
32
C8/144B
Mouse
Monoclonal
Dako
42 ˚C
1:100
32
CD20
L26
Mouse
Monoclonal
Leica Biosystems Newcastle, Ltd
42 ˚C
1:250
20
CD56
123C3
Mouse
Monoclonal
Invitrogen Corporation (Camarillo, CA, USA)
42 ˚C
1:100
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CD169
HSn 7D2
Mouse
Monoclonal
Abcam Inc. (Cambridge, MA, USA)
Room temperature
1:25
60
Osteopontin
OP3N
Mouse
Monoclonal
Leica Biosystems Newcastle, Ltd
42 ˚C
1:50
40
Osteocalcin
OC4-30
Mouse
Monoclonal
Abcam Inc
37 ˚C
1:200
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Osteonectin
15G12
Mouse
Monoclonal
Leica Biosystems Newcastle, Ltd
Room temperature
1:100
60
SDF-1
MM0211-9N26
Mouse
Monoclonal
Abcam Inc
Room temperature
1:40
60
Nestin
196908
Mouse
Monoclonal
R&D System (Boston, MA, USA)
42 ˚C
1:4000
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CD34
Dilution
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ACCEPTED MANUSCRIPT Table 2 Analysis of semiquantitative immunohistochemical staining of bone marrow cells from patients with aplastic anemia (AA), chronic myeloid leukemia (CML), and control subjects AA
CML
Control
CD34
1.36 ± 1.22
14.31 ± 15.44
CD117
2.84 ± 2.06
34.00 ± 21.83
Hematopoietic stem cells
9.49 ± 3.02
0.013
11.17 ± 4.25
<0.01
SC
Lymphocytes, macrophages, and mast cells
P value
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Antigen
18.67 ± 12.88
26.28 ± 10.44
70.83 ± 30.70
<0.01
CD4
1.56 ± 1.25
0.74 ± 1.14
9.87 ± 3.65
<0.01
CD8
12.41 ± 9.79
16.96 ± 6.27
41.17 ± 14.11
<0.01
CD20
1.01 ± 1.07
0.00 ± 0.00
7.37 ± 4.98
<0.01
CD56
0.11 ± 0.12
0.00 ± 0.00
0.00 ± 0.00
<0.01
CD169
1.53 ± 0.96
0.00 ± 0.00
1.05 ± 1.07
<0.01
Mast cells
2.34 ± 1.85
0.00 ± 0.00
0.49 ± 0.41
<0.01
5.79 ± 2.08
0.10 ± 0.32
4.45 ± 1.11
<0.01
113.40 ± 88.54
261.70 ± 323.93
145.20 ± 149.28
0.275
0.40 ± 0.17
2.13 ± 0.73
2.60 ± 1.10
<0.01
2.96 ± 2.76
0.43 ± 0.49
1.94 ± 0.47
<0.01
8.04 ± 8.41
0.00 ± 0.00
3.62 ± 1.21
<0.01
39.20 ± 21.46
186.90 ± 140.95
18.00 ± 7.64
<0.01
Osteocalcin Osteonectin SDF-1
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Osteopontin
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Stromal cell components
Endothelial cells
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AA = aplastic anemia; CML = chronic myeloid leukemia; SDF-1 = stromal cell-derived factor-1 Results are represented as mean ± standard deviation of positive cell number/high power field (×400). Osteocalcin is expressed as mean ± standard deviation of positive peritrabecular line length (µm). Analysis of variance (ANOVA) was used to test for differences between the three types of bone marrow specimens.
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ACCEPTED MANUSCRIPT Decreased T- and B- lymphopoiesis was demonstrated in both AA and CML.
Natural killer cells were observed in AA, but were absent in CML and control.
There were significant differences between of AA and CML stromal cell components.
Alteration of the osteoblasts or sinusoidal endothelium could modify HSC levels.
Changes in the BM microenvironment could lead to AA and/or CML.
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