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Stem Cells, Vol. 16, No. 1, 61-65, January 1998
© 1998 AlphaMed Press

Stromal-Conditioned Medium Synergizes with Thrombopoietin in Stimulating Megakaryocytopoiesis

Mirta Schattner, David Green, Isaac Cohen

Atherosclerosis Program, Rehabilitation Institute of Chicago and Northwestern University, Department of Cell, Molecular and Structural Biology, Chicago, IL, USA

Key Words. Stroma • Conditioned medium • Thrombopoietin • Cytokines • Megakaryocytes • Contact

Dr. Isaac Cohen, Northwestern University Medical School, Rehabilitation Institute of Chicago, 345 East Superior Street, Room 1407, Chicago, IL 60611, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously demonstrated that stromal cells release a soluble factor(s) that enhances thrombopoietin (TPO)-induced megakaryocyte (MK) production. We have now further characterized this enhancing activity of human bone marrow stroma and have also analyzed the role of direct contact of stromal cells with human bone marrow CD34+ cells on MK production. The three- to sixfold stromal-enhancing activity of conditioned media is constitutive and is not influenced by the presence of TPO. The addition of interleukins 3, 6 (IL-3, IL-6), stem cell factor (SCF), and TPO at concentrations found in stroma to a stroma-free system only enhanced by 58% the TPO-stimulated MK production. No influence of heparan sulfates on MK production was observed. In co-cultures of CD34+ cells in direct contact with stromal cells, nonadherent and adherent MK, and proplatelet structures were observed, either in the absence or presence of TPO. Direct contact did not further improve the enhancing activity of stromal cells on MK production induced by TPO. We conclude that the stromal enhancement of MK production is neither accounted for by stromal TPO, IL-3, IL-6, and SCF activities nor modified by physical contact.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The role of marrow stromal cells on hematopoiesis is usually studied in long-term bone marrow cultures [1-4]. In this system, the release of soluble factors from bone marrow stroma as well as the direct interaction between CD34+ cells and extracellular matrix regulate the proliferation and differentiation of hematopoietic stem and progenitor cells [5-8]. The role of bone marrow stroma on megakaryocytopoiesis per se has been less extensively studied. While unactivated human BM microvascular endothelial cells support megakaryocytopoiesis [9], activated human umbilical vein endothelial cells induced adherence of human megakaryocytes (MKs) and increased MK maturation without affecting proliferation [10]. Recently, it has also been demonstrated that although primary mouse stromal cells [11] and the murine MS-5 stromal cell line [12] support proliferation and differentiation of MK progenitors, due to thrombopoietin (TPO) synthesis, proplatelet formation is inhibited [11]. In a previous study, we demonstrated that a soluble factor(s) from human bone marrow stroma enhances TPO-stimulated megakaryocyte development [13]. In the present investigation, additional studies were carried out to further characterize this factor(s). In addition, the role of direct contact of human bone marrow CD34+ cells with stroma on MK production was also investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of Cells
Nonadherent mononuclear cells (MNCs) and CD34+ cells were purified from human bone marrow as described [13]. Bone marrow samples, obtained from the femur of hematologically normal patients having total hip arthroplasty, were collected in accordance with the guidelines of the Institutional Review Board on human subjects.

Human Bone Marrow Stroma
A confluent stroma layer was prepared by adding to the adherent MNCs a medium consisting of Iscove's modified Dulbecco's medium (IMDM) supplemented with 12.5% fetal bovine serum (FBS), 12.5% horse serum, nonessential and essential amino acids, Na pyruvate (100 mM), vitamins, hydrocortisone (5 x 10–6 M) and 2-mercaptoethanol (10–8 M). At confluence, usually reached within a week, the adherent stromal cells were detached by adding trypsin (0.25%)-EDTA (1mM). Following washing with IMDM-base medium with 10% FBS, 200,000 or 800,000 cells/well were plated on 24 or 6 collagen-coated plates, respectively. The following day, the adherent cells were irradiated with a dose of 12 Gy from a 137Cs source (Gammacell 40; Nordion International Inc.; Kanata, Ontario, Canada). Collagen-coated plates were from Collaborative Research (Becton-Dickinson; San Jose, CA).

