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Stem Cells, Vol. 17, No. 2, 107-116, March 1999
© 1999 AlphaMed Press

Stimulation of Adult Human Bone Marrow by Factors Secreted by Fetal Liver Hematopoietic Cells: In Vitro Evaluation Using Semisolid Clonal Assay System

V.P. Kale, L.S. Limaye

National Centre for Cell Science, Ganeshkhind, Pune, India

Key Words. Adult human bone marrow • Fetal liver-derived factors • Clonal assays • TGF-ß1

Dr. Vaijayanti P. Kale, National Centre for Cell Science, Ganeshkhind, Pune 411007, India.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 References
 
Fetal liver infusion (FLI) therapy has been used in various disorders, such as aplastic anemia, leukemia, metabolic disorders, etc., and has been shown to result in stimulation of autologous hematopoiesis in many cases. The aim of the present study was to elucidate the mechanism of stimulation of adult hematopoiesis by fetal liver hematopoietic cells (FLHC) and to identify the factors involved in the process using a clonal assay system in vitro. The effect of FLHC on the clonal growth of bone marrow cells was studied using a co-culture system consisting of mitomycin C-treated FLHC with 2 x 105 bone marrow (BM) mononuclear cells. It was observed that FLHC induced a two- to four-fold increase in the BM colony formation. A further increase in the number of FLHC did not, however, result in an equivalent fold increase in the colony formation, indicating that the number of cells in the BM population responsive to FLHC was perhaps the limiting factor. When the effect of fetal liver cell conditioned medium (FLCM) was examined in a similar fashion, it was observed that the FLCM showed a 1.5- to 4-fold increase in the colony formation when used at 1%-5% along with limiting amounts of growth factors. Higher concentrations of conditioned medium resulted in inhibitory responses. One of the principal factors responsible for the stimulatory activity of FLCM was shown to be transforming growth factor-ß1 (TGF-ß1), by a variety of experiments such as its quantitation in FLCM by enzyme-linked immunosorbent assay, antibody neutralization, and reconstruction experiments using purified TGF-ß1 and normal medium. In these reconstitution experiments, TGF-ß1 stimulated the colony formation when it was applied at 1-50 pg/ml, but at higher concentration it induced an inhibitory effect, mimicking the behavior earlier seen with FLCM. Our data strongly suggest that one of the mechanisms in stimulation of a recipient's hematopoiesis could be mediated by the action of TGF-ß1 secreted by infused FLHC and could provide a rational framework on which FLI therapy can be further evaluated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 References
 
Fetal liver infusion (FLI) therapy has been attempted in certain major disorders, such as immunodeficiency syndrome, aplastic anemia, leukemia, and genetic or metabolic disorders [1-11]. Although the engrafting capacity of transplanted fetal liver (FL) cells has been successfully demonstrated in animal models [1, 12, 13], human subjects have shown extremely variable responses to the treatment, ranging from complete recovery on one hand to no response on the other [1-11]. Most of the animal studies, however, are done with complete pretransplant immune suppression, while in the case of human subjects such preconditioning was not followed routinely. The failure of engraftment, therefore, has been thought to be due to lack of proper preconditioning and, in turn, unavailability of stromal niches. Recently it was shown that preconditioning may not be absolutely essential for successful engraftment to take place [14]. It is also speculated that histocompatibility as well as ontogenic barriers exist which interfere with the engraftment. Experimental results indicate that adult bone marrow (BM) stromal cells induced apoptosis in FL-derived BFU-E, thereby suggesting that FL progenitors may be incapable of differentiating in an adult environment [15]. Although such studies partly explain the failure of engraftment, the mechanism of recovery observed in the patients who responded to the therapy is not well understood. Since the regenerated hematopoiesis is of recipient origin, it is assumed that FL cells secrete some growth factors which stimulated the patients' marrow cells. Several experimental studies carried out in this direction have demonstrated the presence of stimulatory activity in the FL extracts [16-20]. The stimulatory factor(s), however, has not been identified.

