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Stem Cells 2003;21:557-567 www.StemCells.com
© 2003 AlphaMed Press

Neutralization of Autocrine Transforming Growth Factor-ß in Human Cord Blood CD34+CD38-Lin- Cells Promotes Stem-Cell-Factor-Mediated Erythropoietin-Independent Early Erythroid Progenitor Development and Reduces Terminal Differentiation

Salem Akela, Cari Petrow-Sadowskib, Mary J. Laughlinc, Francis W. Ruscettia

a Leukocyte Biology Section, Basic Research Laboratory, Center for Cancer Research, NCI-Frederick, Frederick, Maryland, USA;
b Intramural Research Support Program, SAIC-Frederick, Frederick, Maryland, USA;
c Allogeneic Transplant Program, Ireland Cancer Center, Case Western Reserve University, Cleveland, Ohio, USA

Key Words. Transforming growth factor-ß1 • Serum free • Autocrine regulation • Erythropoiesis • Anti-TGF-ß

Francis W. Ruscetti, Ph.D., Leukocyte Biology Section, Building 567, Room 254, NCI-Frederick, Frederick, Maryland 21702-1201, USA. Telephone: 301-846-1504; Fax: 301-846-7034; e-mail: Ruscettif{at}ncifcrf.gov


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor (TGF)-ß1 exerts autocrine and paracrine effects on hematopoiesis. Here, we have attempted to evaluate the effect of endogenous TGF-ß1 on early erythroid development from primitive human hematopoietic stem cells (HSCs) and to assess the effects of TGF-ß1 on different phases of erythropoiesis. Cord blood CD34+CD38- lineage-marker-negative (Lin-) cells were cultured in serum-free conditions using various combinations of stem cell factor (SCF), erythropoietin (Epo), and TGF-ß-neutralizing antibody. Generation of erythroid progenitors was assessed using colony assay and flow cytometry. Terminal erythroid differentiation was examined when SCF/Epo-stimulated cells were recultured in the presence of Epo with and without TGF-ß1. Anti-TGF-ß augmented the proliferation of CD34+CD38-Lin- cells (day 21) in SCF-stimulated (6.4-fold ± 1.5-fold) and SCF/Epo-stimulated (2.9-fold ± 1.2-fold) cultures. Cells stimulated by SCF/Epo underwent similar levels of erythroid differentiation with and without anti-TGF-ß. While SCF alone stimulated the production of tryptase-positive mast cells, cells stimulated by SCF/anti-TGF-ß were predominantly erythroid (CD36+CD14- and glycophorin A positive). A distinct expansion of erythroid progenitors (CD34+CD36+CD14-) with the potential to form erythroid colonies was seen, revealing early Epo-independent erythroid development. In contrast, the kinetics of erythroid progenitor generation from primitive HSCs indicate that TGF-ß1 is not inhibitory in late erythropoiesis, but it accelerated the conversion of large BFU-E into colony-forming units-erythroid. Finally, TGF-ß1 accelerated Epo-induced terminal erythroid differentiation and resulted in a greater level of enucleation (22% ± 6% versus 7% ± 3%) in serum-free conditions. Serum addition stimulated enucleation (54% ± 18%), which was lower (26% ± 14%) with anti-TGF-ß, suggesting that optimal erythroid enucleation is Epo dependent, requiring serum factors including TGF-ß1.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor (TGF)-ß1 plays a pivotal role in regulating hematopoiesis. This pleiotropic factor can exert either negative or positive effects on proliferation, differentiation, or cell survival, depending on the developmental stage of the target cells and components of the cellular environment [1,2].

