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Murine Bone Marrow Stromal Cells Sustain In Vivo the Survival of Hematopoietic Stem Cells and the Granulopoietic Differentiation of More Mature Progenitors

^a Department of Cytology and Histology,
^b Department of Pathological Anatomy, and
^c Laboratory of Connective Tissues Biology, University of Liège, Liège, Belgium

Key Words. Hematopoiesis • Microenvironment • Stromal cells • MS-5 cell line • In vivo model

Correspondence: Frédérique Hubin, Department of Cytology and Histology, University of Liège, Liège, 4000, Belgium. Telephone: 003-24-366-2403; Fax: 003-24-366-2919; e-mail: F.Hubin{at}ulg.ac.be

	ABSTRACT

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The study of the human hematopoietic system would be facilitated by availability of a relevant animal model. Because the medullar microenvironment is made of different types of cells, interactions between hematopoietic cells and stromal cells are difficult to analyze in detail. As an approach for establishing an in vivo model to dissect these interactions, we grafted murine bone marrow fibroblastic cells (MS-5 cell line) with hematopoietic cells into the kidney capsule of syngenic mice. To identify the origin of cells present in the graft, we used green fluorescent protein–stable transfected MS-5 cells for the transplantation. To analyze the evolution of stromal cells and identify hematopoietic cells able to develop in these conditions, we performed morphology, histochemistry, and immunohistology on tissue sections at different times after transplantation. When injected alone, MS-5 cells differentiate into adipocytes. When injected with a bone marrow suspension or with isolated CD45⁺ cells (leukocytes), the stromal cells keep their fibroblastic morphology and their alkaline phosphatase expression and sustain granulopoiesis. When injected with hematopoietic stem cells called c-kit⁺Sca-1⁺Lin^– suspension, clusters of hematopoietic cells are also observed: They do not present any granulopoietic activity and do not belong to B or T population nor to erythroid lineage. They are quiescent, induce bone marrow recovery and survival of lethally irradiated recipients, are able to form macroscopic colonies in the spleen, and are able to form very few colonies in vitro, suggesting that they are hematopoietic stem cells. In conclusion, our results show that reticular fibroblastic stromal cells MS-5 sustain the survival of stem cells and are not able to induce their differentiation. However, they can control differentiation, proliferation, and/or survival of hematopoietic cells engaged in myeloid lineage.

	INTRODUCTION

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Bone marrow stroma is made of several cell types and an extracellular matrix, which together form a suitable microenvironment modulating quiescence, self-renewal, and commitment of hematopoietic stem cells and the proliferation, maturation, and apoptosis of more mature hematopoietic cells [1–4]. However, it has not been possible so far to correlate these functions with particular cell types of the microenvironment, which consists mainly of macrophages, endothelial cells, fibroblasts, and adipocytes [5]. Hence, the establishment of stromal cell clones has gained importance in the dissection of microenvironmental functions. However, it must be kept in mind that the in vivo situation is much more complex than the in vitro model and that the cultures lack the organized three-dimensional structure of reticular network in the bone marrow. Hence, the development of in vivo models in which the interactions between hematopoietic and microenvironmental cells can be studied is absolutely necessary. Hematopoietic cells can be grown in immunodeficient mice, in which their development can be sustained either by blood-forming tissues of the mouse host itself [6] or by surgically implanted human hematopoietic organs [7]. Yet the overall level of hematopoiesis achieved is still highly variable. It is therefore likely that stem cell/stroma interactions are not optimally reproduced. Furthermore, the cellular complexity of the stroma in these models prevents the understanding of the relationship between stromal cells and hematopoietic cells.

To establish a simplified in vivo situation, we have explored the possibility to graft a bone marrow fibroblastic cell line (MS-5) with hematopoietic cells into the kidney capsule of syngenic mice. MS-5 cell line was derived from the irradiated adherent layer of a Dexter-type long-term culture [8]. Depending on the culture conditions, this cell line provides a permissive environment in vitro for B-cell differentiation and for generation of granulocytes [9] but also supports murine colony-forming unit-spleen (CFU-S), granulocyte macrophage-colony-forming unit (CFU-GM), burst-forming unit-erythroid (BFU-E), and bone marrow cells with reconstituting ability [9–14]. We tried with this model to determine whether hematopoiesis was induced only by a single stromal cell line in vivo without the influence of medium culture and, if so, to clarify putative roles of bone marrow fibroblastic cells in vivo. Furthermore, the potential to produce active bone marrow outside the medullary space could be useful in certain clinical conditions in individuals with irreversible stromal injury.