We have initiated these studies with a view to understanding the mechanism of autologous recovery of adult hematopoiesis in response to FLI [2, 3, 5]. Our contention is that if the mechanism by which FL cells bring about the stimulation of adult BM cells is understood, then it would pave the way for establishing quality control criteria for the "infusion material" and for improving the success rate of FLI therapy.

In the present study, we examined the ability of FL hematopoietic cells (FLHC) to stimulate progenitor cells of normal human adult BM cells using in vitro colony-forming assay. When semisolid assays were set up as a coculture system of BM cells and mitomycin C-treated FLHC (M-FLHC), an enhancement of colony formation was observed. The colony-forming unit (CFU) stimulating ability was related to the transforming growth factor-ß1 (TGF-ß1) secreted by FLHC. The ramifications of clinical applications of FLI therapy are discussed in the light of the present findings.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 References
 
Chemicals and Cell Lines
Mitomycin C, methylcellulose of viscosity 4,000 cps, irrelevant IgG1 antibody, Sigmacoat, and Wright's and Giemsa stains were purchased from Sigma (St. Louis, MO). Recombinant human cytokines, namely erythropoietin (Epo), stem cell factor (SCF), interleukin 3 (IL-3), GM-CSF, and TGF-ß1 were obtained from Boehringer Mannheim (Indianapolis, IN). Iscove's modified Dulbecco's medium (IMDM) and fetal calf serum (FCS) were purchased from GIBCO (Grand Island, NY). TGF-ß1 enzyme-linked immunosorbent assay (ELISA) kit and neutralizing antibody to TGF-ß1, 2, and 3 were purchased from (Genzyme Corporation; Cambridge, MA). Ficoll hypaque ([FH], density = 1.077 gm/ml) was obtained from Pharmacia (Uppsala, Sweden). Amicon filters of molecular weight cut off 10,000 were purchased from Amicon Corporation; (Danver, MA). 5637 bladder carcinoma cell (HTB 9) line purchased from ATCC (Rockville, MD) was used to prepare conditioned medium (CM), as described [21]. The CM was used in the experiments after determining optimal concentration for adult human BM cells. The same batch of CM was used throughout the study.

Collection and Processing of FLHC
Fetuses of 12 to 20 weeks of gestation obtained from MTP cases admitted to local general hospitals were used. The samples were collected on ice and processed soon afterwards. Typically, the liver was dissected out, washed several times with sterile cold saline, and minced, and a single-cell suspension was prepared by gently pressing the tissue pieces against multiple layers of sterile muslin cloth. The cell suspension was allowed to stand on ice for 10 min to allow heavier hepatocytes to settle. The FLHC suspension thus obtained was fractionated on FH to obtain mononuclear FLHC. Samples with more than 98% viability were used further.

Collection and Processing of Human Adult BM Cells
Human adult BM cells were isolated from ribs or sternal bone pieces taken out and discarded as waste material during renal and cardiac surgeries, respectively, or from iliac crest aspirates collected for diagnostic purposes and declared normal. BM cells from ribs and sternal pieces were isolated as described [22]. Mononuclear cells (MNC) from iliac crest aspirates were isolated by FH separation. Protocols for all human studies were approved by the Institutional Review Board.

Cell Viability and Counting
The nucleated cell count of FLHC and BM cells was determined by mixing one part cell suspension with nine parts Turk's solution (0.01% crystal violet in 3% acetic acid) to lyse the RBC [23]. Cells were then counted using a hemocytometer and an inverted microscope. Viability was determined by erythrocin B dye exclusion test.

Mitomycin Treatment of FLHC
FLHC were suspended in complete medium (IMDM supplemented with 20% FCS) at a cell density of 1 x 107 cells/ml and treated with 20 µg/ml mitomycin C. The cells were incubated for 4 h at 37°C and 5% CO2.The cells were then washed extensively to remove the drug. At the end of this treatment, cells were viable but did not form colonies under the assay conditions used in the experiments. A fixed number of BM cells (2 x 105) were mixed with various numbers of M-FLHC in a sterile Eppendorf tube, and the entire contents were plated for CFU assays. Appropriate control plates without M-FLHC or BM cells were included in every experiment.