Previous studies addressing the effect of TGF-ß1, or its neutralization by antibodies or antisense oligonucleotides in association with other cytokines, on the regulation of primitive hematopoietic stem cells (HSCs) have demonstrated that: A) TGF-ß1 has both paracrine and autocrine effects on HSCs [3–6]; B) TGF-ß1 regulates quiescence of HSCs, and C) primitive HSCs, such as human CD34+CD38- lineage-marker-negative (Lin-) and murine Lin- Hoescht 33342low/Rhodamine 123low cells, are more highly sensitive to cell cycle inhibition than their more mature counterparts [6–8]. The ability of TGF-ß1 to maintain primitive HSCs in a quiescent state has been explained by downmodulation of the expressions of various cytokine receptors including: the receptor for stem cell factor (SCF) (c-kit), the thrombopoietin receptor (c-Mpl), the interleukin (IL)-6 receptor, and the Flt-3 ligand receptor [1,9–11]. Moreover, it has been argued that TGF-ß1 may control apoptosis in normal hematopoiesis [11], but recent findings, showing an in vitro reversibility of the cell cycle inhibitory effect exerted by TGF-ß1 on HSCs [8,12–13] and increased HSC survival after TGF-ß neutralization, are not compatible with the induction of cell death [2,14]. Although, the role of TGF-ß1 in regulating HSC proliferation is well established, little is known about the relative contributions of autocrine and paracrine TGF-ß1 to HSC differentiation.

TGF-ß1 is a key regulator of end-stage development of several HSC lineages, such as erythroid. In vitro, it has been identified as a cell cycle inhibitor for early, but not late, erythroid progenitors [15,16]. Other studies, using erythroleukemia cell lines and nontransformed human cells, showed that TGF-ß1 triggered late erythroid differentiation and promoted maturation processes such as hemoglobin (Hb) synthesis [16–20]. The balance between positive and negative cytokine signals regulating the cell cycle may govern the regulation of differentiation in the erythroid compartment [21,22]. This prompted us to investigate the effect of endogenous TGF-ß1 on the growth of primitive HSCs and erythroid development. Recently, Fortunel et al. showed that CD34+ cord blood (CB) progenitors treated with anti-TGF-ß had more in vitro colony formation of most lineages, including BFU-E [23]. The action of anti-TGF-ß on CB CD34+C38-Lin- cell growth and differentiation was explored in a serum-free/erythropoietin (Epo)-free culture system previously shown to allow the proliferation of erythroid progenitors [24]. Here, we show that neutralizing TGF-ß1 in the presence of SCF led to Epo-independent development of early erythroid progenitors that required TGF-ß1 to fully differentiate in vitro.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Growth Factors and Antibodies
The cytokines recombinant human (rh)-Epo (1 U/ml), rh-SCF (100 ng/ml), rh-IL-3 (10 ng/ml), and rh-IL-6 (10 ng/ml), from Peprotech (Rocky Hill, NJ; http://www.peprotech.com), and rh-TGF-ß1 (1 to 10 ng/ml), a gift of Bristol-Myers Squibb (Seattle, WA; http://www.bms.com), were used. Anti-TGF-ß antibody (IgG1, ID11), provided by Hazelton Labs (Rockville, MD), which neutralizes the three mammalian isoforms of TGF-ß [25], and anti-rh-Epo antibody (IgG1; R&D Systems; Minneapolis, MN; http://www.rndsystems.com) were used at 20 µg/ml. Anti-tryptase monoclonal antibody (mAb) was purchased from Chemicon International, Inc. (Temecula, CA; http://www.chemicon.com). Purified mouse antihuman IgG1 isotype control antibody (PharMingen [BD Biosciences]; San Diego, CA http://www.bdbiosciences.com/pharmingen) was used in control cultures when indicated. For flow cytometric analyses, IgG1 isotype control mouse mAbs conjugated to either fluorescein isothiocyanate (FITC), phycoerythrin (PE), or peridinin chlorophyll protein (PerCP) were used along with anti-human specific mAbs (PE-CD117, PerCP-CD34, FITC-CD36, PE-CD61, PE-CD14, FITC-CD13; Becton Dickinson; San Diego, CA; http://www.bd.com) and FITC-conjugated glycophorin-A (GPA) (Immunotech; Marseille, France; http://www.beckmancoulter.com).

