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Successful establishment of long-term bone marrow cultures in 103 patients with myelodysplastic syndromes
Leukemia Research 25 (2001) 941– 954 www.elsevier.com/locate/leukres
Successful establishment of long-term bone marrow cultures in 103 patients with myelodysplastic syndromes Sairah Alvi, Ahmed Shaher, Vilasini Shetty, Benita Henderson, Bruce Dangerfield, Francesca Zorat, Leena Joshi, Shalini Anthwal, Laurie Lisak, Leslie Little, Sefer Gezer, Suneel Mundle, Poluru L. Reddy, Krishnan Allampallam, Xiaoke Huang, Naomi Galili, Raphael Z. Borok, Azra Raza * MDS Center, Section of Myeloid Diseases, Rush Cancer Institute, Rush Uni6ersity, Suite 108, 2242 West Harrison Street, Chicago, IL 60612 -3515, USA Received 5 January 2001; accepted 10 March 2001
Abstract We used bone marrow biopsies instead of mononuclear cells to maintain long-term cultures from 103 patients belonging to all five sub-categories of myelodysplastic syndromes (MDS), as well as 12 normal controls. By week 4, 30 – 50% confluency was reached and could be maintained for up to 12 weeks with 100% confluency. The four prominent cells were fibroblasts, macrophages, endothelial cells and adipocytes. Immunohistochemical and electron microscopic studies provided lineage confirmation. Normal hematopoiesis was well supported by MDS stroma. Neither the FAB nor cytogenetics was co-related with the potency of growth. MDS stroma appears to be both morphologically and functionally normal. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Long-term bone marrow biopsy cultures; Stromal cells; Myelodysplastic syndromes; Apoptosis; Transmission electron microscopy; Functional studies of MDS stroma
1. Introduction The myelodysplastic syndromes (MDS) represent a group of heterogeneous, hematological clonal stem cell disorders characterized by the clinical paradox of variable cytopenias, despite generally hypercellular bone marrow [1]. The diagnostic feature of MDS is dysplasia of erythroid, granulocyte and megakaryocyte lineages [2,3]. There is increased proliferation of the marrow accompanied by an equally increased rate of apoptosis, which may explain some of the cytopenias [4 – 8]. CyAbbre6iations: BMMNC, bone marrow mononuclear cells; BM Bx, bone marrow biopsies; CMMoL, chronic myelomonocytic leukemia; ISEL, in situ end labeling; LTBMC, long-term bone marrow cultures; MDS, myelodysplastic syndromes; RA, refractory anemia; RAEB, RA with excess of blast; RAEB-t, RAEB in transformation; RARS, RA with ringed sideroblast; TEM, transmission electron microscopy. * Corresponding author. Tel.: +1-312-4558474; fax: + 1-3124558479. E-mail address: [email protected] (A. Raza).
tokines derived from marrow mononuclear cells or undefined factors produced by marrow stromal cells, may be extrinsic proteins predisposing to a premature apoptosis of hematopoietic cells [9–11]. Approximately 30% of the patients evolve into acute myeloid leukemia (AML), while 50% of the patients die before transformation due to complications of progressive cytopenias [12]. Abnormalities of chromosome 5 and 7 are fairly common, followed by chromosomal abnormalities affecting chromosomes 8, 20 and 17 [13,14]. While treatment options range from periodic transfusions to stem cell transplantation, for the majority of these patients, the most common therapy continues to be supportive care [1]. Clearly therefore, better therapies are urgently needed and can only be developed by defining the biology of the disease. One pitfall in attempting detailed studies of the marrow cells is related to the fact that large numbers of cells are undergoing apoptosis in MDS marrows and are not available for precise characterization [7]. Attempts to culture bone marrow
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mononuclear cells (BMMNC) from bone marrow (BM) aspirate in vitro have met with variable success, as the progenitor cells obtained from the marrows of the vast majority of patients undergo rapid apoptosis when plated in vitro [15,16]. Since the BMMNC are notoriously difficult to maintain in culture due to excessive in vivo apoptosis, the investigation of MDS progenitor cells in vitro has not yielded consistent results and virtually nothing can be said about the detailed properties of the BM stromal cells in MDS patients [17–22]. In the past, long-term bone marrow cultures (LTBMC), first described by Dexter et al. [23– 25], have been extensively employed as an in vitro model to understand hematopoiesis [26– 29]. In this paper, we report on a novel technique of using core bone marrow biopsies from 103 MDS patients and 12 normal donors in LTBMC as the primary source for stromal cells instead of BMMNC. By culturing the whole bone marrow piece, we believe the close relation between the stroma and parenchyma can be largely preserved in vitro. The bone marrow piece latches onto the dish in the first week of culture, followed by radiating stromal cells. Most of the stem cells and maturing myeloid cells are released from the adherent layer into the growth medium. The main feature of long-term bone marrow culture is the initial establishment of an adherent cellular environment containing four major stromal cell types; epithelial fibroblast like cells, endothelial cells, phagocytic cells and large adipocytes [30]. The MDS patient cultures were maintained in vitro for 3– 6 months and their growth characteristics were carefully studied and compared to hematologically normal subjects. Simultaneously, the phenotype and ultrastructural morphology of stromal cells was also determined. French–American–British (FAB) classification and karyotypic analyses was performed on the diagnostic bone marrow (BM) aspirates of all the patients [2]. In MDS, the complex stromal/hematopoietic cell interaction appears to regulate proliferation, differentiation, cytokine production and apoptosis [10,31– 33]. In future, the MDS LTBMC studies described here may present an ideal in vitro model to study hematopoiesis in various myeloproliferative disorders.
2.1. Bone marrow cultures The bone marrow core biopsy (BM Bx) samples were obtained with informed consent from 103 MDS patients and long-term bone marrow cultures were established. In addition, 12 normal bone marrow biopsies were obtained from informed and consenting hematologically normal individuals. Prior to culture, under sterile conditions, the biopsy piece was washed twice with Hank’s saline, dissected longitudinally and transversely followed by gentle teasing into minute fragments which were distributed in six 35× 10 mm petri dishes. To all petri dishes, 1 ml culture medium, RPMI 1640 (GIBCO-BRL Life Technologies) with 12.5% horse serum, 12.5% fetal bovine serum (GIBCO-BRL), 2.0 mM L-glutamine, 10 − 4 M b-mercaptoethanol, 10 − 6 M hydrocortisone, 200 U/ml penicillin and streptomycin were added. Neither growth factors nor cytokines were added to the culture medium. Cultures were maintained at 37°C in a humidified 5% CO2-in-air environment with replacement of half of the growth medium including non-adherent cells with an equal volume of fresh medium weekly. When cultures were 80–100% confluent, between 8 and 22 weeks, they were treated with 0.25% trypsin (GIBCO) and fixed.
2.2. Parameters used for an objecti6e e6aluation of in 6itro growth patterns The following parameters were used to measure the growth pattern of all the samples over 8–22 weeks in long-term bone marrow cultures. First, the time required for the bone marrow biopsy piece to latch onto the culture plate was recorded. Next, the appearance of radiating stromal cells followed by preadipocytes and adipocytes was observed and lastly, the confluency of the culture plates was evaluated. The growth was termed ‘good growth’ when biopsy piece latched by the second week, the adipocytes appeared by week 4 and the plates appeared 80–90% confluent by 8 weeks. Similarly, the growth was considered ‘fair growth’ if culture was only 40–50% confluent by week 8 and termed ‘poor growth’ when the culture was 10–30% confluent by the eighth week.
2.3. Transmission electron microscopy (TEM) 2. Materials and methods All patients reported here had a confirmed diagnosis of myelodysplastic syndrome and gave their written informed consent prior to a bone marrow examination, as did the control normal volunteers. All marrows were evaluated by hematopathologists at Rush University for diagnostic confirmation. All samples were processed for in vitro studies in Dr. Raza’s laboratory.
To avoid decalcification and cell deformation during the preparation process, a modified technique which preserves the detailed morphology of the adherent stromal layer was used. In short, the adherent layer was fixed with 3% gluteraldehyde in phosphate buffer, post fixed with osmium tetroxide, dehydrated and embedded in situ in Luft’s Epon at 58°C for 48 h. The sections were processed for semithin and ultrathin sections. Semithin sections were stained with Toluidine blue and
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evaluated by light microscopy. Ultrathin sections were contrasted with uranyl acetate and lead citrate and analyzed by TEM (JEOL 200, Japan) [34].
2.4. Immunostaining Cytospin slides from adherent and non-adherent layers were fixed with cold 100% methanol for 10 min at − 40°C. Select set of MDS samples were processed for immunostaining with specific monoclonal mouse antihuman macrophage antibody EBM-11 against the expression of the CD68 antigen to detect monocyte/ macrophage cells in adherent and non-adherent layers of the culture plates. Similarly, specific monoclonal mouse anti-human fibroblast antibody was used against b-subunit of prolyl-4-hydroxylase present in the fibroblast collagen. In addition, immunostaining with monoclonal mouse anti-human von Willebrand factor (factor VIII) antibody was used to detect endothelial cells/ megakaryocytes. The slides were labeled for each of the antibodies as follows: the slides were dehydrated in distilled water for 10 min, incubated with 3% H2O2 for 30 min and then with pronase 1 mg/ml (Calbiochem, La Jolla, CA) for 45 min. They were carefully rinsed with 0.15 M phosphate buffered saline (PBS) (0.15 M sodium chloride in 0.1 M phosphate buffer, pH 7.5) after each incubation. Following the last 0.15 M PBS rinse, they were placed in 0.5 M PBS (0.5 M sodium chloride in 0.1 M phosphate buffer, pH 7.5) for 15 min. The slides were treated with 0.5 M PBS containing 1.5% horse serum for 60 min to block non-specificity. Subsequently, the cells were incubated with the respective monoclonal antibodies: monoclonal mouse anti-human fibroblast 5B5 (Dako), at a dilution of (1:100); or monoclonal mouse anti-human macrophage CD68, EBM-11 (Dako) at a dilution of (1:80) or monoclonal mouse anti-human von Willebrand factor F8/86 (Dako), at a dilution of (1:40) in 0.5 M PBS containing 1.5% horse serum for 60 min. This was followed by incubating the slides with biotinylated anti-mouse IgG (diluted (1:200) in 0.5 M PBS with 1.5% horse serum) for 30 min and with the avidin-biotin complex or (ABC) reagent. The horse serum, biotinylated anti-IgG and ABC complex were reagents in the Vectastain Elite ABC kit (Vector, Burlingham, CA). After each of the
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above incubations, specimens were rinsed in 0.5 M PBS. The color reaction was developed using 0.025%, 3,3%-diamino benzidine tetrachloride (DAB) diluted in 100 ml of 0.5 M Tris buffer and pH 7.5, with 0.01 ml 30% H2O2 for 10 min and rinsed with distilled water. Lastly, the slides were counter-stained and viewed under the light microscope [9,11].