Preparation of Fetal Liver Hematopoietic Cell CM (FLCM)
FLHC were incubated at 37°C in 5% CO2 at a concentration of 1 x 107 cells/ml in IMDM supplemented with 20% FCS. CM was collected after 48 h by centrifugation in a cold centrifuge. CM was concentrated using Amicon filters of 10,000 molecular weight cut off. The higher molecular weight fraction was collected, filtered, and kept frozen in small aliquots in siliconized tubes at -70°C until used. The FLCM was added at various concentrations in the assay plates. In some experiments FLCM was treated with antibodies (20 µg/ml) at 4°C overnight and used in the assays at desired concentration. The same concentration of irrelevant antibody of the same isotype was used as a control.

Semisolid Clonal Assays
BM progenitors were assayed using a modification of the method described by Fausner and Messner [24]. A combination of agar and methylcellulose culture was used. Growth factors used were Epo (1 U/ml) and 5637 CM (5% v/v) to give limiting concentrations. In some experiments, purified IL-3 (10 U/ml ) and GM-CSF (10 U/ml) were used as a substitute for 5637 CM. Plates were incubated at 37°C in 5% CO2 in a humidified atmosphere for 14 days. At the end of the incubation period, the total number of colonies per plate was counted without discriminating between lineage distribution. Counting of the colonies was done using an inverted microscope equipped with flat field objectives and eyepieces (Olympus). Higher magnification (150x) was used to accurately discriminate the colony borders, especially when the number of colonies was high (>500). Only those experiments where accurate scoring could be done were considered for analysis. Separate experiments were set up to identify and assess the effect of FLCM on the CFU-erythroid (CFU-E), BFU-E, granulocyte-macrophage-colony-forming cell (GM-CFC), and granulocyte/erythroid/macrophage/megakaryocyte (GEMM) colony types that formed in these experiments. For these experiments, care was taken to ensure that colonies formed were at low density (<100/dish) [25].

Quantitation of TGF-ß1 in FLCM Using ELISA
TGF-ß1 was estimated in the FLCM by a kit (Genzyme) per the manufacturer's instructions. The values obtained were corrected for by subtracting the background values obtained with the same batch of culture medium incubated under similar conditions.

Calculations and Statistical Analysis
A mean CFU number of at least three replicate experiments was taken, and the values obtained with treatment were compared with those obtained without treatment. One-way repeated measure of variance (one-way RM ANOVA) was used for statistical analysis of CFU numbers using a computerized statistical analysis program, SIGMASTAT (Jandel Scientific Corporation; San Rafael, CA). A p value of <0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 References
 
Effect of Co-culture with M-FLHC on Clonal Growth of Adult BM Cells
We attempted to mimic the possible "in vivo effects of FL infusion" by in vitro semisolid colony formation assays in which premixed M-FLHC and adult BM cells were plated. Since FLHC were capable of forming colonies on their own, we treated them with mitomycin C to abrogate their colony-forming ability. The dose and time of mitomycin C treatment was optimized in preliminary experiments such that the viability of FLHC was not impaired despite their not being able to form colonies.

In initial experiments in which limiting concentrations of 5637 CM and Epo (1 U/ml) were used, 2 x 105 BM cells showed a stimulation of colony formation with various numbers of M-FLHC. The extent of stimulation was variable among different batches of FLHC and even for a given batch of FLHC, the extent of stimulation was observed to be different when the source of BM cells was changed. The stimulatory effect of M-FLHC was discernible at as low as 500 M-FLHC in some of the experiments, and the use of higher numbers of M-FLHC in such cases did not confer much additional advantage. Figure 1 presents representative data for three experiments where different BM as well as different M-FLHC were used. In several experiments, we observed that the stimulation of colony formation was in the range of two- to fourfold with the use of M-FLHC with respect to control experiments without M-FLHC, and this stimulation observed was found to be statistically significant (p < 0.05).