	MATERIALS AND METHODS

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Mice
Mice of C₃H/J strain were purchased from Iffa Credo (Brussels, Belgium), maintained under specific pathogen-free conditions in our animal facility, and given nutritional chow and water ad libitum. Six- to 8-week old female mice were used either as recipients or as bone marrow donors. The experimental work was conducted according to the procedures outlined in the Law for Care and Use of Laboratory Animals (Arrêté Royal, November 14, 1993, Belgium).

Culture of Stromal Cell Lines
Murine hematopoietic-supportive stromal cell line MS-5 (kindly provided by Dr. A. Gothot) was maintained in Iscove’s modified Dulbecco’s medium (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 10% fetal calf serum (FCS) (Gibco) in humidified atmosphere at 37°C and 5% CO₂.

Bone Marrow Cells
Mice were killed by cervical dislocation, and their bone marrow cells were flushed from the femurs into phosphate-buffered saline (PBS) using a syringe and a 26-gauge needle. Residual red blood cells were lysed by an incubation in an hypotonic solution of ammonium chloride. Cell counts were performed, and viability was determined by trypan blue exclusion.

Immunomagnetic Separation of CD45⁺ Cells from Bone Marrow
Murine bone marrow cells were enriched for CD45⁺ using the magnetic-activated cell sorter system (Miltenyi Biotec, Bergisch-Gladbach, Germany, http://www.miltenyibiotec.com), which provides an 80%–95% pure CD45⁺ population. CD45 antibody reacts with antigens found on all cells of hematopoietic origin except erythrocytes. Single-cell suspension (10⁷ cells/100 µl) was incubated for 15 minutes at 4°C (dilution, 1/10) with a monoclonal rat anti-mouse CD45 antibody conjugated to paramagnetic microbeads (Miltenyi Biotec). The cells were then washed three times with PBS and 1% FCS (Sigma, Poole, U.K., http://www.sigmaaldrich.com). CD45⁺ cells were retained in the column matrix in the presence of an external magnetic field while the unlabeled CD45⁺ cells were run through. The column was washed, and the magnetically retained CD45⁺ cells could be eluted as positively selected cell fraction by removing the column from the magnetic field. The purity of CD45⁺ cell suspension was verified by fluorescence-activated cell sorter analysis of the cell suspension with a monoclonal rat anti-mouse CD45 antibody (BD Biosciences, San Diego, http://www.bdbiosciences.com) conjugated with fluorescein isothiocyanate (FITC) that recognizes a different epitope of CD45.

Immunomagnetic Separation of c-kit⁺Sca-1⁺Lin^– Cells from Bone Marrow
Using the lineage cell depletion kit (Miltenyi Biotec), negative cells were isolated from suspension of bone marrow cells by depletion of cells expressing a panel of so-called lineage antigens (CD3, CD45R [B220], CD11b, Gr-1, and Ter-119). Lineage⁺ cells (10⁷ cells/50 µl) are indirectly magnetically labeled using a cocktail of biotin-conjugated monoclonal antibodies (dilution, 1/5) as primary labeling reagent and antibiotin monoclonal antibodies conjugated to microbeads as secondary labeling reagent (dilution, 1/5). The magnetically labeled lineage cells were depleted by retaining them on a magnetized column while the unlabeled lineage-negative cells pass through the column. The lineage-negative cells (10⁸ cells/1 ml) were then incubated with a monoclonal rat anti-mouse Sca1 antibody (dilution, 1/5) conjugated to metal colloid beads (Miltenyi Biotec) and loaded on a column that was placed in the magnetic field of a MACS separator. The magnetically labeled Sca1⁺ cells were retained on the column. After removal of the column from the magnetic field, the magnetically retained Sca1⁺ cells could be eluted as the positively selected cell fraction. The microbeads were then removed from Sca1⁺ cells using Multisort Release reagent to allow magnetic labeling and separation of Sca1⁺ cells according to the expression of CD117. Finally, cell suspensions (10⁷ cells/100 µl) were labeled with a monoclonal rat anti-mouse CD117 (c-kit) antibody (dilution, 1/5) also coupled with microbeads. Positive cells were enriched after separation on column from unlabeled cells that were eluted with PBS and FCS. By these three isolations, we obtained 480 cells per femur (0.004% of total hematopoietic bone marrow).