2.5. Statistics Statistical analysis was performed by using Kruskall–Wallis, Mann–Whitney U, 2-test and Fisher’s exact test.
3. Results Twelve marrow specimens from healthy, normal individuals and 103 specimens from MDS patients are the subject of this report. According to the FAB classification, there were 48 patients with refractory anemia (RA), 17 had RA with ringed sideroblasts (RARS), 25 had RA with excess blasts (RAEB), ten had RAEB in transformation (RAEB-t) and three had chronic myelomonocytic leukemia (CMMoL). Age ranged from 26 to 88 years (median: 68 years). Cytogenetic results were available in all individuals, with 47 patients carrying normal karyotype, while 56 had simple to complex karyotypic abnormalities. These data are shown in Table 1.
3.1. Establishing adherent layer Primary bone marrow biopsy sample were teased and simultaneously cultured in complete medium without any cytokines or growth factors on six plates per patient at 37°C in a humidified 5% CO2 chamber for 8–22 weeks. All the plates were examined weekly under phase contrast microscope before half of the supernatant medium was replaced with fresh medium. The non-adherent cells in the harvested medium were counted for viability and logged routinely. The biopsy pieces latched onto the culture plate in the first week, followed by the generation of stromal layer with the stem cells and maturing myeloid cells being released
Table 1 MDS patients in long-term bone marrow cultures FAB classification
Total number of patients cultured (%)
Patients with normal cytogenetic (%)
Patients with abnormal cytogenetic (%)
RA RARS RAEB RAEB-t CMMoL
48/103 17/103 25/103 10/103 3/103
19/48 (40) 11/17 (65) 11/25 (44) 5/10 (50) 1/3 (33)
29/48 (60.5) 6/17 (35) 14/25 (56) 5/10 (50) 2/3 (66)
(47) (16.5) (24) (10) (3)
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Fig. 1. (a) MDS patient bone marrow biopsy piece observed after 3 weeks in LTBMC with streaming stromal and hematopoietic cells (original magnification × 4). (b) A 2 –3 week old culture plate with 30% confluent adherent layer before the generation of adipocytes (original magnification ×4). (c) Bone marrow biopsy of RA patient after 4 weeks in LTBMC with 50% confluent bipolar or tripolar monolayers and adipocytes (original magnification ×10). (d) In LTBMC of MDS patient clearly visible hematopoietic cells, adipocytes, fibroblasts and macrophages released from the bone marrow compartment into the growth medium (original magnification × 40).
into the growth medium. The most interesting finding in this context was that of layer upon layer of cells streaming out of bony pieces in various areas of the plates (Fig. 1). By week 4, the plates were 30–50% confluent with bipolar, tripolar or multipolar monolayers and adipocytes, followed by 80– 100% confluency in the 8 –10th week with compact parallel multiple layers of fibroblasts, macrophages and adipocytes. In addition, preadipocytes and some adipocytes were routinely observed in close proximity of the bone pieces, over the monolayer. Cells in the area close to the bone achieved contact inhibition of growth earlier than those at the expanding peripheral margins and morphologically ap-
peared epithelial-like under the phase contrast microscope. On the other hand, giant adipocyte colonies were scattered across the plate. The plates were sacrificed between 8 and 22 weeks, with at least one plate sub-cultured under the same conditions.
3.2. Ultrastructural characteristics The electron microscopic studies were carried out on the bone marrow biopsy cultures that were sacrificed between 8 and 22 weeks. The adherent layer was in situ fixed in 3% gluteraldehyde, post fixed in osmium tetroxide, dehydrated, embedded and sectioned for TEM.
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Under TEM, the different types of stromal elements were identified and characterized, namely the fibroblasts, endothelial cells, monocytes/macrophages and the adipocytes [25,34]. Overall, the stromal compartment appeared very healthy and certainly without any significant apoptosis. The following describes the prominent and easily identifiable cell types.
3.3. Adipocytes The week 4 cultures, when observed under the light microscope, showed flat fibroblast like cells with refractile lipid vacuoles accumulating in the perinuclear region. This is the characteristic feature of the preadipocyte stage followed by adipocyte development. The fat vesicles would eventually enlarge in the proximity of the nuclei, round up as a giant fatty vacuole and gradually progress into the cytoplasm. Not all
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preadipocytes developed into large adipocytes, with some remaining at the early stage for the entire duration of the culture. Fig. 2 shows a typical plate containing large quantities of adipocytes.
3.4. Fibroblasts Vigorous growth of fibroblasts was noted in essentially all samples cultured with the new technique of using partially disaggregated bone biopsies. Fig. 3 shows a 3-week plate from an MDS patient with robust fibroblast growth. The fibroblast-like cells under the TEM often appeared as homogeneous elongated, structures with cytoplasmic region containing dilated endoplasmic reticulum and numerous microfilaments. Vertical sections through the stromal layer showed profiles of endoplasmic reticulum carrying numerous primary and secondary lysosomes (Fig. 4). The mitochondria were always present along with the well-devel-
Fig. 2. (a) RAEB patient in LTBMC after 5 weeks exhibiting 80% confluent adherent layer with preadipocytes and adipocytes (original magnification × 20). (b) Typical 5-week culture plate with large number of fleshy corpulent adipocytes (original magnification × 40).
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Fig. 3. (a,b) Confluent plate with compact parallel multiple layers of fibroblasts in LTBMC after 8 – 10 weeks (original magnification ×10 and ×20, respectively). (c) Maturing hematopoietic cells of MDS patient after exiting the bone marrow biopsy piece were observed seeding upon the streaming layer of fibroblasts and macrophages in LTBMC (original magnification ×40).
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Fig. 4. In long-term multilayer bone marrow cultures, spindle shaped fibroblasts with euchromatin nucleus and one or more prominent nucleolus were observed (uranyl acetate and lead citrate, original magnification ×3300).
oped Golgi complex. The nuclei showed sparse heterochromatin and prominent nucleoli. No phagocytosed material was seen in the fibroblast.
3.5. Endothelial cells Unlike fibroblasts, very few endothelial cells were observed. The surface of the nucleus in these cells was undulated and, ergastroplasm and glycogen rosettes, as shown in Fig. 5 surrounded the Golgi bodies.
3.6. Macrophages In addition to fibroblasts, large numbers of macrophages were observed in the adherent layer of long-term bone marrow cultures. They possessed extremely active and complex cell surface projections lobed nuclei with heterochromatin and extensive cytoplasm that was very rich in all the organelles. A majority of these macrophages contained cellular debri or residual bodies, as well as primary and secondary lysosomes in their cytoplasm. Different stages of phagocytosis in the erythrocytes were predominantly seen in the cytoplasm, as seen in Fig. 6.
3.7. In6estigation of stromal cell function: co-culture with CD34+ or cord blood cells The confluent plates (8– 12th week cultures) of 12 MDS patients in LTBMC were co-cultured with
CD34 + cells separated from human cord blood samples, donated by healthy pregnant female volunteers following delivery. The MDS adherent layer appeared to support the growth of these progenitor cells in a normal, expected manner. The viability count of the non-adherent layer after 1 week of co-culture was on average 85% and dropped to only 80% by the second week. The ultra structure morphology of MDS co-culture was similar to the original culture except that macrophages were actively surrounded by CD34+ cells, though no CD34+ apoptotic cells were observed. These studies appeared to suggest that the MDS stromal cells have retained their ability to support the growth of normal hematopoietic progenitors.
3.8. Immunohistochemical characteristics After 8–22 weeks of culture, the plates were sacrificed, the adherent layer trypsinized and cytospin slides prepared for immunostaining. Specific monoclonal mouse anti-human macrophage antibody (EBM11, Dako) against the expression of the CD68 antigen was used to confirm and estimate the monocyte/ macrophage population in the culture (Fig. 7). In 15 RA patients with normal or abnormal cytogenetics, 25% of the stroma consisted of macrophages. Interestingly, in eight RARS patients, 44% of the stromal cells belonged to monocyte/macrophages lineage, that is almost twice the number when compared to the RA group. In the 16 RAEB patients, :18% of the stromal
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cell population was composed of macrophages, whereas 35% of the cells in the two RAEB-t patients were macrophages. While in three CMMoL patients 31% of the cells were positively stained for macrophages, that is again much higher when compared to the RAEB group. The median percentage of macrophages was ten for RA, eight for RAEB and 50 for RARS. These differences reached a statistical significance among the three MDS FAB categories (P =0.0431). Similarly, a specific monoclonal mouse anti-human fibroblast antibody against b-subunit of prolyl-4-hydroxylase present in the collagen was used to identify and confirm the fibroblast population in the cultured adherent layer with immunostaining (Fig. 8). The antibody shows very restrictive positive staining under the stringent laboratory conditions and does not cross react with lymphocytes, monocytes, dendritic cells and granulocytes. In the 14 RA patients, : 13% cells were positively stained with the antibody, while eight RARS had 31% and 17 RAEB had 13% positive cells. The three RAEB-t and two CMMoL had 10 and 8% positively stained cells, respectively. It appears that RARS had maximum number of positively stained fibroblast cells and both RA and RAEB had less than half the number of cells belonging to fibroblast lineage compared to RARS and the difference almost reached statistical significance using non parametric analysis (P= 0.06) [35]. In addition, immunostaining with monoclonal mouse anti-human von Willebrand factor (factor VIII) antibody was performed on adherent layer to detect en-
dothelial cells/megakaryocytes. Under the light microscope, only few cells were stained positively, hence it was concluded that the number of endothelial cells present in the culture plates might be very low as the culture aged.
3.9. Growth characteristics of MDS long-term bone marrow cultures Of the 103 MDS patients that were cultured in LTBMC, 97 patients (RA 45, RARS 17, RAEB 23, RAEB-t nine and CMMoL three) were statistically evaluable. The basic clinical characteristics are presented in Table 2. Growth characteristics of normal healthy volunteers as well as the various FAB categories of MDS patients are presented in Table 3(A–E). Among normal marrows, a third had good growth, a third had fair growth and a third had poor growth in vitro (Table 3A). On the other hand, among the 48 RA patients, 54% had good growth, 16% had fair growth and 29% had poor growth overall. RA patients with an abnormal karyotype appeared to fare better in vitro, with 80% showing a good or fair growth (Table 3B), but these differences were not statistically significant. Half of the RARS patients had good and the other half had poor growth in vitro, without any differences related to cytogenetics (Table 3C). Among the RAEB and/or RAEB-t patients, :40% patients had good growth, and cytogenetics did not affect the ability of the cells to grow well in vitro (Table 3D,E). Finally, all three CMMoL patients showed good growth in vitro.
Fig. 5. Endothelial cells with classical morphology were observed in culture plates (uranyl acetate and lead citrate, original magnification × 6600).
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Fig. 6. Macrophages were frequently observed engulfing partly degraded erythyroid cells in long-term bone marrow cultures. Note the cross-sectional view showing multiple layers in culture (uranyl acetate and lead citrate, original magnification ×6600).