Isolation of Hematopoietic Stem/Progenitor Cells
CB samples were collected after normal deliveries according to approved guidelines of Case Western Reserve University. From mononuclear cells (MNCs), CD34+ cells were isolated by two cycles of positive selection using a magnetic cell-sorting system, MACS (CD34 isolation kit; Miltenyi Biotech; Auburn, CA; http://www.miltenyibiotec.com). CD36+ cells were positively selected from cultures using magnetic beads. Separation of CD34+CD38-Lin- cells was achieved in two steps. First, primitive cells were enriched by depleting committed progenitors and differentiated cells using a primitive lineage depletion cocktail (Stem Cell Technologies; Vancouver, Canada; http://www.stemcell.com). Next, CD34+ cells were positively selected to a purity of >90%.

Generation of Erythroid Progenitors from Primitive HSCs in Liquid Culture (Phase I)
CD34+ or CD34+CD38-Lin- cells were suspended at 105/ml in serum-free medium (Iscove’s modified Dulbecco’s medium [IMDM]; Life Technologies; Rockville, MD; http://www.lifetech.com) supplemented with 20% BIT 9500 (detoxified bovine serum albumin, charcoal filtered to remove steroids and cytokines [26], iron-saturated transferrin, and insulin; Stem Cell Technologies), 100 U/ml penicillin-streptomycin, and 2 mM L-glutamine. Cytokines and antibodies were added as described. Cell counts were maintained below 106/ml by repeated cell dilution in fresh medium containing cytokines and/or antibodies.

Terminal Erythroid Differentiation in Liquid Culture (Phase II)
Washed cells obtained after 10 days of SCF/Epo stimulation were cultured at 2 x 105/ml in a second phase of erythroid culture (PII, 14 days) in IMDM containing 20% BIT, 10-5 M ß-mercaptoethanol (ß-ME), 10-6 M dexamethasone, and 1 U/ml rh-Epo. TGF-ß1 (5 ng/ml) was added on day 0, 5, or 10 of PII. In some cultures, we used 30% fetal bovine serum (FBS) (GIBCO/BRL; Grand Island, NY; http://www.invitrogen.com) with and without anti-TGF-ß. Hb-containing cells were identified by the acetic acid-benzidine peroxide procedure [27], and erythroid maturation was assessed microscopically on cytocentrifuge slides stained with Giemsa.

Epo-Independent Liquid Culture of Erythroid Progenitors
Freshly isolated peripheral blood (PB) MNCs from normal adults were obtained from the National Institutes of Health blood bank following informed consent. Cells were cultured as described by Fibach et al. [28]. In brief, cells were grown at 106/ml in IMDM, 10% FBS, 1 µg/ml cyclosporin A, and 10% conditioned medium from the K5637 bladder carcinoma cell line (a source of burst promoting activity [BPA]) with and without TGF-ß1 (5 ng/ml). After various days of culture, the types and contents of erythroid colony-forming cells (CFCs) were determined.

Colony Assays
Cells were plated at 1-5 x 104/ml in triplicate in 1 ml of IMDM containing 0.9% methylcellulose, 30% FBS, 1% bovine serum albumin, 10-4 M ß-ME, 3 U/ml rh-Epo, 50 ng/ml rh-SCF, and 10 ng/ml rh-IL-3. Colony-forming units-granulocyte-macrophage (CFU-GM) were assayed in the presence of rhGM-CSF and IL-3. After 12-14 days of incubation, colonies were counted and classified, as reported elsewhere [16,20,29].

Flow Cytometry and Immunocytochemical Staining
Slides from cytocentrifuged cells, fixed in Cornoy’s fluid and blocked with normal rabbit serum, were reacted with anti-tryptase mAb for 30 minutes. The reaction was detected using the APAAP method (APAAP kit system; DAKO; Carpenteria, CA; http://us.dakocytomation.com). Cells were counterstained with hematoxylin, and 300-500 cells were examined microscopically. For flow cytometry, cells were washed in 2% normal human AB serum in phosphate-buffered saline (PBS), blocked with 5 µl of a mixture of normal human and mouse sera, washed, and incubated with 15 µl of each of the mAbs and isotype controls at 4°C for 30 minutes. After washing, cells were suspended in equal volumes of PBS and 4% paraformaldehyde. Analysis was carried out using a FACScan cytometer (Becton Dickinson) and WinMIDI 2.8 software developed by Dr. Joe Trotter, Purdue University, West Lafayette, IN.