Flow Cytometry Analysis
Cells were stained with anti-CD45 FITC (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) by incubating cells (10⁶ cells/10 µl) for 30 minutes on ice. Cells used for isotype control were incubated under the same conditions (10⁶ cells/10 µl) with an immunoglobulin G control. After incubation, the cells were washed once in PBS and 0.5% bovine serum albumin and resuspended in 0.3 ml PBS. Analysis was performed on a FACStar Plus (Becton Dickinson, Immunocytometry Systems, San Jose, CA, http://www.bd.com) equipped with argon and helium neon lasers. Specific fluorescence of FITC excited at 488 nm, as well known forward and orthogonal light-scattering properties of normal murine bone marrow cells, was used to establish gates. Data acquisition and analysis were performed using Cellquest software (Becton Dickinson).

Transplantation
Mice were anesthetized during the transplantation procedure with 10 mg/kg of xylazine (Rompun; Bayer, Leverkusen, Germany, http://www.bayer.com) and 100 mg/kg of ketamine (Kétalar; Pfizer, New York, http://www.pfizer.com) injected intraperitoneally. Either stromal (MS-5) or bone marrow cells or a mixture of MS-5 and bone marrow cells was injected into the kidney capsule of recipient mice. A small incision was made on the back of the animal, 50 µl of the cell suspension was injected with a 26-gauge needle into the kidney capsule, and the incised region was closed using a stapler (Becton Dickinson). The site of injection was stained with black ink (Pelican). Each mouse received 5 x 10⁶ MS-5 cells alone, 1 x 10⁶ bone marrow cells alone, or 5 x 10⁶ MS-5 cells and 1 x 10⁶ bone marrow cells together. When murine stem cells were engrafted, only 1,000 c-kit⁺Sca-1⁺Lin^– cells were coinjected with MS-5 cells.

Tissue Processing for Histology
C₃H/J mice were euthanized at 5, 10, 15, 30, and 60 days after transplantation of stromal cells or bone marrow cells. Kidneys were collected, fixed in 4% paraformaldehyde (Polysciences Inc., Warrington, PA, http://www.polysciences.com), and paraffin-embedded or dehydrated in acetone (Merck, Darmstadt, Germany, http://www.merck.com) and embedded in a JB-4 solution (Polysciences Inc.). Engrafted kidneys were also embedded in Tissue-tek (Sakura Finetek Europe B.V, Zoeterwoude, The Netherlands, http://www.sakuraeu.com) and frozen at –80°C. Four-micrometer paraffin sections and 2-µm plastic sections were cut and stained with hematoxylin and eosin (H&E).

Alkaline Phosphatase Activity Detection
For alkaline phosphatase activity, 2-µm JB-4–embedded sections were incubated for 45 minutes at 37°C in a reaction medium containing 1 mg/ml^–1 fast blue BB salt, 0.3 mg/ml^–1 naphtol-AS-phosphate (Sigma), and 0.5% N,N-dimethylformamide in 0.2 M Tris buffer (pH 9.1). Negative control was incubated with the naphtol-AS-phosphate solution without fast blue BB salt.

Acid Phosphatase Activity Detection
For acid phosphatase activity, 2-µm JB-4–embedded sections were incubated for 1.5 hours at 37°C in a reaction medium containing 0.5 mg/ml^–1 naphtol-ASBi-phosphate (Sigma), 4% hexazotized pararosaniline, and 0.5% N,N-dimethylformamide in 0.15 M Veronal buffer (pH 5) (VWR, Leuven, Belgium, http://www.vwrsp.com). Negative control was incubated with the naphtol-ASBi-phosphate solution without hexazotized pararosaniline.

Fat Cell Detection
Cryostat sections (5-µm) were used to test the presence of fat cells. The sections were stained for 10 minutes with oil red O solution (VWR) prepared by dissolving 250 mg oil red O in 100 ml absolute isopropanol. This solution was mixed with distilled water (6:4 vol/vol), left for 10 minutes, and filtered before use.

Chloroacetate Esterase Activity Detection
For chloroacetate esterase activity, 2-µm JB-4–embedded sections were incubated for 1 hour at 30°C in a reaction medium containing 4% hexazotized pararosaniline, 0.3 mg/ml^–1 naphtol-ASD-chloroacetate (Sigma), and 1% N,N-dimethylformamide in 0.15 M Veronal buffer (pH 6.3) (VWR).