Thus, it can be concluded that neither the pre-culture FAB type (P =0.761) nor the cytogenetic results (P= 0.633) affected the growth of stromal cells in these MDS patients.
3.10. Effect of adipocytes on growth of MDS stromal cells in 6itro Under the light microscope, the morphology of stromal layer from MDS patients could be broadly categorized into three cell types; fibroblasts, macrophages and adipocytes. In one half (47/97) of cultured MDS patients it was observed that stroma predominately consisted of fibroblast/macrophages with very few adipocytes scattered across the culture plates. In three of these 47 patients, there was no significant adherent layer, in 13 samples there was fair growth and in 29 patients, poor growth was observed. In the other 47 patients, 30–100% confluent stromal layer of fibro-
blasts/macrophages was accompanied by a very high number of adipocytes. The number of adipocytes therefore consistently appeared to predict a good culture growth (PB0.0001). The time to reach optimum confluency in the cultured samples showed that 54 patients had very slow stromal proliferation over the 4-week period, while 40 patients had rapid growth and reached confluency (80– 100%) in a significantly shorter time, i.e. B 4 weeks (PB 0.001).
4. Discussion In this paper, we report upon the use of bone marrow biopsy material to obtain long-term in vitro growth of stromal cells from 103 patients with myelodysplastic syndromes and 12 normal controls. The salient findings described in this paper can be summarized here:
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Bone marrow biopsy tissue was successfully used to generate robust stromal cells in the vast majority of MDS patients. The stromal cells appear to be morphologically normal when examined by light microscopy or by transmission electron microscopy. Presence of apoptotic stromal cells that are frequently seen in MDS marrow biopsy sections examined by in situ end labeling (ISEL) could not be confirmed in the stromal cells which were studied in vitro following long-term cultures. Two probable reasons for this among many possible explanations are that either the apoptotic stromal cells died right away following initiation of in vitro cultures or the apoptosis inducing cytokine milieu was not replicated adequately in the long-term culture environment. Neither the FAB type nor the cytogenetics were co-related with the potency of in vitro growth of stromal cells. Given that both cytogenetics and FAB typing is performed strictly on parenchymal cells, it is no surprise that the vigor of growth seen in stromal cells is quite independent of their parenchymal neighbors. The MDS stromal cells appear to be functionally normal by virtue of their ability to support the proliferation of normal cord blood cell derived CD34 + cells. Large numbers of stromal cells can be generated from MDS marrow biopsies for further detailed characterization and future studies.
Given that the treatment options for most patients with MDS are few because of their advanced age and our lack of understanding regarding the etiology of their variable cytopenias, there is an urgency to investigate the biology of these disorders. One major stumbling block in this area has been a failure to generate large numbers of hematopoietic parenchymal or stromal cells in vitro for detailed characterization. More recently, it has been shown that the failure to generate adequate colony growth from MDS patients in vitro may be related to an excessive intramedullary apoptotic death of these cells. As a result of these difficulties, very few studies exist which actually describe colony growth in MDS patients and as far as stromal cells are concerned, such studies are even fewer. In order to circumvent these problems, bone marrow biopsy tissue was used directly to generate stromal cell cultures. Studies described in this paper demonstrate that such a technique can be successfully applied for long-term growth of MDS marrows. This is an important finding in itself, since one can examine the functional characteristics of these cells. The stromal cells in MDS patients appear to be both morphologically and functionally normal. This is no surprise since MDS has never been regarded as a disease of bone marrow stroma. The surprise was to find no apoptotic stromal cells in culture, since we have frequently observed and reported ISEL positive fibroblasts, endothelial cells and macrophages in BM biopsy sections obtained from MDS patients belonging to all
Fig. 7. Specific monoclonal macrophage antibody (EBM-11) against the expression of CD68 antigen was used to identify the monocyte/ macrophage population in the LTBMC. Darkly stained positive cells constituting 16% of the total cells from RA patient were estimated under the light microscope (original magnification × 40).
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Fig. 8. Specific monoclonal fibroblast antibody against the collagen was used to identify the fibroblast population in the LTBMC. Darkly stained 68% positive cells from RA patient were estimated under the light microscope (original magnification ×40).
FAB categories. Obviously, the in vitro cultures only represent stromal cells, while in vivo BM biopsies contain an intimately interactive stroma and parenchyma and therefore, the two situations cannot be directly compared. It is possible that the cells, which are the major source of pro-apoptotic cytokines in vivo, such as monocytes are absent from the cultures [36]. Alternatively, it may be that the apoptotic stromal cells died early on in the culture environment while the long-term stromal elements were descended from healthy, nonapoptotic parent cells. Nonetheless, these findings underscore the very basic artificiality of in vitro systems that at best represent one class of cells, if not one lineage. So for example, while in vivo, we could see a number of apoptotic stromal cells in MDS marrow biopsies, the in vitro cultures did not show such cells due to the absence of neighboring cells mediating that apoptosis. An important conclusion of these observations is that inherently, the stromal cells may not be diseased in the majority of MDS patients, a finding which is further confirmed by their functional adequacy in supporting the proliferation of cord blood derived CD34+ cells. It must be remembered however that MDS is a heterogeneous group of disorders and thus, the same pathophysiology will not uniformly apply to all cases. Therefore, it is possible that the marrow stroma is equally or primarily diseased in some patients with MDS. Another possibility is that the stromal growth obtained in vitro selectively originates in the healthy stromal progenitor, while the MDS stromal progenitor cells are unable to proliferate and form colonies in
vitro. If this were the case then the present report at least shows that there are normal residual stromal cells in MDS patients that can give rise to rich colony formation in vitro. In the past, stromal cells have been generated from BM aspirate cells at least in one study and these showed a defective support of normal hematopoietic cells [37]. Even in their study, the inadequate support of hematopoietic growth was only seen in a fraction of their 11 total cases studied. Another study of stromal cell function in MDS patients also found two broad patterns of growth [38]. In one group, hematopoietic cell numbers steadily declined while they were well-maintained in the second. Given the small number of cases studied to date, it is impossible to draw any conclusions regarding the functional characteristics of stromal cells in a disease as complex and heterogeneous as MDS. What can be concluded on the basis of previous reports and our own studies, is that in each report, there are stromal cell cultures from MDS
Table 2 Clinical characteristics of MDS patients under study Clinical variables
Mean
Median
Hgb (n =92) WBC (n =91) ANC (n =20) Platelets (n =92) BM Bx cellularity (n = 88) BM Asp cellularity (n =75) BM Asp blasts (n = 75)
9.69 5.13 1.78 139.72 65.53 2.44 6.49
9.5 3.08 1.39 80.5 70 3 3
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Table 3 Growth characteristics in healthy 6olunteers Number of normal bone marrow Good growth (80–90%) stroma biopsies from healthy volunteers confluent by week 8 12 4/12 (33%) Growth characteristics in RA RA 48 patients Normal cytogenetic 19/48 Abnormal cytogenetic 29/48 Growth characteristics in RARS RARS 17 patients Normal cytogenetic 11/17 Abnormal cytogenetic 6/17 Growth characteristics in RAEB RAEB 25 patients Normal cytogenetic 11/25 Abnormal cytogenetic 14/25 Growth characteristics in RAEB-t RAEB-t 10 patients Normal cytogenetic 5/10 Abnormal cytogenetic 5/10
Fair growth (40–50%) stroma confluent by week 8 4/12 (33%)
Poor growth (10–30%) stroma confluent by week 8 4/12 (33%)
Good growth (80–90%) stroma confluent by week 8 26/48 (54%) 9/19 (47%) 17/29 (59%)
Fair growth (40–50%) stroma confluent by week 8 8/48 (17%) 2/19 (11%) 6/29 (21%)
Poor growth (10–30%) stroma confluent by week 8 14/48 (29%) 8/19 (42%) 6/29 (21%)
Good growth (80–90%) stroma confluent by week 8 9/17 (53%) 6/11 (55%) 3/6 (50%)
Poor growth (10–30%) stroma confluent by week 8 8/17 (47%) 5/11 (45%) 3/6 (50%)
Good growth (80–90%) stroma confluent by week 8 11/25 (44%) 4/11 (36%) 7/14 (50%)
Fair growth (40–50%) stroma confluent by week 8 5/25 (20%) N/A 5/14 (36%)
Poor growth (10–30%) stroma confluent by week 8 9/25 (36%) 7/11 (64%) 2/14 (14%)
Good growth (80–90%) stroma confluent by week 8 4/10 (40%) 2/5 (40%) 2/5 (40%)
Fair growth (40–50%) stroma confluent by week 8 4/10 (40%) 2/5 (40%) 2/5 (40%)
Poor growth (10–30%) stroma confluent by week 8 2/10 (20%) 1/5 (20%) 1/5 (20%)
patients which provide adequate support for hematopoietic cell growth. The four major stromal cells that were consistently observed in the cultures we report are bone marrow fibroblasts, adipocytes, rare endothelial cells and abundant macrophages. The fibroblast and adipocytes have a similar mesenchymal stem cell origin while the marrow macrophages are derived from hematopoietic system. Endothelial cells, on the other hand, constitute a distinct stromal cell lineage [30]. The fibroblast and macrophage-like cells proliferated to a confluent monolayer and tended to become contact-inhibited. Simultaneously, areas close to the bone marrow biopsy developed multi-layer growth pattern with adipocyte colonies abutting on the stromal layers. Two interesting observations related to these cultures are worth mentioning. First, the appearance of adipocytes was not associated with a rapid termination of culture growth. In fact, the presence of large numbers of fleshy, corpulent adipocytes generally foretold the maintenance of a thriving culture likely to last 10– 12 weeks without colony death. Second, when bone marrow biopsy pieces were removed from confluent plates after 10– 12 weeks of culture, washed and sub-cultured in fresh plates, ample stromal colonies were once again generated. This was especially true for the plates that contained an abundance of adipocytes. Furthermore, these sub-cul-
tures after 12 weeks of primary cultures did not show any sign of aging or degeneration. The sub-cultures were grown for another 4–5 weeks before they were sacrificed and then select samples were further sub-cultured successfully. The same biopsy pieces could be used for the initiation of fresh cultures serially for up to 8–9 months without compromising the quality of stromal cells generated in a number of MDS cases as well as normal controls. Another interesting feature of colony forming unit fibroblast (CFU-F) in MDS is the appearance of adipocyte colonies as early as 2 weeks in culture that has not been observed in normal donor marrow cultures. Castoldi et al. have reported that the BMMNC LTBMC of MDS patients with RA and RARS produce normal stromal layers but RAEB and RAEB-t patients produce incomplete stromal confluency with reduced fat cells [39]. On the other hand, we do not observe any significant difference in stromal confluency or number of adipocytes among the FAB sub types as 66% of low risk and 68% of high risk patients had between 50 and 100% confluent plates [40]. In addition, no progressive impairment in the stromal progenitor number or in the proliferative capacity beginning from the less aggressive MDS to RAEB-t was observed. Most likely, these discrepancies result from the different sources from which cells were derived for the initiation of stromal
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cell cultures. We used BM biopsy tissue, whereas Castoldi et al. used BM aspirate cells. In summary, therefore, we report upon the use of BM biopsies for generation of large numbers of stromal cells in vitro from MDS patients. Material thus obtained can be used for detailed molecular, genetic and functional characterization of these heretofore rather elusive cells.