Conditioned Media
Conditioned medium (CM) was prepared by adding IMDM containing 1% human serum albumin (HSA), 2.5% normal serum (NS) and 50 U/ml TPO (CM-TPO) to six-well-stroma-coated plates. After 48-72 h supernatant was collected and centrifuged at 260g for 10 min at 4°C. In some experiments, CM was prepared without TPO.

Culture Conditions
Cells were cultured for 12-14 days at 37°C in a 5% CO2 fully humidified atmosphere in an IMDM-based medium with 1% HSA and 2.5% NS. To prevent the inhibitory effects on MK growth of transforming growth factor-ß, ß-thromboglobulin, and platelet factor 4 released from activated platelets [14-16], serum was obtained by recalcification of citrated platelet-poor plasma. The effect of stroma from human bone marrow was evaluated by culturing purified CD34+ cells in the presence or absence of TPO (Zymogenetics Corp.; Seattle, WA), either in direct contact with stroma (contact) or in inserts (Collaborative Research) separated from the stroma by a 0.41 µm pore membrane (noncontact). In control experiments, CD34+ cells were cultured in inserts placed over stroma-free wells (stroma-free). Additional interleukin 3 (IL-3), IL-6, and stem cell factor (SCF), when used, were from R&D Systems (Minneapolis, MN). Heparan sulfates (HSs) from bovine kidney and bovine intestinal mucosa were from Sigma (St. Louis, Mo). Completely desulfated-N-acetylated heparin (CDSNAc), completely desulfated-N-sulfated heparin (CDSNS) and N-desulfated-N-acetylated heparin (NDSNAc) were from Seikagaku America, Inc.; Ijamamsville, MD.

Immunocytochemistry
Adherent MKs were scored following immunochemical staining. The well plates were washed twice with 0.05 M Tris, 0.15 M NaCl pH 7.6 tris-buffered saline (TBS). After fixation for two min with methanol-acetone (1:1), sequential incubations, interspersed by washing with TBS, were carried out with blocking solution (5% human serum in TBS, 15 min), a cocktail of monoclonal antibodies (mAbs) antiGPIIb (CD41) and GPIb (CD42b) (2 µg/ml, one h) and a secondary antibody (rabbit anti-mouse IgG coupled to alkaline phosphatase [Sigma], diluted 1:200 with 15% human serum in TBS, 30 min). Plates were then washed and incubated for 15 min with 0.1 M Tris pH 8.2, followed by a 20-min incubation with alkaline phosphatase substrate Fast red (Sigma). The reaction was stopped by rinsing with water.

Cell Labeling and Flow Cytometric Analysis
Phenotypic analysis for nonadherent CD34+ cells and MKs was performed using anti-CD34-phycoerythrin (Becton-Dickinson) and anti-CD41-FITC (Coulter-Immunotech; Westbrook, ME), respectively, as previously described [13].

Immunoenzymatic Assays
Stroma (confluence at 800,000 cells) in the absence or presence of TPO (50 U/ml) was incubated with IMDM-base medium containing 1% HSA and 2.5% NS for 12-14 days at 37°C in a 5% CO2 fully humidified atmosphere. The supernatant was then centrifuged to eliminate cell debris and samples were frozen (-70°C) until assayed. IL-6, IL-3, and SCF were measured using enzyme-linked immunosorbent assays (ELISA) kits from R&D Systems in accordance with the manufacturer's recommendation. The control sample was IMDM-based medium with 1% HSA and 2.5% NS and the values of IL-6, IL-3, and SCF found were subtracted from the sample values.