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  		Figure 1. Presence of M-FLHC in the semisolid media stimulates colony formation of human BM MNC. Clonal cultures were established in methylcellulose medium with limiting concentrations of 5637 CM and Epo. 2 x 105 BM MNC were mixed with the indicated number of M-FLHC. The cellular mixture was plated for colony formation. The results are expressed as the mean of three replicate sets per experiment. The bars indicate the standard error. The colony numbers without M-FLHC were compared with the results obtained with various amounts of M-FLHC used. Results from three different experiments making use of different BM cells and different M-FLHC are shown. * Indicates p < 0.05.

 
Effect of Addition of FLCM on Clonal Growth of Adult BM Cells
In the aforementioned experiments, we noted indications that M-FLHC were capable of stimulating colony formation of adult BM cells; however, the mechanism of stimulation was not apparent. We hypothesized that the factors secreted from M-FLHC were responsible and, therefore, subjected the CM prepared from two FLHC samples to test their possible stimulatory effects on the colony formation of the adult BM cells. Both the FLCM inhibited colony formation from adult BM cells when used at higher concentrations (10%-20%). By contrast, both showed stimulatory effects on the colony formation when applied at lower concentrations (1%-5%). Figure 2 shows data from two representative experiments. As in the case of intact cells, the conditioned media could again stimulate colony formation of BM cells. We, therefore, conclude that some factor secreted by FLHC was responsible, at least in part, for the stimulation observed. The inhibition of colony formation seen at 10%-20% input of FLCM indicated either that the secreted factor(s) may have had inhibitory activity at higher concentrations or that the FLCM consisted of a cocktail of stimulatory and inhibitory factors of which the stimulatory factor became functionally dominant at lower concentrations of FLCM.



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  		Figure 2. Effect of various concentrations of FLCM on the colony formation of human BM MNC. Clonal cultures were established for 2 x 105 BM MNC per plate with limiting concentrations of 5637 CM and Epo. FLCM was added in various concentrations as indicated in the figure. Mean colony number obtained with and without addition of FLCM was used to calculate the fold increase. The figure shows results from the use of two different FLCM on two different BM MNC preparations.

 
We next examined whether the stimulation seen was of a general nature encompassing the colony formation along different lineages or was specifically directed towards a particular lineage. For this purpose we carried out experiments under conditions such that discrete and morphologically distinguishable colonies formed for accurate enumeration of CFU-E, BFU-E, GM, and GEMM type colonies. The low density of colonies formed per plate (<100) ensured that there was no physical mixing of the colonies. These colonies were identified by their morphological criteria [26] in five to six replicate experiments per set, and the data were subjected to statistical evaluation for significance. In these experiments, we used IL-3 (10 U/ml) and GM-CSF (10 U/ml) along with Epo (1U/ml) to rule out the possibility that the 5637 CM could interact with FLCM in an unknown manner, which resulted in the observed effect. As shown in Figure 3, it is clear that the stimulatory effect seen was of a general type since both the erythroid and GM compartments were found to be stimulated nearly to the same extent. In these experiments, there was a stimulatory effect seen on the GEMM compartment as well, but in view of the very low number of GEMM colonies obtained in these experiments, further work will be necessary to reliably determine whether this stimulation was significant.



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  		Figure 3. FLCM-mediated stimulation of various types of progenitors derived from human BM MNC. Clonal cultures were established for 2 x 104 BM MNC per plate with IL-3, GM-CSF, and Epo. FLCM was added at concentrations indicated. Different types of colonies belonging to CFU-E, BFU-E, GM-CFC, and GEMM-CFC were scored. Mean colony numbers with and without the addition of FLCM were compared for each colony type. * Indicates p < 0.05.