[7]
[8]
Acknowledgements This work has been supported by a grant from the National Cancer Institute (PO1 CA 75606) and the Dr Roy Ringo Grant for basic research in MDS and Rush Presbyterian St Luke’s Medical Center Woman’s Board Time Center grant. The authors wish to thank their devoted laboratory colleagues for their constant support and dedication and Lakshmi Venugopal and Sandra Howery for excellent administrative/secretarial assistance. Authors contributions as follows: Conception and design: S Alvi, A Shaher, V Shetty, B Henderson, B Dangerfield, F Zorat, S Mundle, P Reddy, K Allampallam, X Huang, N Galili, R Borok, A Raza. Analysis and interpretation of data: S Alvi, A Shaher, V Shetty, B Henderson, R Borok, A Raza. Drafting the article: S Alvi, L Joshi, A Raza. Critical revision of the article for important intellectual content: S Alvi, S Anthwal, S Mundle, P Reddy, K Allampallam, N Galili, R Borok, A Raza. Final approval of article: S Alvi, S Anthwal, A Raza. Provision of study materials or patients: L Lisak, L Little, S Gezer, A Raza. Statistical expertise: R Borok. Obtaining funding: A Raza. Administrative, technical or logistic support: A Shaher, V Shetty, B Henderson, B Dangerfield, F Zorat, L Lisak, L Little, S Gezer, X Huang, A Raza. Collection or assembly of data: S Alvi, A Shaher, V Shetty, B Henderson, A Raza.
[9]
[10] [11]
[12] [13]
[14]
[15]
[16]
[17]
References [1] Resegotti L. The nature and natural history of myelodysplasia. Haematologica 1993;25:191. [2] Verhoef GEG, Pittaluga S, De Wolf-Peters C, Boogaerts MA. FAB classification of myelodysplastic syndromes: merits and controversies. Ann Hematol 1995;71:3. [3] Bennett JM, Catovsky D, Flandrin DMT, Galton GDAG, Gralnick HR, Sultan C. Proposal for the classification of the myelodysplastic syndromes. Br J Haematol 1982;51:189. [4] Clark DM, Lampert IA. Apoptosis is a common histopathological finding in myelodysplasia: the correlate of ineffective hematopoiesis. Leuk Res 1990;2:415. [5] Yoshida Y. Apoptosis may be the mechanism responsible for the premature intramedullary cell death in myelodysplastic syndromes. Leukemia 1993;7:144. [6] Raza A, Mundle S, Iftikhar A, Gregory S, Marcus B, Khan Z, Alvi S, Shetty V, Dameron S, Wright V, Adler S, Loew J, Scott
[18]
[19]
[20]
[21]
[22]
953
S, Ali SN, Preisler HD. Simultaneous assessment of cell kinetics and programmed cell death in bone marrow biopsies of myelodysplastics reveals extensive apoptosis as the probable basis for ineffective hematopoiesis. Am J Hematol 1995;48:143. Raza A, Gezer S, Mundle S, Gao XZ, Alvi S, Borok R, Rifkin S, Iftikhar A, Shetty V, Parcharidou A, Loew J, Marcus B, Khan Z, Chaney C, Showel J, Gregory S, Preisler HD. Apoptosis in bone marrow biopsy samples involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes. Blood 1996;86:268. Parker JE, Mufti GJ. Ineffective hematopoiesis and apoptosis in myelodysplastic syndromes. Br J Hematol 1998;101:220. Mundle S, Ali A, Cartlidge J, Reza S, Alvi S, Showel M, Mativi BY, Shetty V, Venugopal P, Gregory S, Raza A. Evidence for involvement of tumor necrosis factor a in apoptotic death of bone marrow cells in myelodysplastic syndromes. Am J Hematol 1999;60:36. Greenburg PL. Biologic nature of the myelodysplastic syndromes. Acta Haematol 1987;78:94. Shetty V, Mundle S, Alvi S, Showel M, Broady-Robinson L, Dar S, Borok R, Showel J, Greagory S, Rifkin S, Gezer S, Parcharidou A, Venugopal P, Shah R, Hernandez B, Klein M, Alston D, Robin E, Dominguez C, Raza A. Measurement of apoptosis, proliferation and three cytokine in 46 patients with myelodysplastic syndromes. Leuk Res 1996;20:891. Yanuck MD, Saleem A. Leukemic transformation in myelodysplastic syndrome: a review. Ann Clin Lab Sci 1991;3:171. Pedersen-Bjergaard J, Philip P, Larsen SO, Anderson M, Daugaard G, Ersboll J, Hansen SW, Hou-Jensen K, Nielsen D, Sigsgaard TC, Specht L, Osterlind K. Therapy related myelodysplastic syndromes and acute myeloid leukemia. Cytogenetic characteristics of 115 consecutive cases and risk in seven cohorts of patients treated intensively for malignant diseases in the Copenhagen series, Leukemia 1993;7:1975 – 86. Vallespi T, Imbert M, Mecucci C, Preudhomme C, Fenaux P. Diagnosis, classification, and cytogenetics of myelodysplastic syndromes. Haematologica 1998;83:248. Ohmori M, Ohmori S, Ueda Y, Yoshida Y, Okuma M. Ineffective hematopoiesis in myelodysplastic syndromes (MDS) as studied by daily in situ observation of colony-cluster formation. Int J Cell Clon 1991;5:521. Aizawa S, Nakano M, Iwase O, Yaguchi M, Hiramoto M, Hoshi H, Nabeshima R, Shima D, Handa H, Toyama K. Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD 34-positive cell proliferation and differentiation in vitro. Leuk Res 1999;23:239. Sawada K. Proliferation and differentiation of myelodysplastic CD34+ cells. Leuk Lymph 1996;1 – 2:43. Tennant GB, Walsh V, Truran LN, Edwards P, Mills KI, Burnett AK. Abnormalities of adherent layers grown from bone marrow of patients with myelodysplasia. Br J Haematol 2000;3:853. Richert-Boe KE, Bagby CG Jr. In vitro hematopoiesis in myelodysplasia: liquid and soft-gel culture studies. Hematol Oncol Clin North Am 1992;6:543. Flores-Figueroa E, Gutierrez-Espindola G, Guerrero-Rivera S, Pizzuto-Chavez J, Mayani H. Hematopoietic progenitor cells from patients with myelodysplastic syndromes: in vitro colony growth and long term proliferation. Leuk Res 1999;23:385. Sato T, Kim S, Selleri C, Young NS, Maciejewski JP. Measurement of secondary colony formation after 5 weeks in long-term cultures in patients with myelodysplastic syndrome. Leukemia 1998;12:1187. Roberts IAG, McMullin MF. Apractical miniature long-term bone marrow culture system for investigating early myelodysplasia. Leuk Res 1992;16:737.
954
S. Al6i et al. / Leukemia Research 25 (2001) 941–954
[23] Dexter TM. Haemopoiesis in long term bone marrow cultures. Acta Haematol 1979;62:299. [24] Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of haempoietic stem cells in vitro. J Cell Physiol 1977;91:335. [25] Lanotte M, Allen TD, Dexter MT. Histochemical and ultrastructural characteristics of a cell line from human bone marrow stroma. J Cell Sci 1981;50:281. [26] Greenburg PL. In vitro marrow culture studies in the myelodysplastic syndromes. Semin Oncol 1992;1:34. [27] Blazsek I, Liu XH, Anjo A, Quittet P, Comisso M, Kim-Triana B, Misset JL. The hematon, a morphogenetic functional complex in mammalian bone marrow, involves erythroblastic islands and granulocytic cobblestones. Exp Hematol 1995;23:309. [28] Chang J, Dexter TM. Long-term cultures: in vitro purging of leukemic cells. Baillieres Clin Haematol 1991;3:775. [29] Chang J, Allen TD, Dexter TM. Long-term bone marrow cultures: their use in autologous marrow transplantation. Cancer Cells 1989;1:17. [30] Duhrsen U, Hossfeld DK. Stromal abnormalities in neoplastic bone marrow diseases. Ann Hematol 1996;73:53. [31] Sawada K. Growth characteristics of myelodysplastic CD34 + cells. Leuk Lymph 1998;12:49. [32] Gidali J, Feher I, Hollan SR. Blast colony forming cell-binding capacity of bone marrow stroma from myelodysplastic patients. Stem Cells 1996;14:577. [33] Ohmori M, Ueda Y, Masutani H, Hirama T, Anzai N, Yoshida Y, Okuma M. Myelodysplastic syndrome (MDS)-associated inhibitory activity on haematopoietic progenitor cells: Contribution of monocyte-derived lipid containing macrophages (MDLM). Br J Haematol 1992;81:67.
[34] Payne CM, Greenberg B, Cromey D, Woo L. Morphological evidence of an altered bone marrow microenvironment in patients with acute nonlymphoblastic leukemia and myelodysplastic disorders. Exp Hematol 1987;15:143. [35] Zhang W, Knieling G, Vohwinkel G, Martinez T, Kuse R, Hossfeld DK, Duhrsen U. Origin of stroma cells in long term bone marrow cultures from patients with acute myeloid leukemia. Ann Hematol 1999;78:305. [36] Deeg HJ, Beckham C, Loken MR, Bryant E, Lesnikova M, Shulman HM, Gooley T. Negative regulators of hematopoiesis and stroma function in patients with myelodysplastic syndromes. Leuk Lymph 2000;37:405. [37] Aizawa S, Hiramoto M, Hoshi H, Toyama K, Shima D, Handa H. Establishment of stromal cell line from an MDS RA patient which induced an apoptotic change in hematopoietic and leukemic cells in vitro. Exp Hematol 2000;28:148. [38] Coutinho LH, Geary CG, Chang J, Harrison C, Testa NG. Functional studies of bone marrow haemopoietic and stromal cells in the myelodysplastic syndromes (MDS). Br J Haematol 1990;75:16. [39] Diana C, Dominici M, Lanza F, Punturieri M, Pauli S, Tieghi A, Dabusti M, Scapoli G, Castoldi G. Impairment of stromal cells compartment in hypoplastic myelodysplastic syndromes. Blood 2000;96:357a Abstract no. 1541. [40] Alvi S, Shaher A, Henderson B, Dar S, Zorat F, Broderick E, Lisak L, Du Randt M, Reddy P, Mundle S, Galili N, Borok RZ, Raza A. Improved growth of stromal cells in long term bone marrow cultures (LTBMC) of myelodysplastic syndrome (MDS) patients treated with thalidomide. Blood 2000;96:359a Abstract no. 1547.