Immunohistology
Endogenous peroxidase activity was inhibited by incubating the sections for 10 minutes in 3% H₂O₂. For detection of B lymphocytes, parallel sections were incubated for 2 hours at room temperature with a monoclonal rat anti-mouse biotin CD45R/B220 antibody (Pharmingen) titrated with reference tissues and used at saturating concentrations and then with an avidin-biotin-peroxydase reagent (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) for 1 hour. After incubation in diaminobenzidine (DAB) (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com), sections were counterstained with H&E (Surgipath, Richmond, IL, http://www.surgipath.com), mounted with entellan (Merck), and observed with a light microscope. For detection of erythroid cells, sections were incubated for 2 hours at room temperature with a monoclonal rat anti-mouse biotin Ter-119 antibody (Pharmingen) used at optimal dilutions and then with an avidin-biotin-peroxydase reagent for 1 hour. After incubation in DAB, sections were counterstained with H&E, mounted with entellan, and observed with a light microscope. Positive cells were stained in brown. For detection of T lymphocytes, sections were incubated for 2 hours at room temperature with a monoclonal hamster anti-mouse biotin CD3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) used at optimal dilutions and then with an avidin-biotin-peroxydase reagent for 1 hour. After incubation in DAB, sections were counterstained with H&E, mounted with entellan, and observed with a light microscope. Positive cells were stained in brown. For detection of proliferative cell staining, paraffin sections were pretreated with EDTA and incubated for 1 hour at room temperature with a polyclonal goat anti-mouse biotin Ki-67 antibody (Santa Cruz Biotechnology) used at optimal dilutions, with a secondary anti-goat antibody for 1 hour at room temperature, and then with a avidin-biotin-peroxydase reagent for 1 hour. After incubation in DAB, sections were counterstained with H&E, mounted with entellan, and observed with a light microscope. Positive cells were stained in brown.

Transfection Procedure
A plasmid expressing the green fluorescent protein (pIRES-puro-GFP, kindly provided by C. Lambert) was used for the transfection procedure. MS-5 cells were transfected by lipofection following the manufacturer’s protocol (Fugene6; Roche, Basel, Switzerland, http://www.roche-applied-science.com) of pIRES-puro-GFP. Plasmid concentrations, optimized for the transfection studies described, were 1 µg for 3 µl of Fugene6. Forty-eight hours after transfection, cells were observed under fluorescent microscope. Stable transfectants of MS-5 cells were selected in culture for resistance to puromycin and screened for expression of green fluorescent protein (GFP) by flow cytometry.

Capture and Analysis of Images
Images were taken with a light microscope (Leica, Van Hopplynus, Germany, http://www.leica.com) linked to a computer. These images were analyzed using an image-analysis program (Spot Imaging Software; Diagnostic Instruments, Sterling Heights, MI, http://www.diaginc.com).

Colony Formation in Semisolid Media
Methylcellulose assay was performed to test the presence of progenitor cells. The murine c-kit⁺Sca-1⁺Lin^– cells (10 x 10⁴) were plated in a volume of 2.5 ml of methylcellulose (Methocult GF M3434; Stem Cell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com), and cells isolated from the graft under the kidney’s capsule (20 x 10⁴ and 50 x 10⁴) were also plated following the same conditions. After 14 days of culture at 37°C and 5% of CO₂ in air, colonies consisting of more than 50 cells were counted in triplicate as colony-forming units in culture derived from CFU-GM, CFU-G, CFU-M, BFU-E, and mixed granulo-erythropoietic colony-forming units (CFU-Mix) according to established criteria using an inverted microscope.

Transplantation
In preliminary experiments, all mice that had received irradiation at 6 Gy died within 4 days. To verify the reconstitution potential of cells isolated from the graft, mice were lethally irradiated at 6 Gy, and 1,000 cells isolated from the kidney capsule were intravenously injected into the retro-orbital sinus. The animals were maintained in a pathogen-free environment, and aqueous antibiotic was added to their drinking water 2 days before irradiation and for 6 weeks after cell transplantation (15 mice). As controls, 6 Gy–irradiated mice were transplanted with 100 c-kit⁺Sca-1⁺Lin^–. The two groups of mice were monitored daily for survival. Five mice from each group were euthanized 15 days after transplantation; marrow cellularity was evaluated, and the CFU-S was counted by determination of the number of macroscopic colonies per spleen fixed into Bouin’s solution.