Statistical Analyses
Results are expressed as a mean value ± standard deviation (SD). The paired Student’s t-test was used to determine the statistical significance of differences between means, which were calculated from at least three experiments.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of TGF-ß Neutralization on Proliferation/Differentiation of CB CD34+ Cells Stimulated by SCF and/or Epo
CB CD34+ cells were grown in serum-free conditions with growth factors and anti-TGF-ß for 21 days. As shown in Figure 1A, 1B, and 1CGo, differential proliferations of CD34+ cells were observed in the presence of Epo, SCF, and SCF/Epo (approximately 9-, 24-, and 1,650-fold increases, respectively). In the case of Epo, cells were mainly erythroid (data not shown). Culture with SCF alone led to the appearance of an increasing proportion of cells with tryptase activity (Table 1Go), indicating their mast cell nature [30]. The synergistic effect of Epo with SCF induced erythroid differentiation. Cells were 82% ± 9% CD36+, 76% ± 4% GPA+, 2% ± 2% tryptase positive, and 5% ± 2% CD14+. Inclusion of anti-TGF-ß had a significant (p < 0.01) effect on the proliferation of CD34+ cells stimulated by SCF/Epo but not by SCF or Epo (Fig. 1Go). In the same cultures, anti-TGF-ß did not have a significant effect on cell differentiation. In serum-free cultures without added cytokines, anti-TGF-ß did not have any effect on cell growth or differentiation (data not shown).



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  		Figure 1. The effect of anti-TGF-ß antibody on proliferation of CD34+ (A, B, and C) and CD34+CD38-Lin- (D, E, and F) cells. Cells were seeded at 105/ml in serum-free liquid culture in the presence of Epo (A and D), SCF (B and E), and SCF/Epo (C and F). Cell growth with the addition of anti-TGF-ß or control Ab was determined at days 7, 10, 14, and 21 by counting viable cells using trypan blue exclusion stain. Results are expressed as total cell count per ml of initial culture and represent mean ± SD of three independent experiments.

 

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  		Table 1. Tryptase-positive cells in serum-free cultures of CD34+ and CD34+CD38-Lin- cells stimulated by SCF with and without anti-TGF-ß
 
Neutralization of TGF-ß Augments Proliferation of CB CD34+CD38-Lin- Cells Grown in SCF and Reveals an Early Epo-Independent Erythroid Development
Next, we determined whether the more primitive CD34+CD38-Lin- cell population, which contains an enriched population of stem cells, responded to the cytokine/anti-TGF-ß treatment in the same manner as did CD34+ cells (Fig. 1D, 1E, and 1FGo). After 21 days, approximately 3-, 110-, and 6,800-fold increases in cell numbers were found in cultures stimulated with Epo, SCF, and Epo/SCF, respectively. The inclusion of anti-TGF-ß resulted in a significantly greater (p < 0.01) cell proliferation in SCF-stimulated (6.4-fold ± 1.5-fold) and SCF/Epo-stimulated (2.9-fold ± 1.2-fold) cultures (day 21). The greater proliferation was due to a greater production of erythroid cells in SCF/Epo-stimulated cultures (Fig. 2BGo). Unlike cultures of CD34+ cells, cells produced by the SCF/ anti-TGF-ß treatment of CD34+ CD38-Lin- cells did not feature mast cell properties; they were predominantly tryptase negative (Table 1Go), and their vast majority exhibited an erythroid phenotype (CD36+CD14- and, to a lesser extent, GPA+) (Fig. 2AGo). In the same cultures, the addition of anti-TGF-ß resulted in a nonsignificant (p > 0.05) lower number of mast cells (Table 1Go). Due to the presence of CD36 on several hematopoietic populations [24,31], CD34+CD36+ cells were isolated from day 7 cultures. The erythroid nature of these cells was confirmed in colony assays, where 91% of the colonies were BFU-E/CFU-E and 7% were CFU-GM (Table 2Go). Erythroid differentiation was also confirmed by reculturing day-14 SCF/anti-TGF-ß-stimulated cells in the presence of Epo alone (PII). Within 5 days, cells underwent two to three divisions and were mostly (>70%) benzidine positive, while cells grown initially in SCF alone were benzidine negative (>90%) (Fig. 3Go).