Successful establishment of long-term bone marrow cultures in 103 patients with myelodysplastic syndromes Sairah Alvi, Ahmed Shaher, Vilasini Shetty, Benita Henderson, Bruce Dangerfield, Francesca Zorat, Leena Joshi, Shalini Anthwal, Laurie Lisak, Leslie Little, Sefer Gezer, Suneel Mundle, Poluru L. Reddy, Krishnan Allampallam, Xiaoke Huang, Naomi Galili, Raphael Z. Borok, Azra Raza * MDS Center, Section of Myeloid Diseases, Rush Cancer Institute, Rush Uni6ersity, Suite 108, 2242 West Harrison Street, Chicago, IL 60612 -3515, USA Received 5 January 2001; accepted 10 March 2001
Abstract We used bone marrow biopsies instead of mononuclear cells to maintain long-term cultures from 103 patients belonging to all five sub-categories of myelodysplastic syndromes (MDS), as well as 12 normal controls. By week 4, 30 – 50% confluency was reached and could be maintained for up to 12 weeks with 100% confluency. The four prominent cells were fibroblasts, macrophages, endothelial cells and adipocytes. Immunohistochemical and electron microscopic studies provided lineage confirmation. Normal hematopoiesis was well supported by MDS stroma. Neither the FAB nor cytogenetics was co-related with the potency of growth. MDS stroma appears to be both morphologically and functionally normal. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Long-term bone marrow biopsy cultures; Stromal cells; Myelodysplastic syndromes; Apoptosis; Transmission electron microscopy; Functional studies of MDS stroma
1. Introduction The myelodysplastic syndromes (MDS) represent a group of heterogeneous, hematological clonal stem cell disorders characterized by the clinical paradox of variable cytopenias, despite generally hypercellular bone marrow [1]. The diagnostic feature of MDS is dysplasia of erythroid, granulocyte and megakaryocyte lineages [2,3]. There is increased proliferation of the marrow accompanied by an equally increased rate of apoptosis, which may explain some of the cytopenias [4 – 8]. CyAbbre6iations: BMMNC, bone marrow mononuclear cells; BM Bx, bone marrow biopsies; CMMoL, chronic myelomonocytic leukemia; ISEL, in situ end labeling; LTBMC, long-term bone marrow cultures; MDS, myelodysplastic syndromes; RA, refractory anemia; RAEB, RA with excess of blast; RAEB-t, RAEB in transformation; RARS, RA with ringed sideroblast; TEM, transmission electron microscopy. * Corresponding author. Tel.: +1-312-4558474; fax: + 1-3124558479. E-mail address: [email protected] (A. Raza).
tokines derived from marrow mononuclear cells or undefined factors produced by marrow stromal cells, may be extrinsic proteins predisposing to a premature apoptosis of hematopoietic cells [9–11]. Approximately 30% of the patients evolve into acute myeloid leukemia (AML), while 50% of the patients die before transformation due to complications of progressive cytopenias [12]. Abnormalities of chromosome 5 and 7 are fairly common, followed by chromosomal abnormalities affecting chromosomes 8, 20 and 17 [13,14]. While treatment options range from periodic transfusions to stem cell transplantation, for the majority of these patients, the most common therapy continues to be supportive care [1]. Clearly therefore, better therapies are urgently needed and can only be developed by defining the biology of the disease. One pitfall in attempting detailed studies of the marrow cells is related to the fact that large numbers of cells are undergoing apoptosis in MDS marrows and are not available for precise characterization [7]. Attempts to culture bone marrow
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mononuclear cells (BMMNC) from bone marrow (BM) aspirate in vitro have met with variable success, as the progenitor cells obtained from the marrows of the vast majority of patients undergo rapid apoptosis when plated in vitro [15,16]. Since the BMMNC are notoriously difficult to maintain in culture due to excessive in vivo apoptosis, the investigation of MDS progenitor cells in vitro has not yielded consistent results and virtually nothing can be said about the detailed properties of the BM stromal cells in MDS patients [17–22]. In the past, long-term bone marrow cultures (LTBMC), first described by Dexter et al. [23– 25], have been extensively employed as an in vitro model to understand hematopoiesis [26– 29]. In this paper, we report on a novel technique of using core bone marrow biopsies from 103 MDS patients and 12 normal donors in LTBMC as the primary source for stromal cells instead of BMMNC. By culturing the whole bone marrow piece, we believe the close relation between the stroma and parenchyma can be largely preserved in vitro. The bone marrow piece latches onto the dish in the first week of culture, followed by radiating stromal cells. Most of the stem cells and maturing myeloid cells are released from the adherent layer into the growth medium. The main feature of long-term bone marrow culture is the initial establishment of an adherent cellular environment containing four major stromal cell types; epithelial fibroblast like cells, endothelial cells, phagocytic cells and large adipocytes [30]. The MDS patient cultures were maintained in vitro for 3– 6 months and their growth characteristics were carefully studied and compared to hematologically normal subjects. Simultaneously, the phenotype and ultrastructural morphology of stromal cells was also determined. French–American–British (FAB) classification and karyotypic analyses was performed on the diagnostic bone marrow (BM) aspirates of all the patients [2]. In MDS, the complex stromal/hematopoietic cell interaction appears to regulate proliferation, differentiation, cytokine production and apoptosis [10,31– 33]. In future, the MDS LTBMC studies described here may present an ideal in vitro model to study hematopoiesis in various myeloproliferative disorders.
2.1. Bone marrow cultures The bone marrow core biopsy (BM Bx) samples were obtained with informed consent from 103 MDS patients and long-term bone marrow cultures were established. In addition, 12 normal bone marrow biopsies were obtained from informed and consenting hematologically normal individuals. Prior to culture, under sterile conditions, the biopsy piece was washed twice with Hank’s saline, dissected longitudinally and transversely followed by gentle teasing into minute fragments which were distributed in six 35× 10 mm petri dishes. To all petri dishes, 1 ml culture medium, RPMI 1640 (GIBCO-BRL Life Technologies) with 12.5% horse serum, 12.5% fetal bovine serum (GIBCO-BRL), 2.0 mM L-glutamine, 10 − 4 M b-mercaptoethanol, 10 − 6 M hydrocortisone, 200 U/ml penicillin and streptomycin were added. Neither growth factors nor cytokines were added to the culture medium. Cultures were maintained at 37°C in a humidified 5% CO2-in-air environment with replacement of half of the growth medium including non-adherent cells with an equal volume of fresh medium weekly. When cultures were 80–100% confluent, between 8 and 22 weeks, they were treated with 0.25% trypsin (GIBCO) and fixed.
2.2. Parameters used for an objecti6e e6aluation of in 6itro growth patterns The following parameters were used to measure the growth pattern of all the samples over 8–22 weeks in long-term bone marrow cultures. First, the time required for the bone marrow biopsy piece to latch onto the culture plate was recorded. Next, the appearance of radiating stromal cells followed by preadipocytes and adipocytes was observed and lastly, the confluency of the culture plates was evaluated. The growth was termed ‘good growth’ when biopsy piece latched by the second week, the adipocytes appeared by week 4 and the plates appeared 80–90% confluent by 8 weeks. Similarly, the growth was considered ‘fair growth’ if culture was only 40–50% confluent by week 8 and termed ‘poor growth’ when the culture was 10–30% confluent by the eighth week.
2.3. Transmission electron microscopy (TEM) 2. Materials and methods All patients reported here had a confirmed diagnosis of myelodysplastic syndrome and gave their written informed consent prior to a bone marrow examination, as did the control normal volunteers. All marrows were evaluated by hematopathologists at Rush University for diagnostic confirmation. All samples were processed for in vitro studies in Dr. Raza’s laboratory.
To avoid decalcification and cell deformation during the preparation process, a modified technique which preserves the detailed morphology of the adherent stromal layer was used. In short, the adherent layer was fixed with 3% gluteraldehyde in phosphate buffer, post fixed with osmium tetroxide, dehydrated and embedded in situ in Luft’s Epon at 58°C for 48 h. The sections were processed for semithin and ultrathin sections. Semithin sections were stained with Toluidine blue and
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evaluated by light microscopy. Ultrathin sections were contrasted with uranyl acetate and lead citrate and analyzed by TEM (JEOL 200, Japan) [34].
2.4. Immunostaining Cytospin slides from adherent and non-adherent layers were fixed with cold 100% methanol for 10 min at − 40°C. Select set of MDS samples were processed for immunostaining with specific monoclonal mouse antihuman macrophage antibody EBM-11 against the expression of the CD68 antigen to detect monocyte/ macrophage cells in adherent and non-adherent layers of the culture plates. Similarly, specific monoclonal mouse anti-human fibroblast antibody was used against b-subunit of prolyl-4-hydroxylase present in the fibroblast collagen. In addition, immunostaining with monoclonal mouse anti-human von Willebrand factor (factor VIII) antibody was used to detect endothelial cells/ megakaryocytes. The slides were labeled for each of the antibodies as follows: the slides were dehydrated in distilled water for 10 min, incubated with 3% H2O2 for 30 min and then with pronase 1 mg/ml (Calbiochem, La Jolla, CA) for 45 min. They were carefully rinsed with 0.15 M phosphate buffered saline (PBS) (0.15 M sodium chloride in 0.1 M phosphate buffer, pH 7.5) after each incubation. Following the last 0.15 M PBS rinse, they were placed in 0.5 M PBS (0.5 M sodium chloride in 0.1 M phosphate buffer, pH 7.5) for 15 min. The slides were treated with 0.5 M PBS containing 1.5% horse serum for 60 min to block non-specificity. Subsequently, the cells were incubated with the respective monoclonal antibodies: monoclonal mouse anti-human fibroblast 5B5 (Dako), at a dilution of (1:100); or monoclonal mouse anti-human macrophage CD68, EBM-11 (Dako) at a dilution of (1:80) or monoclonal mouse anti-human von Willebrand factor F8/86 (Dako), at a dilution of (1:40) in 0.5 M PBS containing 1.5% horse serum for 60 min. This was followed by incubating the slides with biotinylated anti-mouse IgG (diluted (1:200) in 0.5 M PBS with 1.5% horse serum) for 30 min and with the avidin-biotin complex or (ABC) reagent. The horse serum, biotinylated anti-IgG and ABC complex were reagents in the Vectastain Elite ABC kit (Vector, Burlingham, CA). After each of the
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above incubations, specimens were rinsed in 0.5 M PBS. The color reaction was developed using 0.025%, 3,3%-diamino benzidine tetrachloride (DAB) diluted in 100 ml of 0.5 M Tris buffer and pH 7.5, with 0.01 ml 30% H2O2 for 10 min and rinsed with distilled water. Lastly, the slides were counter-stained and viewed under the light microscope [9,11].
2.5. Statistics Statistical analysis was performed by using Kruskall–Wallis, Mann–Whitney U, 2-test and Fisher’s exact test.