Statistical Analysis
Statistical analysis was performed using the two-tailed Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of Stromal Cytokines on MK Production
To evaluate whether the stromal enhancing activity on MK production was related to TPO release by stromal cells, we analyzed the effect of stroma on CD34+ cultures supplemented or not with exogenous TPO. The addition of TPO to CD34+ cells either in the presence or absence of stroma significantly stimulated MK development ( Table 1). In the absence of exogenous TPO, MK formation (2 x 103) which was observed only in the presence of stroma, was decreased by 50% following the addition of soluble c-mpl receptor (10 µg/ml). The endogenous TPO, presumably responsible for this relatively small stroma-induced MK production, corresponded to a level of 0.8-1.5 U/ml TPO in our culture conditions. Such a small TPO concentration did not enhance MK production stimulated by 50 U/ml TPO in a stroma-free system.


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  		Table 1. Effect of stroma on MK production
 
We next evaluated the stromal release of different pleiotropic growth factors that are known to modulate MK development [13, 17-19] . The concentrations of IL-3, SCF, and IL-6 released in the culture medium were not significantly different in the presence or absence of TPO (5.5 ± 2, 962 ± 423 and 306 ± 113 pg/ml of IL-3, SCF, and IL-6, respectively, without TPO and 4 ± 1, 940 ± 269, and 347 ± 94 pg/ml of IL-3, SCF, and IL-6, respectively, with TPO). Addition of IL-3, IL-6, and SCF, in concentrations secreted by stroma, enhanced by 58% TPO-stimulated MK production in stroma-free conditions (3.8 x 105 ± 4 x 104 SEM MKs in presence of TPO/IL-3/SCF/IL-6 versus 2.45 x 105 ± 5.7 x 104 SEM MKs in presence of TPO alone, n = 4, p < 0.02).

The constitutive or inducible nature of the MK-enhancing activity of stroma was investigated using conditioned medium. As can be seen in Figure 1 , a sixfold stromal-enhancing effect on MK development was obtained in the presence of stroma-conditioned medium obtained either in the presence or absence of TPO.



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  		Figure 1. Effect of stromal conditioned medium (CM) on MK production. 70 x 103 CD34+ cells/ml were seeded in a 2 ml final volume culture medium in the presence and absence of CM or TPO. CM+TPO, CM obtained in the presence of 50 U/ml TPO; CM(+TPO), CM obtained in the absence of TPO with 50 U/ml TPO added in culture with CD34+ cells. The difference between MK numbers obtained with CM+TPO and CM(+TPO) was not statistically significant.

 
Role of HSs on MK Production
The effect of various TPO/HS combinations on enhancement of MK production was investigated. HS from both bovine intestinal mucosa and bovine kidney as well as modified heparins (CDSNAc, CDSNS and NDSNAc) at concentrations ranging from 0.5 to 50 µg/ml did not have any enhancing effect on MK development stimulated by TPO and stromal concentrations of IL-3, IL-6, and SCF.

Effect of Direct Contact of CD34+ Cells with Stroma on MK Production
Culture of CD34+ cells in the absence of stroma and TPO did not result in MK formation. In the presence of stroma, while TPO enhanced MK production, adherent MKs (AMKs), nonadherent MKs (NAMKs) and proplatelet structures occurred whether in the presence or absence of TPO. In all cases, the majority of MKs (89%) were in the nonadherent phase ( Table 2). Similar numbers of MKs were obtained in the presence of TPO, whether noncontact or contact systems (AMKs + NAMKs) were used (1.9 x 104 ± 0.9 x 104 SEM MKs in noncontact versus 1.4 x 104 ± 0.7 x 104 SEM MKs in contact system, n = 3, p > 0.1).