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  		Figure 2. Effect of anti-TGF-ß antibody on the phenotypes of cells formed in cultures of CD34+CD38-Lin- cells. Cells were seeded at 105/ml in serum-free medium in the presence of SCF with anti-TGF-ß or control Ab (A) and SCF/Epo with anti-TGF-ß or control Ab (B). Aliquots of cultured cells were harvested at day 14 and subjected to fluorescence-activated cell sorter analysis for CD36, GPA, CD14, CD13, and CD61 markers. Total numbers of erythroid (CD36+CD14- or GPA+), monocytic/macrophage (CD36+CD14+), myeloid (CD13+), and megakaryocytic (CD61+) cells were estimated and expressed per ml of initial culture. Results represent mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01 (versus control cultures without anti-TGF-ß).

 

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  		Table 2. Composition of CFCs in CD34+CD36+ cells isolated from serum-free cultures of CD34+CD38-Lin- cells stimulated by SCF with and without anti-TGF-ß
 


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  		Figure 3. Morphologies of cells initially developed from CD34+CD38-Lin- cells in the presence of SCF with and without anti-TGF-ß antibody and stimulated later by Epo. Cells were stimulated for 14 days with SCF + control Ab (left) and SCF + anti-TGF-ß (right). Cells (2 x 105/ml) were transferred into PII of erythroid culture in the presence of Epo. After 7 days, cell morphologies were assessed on cytocentrifuged slides stained with benzidine and Harris hematoxylin (magnification = 1,000x).

 
Inclusion of anti-Epo (20 mg/ml) in cultures containing SCF/anti-TGF-ß had no significant effect on proliferation/differentiation of CD34+CD38-Lin- cells (p > 0.2) (Table 3Go). Conversely, the addition of anti-Epo to SCF/Epo-treated cultures resulted in an 87% lower cell expansion and blocked erythroid differentiation (p < 0.01) (Table 3Go). Thus, the early erythroid production mediated by anti-TGF-ß in SCF-stimulated cultures was likely not related to traces of Epo contaminating culture gradients or endogenous Epo secreted from cultured cells [32].


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  		Table 3. Effects of anti-Epo antibody on erythropoiesis in serum-free cultures of CD34+CD38-Lin- cells stimulated by SCF with and without EPO and with and without anti-TGF-ß
 
Effect of Anti-TGF-ß on Kinetics of Erythroid Progenitors Generated from CD34+CD38-Lin- Cells by SCF and/or Epo
The greater production of erythroid cells by the addition of anti-TGF-ß to cultures stimulated with SCF with and without Epo (Fig. 2Go) suggests that inhibition of TGF-ß1 influences the generation of erythroid progenitors from uncommitted (CD34+CD38-Lin-) HSCs. Anti-TGF-ß induced a significantly greater (p < 0.01) expansion of erythroid CD34+CD36+ cells (about eightfold, day 10) in cultures stimulated with SCF (Fig. 4AGo), whereas, inhibition of TGF-ß had a significant (p < 0.05), but little (1.2-fold), effect on the expansion of CD34+CD36+ cells in cultures stimulated with SCF/Epo (Fig 4BGo).



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  		Figure 4. Effect of anti-TGF-ß antibody on production of CD34+ and CD34+CD36+ cells from CD34+CD38-Lin- cells. Cells were harvested from the same cultures described in Figure 1Go. Cells grown in the presence of SCF with anti-TGF-ß or control Ab (A) and SCF/Epo with anti-TGF-ß or control Ab (B) for 10 days were subjected to FACS analysis for CD34+ and CD34+CD36+ (erythroid progenitor) cells. The total numbers of produced progenitors were calculated from results of fluorescence-activated cell sorter and cell counts and are expressed per ml of initial seeded cells, i.e., per 105 CD34+CD38-Lin- cells. * p < 0.05 and** p < 0.01 (versus control cultures without anti-TGF-ß).