	RESULTS

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Differentiation of Murine Stromal Cells MS-5 into Adipocytes
In vitro, MS-5 stromal cells present a fibroblastic morphology with long spindle-shaped processes. They are alkaline phosphatase–positive and acid phosphatase–negative and contain discrete lipidic droplets stained by oil red O. When 5 x 10⁶ cells are transplanted into kidney capsule, most of them adopt an adipocytic morphology as soon as 5 days after injection, and some of them keep a fibroblastic morphology. These cells are intensely stained by oil red O (Fig. 1A) but lack alkaline phosphatase activity (Fig. 1B). Hematopoietic cells are not detected in the graft. The experiment was performed three times on a total of 22 mice; the results were identical at each tested delay (5, 10, 15, and 30 days). They indicate that MS-5 stromal cells in vivo differentiate into adipocytes and suggest that they do not exert any chemoattractive activity upon hematopoietic stem cells.

View larger version (61K): [in this window] [in a new window]   Figure 1. Murine stromal cells (MS-5) injected underneath the kidney’s capsule differentiate into adipocytes. Oil red O staining in red confirms the presence of lipid droplets (A) (x200), whereas alkaline phosphatase activity of the stromal cells has disappeared (B) (x200).

View larger version (127K): [in this window] [in a new window]   Figure 2. Bone marrow mononuclear cells grafted underneath the kidney’s capsule display a fibroblastic morphology. These cells are alkaline phosphatase–negative (x200).

View larger version (101K): [in this window] [in a new window]   Figure 3. Coinjection of stromal and bone marrow mononuclear cells underneath the kidney’s capsule gives rise to transient granulopoiesis. (A): Chloroacetate esterase activity identifies clusters of granulopoietic cells (x200). A network of (B) alkaline phosphatase fibroblastic cells (x200) and of (C) collagen III fibers (x200) surrounds these clusters of granulopoiesis. (D): The lack of acid phosphatase activity excludes the possibility that macrophages were present in the graft (x200). (E): Three months after transplantation, granulopoiesis disappeared and graft was comprised of adipocytes (x200).

One month after transplantation, granulopoietic cells had disappeared, and the graft was almost completely comprised of adipocytes; 3 months after transplantation, only adipocytes were found in the graft (Fig. 3E).

Identification of Engrafting Cells Using Stable Transfected GFP-MS-5 Cells
To assess the origin of alkaline phosphatase–positive fibroblastic cells in the graft, MS-5 stromal cells were stably transfected with a plasmid expressing GFP. Fluorescence microscopy of the grafts (34 mice, four different experiments) revealed that 15 days after grafting, clusters of granulopoiesis are surrounded by a network of green fluorescent MS-5 cells (Figs. 4A, 4B), whereas 1 month after, only green fluorescent adipocytes are observed (five of six mice; Fig. 4C).

View larger version (58K): [in this window] [in a new window]   Figure 4. After coinjection of green fluorescent protein (GFP)–transfected MS-5 cells and bone marrow mononuclear cells, fluorescence microscopy reveals that (A) clusters of granulopoiesis (x200) are surrounded by (B) a network of fibroblastic cells expressing GFP (15 days, x200). (C): At 30 days, only green adipocytes are observed (x200).

View larger version (47K): [in this window] [in a new window]   Figure 5. Isolated CD45+ cells coinjected with green fluorescent protein (GFP)–transfected MS-5 cells underneath the kidney’s capsule give rise to (A) clusters of granulopoiesis (x200) surrounded by (B) a network of fibroblastic cells expressing GFP (15 days, x200).

View larger version (46K): [in this window] [in a new window]   Figure 6. Murine c-kit+Sca-1+Lin– cells coinjected with green fluorescent protein (GFP)–transfected MS-5 cells underneath the kidney’s capsule give rise to clusters that do not belong to the granulocytic lineage as assessed by (A) the absence of chloroacetate esterase (x200) and that are surrounded by (B) a network of fibroblastic cells expressing GFP (15 days, x200).

View larger version (101K): [in this window] [in a new window]   Figure 7. Hematopoietic cells obtained after coinjection of c-kit+Sca-1+Lin– cells and green fluorescent protein–transfected MS-5 cells do not express (A) B2220 (10 days, x500), (B) CD3 (10 days, x500), (C) Ter-119 (10 days, x200), and (D) Ki-67 (10 days, x500). (E–H): Positive controls, respectively, for B220 (lymph node), CD3 (lymph node), Ter-119 (bone marrow), and Ki-67 (bone marrow).

Both groups of mice were still alive 30 days after transplantation. At day 15 after transplantation, the femurs of transplanted mice contained an average of 3 x 10⁶ hematopoietic cells, and macroscopic colonies (CFU-S) were observed in their spleens.