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  		Table 2. Adherent and nonadherent MK formation in presence of stroma
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies on the role of stroma on hematopoiesis have primarily used long-term cultures [1-8]. This culture system promotes mainly myelopoiesis and lymphopoiesis while megakaryocytopoiesis is markedly suppressed due to the presence of transforming growth factor-ß and ß-thromboglobulin [20]. Using short-term cultures and changing culture medium conditions, we have previously demonstrated that human bone marrow stroma enhanced TPO-induced MK growth by releasing a soluble factor(s) [13]. In the present study, we observed that the MK growth-promoting activity of stroma is partly due to endogenous secretion of TPO since treatment of stroma with the c-mpl soluble receptor decreased production of nonadherent MKs. However, the small amount of TPO synthesized by stromal cells could not explain the marked enhancement of MK production in the presence of stroma and exogenous TPO. To determine whether exogenous TPO modulates the stromal enhancement activity, stromal CMs were prepared in the presence or absence of TPO. No significant difference was observed, indicating the constitutive nature of this activity.

Although TPO is the main regulator of the different stages of megakaryocytopoiesis [21-25], several studies demonstrated that other pleiotropic growth factors enhance the activity of TPO on MK progenitor proliferation and maturation. We therefore investigated whether IL-3, IL-6, or SCF contributed to the stromal enhancement of TPO-stimulated megakaryocytopoiesis. Our results showed that IL-3, IL-6, and SCF, constitutively synthesized by bone marrow stroma, only increased by 58% the TPO-stimulated MK production when added at stromal concentrations. This enhancement was too modest to account for the total three- to sixfold enhancement of stromal stimulation of MK production. However, we do not rule out the possibility that the concerted action of either more cytokines or different cytokine combinations could account for the synergistic effect of stromal cells on TPO-mediated MK growth.

Since HS in combination with cytokines plays a positive role in long-term bone marrow culture-initiating cell maintenance [26, 27] and megakaryocytopoiesis [28], we investigated the effect of various HSs and modified heparins on MK production stimulated by TPO and stromal concentrations of IL-3, IL-6, and SCF. The presence of the different heparins did not modify MK production induced by TPO and IL-3, IL-6, and SCF. We therefore could not confirm the reported stimulation of megakaryocytopoiesis by HSs [28]. The positive effect of HSs in combination with cytokines for the maintenance of long-term culture-initiating cells [27] is probably due to inhibition of differentiation stages, which may explain their lack of effect on megakaryocytopoiesis.

Using human bone marrow stroma and CD34+ cells in a contact system showed that proplatelet structures and nonadherent and adherent MKs occur in the presence and in the absence of exogenous TPO. The frequency of nonadherent MKs was overwhelmingly larger than that of adherent MKs. Moreover, nonadherent MK production was similar when CD34+ cells were cultured either in transwell systems separated from the stroma or in direct contact with the stroma. These results indicate that A) direct contact of CD34+ cells with stroma did not further improve the enhancing effect of stroma-derived soluble factor(s) on TPO-induced MK growth and B) contact does not appear to have an important influence on MK production. The latter interpretation does not preclude a critical role of stromal contact in an in vivo microenvironment whereby, in the absence of high concentrations of exogenous cytokines, it would modulate adhesion, proliferation, and differentiation of MKs by physical contact and secretion of cytokines.

In conclusion, stroma-secreted TPO, IL-3, SCF, and IL-6 do not entirely explain the enhancement of MK production by exogenous TPO. The stromal MK enhancement factor(s) is constitutive, and direct contact of progenitors with the stroma did not further increase the enhancement effect of stroma on TPO-induced MK growth. These results have a direct implication on ex vivo expansion of MKs, which may be used in a clinical setting to offset post-transplant thrombocytopenia. While stroma significantly enhances TPO-stimulated megakaryocytopoiesis, contact with stroma is not necessary, and CM derived from stroma may be used in culture. Elucidation of the nature of the stroma enhancing factor(s) awaits further investigation and will be useful for the design of ex vivo expansion systems.


   Acknowledgments
 
This work was supported in part by a grant from the U.S. Army Medical Research and MaterielCommand (DAMD17-94-J-4465) and the Rehabilitation Institute of Chicago. We thankDr. Don Foster (Zymogenetics; Seattle, WA) for providing TPO and the soluble mpl receptor,Dr. Richard L. Wixson for the availability of bone marrow samples, and Dr. A. Rademaker for the statistical analysis.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication October 2, 1997.



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