 
Employing colony assays, anti-TGF-ß enhanced, with time, the production of both early (large BFU-E) and late (small BFU-E/CFU-E) erythroid progenitors from CD34+CD38-Lin- cells stimulated with SCF alone (Fig. 5A and 5BGo). The enhancement was exponential between day 10 and day 14, but gradually declined after that (data not shown). Conversely, in cultures stimulated with SCF/Epo, the addition of anti-TGF-ß had no significant (p > 0.1) effect on the development of large BFU-E (Fig. 5CGo) but stimulated a delayed production of small BFU-E/CFU-E between days 10 and 14 (p < 0.01) (Fig. 5DGo). Interestingly, the number of erythroid CFCs formed in cultures supported with SCF/anti-TGF-ß for 14 days was comparable with those found in cultures supported with SCF/Epo for 10 days. Since results shown in Figure 5Go are per original cell input, these findings suggest that erythroid CFCs capable of surviving in the presence of SCF/Epo can survive as well with SCF/anti-TGF-ß.



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  		Figure 5. Kinetics of erythroid CFC production from CD34+CD38-Lin- cells. Cells were harvested at days 10, 12, and 14 from the same cultures described in Figure 1Go. Cells were plated at 104 cells/ml for colony formation. Colonies were classified as large BFU-E (left) and CFU-E/ small BFU-E (right), and their total numbers were expressed per ml of initial liquid culture, i.e., per 105 CD34+CD38-Lin- cells.

 
TGF-ß1 Accelerates/Expands the Generation of Late Erythroid Progenitors in the Absence of Epo
Next, we tested the role of TGF-ß1 on the development of erythroid progenitors in the absence of exogenous Epo. Cultures were initiated with PB MNCs as a rich source of human early erythroid progenitors (large BFU-E) [28,33]. Production of erythroid CFCs in this system was maximal at day 7, and most of the generated CFCs represented the more mature progenitors (small BFU-E and CFU-E) (Fig. 6Go). Treatment with TGF-ß1 yielded a twofold, significantly greater (p < 0.05) total number of colonies (day 7, versus control cultures). This greater number of CFCs in TGF-ß1-treated cultures was transient, and colonies formed in semisolid cultures were smaller but more hemoglobinized than control-culture-derived colonies. By day 10, the number of produced small BFU-E/CFU-E was 40% lower (versus day 6) and was comparable with the number determined from control cultures (p > 0.08) (Fig. 6Go). This suggests that TGF-ß1 accelerates/expands the production of late erythroid progenitors from their precursors in the absence of exogenous Epo. TGF-ß1 (<5 ng/ml) did not inhibit the growth of large BFU-E in the same cultures (p > 0.2 versus control cultures).



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  		Figure 6. Effect of TGF-ß 1 on the development of erythroid CFCs in Epo-independent erythroid cultures of adult PB MNCs. Peripheral blood MNCs (1 x 106cells/ml) were cultured (10 days) in suspension in the absence of Epo as described in Materials and Methods. At the day of isolation (day 0) and after 4, 7, and 10 days of culture, aliquots of cells were harvested and plated in the clonogenic assay at 5 x 104/ml. Erythroid colonies formed after 12-14 days were counted and classified as large and small BFU-E and CFU-E. Total estimates of colonies per 106 of initially suspended cells were calculated in control cultures (A) and in cultures treated with 5 ng/ml of TGF-ß1 (B). Results represent mean ± SD of three independent experiments. *p < 0.05 and ** p < 0.01 (versus control cultures without TGF-ß1).