 
FLCM Contains Substantial Amounts of TGF-ß1
The inhibitory effects seen with higher doses of FLCM on colony formation were reminiscent of similar effects of TGF-ß1 reported in the literature. We were interested, therefore, in trying to determine whether TGF-ß1 was present in FLCM. It was envisaged that the removal/neutralization of such inhibitory components as TGF-ß1 from FLCM might improve the efficacy of FLCM for stimulation. Accordingly, four different FLCM preparations were subjected to quantitation of TGF-ß1 content by ELISA. All four of them indeed contained TGF-ß1 by this assay in the range of 651-2,387 pg/ml ( Fig. 4), confirming the actual presence of this known inhibitor of colony formation. Two of these were tested further to see if they stimulated the BM colony formation. These two samples, corresponding to FLCM-2 and FLCM-3 in Figure 4, contained 1,302 and 2,387 pg/ml of TGF-ß1, respectively. Both FLCM-2 and FLCM-3 showed stimulation in colony formation assays when they were applied at the input of 1% v/v to the extent of 2.7- and nearly threefold, respectively. These results showed clearly that at least one known negative regulator, TGF-ß1, was present in the FLCM which could account for the inhibition of colony formation seen with higher doses of FLCM. Interestingly, FLCM-3 had a TGF-ß1 content nearly 1.8 times higher than that of FLCM-2 and yet showed a better stimulation of colony formation. These data, however, cannot distinguish between the two possibilities: A) that a single secreted factor was responsible for both the stimulation and inhibition depending on its content in the FLCM and B) that at least two factors, one stimulatory and the other inhibitory, were present in the FLCM, and the overall effect seen in these experiments was a reflection of their relative contribution to the colony formation.



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  		Figure 4. Presence of TGF-ß1 in FLCM samples as detected by ELISA. Four independently prepared CM using different FL samples were used in this experiment. The ELISA kit (Genzyme) was used per the instructions of the vendor and included an activation step. The sample values were corrected for the background by using the same culture medium as used in the preparation of the FLCM.

 
Effect of Neutralizing Antibody to TGF-ß1 on FLCM Activity
One way to rationalize the mode of action and to delineate the contribution of TGF-ß1 on the colony formation was to abrogate its biological activity in FLCM while it is being applied on colony assays. We, therefore, chose a CM pretested for its stimulatory activity on colony formation when applied at the 1% level. The FLCM was then treated with a neutralizing antibody (Genzyme), and this preparation was evaluated in the colony formation assays again. As seen from Figure 5, the CM treated with an antibody irrelevant to TGF-ß1 clearly showed the stimulation of colony formation nearly to the extent of fourfold. By contrast, when neutralizing antibody to TGF-ß1 was used, the extent of stimulation seen in parallel experiments dropped to less than twofold. These results were surprising in that an actual inhibition in the colony formation was observed rather than the stimulation that was expected. The data, therefore, exclude the possibility that there were colony-stimulating factors in the FLCM that acted to oppose the inhibitory action of TGF-ß1. Alternatively, these results emphasize clearly that the TGF-ß1 present in the FLCM had a positive stimulatory effect on the colony formation which could be abrogated to a significant extent by using an antibody neutralizing to TGF-ß1, and the hypothesis that a single secreted factor is responsible for both the stimulation and inhibition became more likely to be true.



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  		Figure 5. A neutralizing antibody (AB) to TGF-ß1 abrogates the stimulatory property of FLCM. Clonal cultures were established with FLCM (1%) with or without neutralizing AB to TGF-ß1 along with limiting concentrations of 5637 CM and Epo. Mean of replicate experiments was calculated. The mean colony number obtained from TGF-ß1-specific AB was compared with results of similar experiments where either a control AB or no specific AB was used.

 
Stimulatory Effect of Purified TGF-ß1 on Colony Formation
Although a convincing role of TGF-ß1 in the stimulation of colony formation was apparent from the experiments described above, it was not clear whether other components of the FLCM were also involved in addition to TGF-ß1 for its stimulatory role. To address this issue, we conducted experiments to directly evaluate if the purified TGF-ß1 alone was sufficient to stimulate colony formation. Our previous experiments which measured the TGF-ß1 content of FLCM by ELISA had indicated that TGF-ß1 was found in several FLCM preparations in the range of 651-2,387 pg/ml, and further, these media when applied at 1% (i.e., equivalent TGF-ß1 in the range of 6.5-23.8 pg/ml) could effectively stimulate colony formation. We, therefore, chose to apply purified TGF-ß1 in the range of 0-50 pg/ml to test for its possible stimulatory role in colony formation. These data are presented in Figure 6. TGF-ß1 indeed stimulated colony formation in a dose-dependent manner up to a concentration of 50 pg/ml. The stimulatory effects of TGF-ß1 on colony formation were discernible at a dose as low as 1 pg/ml in at least three independent experiments ( Figs. 6 -8). These results, therefore, strongly suggest that TGF-ß1 alone is both necessary and sufficient in the colony formation observed in the series of experiments involving FLCM.