3. Results Twelve marrow specimens from healthy, normal individuals and 103 specimens from MDS patients are the subject of this report. According to the FAB classification, there were 48 patients with refractory anemia (RA), 17 had RA with ringed sideroblasts (RARS), 25 had RA with excess blasts (RAEB), ten had RAEB in transformation (RAEB-t) and three had chronic myelomonocytic leukemia (CMMoL). Age ranged from 26 to 88 years (median: 68 years). Cytogenetic results were available in all individuals, with 47 patients carrying normal karyotype, while 56 had simple to complex karyotypic abnormalities. These data are shown in Table 1.
3.1. Establishing adherent layer Primary bone marrow biopsy sample were teased and simultaneously cultured in complete medium without any cytokines or growth factors on six plates per patient at 37°C in a humidified 5% CO2 chamber for 8–22 weeks. All the plates were examined weekly under phase contrast microscope before half of the supernatant medium was replaced with fresh medium. The non-adherent cells in the harvested medium were counted for viability and logged routinely. The biopsy pieces latched onto the culture plate in the first week, followed by the generation of stromal layer with the stem cells and maturing myeloid cells being released
Table 1 MDS patients in long-term bone marrow cultures FAB classification
Total number of patients cultured (%)
Patients with normal cytogenetic (%)
Patients with abnormal cytogenetic (%)
RA RARS RAEB RAEB-t CMMoL
48/103 17/103 25/103 10/103 3/103
19/48 (40) 11/17 (65) 11/25 (44) 5/10 (50) 1/3 (33)
29/48 (60.5) 6/17 (35) 14/25 (56) 5/10 (50) 2/3 (66)
(47) (16.5) (24) (10) (3)
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Fig. 1. (a) MDS patient bone marrow biopsy piece observed after 3 weeks in LTBMC with streaming stromal and hematopoietic cells (original magnification × 4). (b) A 2 –3 week old culture plate with 30% confluent adherent layer before the generation of adipocytes (original magnification ×4). (c) Bone marrow biopsy of RA patient after 4 weeks in LTBMC with 50% confluent bipolar or tripolar monolayers and adipocytes (original magnification ×10). (d) In LTBMC of MDS patient clearly visible hematopoietic cells, adipocytes, fibroblasts and macrophages released from the bone marrow compartment into the growth medium (original magnification × 40).
into the growth medium. The most interesting finding in this context was that of layer upon layer of cells streaming out of bony pieces in various areas of the plates (Fig. 1). By week 4, the plates were 30–50% confluent with bipolar, tripolar or multipolar monolayers and adipocytes, followed by 80– 100% confluency in the 8 –10th week with compact parallel multiple layers of fibroblasts, macrophages and adipocytes. In addition, preadipocytes and some adipocytes were routinely observed in close proximity of the bone pieces, over the monolayer. Cells in the area close to the bone achieved contact inhibition of growth earlier than those at the expanding peripheral margins and morphologically ap-
peared epithelial-like under the phase contrast microscope. On the other hand, giant adipocyte colonies were scattered across the plate. The plates were sacrificed between 8 and 22 weeks, with at least one plate sub-cultured under the same conditions.
3.2. Ultrastructural characteristics The electron microscopic studies were carried out on the bone marrow biopsy cultures that were sacrificed between 8 and 22 weeks. The adherent layer was in situ fixed in 3% gluteraldehyde, post fixed in osmium tetroxide, dehydrated, embedded and sectioned for TEM.
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Under TEM, the different types of stromal elements were identified and characterized, namely the fibroblasts, endothelial cells, monocytes/macrophages and the adipocytes [25,34]. Overall, the stromal compartment appeared very healthy and certainly without any significant apoptosis. The following describes the prominent and easily identifiable cell types.
3.3. Adipocytes The week 4 cultures, when observed under the light microscope, showed flat fibroblast like cells with refractile lipid vacuoles accumulating in the perinuclear region. This is the characteristic feature of the preadipocyte stage followed by adipocyte development. The fat vesicles would eventually enlarge in the proximity of the nuclei, round up as a giant fatty vacuole and gradually progress into the cytoplasm. Not all
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preadipocytes developed into large adipocytes, with some remaining at the early stage for the entire duration of the culture. Fig. 2 shows a typical plate containing large quantities of adipocytes.
3.4. Fibroblasts Vigorous growth of fibroblasts was noted in essentially all samples cultured with the new technique of using partially disaggregated bone biopsies. Fig. 3 shows a 3-week plate from an MDS patient with robust fibroblast growth. The fibroblast-like cells under the TEM often appeared as homogeneous elongated, structures with cytoplasmic region containing dilated endoplasmic reticulum and numerous microfilaments. Vertical sections through the stromal layer showed profiles of endoplasmic reticulum carrying numerous primary and secondary lysosomes (Fig. 4). The mitochondria were always present along with the well-devel-
Fig. 2. (a) RAEB patient in LTBMC after 5 weeks exhibiting 80% confluent adherent layer with preadipocytes and adipocytes (original magnification × 20). (b) Typical 5-week culture plate with large number of fleshy corpulent adipocytes (original magnification × 40).
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Fig. 3. (a,b) Confluent plate with compact parallel multiple layers of fibroblasts in LTBMC after 8 – 10 weeks (original magnification ×10 and ×20, respectively). (c) Maturing hematopoietic cells of MDS patient after exiting the bone marrow biopsy piece were observed seeding upon the streaming layer of fibroblasts and macrophages in LTBMC (original magnification ×40).
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Fig. 4. In long-term multilayer bone marrow cultures, spindle shaped fibroblasts with euchromatin nucleus and one or more prominent nucleolus were observed (uranyl acetate and lead citrate, original magnification ×3300).
oped Golgi complex. The nuclei showed sparse heterochromatin and prominent nucleoli. No phagocytosed material was seen in the fibroblast.
3.5. Endothelial cells Unlike fibroblasts, very few endothelial cells were observed. The surface of the nucleus in these cells was undulated and, ergastroplasm and glycogen rosettes, as shown in Fig. 5 surrounded the Golgi bodies.
3.6. Macrophages In addition to fibroblasts, large numbers of macrophages were observed in the adherent layer of long-term bone marrow cultures. They possessed extremely active and complex cell surface projections lobed nuclei with heterochromatin and extensive cytoplasm that was very rich in all the organelles. A majority of these macrophages contained cellular debri or residual bodies, as well as primary and secondary lysosomes in their cytoplasm. Different stages of phagocytosis in the erythrocytes were predominantly seen in the cytoplasm, as seen in Fig. 6.
3.7. In6estigation of stromal cell function: co-culture with CD34+ or cord blood cells The confluent plates (8– 12th week cultures) of 12 MDS patients in LTBMC were co-cultured with
CD34 + cells separated from human cord blood samples, donated by healthy pregnant female volunteers following delivery. The MDS adherent layer appeared to support the growth of these progenitor cells in a normal, expected manner. The viability count of the non-adherent layer after 1 week of co-culture was on average 85% and dropped to only 80% by the second week. The ultra structure morphology of MDS co-culture was similar to the original culture except that macrophages were actively surrounded by CD34+ cells, though no CD34+ apoptotic cells were observed. These studies appeared to suggest that the MDS stromal cells have retained their ability to support the growth of normal hematopoietic progenitors.
3.8. Immunohistochemical characteristics After 8–22 weeks of culture, the plates were sacrificed, the adherent layer trypsinized and cytospin slides prepared for immunostaining. Specific monoclonal mouse anti-human macrophage antibody (EBM11, Dako) against the expression of the CD68 antigen was used to confirm and estimate the monocyte/ macrophage population in the culture (Fig. 7). In 15 RA patients with normal or abnormal cytogenetics, 25% of the stroma consisted of macrophages. Interestingly, in eight RARS patients, 44% of the stromal cells belonged to monocyte/macrophages lineage, that is almost twice the number when compared to the RA group. In the 16 RAEB patients, :18% of the stromal
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cell population was composed of macrophages, whereas 35% of the cells in the two RAEB-t patients were macrophages. While in three CMMoL patients 31% of the cells were positively stained for macrophages, that is again much higher when compared to the RAEB group. The median percentage of macrophages was ten for RA, eight for RAEB and 50 for RARS. These differences reached a statistical significance among the three MDS FAB categories (P =0.0431). Similarly, a specific monoclonal mouse anti-human fibroblast antibody against b-subunit of prolyl-4-hydroxylase present in the collagen was used to identify and confirm the fibroblast population in the cultured adherent layer with immunostaining (Fig. 8). The antibody shows very restrictive positive staining under the stringent laboratory conditions and does not cross react with lymphocytes, monocytes, dendritic cells and granulocytes. In the 14 RA patients, : 13% cells were positively stained with the antibody, while eight RARS had 31% and 17 RAEB had 13% positive cells. The three RAEB-t and two CMMoL had 10 and 8% positively stained cells, respectively. It appears that RARS had maximum number of positively stained fibroblast cells and both RA and RAEB had less than half the number of cells belonging to fibroblast lineage compared to RARS and the difference almost reached statistical significance using non parametric analysis (P= 0.06) [35]. In addition, immunostaining with monoclonal mouse anti-human von Willebrand factor (factor VIII) antibody was performed on adherent layer to detect en-
dothelial cells/megakaryocytes. Under the light microscope, only few cells were stained positively, hence it was concluded that the number of endothelial cells present in the culture plates might be very low as the culture aged.
3.9. Growth characteristics of MDS long-term bone marrow cultures Of the 103 MDS patients that were cultured in LTBMC, 97 patients (RA 45, RARS 17, RAEB 23, RAEB-t nine and CMMoL three) were statistically evaluable. The basic clinical characteristics are presented in Table 2. Growth characteristics of normal healthy volunteers as well as the various FAB categories of MDS patients are presented in Table 3(A–E). Among normal marrows, a third had good growth, a third had fair growth and a third had poor growth in vitro (Table 3A). On the other hand, among the 48 RA patients, 54% had good growth, 16% had fair growth and 29% had poor growth overall. RA patients with an abnormal karyotype appeared to fare better in vitro, with 80% showing a good or fair growth (Table 3B), but these differences were not statistically significant. Half of the RARS patients had good and the other half had poor growth in vitro, without any differences related to cytogenetics (Table 3C). Among the RAEB and/or RAEB-t patients, :40% patients had good growth, and cytogenetics did not affect the ability of the cells to grow well in vitro (Table 3D,E). Finally, all three CMMoL patients showed good growth in vitro.
Fig. 5. Endothelial cells with classical morphology were observed in culture plates (uranyl acetate and lead citrate, original magnification × 6600).
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Fig. 6. Macrophages were frequently observed engulfing partly degraded erythyroid cells in long-term bone marrow cultures. Note the cross-sectional view showing multiple layers in culture (uranyl acetate and lead citrate, original magnification ×6600).