The ability of the cells isolated from the graft to form colonies in vitro was compared with that of a c-kit⁺Sca-1⁺Lin^– suspension; c-kit⁺Sca-1⁺Lin^– cells form CFU-Mix (3 colonies), CFU-GM (37 colonies), CFU-G (14 colonies), CFU-M (8 colonies), and BFU-E (7 colonies), whereas only one CFU-Mix, CFU-GM, and CFU-G was detected after plating the cells isolated from the graft.

	DISCUSSION

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The observations we present provide a new model for the study of relations between stromal cells and hematopoietic cells and of the differentiation of cells of the bone marrow microenvironment. The MS-5 stromal cells differentiate into adipocytes in the absence of hematopoietic cells. Prominent fat cells are features of aplastic bone marrow in vivo [15, 16]. The observation that marrows from patients with chemotherapy-induced hypoplasia can also form fat cells spontaneously supports the idea that fat cell formation may be a physiological response to a hematological deficit. Differentiated human adventitial reticular cells can rapidly accumulate fat and become adipocytes upon myelosuppression in vivo [15–17]. Our results and these observations indicate that marrow microenvironment has the potential to respond to levels of hematopoietic activity. They emphasize the need to distinguish between physiological and pathological responses in the hematopoietic microenvironment in hematological disease and treatment.

Surprisingly, hematopoiesis does not develop when bone marrow cells are grafted without MS-5 cells, although the grafted cell suspension probably contains hematopoietic and stromal cells. Grafted cells adopt a fibroblastic morphology but do not express alkaline phosphatase activity characteristic of Western Bainton fibroblastic reticular cells assumed to represent granulopoiesis-supporting stromal cells [16–20]. It is therefore likely that stem cell stroma interactions are not reproduced in the grafts.

In vitro MS-5 cell line is described as supporting CFU-GM and granulocytopoiesis [8] and as maintaining long-term bone marrow–repopulating cells, day-12 and day-8 spleen colony-forming units (CFU-S12 and CFU-S8), and BFU-E [11] without relation to the expression of the major hematopoietic cytokines [11]. This cell line also provides a permissive environment for B-cell differentiation and for the generation of granulocytes depending on the culture conditions [9]. In vivo, hematopoietic cells are observed in the grafts of MS-5 and bone marrow. These cells are granulocytic cells, as determined by their morphology and chloroacetate esterase activity. Hematopoietic cells were observed surrounded by alkaline phosphatase–positive cytoplasmic processes of MS-5 cells. This morphology mirrors the in situ bone marrow appearance; differentiated alkaline phosphatase–positive fibroblastic reticular cells possess thin cytoplasmic processes enveloping immature granulocytic cells, which suggests that they are functional interactions between the two cell types [16–20].

MS-5 does not seem to have the ability to attract or home hematopoietic stem cells or early progenitors from the host animal. Alternatively, it could induce injected stromal cells to produce a microenvironment suitable for the growth of hematopoiesis; this hypothesis can be excluded by the observation of granulocytopoiesis development when stromal cells are eliminated from the injected cell suspension after CD45⁺ cell purification.

Neither granulopoiesis, erythropoiesis, nor lymphopoiesis is observed when MS-5 cells are injected with c-kit⁺Sca-1⁺Lin^– cells. From these results, we assume that MS-5 provides an environment for immature cell survival. Hematopoietic cells isolated from the graft are likely hematopoietic stem cells. First, they induce the survival of lethally irradiated mice and support bone marrow recovery. Second, they are able to form hematopoietic colonies in the spleens. Third, they give rise to a limited number of colonies when plated in methylcellulose.

Our interpretation is that MS-5 is unable to induce the differentiation of hematopoietic stem cells into granulocyte precursors but enhances one or more of the following: proliferation rate, survival or maturation rate of granulocytic progenitors/precursors, and survival of end-stage polymorphonuclear granulocytes.

The wave of granulocytopoiesis was of limited duration, however. Thus, other cells or factors are likely necessary for long-term hematopoiesis. In this context, macrophages are a major source of interleukin-1 and tumor necrosis factor, factors that greatly upregulate the production of several cytokines by bone marrow stromal cells [21–24].

	ACKNOWLEDGMENTS

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This work was supported by a grant of the Fond National de la Recherche Scientifique. Frédérique Hubin and Zakia Belaid are Télévie fellows. The technical assistance of Marie-Christine Petit and Delphine Delneuville is acknowledged. We also thank Charles Lambert and Philippe Ruggeri for their contribution in the transfection procedures.

DISCLOSURES
The authors indicate no potential conflicts of interest.

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