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  		Figure 6. Stimulation of BM MNC colony formation by purified TGF-ß1. Purified TGF-ß1 was applied in a standard stimulation assay as described in legend to Figure 2. The mean colony number obtained from three experiments per dose were compared pair-wise, with the experiment without TGF-ß1. * Indicates p < 0.05.

 


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  		Figure 7. The stimulation induced by TGF-ß1 is not lineage-specific. Clonal cultures were established from 2 x 104 BM MNC in the presence of IL-3, GM-CSF, and Epo. TGF-ß1 at 1 pg/ml was added to the plates. Colonies of various lineages belonging to CFU-E, BFU-E, GM-CFC, and GEMM-CFC were scored in six replicate experiments. Mean number of colonies with or without TGF-ß1 was compared for each type of colony. * Indicates p < 0.05.

 


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  		Figure 8. TGF-action is independent of SCF. TGF-ß1 was applied at various concentrations as indicated in a standard stimulation assay as described in Figure 2 with the addition of SCF at 500 pg/ml. Plates without SCF and without TGF-ß1 were also set. Mean of three replicate experiments was calculated. The mean colony number from plates with the addition of TGF-ß1 and SCF was individually compared with the mean colony number from plates with SCF alone without TGF-ß1. * Indicates p < 0.05.

 
We carried out separate experiments to examine whether the stimulation of colony formation by TGF-ß1 was of a general nature or was directed towards a specific lineage of cells. These experiments were done under the same conditions as described earlier for FLCM, using purified growth factors and plating fewer BM cells to achieve small numbers of discrete and non-overlapping colonies. Figure 7 presents the data as a mean of six replicate experiments using a single source of BM cells. The results clearly show that the TGF-ß1 exerted a stimulatory effect on the colony formation, which was of a general nature. As shown in Figure 7, it is clear that TGF-ß1 exerted a stimulatory effect on colony formation of a general nature, since both erythroid and GM compartments were stimulated to a similar extent.

Despite the observation that TGF-ß1 alone was sufficient to stimulate colony formation, it was still possible that TGF-ß1 action could be amenable to modulation by other growth factors. It has recently been observed that some of the FLCM samples contained SCF in the range of 50-500 pg/ml [27], and it was possible that SCF could work in concert with TGF-ß1 to further enhance/inhibit the colony formation. The purified SCF (500 pg/ml) was, therefore, evaluated for its potential ability to stimulate colony formation in the semisolid assays, but experiments clearly showed that SCF had neither a stimulatory nor an inhibitory effect on the colony formation. We evaluated next whether the addition of exogenous TGF-ß1 to medium with SCF (500 pg/ml) will still show the stimulatory profile observed earlier. As shown in Figure 8, the TGF-ß1 indeed stimulated the colony formation in the presence of SCF in a dose-dependent manner. As the TGF-ß1 concentration was increased beyond 50 pg/ml, the inhibitory effects of TGF-ß1 became noticeable. These results showed that SCF, if it were to be present at 500 pg/ml in FLCM, could not have significantly influenced the stimulation of colony formation in our experiments.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 References
 
Present work was carried out to elucidate the mechanism of autologous recovery observed in some patients treated with FLI therapy, with a special focus on variable responses obtained even in a single clinical group [1-11]. As a first step towards this goal, we carried out experiments to check whether FLHC themselves could stimulate normal adult human BM progenitors, and if they do, then by what mechanism. Although it is a well-accepted fact that FLHC have the ability to secrete growth factors, and clinical reports of hematopoietic recovery following FLI therapy as well as experimental reports describing stimulatory activity secreted by FL cells support this contention, the putative factor(s) has not been identified [16-20]. FL-derived hepatocytes as well as stromal cells have been shown to produce hematopoietic stimulatory activity [16, 18]. These data perhaps explain the reason behind active hematopoiesis during the second trimester. Since clinical FLI therapy generally involves infusion of hematopoietic cells, we have concentrated only on this component of FL. But the presence of a few contaminating stromal cells and mononuclear hepatocytes in our preparation cannot be ruled out, as we have not attempted rigorous purification of any cell type.