Thus, it can be concluded that neither the pre-culture FAB type (P =0.761) nor the cytogenetic results (P= 0.633) affected the growth of stromal cells in these MDS patients.
3.10. Effect of adipocytes on growth of MDS stromal cells in 6itro Under the light microscope, the morphology of stromal layer from MDS patients could be broadly categorized into three cell types; fibroblasts, macrophages and adipocytes. In one half (47/97) of cultured MDS patients it was observed that stroma predominately consisted of fibroblast/macrophages with very few adipocytes scattered across the culture plates. In three of these 47 patients, there was no significant adherent layer, in 13 samples there was fair growth and in 29 patients, poor growth was observed. In the other 47 patients, 30–100% confluent stromal layer of fibro-
blasts/macrophages was accompanied by a very high number of adipocytes. The number of adipocytes therefore consistently appeared to predict a good culture growth (PB0.0001). The time to reach optimum confluency in the cultured samples showed that 54 patients had very slow stromal proliferation over the 4-week period, while 40 patients had rapid growth and reached confluency (80– 100%) in a significantly shorter time, i.e. B 4 weeks (PB 0.001).
4. Discussion In this paper, we report upon the use of bone marrow biopsy material to obtain long-term in vitro growth of stromal cells from 103 patients with myelodysplastic syndromes and 12 normal controls. The salient findings described in this paper can be summarized here:
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Bone marrow biopsy tissue was successfully used to generate robust stromal cells in the vast majority of MDS patients. The stromal cells appear to be morphologically normal when examined by light microscopy or by transmission electron microscopy. Presence of apoptotic stromal cells that are frequently seen in MDS marrow biopsy sections examined by in situ end labeling (ISEL) could not be confirmed in the stromal cells which were studied in vitro following long-term cultures. Two probable reasons for this among many possible explanations are that either the apoptotic stromal cells died right away following initiation of in vitro cultures or the apoptosis inducing cytokine milieu was not replicated adequately in the long-term culture environment. Neither the FAB type nor the cytogenetics were co-related with the potency of in vitro growth of stromal cells. Given that both cytogenetics and FAB typing is performed strictly on parenchymal cells, it is no surprise that the vigor of growth seen in stromal cells is quite independent of their parenchymal neighbors. The MDS stromal cells appear to be functionally normal by virtue of their ability to support the proliferation of normal cord blood cell derived CD34 + cells. Large numbers of stromal cells can be generated from MDS marrow biopsies for further detailed characterization and future studies.
Given that the treatment options for most patients with MDS are few because of their advanced age and our lack of understanding regarding the etiology of their variable cytopenias, there is an urgency to investigate the biology of these disorders. One major stumbling block in this area has been a failure to generate large numbers of hematopoietic parenchymal or stromal cells in vitro for detailed characterization. More recently, it has been shown that the failure to generate adequate colony growth from MDS patients in vitro may be related to an excessive intramedullary apoptotic death of these cells. As a result of these difficulties, very few studies exist which actually describe colony growth in MDS patients and as far as stromal cells are concerned, such studies are even fewer. In order to circumvent these problems, bone marrow biopsy tissue was used directly to generate stromal cell cultures. Studies described in this paper demonstrate that such a technique can be successfully applied for long-term growth of MDS marrows. This is an important finding in itself, since one can examine the functional characteristics of these cells. The stromal cells in MDS patients appear to be both morphologically and functionally normal. This is no surprise since MDS has never been regarded as a disease of bone marrow stroma. The surprise was to find no apoptotic stromal cells in culture, since we have frequently observed and reported ISEL positive fibroblasts, endothelial cells and macrophages in BM biopsy sections obtained from MDS patients belonging to all
Fig. 7. Specific monoclonal macrophage antibody (EBM-11) against the expression of CD68 antigen was used to identify the monocyte/ macrophage population in the LTBMC. Darkly stained positive cells constituting 16% of the total cells from RA patient were estimated under the light microscope (original magnification × 40).
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Fig. 8. Specific monoclonal fibroblast antibody against the collagen was used to identify the fibroblast population in the LTBMC. Darkly stained 68% positive cells from RA patient were estimated under the light microscope (original magnification ×40).
FAB categories. Obviously, the in vitro cultures only represent stromal cells, while in vivo BM biopsies contain an intimately interactive stroma and parenchyma and therefore, the two situations cannot be directly compared. It is possible that the cells, which are the major source of pro-apoptotic cytokines in vivo, such as monocytes are absent from the cultures [36]. Alternatively, it may be that the apoptotic stromal cells died early on in the culture environment while the long-term stromal elements were descended from healthy, nonapoptotic parent cells. Nonetheless, these findings underscore the very basic artificiality of in vitro systems that at best represent one class of cells, if not one lineage. So for example, while in vivo, we could see a number of apoptotic stromal cells in MDS marrow biopsies, the in vitro cultures did not show such cells due to the absence of neighboring cells mediating that apoptosis. An important conclusion of these observations is that inherently, the stromal cells may not be diseased in the majority of MDS patients, a finding which is further confirmed by their functional adequacy in supporting the proliferation of cord blood derived CD34+ cells. It must be remembered however that MDS is a heterogeneous group of disorders and thus, the same pathophysiology will not uniformly apply to all cases. Therefore, it is possible that the marrow stroma is equally or primarily diseased in some patients with MDS. Another possibility is that the stromal growth obtained in vitro selectively originates in the healthy stromal progenitor, while the MDS stromal progenitor cells are unable to proliferate and form colonies in
vitro. If this were the case then the present report at least shows that there are normal residual stromal cells in MDS patients that can give rise to rich colony formation in vitro. In the past, stromal cells have been generated from BM aspirate cells at least in one study and these showed a defective support of normal hematopoietic cells [37]. Even in their study, the inadequate support of hematopoietic growth was only seen in a fraction of their 11 total cases studied. Another study of stromal cell function in MDS patients also found two broad patterns of growth [38]. In one group, hematopoietic cell numbers steadily declined while they were well-maintained in the second. Given the small number of cases studied to date, it is impossible to draw any conclusions regarding the functional characteristics of stromal cells in a disease as complex and heterogeneous as MDS. What can be concluded on the basis of previous reports and our own studies, is that in each report, there are stromal cell cultures from MDS
Table 2 Clinical characteristics of MDS patients under study Clinical variables
Mean
Median
Hgb (n =92) WBC (n =91) ANC (n =20) Platelets (n =92) BM Bx cellularity (n = 88) BM Asp cellularity (n =75) BM Asp blasts (n = 75)
9.69 5.13 1.78 139.72 65.53 2.44 6.49
9.5 3.08 1.39 80.5 70 3 3
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Table 3 Growth characteristics in healthy 6olunteers Number of normal bone marrow Good growth (80–90%) stroma biopsies from healthy volunteers confluent by week 8 12 4/12 (33%) Growth characteristics in RA RA 48 patients Normal cytogenetic 19/48 Abnormal cytogenetic 29/48 Growth characteristics in RARS RARS 17 patients Normal cytogenetic 11/17 Abnormal cytogenetic 6/17 Growth characteristics in RAEB RAEB 25 patients Normal cytogenetic 11/25 Abnormal cytogenetic 14/25 Growth characteristics in RAEB-t RAEB-t 10 patients Normal cytogenetic 5/10 Abnormal cytogenetic 5/10
Fair growth (40–50%) stroma confluent by week 8 4/12 (33%)
Poor growth (10–30%) stroma confluent by week 8 4/12 (33%)
Good growth (80–90%) stroma confluent by week 8 26/48 (54%) 9/19 (47%) 17/29 (59%)
Fair growth (40–50%) stroma confluent by week 8 8/48 (17%) 2/19 (11%) 6/29 (21%)
Poor growth (10–30%) stroma confluent by week 8 14/48 (29%) 8/19 (42%) 6/29 (21%)
Good growth (80–90%) stroma confluent by week 8 9/17 (53%) 6/11 (55%) 3/6 (50%)
Poor growth (10–30%) stroma confluent by week 8 8/17 (47%) 5/11 (45%) 3/6 (50%)
Good growth (80–90%) stroma confluent by week 8 11/25 (44%) 4/11 (36%) 7/14 (50%)
Fair growth (40–50%) stroma confluent by week 8 5/25 (20%) N/A 5/14 (36%)
Poor growth (10–30%) stroma confluent by week 8 9/25 (36%) 7/11 (64%) 2/14 (14%)
Good growth (80–90%) stroma confluent by week 8 4/10 (40%) 2/5 (40%) 2/5 (40%)
Fair growth (40–50%) stroma confluent by week 8 4/10 (40%) 2/5 (40%) 2/5 (40%)
Poor growth (10–30%) stroma confluent by week 8 2/10 (20%) 1/5 (20%) 1/5 (20%)
patients which provide adequate support for hematopoietic cell growth. The four major stromal cells that were consistently observed in the cultures we report are bone marrow fibroblasts, adipocytes, rare endothelial cells and abundant macrophages. The fibroblast and adipocytes have a similar mesenchymal stem cell origin while the marrow macrophages are derived from hematopoietic system. Endothelial cells, on the other hand, constitute a distinct stromal cell lineage [30]. The fibroblast and macrophage-like cells proliferated to a confluent monolayer and tended to become contact-inhibited. Simultaneously, areas close to the bone marrow biopsy developed multi-layer growth pattern with adipocyte colonies abutting on the stromal layers. Two interesting observations related to these cultures are worth mentioning. First, the appearance of adipocytes was not associated with a rapid termination of culture growth. In fact, the presence of large numbers of fleshy, corpulent adipocytes generally foretold the maintenance of a thriving culture likely to last 10– 12 weeks without colony death. Second, when bone marrow biopsy pieces were removed from confluent plates after 10– 12 weeks of culture, washed and sub-cultured in fresh plates, ample stromal colonies were once again generated. This was especially true for the plates that contained an abundance of adipocytes. Furthermore, these sub-cul-
tures after 12 weeks of primary cultures did not show any sign of aging or degeneration. The sub-cultures were grown for another 4–5 weeks before they were sacrificed and then select samples were further sub-cultured successfully. The same biopsy pieces could be used for the initiation of fresh cultures serially for up to 8–9 months without compromising the quality of stromal cells generated in a number of MDS cases as well as normal controls. Another interesting feature of colony forming unit fibroblast (CFU-F) in MDS is the appearance of adipocyte colonies as early as 2 weeks in culture that has not been observed in normal donor marrow cultures. Castoldi et al. have reported that the BMMNC LTBMC of MDS patients with RA and RARS produce normal stromal layers but RAEB and RAEB-t patients produce incomplete stromal confluency with reduced fat cells [39]. On the other hand, we do not observe any significant difference in stromal confluency or number of adipocytes among the FAB sub types as 66% of low risk and 68% of high risk patients had between 50 and 100% confluent plates [40]. In addition, no progressive impairment in the stromal progenitor number or in the proliferative capacity beginning from the less aggressive MDS to RAEB-t was observed. Most likely, these discrepancies result from the different sources from which cells were derived for the initiation of stromal
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cell cultures. We used BM biopsy tissue, whereas Castoldi et al. used BM aspirate cells. In summary, therefore, we report upon the use of BM biopsies for generation of large numbers of stromal cells in vitro from MDS patients. Material thus obtained can be used for detailed molecular, genetic and functional characterization of these heretofore rather elusive cells.