 
TGF-ß1 Enhanced Terminal Erythroid Differentiation and Red Cell Enucleation
In agreement with previous studies [18,20], the enhanced development of late erythroid progenitors induced by TGF-ß1 suggests that it plays a role in late erythroid maturation. To further test this, CD34+CD38-Lin- cells grown in SCF/Epo for 10 days were transferred into PII of serum-free liquid culture supplemented with Epo. Within 5 days, the cells differentiated into more mature erythroid normoblasts. After that, benzidine-positive orthochromatic normoblasts increased gradually with time (Table 4Go). At day 14, the cells were 72% orthochromatic, >90% benzidine positive, and 7% enucleated red cells. Employing this system for terminal erythroid cell differentiation, TGF-ß1 was added at various times (day 0, 5, or 10) of PII. TGF-ß1, at a final concentration up to 5 ng/ml, had no significant effect on cell proliferation (data not shown). In contrast, TGF-ß1 mildly accelerated erythroid differentiation and significantly improved red cell enucleation (p < 0.05) (Table 4Go). TGF-ß1 was significantly more effective (p < 0.01) in stimulating enucleation when added at day 5 of culture. These serum-free cultures were devoid of macrophages. The addition of FBS to these cultures stimulated production of macrophages and optimal erythroid enucleation. In three independent experiments, neutralization of serum TGF-ß1 resulted in a lower percentage of enucleated red cells (26% ± 14% versus 54% ± 18%), supporting a role for TGF-ß1 in terminal erythroid differentiation and enucleation.


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  		Table 4. Effect of TGF-ß1 on terminal erythroid differentiation
 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here, neutralization of autocrine TGF-ß in serum-free liquid culture resulted in a significantly greater proliferation of CB CD34+CD38-Lin- cells in the presence of SCF and/or Epo. This growth stimulatory effect was previously reported in other culture systems [32,34–37]. Possible mechanism(s) cited for the ability of anti-TGF-ß to activate quiescent cells include upregulation of SCF and/or Epo receptors [1,9–11]; however, in our cultures, anti-TGF-ß-mediated proliferation of CD34+CD38-Lin- cells was not preceded by the upregulation of c-kit expression, as measured by flow cytometry (data not shown). Because TGF-ß1 regulates the production and/or function of the cell cycle inhibitors p21 and p27 [38–41], our results are consistent with the concept that neutralization of endogenous TGF-ß1 leads to greater cell cycle progression of quiescent cells.

SCF has been shown to act as a major growth and differentiation factor for mast cell development [30]. Here, serum-free cultures of CB CD34+ cells and their more primitive counterparts, CD34+CD38- Lin- cells, showed that SCF alone induced a selective growth of mast cells with high purity. Recently, Sawai et al. [42] showed that mast cells originate from bone marrow multilineage CFCs that had the potential to differentiate into neutrophil/macrophage/mast cell/erythroid lineages.

Here, we show for the first time that neutralization of TGF-ß in CB CD34+CD38-Lin- cells made it possible for SCF to markedly increase the proliferation of these cells in serum-free cultures, leading to a predominant early erythroid production while leaving mast cells essentially unaffected. This greater number of erythroid progenitors in cultures devoid of Epo reveals an Epo-independent early event in erythropoiesis and suggests that neutralization of endogenous TGF-ß has positive effects on early erythroid progenitor production. It remains unresolved whether neutralization of endogenous TGF-ß enables already existing early erythroid progenitors to increase their production or induces uncommitted cells to differentiate down the erythroid lineage. At the moment, it is not possible to distinguish between these two possibilities, although the neutralization of endogenous TGF-ß and SCF in purified murine stem cells rapidly induces differentiation of erythroid and other lineages [2]. This effect may be explained by the ability of anti-TGF-ß to delay the differentiation of CD34+CD38-Lin- (pre-BFU-E) cells, allowing larger numbers of large BFU-E to be produced and finally leading to an enhanced total erythroid cell production. Since mast cell formation was not significantly affected, it is difficult to conclude that neutralization by anti-TGF-ß switched differentiation of uncommitted progenitors from mast cells to the erythroid lineage. Moreover, based on our results, it is unlikely that production of erythroid cells by anti-TGF-ß is related to the direct effect on proliferation of large BFU-E that are formed during culture because: A) anti-TGF-ß added to SCF-stimulated CB CD34+ cells (known to harbor BFU-E) [24,43] did not induce erythroid cell production but rather enhanced mast cell formation, and B) the inclusion of TGF-ß1 in Epo-independent cultures of PB MNCs cells did not inhibit proliferation of BFU-E.