In order to study the effect of FLHC on the hematopoietic system, we have used normal human BM population as the test system. Although the use of clinical material from disorders such as aplastic anemia would have been more relevant, the heterogeneity in the diseases themselves [28] may interfere with the interpretation of the results. We felt that it would be more fruitful to take up such studies with well-defined clinical samples after optimizing the experimental system with normal cells.

In part of our study, we have used a system of FLHC co-culture in the assay system. The FLHC were treated with mitomycin C such that they were perfectly viable but could not form colonies under the assay conditions used. We have shown that the cells produced a two- to fourfold increase in colony formation. As the FLHC did not form any colonies by themselves, the increase in colony number was attributed to stimulation of BM cells by FLHC. Since the addition of a higher number of FLHC did not result in a parallel fold increase in colony formation, we feel that the number of target cells in the test sample itself might be the limiting factor. This situation could also be occurring in the case of patients receiving FLI therapy. If the target cells responsive to FLI treatment are absent or in some way compromised, the therapy may not result in expected outcome. It is therefore necessary to identify and quantitate the target population of FLHC action. We plan to carry out further experiments in this direction.

In the experiments carried out with various concentrations of FLCM to examine whether FLHC stimulated colony formation by secretion of soluble factors, we observed that the CM stimulated colony formation only when used at low concentration (1%-5%). At higher concentrations (10%-20%), a clear inhibition was seen. Since TGF-ß is a known hematopoietic inhibitor shown to be associated with active sites of hematopoiesis such as FL [29, 30], we carried out ELISA experiments to quantitate TGF-ß1 in the FLCM. We found that all four CM preparations tested showed the presence of substantial but variable levels of TGF-ß1. The ELISA kit which we have used is designed, however, to detect both active and inactive precursor forms of TGF-ß1, since an acid activation step in the procedure is involved. The exact amount of active TGF-ß1 component in native FLCM could not, therefore, be determined by these assays, and it is likely that these components of TGF-ß1 in various FLCM were lower than the values obtained after acid activation in these experiments.

Our observation that a neutralization antibody specific to TGF-ß1 impaired rather than stimulated the colony formation by the BM cells was unexpected and pointed to the possibility that TGF-ß1 may have had a positive stimulatory role in the colony formation. This observation could be contrasted with the previous observations which had shown that the downregulation of TGF-ß1 activity in long-term BM cultures had a stimulatory effect on the progenitor output [31-33]. The notion that low concentrations of TGF-ß1 was stimulatory was further borne out by the use of purified growth factor and demonstration of a dose-dependent stimulation of colony formation. The dose-dependent stimulation was a bell-shaped curve indicating that TGF-ß1 was stimulatory at low concentrations (1-50 pg/ml), whereas its inhibitory activity became manifest gradually beyond 50 pg/ml ( Fig. 8). This observation clearly points to the duality in the actions of TGF-ß1 which are mutually opposing. Our novel finding that low concentrations of TGF-ß1 can stimulate colony formation may provide some new insight into the role of TGF-ß1 in the hematopoietic process.

We have provided convincing evidence that the stimulation of colony formation was of a general nature. Our demonstration that CFU-E, BFU-E, GM-CFC, and GEMM-CFC were all stimulated at the same time implicates the primitive progenitor cells as a putative target of TGF-ß1 action.