[7]
[8]
Acknowledgements This work has been supported by a grant from the National Cancer Institute (PO1 CA 75606) and the Dr Roy Ringo Grant for basic research in MDS and Rush Presbyterian St Luke’s Medical Center Woman’s Board Time Center grant. The authors wish to thank their devoted laboratory colleagues for their constant support and dedication and Lakshmi Venugopal and Sandra Howery for excellent administrative/secretarial assistance. Authors contributions as follows: Conception and design: S Alvi, A Shaher, V Shetty, B Henderson, B Dangerfield, F Zorat, S Mundle, P Reddy, K Allampallam, X Huang, N Galili, R Borok, A Raza. Analysis and interpretation of data: S Alvi, A Shaher, V Shetty, B Henderson, R Borok, A Raza. Drafting the article: S Alvi, L Joshi, A Raza. Critical revision of the article for important intellectual content: S Alvi, S Anthwal, S Mundle, P Reddy, K Allampallam, N Galili, R Borok, A Raza. Final approval of article: S Alvi, S Anthwal, A Raza. Provision of study materials or patients: L Lisak, L Little, S Gezer, A Raza. Statistical expertise: R Borok. Obtaining funding: A Raza. Administrative, technical or logistic support: A Shaher, V Shetty, B Henderson, B Dangerfield, F Zorat, L Lisak, L Little, S Gezer, X Huang, A Raza. Collection or assembly of data: S Alvi, A Shaher, V Shetty, B Henderson, A Raza.
[9]
[10] [11]
[12] [13]
[14]
[15]
[16]
[17]
References [1] Resegotti L. The nature and natural history of myelodysplasia. Haematologica 1993;25:191. [2] Verhoef GEG, Pittaluga S, De Wolf-Peters C, Boogaerts MA. FAB classification of myelodysplastic syndromes: merits and controversies. Ann Hematol 1995;71:3. [3] Bennett JM, Catovsky D, Flandrin DMT, Galton GDAG, Gralnick HR, Sultan C. Proposal for the classification of the myelodysplastic syndromes. Br J Haematol 1982;51:189. [4] Clark DM, Lampert IA. Apoptosis is a common histopathological finding in myelodysplasia: the correlate of ineffective hematopoiesis. Leuk Res 1990;2:415. [5] Yoshida Y. Apoptosis may be the mechanism responsible for the premature intramedullary cell death in myelodysplastic syndromes. Leukemia 1993;7:144. [6] Raza A, Mundle S, Iftikhar A, Gregory S, Marcus B, Khan Z, Alvi S, Shetty V, Dameron S, Wright V, Adler S, Loew J, Scott
[18]
[19]
[20]
[21]
[22]
953
S, Ali SN, Preisler HD. Simultaneous assessment of cell kinetics and programmed cell death in bone marrow biopsies of myelodysplastics reveals extensive apoptosis as the probable basis for ineffective hematopoiesis. Am J Hematol 1995;48:143. Raza A, Gezer S, Mundle S, Gao XZ, Alvi S, Borok R, Rifkin S, Iftikhar A, Shetty V, Parcharidou A, Loew J, Marcus B, Khan Z, Chaney C, Showel J, Gregory S, Preisler HD. Apoptosis in bone marrow biopsy samples involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes. Blood 1996;86:268. Parker JE, Mufti GJ. Ineffective hematopoiesis and apoptosis in myelodysplastic syndromes. Br J Hematol 1998;101:220. Mundle S, Ali A, Cartlidge J, Reza S, Alvi S, Showel M, Mativi BY, Shetty V, Venugopal P, Gregory S, Raza A. Evidence for involvement of tumor necrosis factor a in apoptotic death of bone marrow cells in myelodysplastic syndromes. Am J Hematol 1999;60:36. Greenburg PL. Biologic nature of the myelodysplastic syndromes. Acta Haematol 1987;78:94. Shetty V, Mundle S, Alvi S, Showel M, Broady-Robinson L, Dar S, Borok R, Showel J, Greagory S, Rifkin S, Gezer S, Parcharidou A, Venugopal P, Shah R, Hernandez B, Klein M, Alston D, Robin E, Dominguez C, Raza A. Measurement of apoptosis, proliferation and three cytokine in 46 patients with myelodysplastic syndromes. Leuk Res 1996;20:891. Yanuck MD, Saleem A. Leukemic transformation in myelodysplastic syndrome: a review. Ann Clin Lab Sci 1991;3:171. Pedersen-Bjergaard J, Philip P, Larsen SO, Anderson M, Daugaard G, Ersboll J, Hansen SW, Hou-Jensen K, Nielsen D, Sigsgaard TC, Specht L, Osterlind K. Therapy related myelodysplastic syndromes and acute myeloid leukemia. Cytogenetic characteristics of 115 consecutive cases and risk in seven cohorts of patients treated intensively for malignant diseases in the Copenhagen series, Leukemia 1993;7:1975 – 86. Vallespi T, Imbert M, Mecucci C, Preudhomme C, Fenaux P. Diagnosis, classification, and cytogenetics of myelodysplastic syndromes. Haematologica 1998;83:248. Ohmori M, Ohmori S, Ueda Y, Yoshida Y, Okuma M. Ineffective hematopoiesis in myelodysplastic syndromes (MDS) as studied by daily in situ observation of colony-cluster formation. Int J Cell Clon 1991;5:521. Aizawa S, Nakano M, Iwase O, Yaguchi M, Hiramoto M, Hoshi H, Nabeshima R, Shima D, Handa H, Toyama K. Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD 34-positive cell proliferation and differentiation in vitro. Leuk Res 1999;23:239. Sawada K. Proliferation and differentiation of myelodysplastic CD34+ cells. Leuk Lymph 1996;1 – 2:43. Tennant GB, Walsh V, Truran LN, Edwards P, Mills KI, Burnett AK. Abnormalities of adherent layers grown from bone marrow of patients with myelodysplasia. Br J Haematol 2000;3:853. Richert-Boe KE, Bagby CG Jr. In vitro hematopoiesis in myelodysplasia: liquid and soft-gel culture studies. Hematol Oncol Clin North Am 1992;6:543. Flores-Figueroa E, Gutierrez-Espindola G, Guerrero-Rivera S, Pizzuto-Chavez J, Mayani H. Hematopoietic progenitor cells from patients with myelodysplastic syndromes: in vitro colony growth and long term proliferation. Leuk Res 1999;23:385. Sato T, Kim S, Selleri C, Young NS, Maciejewski JP. Measurement of secondary colony formation after 5 weeks in long-term cultures in patients with myelodysplastic syndrome. Leukemia 1998;12:1187. Roberts IAG, McMullin MF. Apractical miniature long-term bone marrow culture system for investigating early myelodysplasia. Leuk Res 1992;16:737.
954
S. Al6i et al. / Leukemia Research 25 (2001) 941–954
[23] Dexter TM. Haemopoiesis in long term bone marrow cultures. Acta Haematol 1979;62:299. [24] Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of haempoietic stem cells in vitro. J Cell Physiol 1977;91:335. [25] Lanotte M, Allen TD, Dexter MT. Histochemical and ultrastructural characteristics of a cell line from human bone marrow stroma. J Cell Sci 1981;50:281. [26] Greenburg PL. In vitro marrow culture studies in the myelodysplastic syndromes. Semin Oncol 1992;1:34. [27] Blazsek I, Liu XH, Anjo A, Quittet P, Comisso M, Kim-Triana B, Misset JL. The hematon, a morphogenetic functional complex in mammalian bone marrow, involves erythroblastic islands and granulocytic cobblestones. Exp Hematol 1995;23:309. [28] Chang J, Dexter TM. Long-term cultures: in vitro purging of leukemic cells. Baillieres Clin Haematol 1991;3:775. [29] Chang J, Allen TD, Dexter TM. Long-term bone marrow cultures: their use in autologous marrow transplantation. Cancer Cells 1989;1:17. [30] Duhrsen U, Hossfeld DK. Stromal abnormalities in neoplastic bone marrow diseases. Ann Hematol 1996;73:53. [31] Sawada K. Growth characteristics of myelodysplastic CD34 + cells. Leuk Lymph 1998;12:49. [32] Gidali J, Feher I, Hollan SR. Blast colony forming cell-binding capacity of bone marrow stroma from myelodysplastic patients. Stem Cells 1996;14:577. [33] Ohmori M, Ueda Y, Masutani H, Hirama T, Anzai N, Yoshida Y, Okuma M. Myelodysplastic syndrome (MDS)-associated inhibitory activity on haematopoietic progenitor cells: Contribution of monocyte-derived lipid containing macrophages (MDLM). Br J Haematol 1992;81:67.
[34] Payne CM, Greenberg B, Cromey D, Woo L. Morphological evidence of an altered bone marrow microenvironment in patients with acute nonlymphoblastic leukemia and myelodysplastic disorders. Exp Hematol 1987;15:143. [35] Zhang W, Knieling G, Vohwinkel G, Martinez T, Kuse R, Hossfeld DK, Duhrsen U. Origin of stroma cells in long term bone marrow cultures from patients with acute myeloid leukemia. Ann Hematol 1999;78:305. [36] Deeg HJ, Beckham C, Loken MR, Bryant E, Lesnikova M, Shulman HM, Gooley T. Negative regulators of hematopoiesis and stroma function in patients with myelodysplastic syndromes. Leuk Lymph 2000;37:405. [37] Aizawa S, Hiramoto M, Hoshi H, Toyama K, Shima D, Handa H. Establishment of stromal cell line from an MDS RA patient which induced an apoptotic change in hematopoietic and leukemic cells in vitro. Exp Hematol 2000;28:148. [38] Coutinho LH, Geary CG, Chang J, Harrison C, Testa NG. Functional studies of bone marrow haemopoietic and stromal cells in the myelodysplastic syndromes (MDS). Br J Haematol 1990;75:16. [39] Diana C, Dominici M, Lanza F, Punturieri M, Pauli S, Tieghi A, Dabusti M, Scapoli G, Castoldi G. Impairment of stromal cells compartment in hypoplastic myelodysplastic syndromes. Blood 2000;96:357a Abstract no. 1541. [40] Alvi S, Shaher A, Henderson B, Dar S, Zorat F, Broderick E, Lisak L, Du Randt M, Reddy P, Mundle S, Galili N, Borok RZ, Raza A. Improved growth of stromal cells in long term bone marrow cultures (LTBMC) of myelodysplastic syndrome (MDS) patients treated with thalidomide. Blood 2000;96:359a Abstract no. 1547.