Previous studies have shown that erythroid progenitors produce Epo, suggesting that endogenous Epo plays a role in erythroid differentiation [44,45]. Sato et al. [32] showed that tenfold more anti-Epo antibody was necessary to block the effects of endogenous Epo compared with exogenous Epo. At these concentrations, in our study, anti-Epo blocked the enhanced proliferation and erythroid development in SCF/Epo-stimulated cultures but had no significant effect on growth or differentiation in SCF/anti-TGF-ß serum-free cultures. Thus, early erythroid development in the anti-TGF-ß cultures occurred in the absence of endogenous Epo.

Kinetics of erythroid CFC production from CD34+ CD38-Lin- cells indicate that anti-TGF-ß delayed production of late erythroid progenitors in SCF/Epo-stimulated cultures. Krystal et al. [20] demonstrated that TGF-ß1 triggers the conversion of SCF/Epo-stimulated BFU-E into CFU-E and, thus, induces premature erythroid differentiation and reduces the overall number of mature erythrocytes. In contrast with Krystal et al., our results suggest that neutralization of TGF-ß1 performed the opposite function and precluded conversion of large BFU-E into CFU-E, thus allowing more cell division and contributing to the greater overall number of erythroid cells. In the absence of Epo, TGF-ß1 synergized with BPA and accelerated/expanded the generation of small BFU-E/CFU-E from their precursors.

Erythroid progenitors were directed to terminal differentiation using Epo. Enucleation of erythrocytes was observed in serum-free medium in the absence of macrophages. TGF-ß1 did not inhibit the growth of normoblasts at concentrations up to 5 ng/ml and induced significant enucleation in the absence of macrophages. Recently, Zermati et al. [46] suggested that TGF-ß1-accelerated differentiation, including enucleation, was caused by the ability of TGF-ß1 to arrest the cell cycle of immature erythroid cells and, thus, to reduce the rate of self-renewal and proliferation in favor of differentiation. Here, treatment with TGF-ß1 did not reduce the number of viable cells formed in cultures over 14 days, suggesting that acceleration of differentiation was not a consequence of apoptosis or cell cycle arrest. Enucleation was maximal when TGF-ß1 was added at day 5 of PII, indicating that TGF-ß1 most likely affected intermediate and late normoblasts. Nevertheless, TGF-ß1 did not induce the optimal levels of enucleation observed in the presence of serum. Furthermore, neutralization of serum TGF-ß1 only partially blocked serum-induced enucleation, revealing that other serum factors contribute to optimal erythrocyte enucleation.

In vivo, defective hematopoiesis was found in TGF-ß1 knockout mice, resulting in a reduced formation of mature erythroid cells and anemia [1,47,48]. Recently, Larsson et al. reported that there were no mature erythroid or myeloid cells in the yolk sacs of TGF-ßRI null mice, but there were three times the number of erythroid progenitors [49]. Thus, in agreement with our in vitro results, it seems that defective erythropoiesis and anemia in mice lacking TGF-ß1 signaling are not consequences of an impaired growth of primitive stem cells and early progenitors, but most likely are due to an inadequate proliferation of late erythroid progenitors and terminal maturation of erythroid cells.

In conclusion, neutralization of TGF-ß not only increased the proliferation of SCF-treated quiescent human CB CD34+CD38-Lin- cells but also influenced their differentiation by markedly enhancing SCF-mediated growth into erythroid progenitors in the absence of Epo. Moreover, TGF-ß1 was not inhibitory in late erythropoiesis, but accelerated conversion of large BFU-E into CFU-E both in the presence and in the absence of Epo. Finally, TGF-ß1 played a role in terminal erythroid differentiation and red cell enucleation.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors thank Dr. Lis Welniak and Kathy Daum-Woods, for many useful discussions, and Dan Bertolette, for his assistance in the preparation of this manuscript. This project was funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000.

The content of this publication does not reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received February 28, 2003; accepted for publication June 2, 2003.



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