Since our assays involve a mixed population of FL-derived MNC, it has not been possible to identify the source from which TGF-ß1 is derived in the FLCM. Previous experiments have documented that hematopoietic progenitor cells themselves produce TGF-ß1 [33, 34]. Further, monocytes present in this population are also known to release TGF-ß1 under a variety of experimental conditions. The constitutive expression of TGF-ß1 is also known to take place in the BM stromal cells [35], but in our experiments, these types of cells are absent or may be present at very low levels. The observation that TGF-ß1 was consistently associated with hematopoietically active tissues had clear implications of its involvement in hematopoiesis. It has even been observed that TGF-ß1 could be an activator [36-38] or inhibitor [32, 33, 39-45] of hematopoiesis, depending on the target population. The activatory role of TGF-ß1 in these experiments was confined only to GM progenitors and required that higher concentrations of TGF-ß1 (ng/ml) be applied. Our experiments are different from these at least in three respects in that A) we have used semisolid clonal assays with limited amounts of CSF to monitor the stimulation of colony formation; B) the amount of TGF-ß1 applied is significantly less (pg/ml), and C) the nature of stimulation seen in the colony formation was more general in nature and was not restricted to the GM compartment alone. It is therefore possible that the combination of very low levels of TGF-ß1 and limited amounts of growth factors used in our experiments led to the detection of the stimulatory activity. The molecular mechanism underlying this stimulation remains unknown and needs further examination.

TGF-ß1 is a pleiotropic growth factor, and therefore, its action on hematopoiesis may well transcend the effects suggested above through its actions on other accessory cells present in the environment. For example, TGF-ß1 has been shown to induce monocyte differentiation [46], and therefore, it is possible that the monocytes formed in response to TGF-ß1 may release growth factors. In some cases of successful FLI therapy, both clinical and experimental, a predominant erythroid development has been reported [1, 47, 48]. Although we did not observe such a bias towards erythroid lineage in our experiments, it is still possible that the infused FLHC secrete Epo differentially depending on the context of the host environment [49], and that may determine an apparent bias towards erythroid development. Another distinct mechanism by which TGF-ß1 can play a positive modulatory role in hematopoiesis is unraveled by the recent observation that it can abrogate Fas-mediated apoptosis of Lin– BM cells [50]. Interestingly, activation of Fas has been reported in CD34+ progenitor cells from aplastic anemia patients [51]. Since the utility of FLI therapy in disorders such as aplastic anemia has been underscored by clinical as well as experimental studies [1-11], it would be important to check whether FLHC treatment and the attendant success in aplastic anemia correlates to the Fas activation profiles on their progenitor cells.

The already known pleiotropic nature of TGF-ß1, its previously demonstrated colony-inhibiting property, and our present data suggesting that it stimulates BM colony formation at low concentrations all warrant a cautious approach in FLI therapy. Administration of high doses of TGF-ß1 can result in inhibition of hematopoiesis and secretion of TNF-{alpha}, which may have additional deleterious effects [52]. Our observation that the TGF-ß1 level in different FLCM can vary within a wide range although they were derived from a fixed number of FLHC indicates that the dose of FL cells to be employed for clinical use needs to be calibrated case by case and carefully. It is possible that those cases where no significant hematopoietic recovery was observed might have inadvertently received either too few or too many cells in terms of their TGF-ß1-secreting ability. Our results, therefore, provide an experimental framework in which the FL cells can first be categorized in terms of their TGF-ß1-secreting ability and then tried for clinical evaluation.


   Summary
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
References
 
Our results show that FL hematopoietic cells stimulate adult BM-derived progenitors by secretion of soluble factors. The data further indicate that TGF-ß1 could be one of the factors responsible for the observed stimulation.


   Acknowledgments
 
The authors wish to thank Drs. U. V. Wagh and G. C. Mishra for support; Dr. Vinod Kochupillai for useful discussions; Dr. Bharucha, Dr. A. V. Jamkar, Dr. R. L. Marathe, and Dr. A. Choukar for supply of experimental samples; and Mr. George Fernandes and Mr. Sarang Sattoor for excellent technical assistance. We are grateful to the Department of Biotechnology, Government of India for financial support.

This work was supported by the grants given to VPK by Department of Biotechnology, Government of India, in collaboration with Dr. V. Kochupillai, I.R.C.H., New Delhi. The work was carried out independently at both centers.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 References
 

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accepted for publication January 18, 1999.




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