TY - JOUR
AU - Matthew, Howard W. T.
AB - Abstract Recent studies have indicated that glycosaminoglycan (GAG) interactions with hematopoietic progenitors play a significant role in the regulation of hematopoiesis. However, the details of these interactions are not clear. In this study, we examined the role of soluble and immobilized GAGs in the proliferation of CD34+ cells. Chitosan, a cationic polysaccharide, was used to immobilize GAGs in ionic complex membranes. The GAGs studied were heparin, hyaluronate, and chondroitin sulfates A, B, and C. CD34-enriched umbilical cord blood cells were seeded onto tissue culture plates coated with the GAG-chitosan complex membranes. Cultures were maintained in medium supplemented with stem cell factor and interleukin 3 for up to six weeks, during which total and CD34+ cell numbers were determined by flow cytometry. Total cell number expansion ranged from 25-fold to 40-fold after six weeks. However, only heparin and chondroitin sulfate B (CSB) surfaces retained a significant CD34+ fraction. All other surfaces exhibited declines in CD34 expression, with negligible CD34+ percentages remaining after four weeks. In contrast, heparin and CSB surfaces exhibited CD34+ fractions as high as 90% after four weeks. GAG desorption studies indicated that the observed effects were partly mediated by desorbed GAGs in a concentration dependent manner. Subsequent studies showed that sustained high (160 μg/ml) heparin levels had toxic effects, while the same concentration of CSB exhibited more rapid early proliferation of CD34+ cells. In conclusion, this culture system has demonstrated the ability to produce simultaneous proliferation and CD34+ cell enrichment of a partially purified cord blood population by controlling the nature and levels of GAG moieties to which the cells are exposed. The results indicate that specific GAGs can significantly influence the growth and differentiation characteristics of cultured CD34+ cells. Glycosaminoglycan, Heparin, Chondroitin sulfate, CD34+ cells, Chitosan, Proliferation Introduction The hematopoietic microenvironment plays a major role in controlling proliferation and lineage differentiation during hematopoiesis [1-5]. Many studies have demonstrated that the activities of hematopoietic progenitors are regulated by multiple receptor-ligand interactions with soluble, cell-surface-bound, and extracellular-matrix (ECM)-bound cytokines. However, there is a paucity of information regarding the detailed mechanisms of these interactions. In fact, the evidence shows that many important hematopoietic cytokines bind with varying affinities to a variety of ECM molecules [5-9]. The specific effects of these binding interactions vary widely depending on the cytokine and ECM molecules involved. Such effects may range from activity reductions to activity enhancement. In addition, the nature of the effect (enhancement or inhibition) may also depend on the relative concentrations of participating molecules. Proteoglycans and the ECM polysaccharides termed glycosaminoglycans (GAGs) are major components of the hematopoietic matrix [10, 11]. A number of studies have indicated that glycosaminoglycans bind and present cytokines to hematopoietic cells in a more highly active form [6, 7, 9, 12-14]. Most notably, the results show that heparan sulfate is required for maintenance of long-term culture-initiating cells (LTC-ICs) [9]. The mechanism of action seems to be via selective enhancement of certain activities of early-acting cytokines. Additionally, it has been shown that heparin (a more highly sulfated version of heparan sulfate) can directly activate some growth factor receptors. However, the detailed mechanisms of these effects are poorly understood. In particular, the degree of specificity of the GAG-cytokine interactions and questions of opposing effects on different progenitors and lineages remain largely unresolved. In light of this uncertainty, culturing hematopoietic cells on ECM polysaccharide surfaces should provide more insight into the roles of these molecules in hematopoiesis. Within the ECM, GAGs are immobilized by combinations of covalent, ionic, and hydrogen bonding interactions. In this interfacial environment, the three-dimensional nature of GAG-growth factor interactions may be somewhat different from the corresponding interactions in solution. However, most in vitro culture systems either incorporate GAGs with other complicating factors (i.e., stroma-based systems), or supply GAGs in purely soluble form (cytokine-based, stroma-free systems). In this study, the effects of specific GAGs on the expansion of CD34+ cord blood cells were studied in stroma-free cultures using a model GAG surface. GAGs were immobilized onto culture surfaces by forming insoluble ionic complexes with the amino-polysaccharide chitosan [15-17]. Human umbilical cord blood CD34+ cells were seeded onto the complex surfaces, and their proliferation and retention of CD34 expression were followed. Materials and Methods Chitosan (66% deacetylated), glacial acetic acid, hyaluronic acid (HA) from human umbilical cord, heparin from porcine intestinal mucosa, chondroitin sulfate A (CSA) from bovine articular cartilage, chondroitin sulfate B (CSB) from bovine intestinal mucosa, and chondroitin sulfate C (CSC) from shark cartilage, were purchased from Sigma Chemical Co. (St. Louis, MO). Type I collagen was extracted from rat tail tendon by the method of Elsdale and Bard as modified by Dunn [18]. Alcian blue 8GX and toluidine blue were obtained from Polysciences, Inc. (Warrington, PA). All culture medium components were purchased from Life Technologies (Rockville, MD). Preparation of Immobilized GAG Culture Membranes Polysaccharide complex membranes were deposited onto tissue culture polystyrene dishes using the ionic interaction between a GAG solution and a pre-deposited chitosan-acetic acid membrane. To prepare sterile chitosan solution (2 wt% in 0.2 M acetic acid), 2 g of chitosan was autoclaved in 100 ml of water and then dissolved by adding 1.2 g of sterile filtered glacial acetic acid and stirring for 2 h. Sterile GAG solutions (1 wt%) were prepared by dissolving the GAG in saline (0.9% NaCl) buffered with 50 mM HEPES (pH 7.4) and then autoclaving. The GAGs evaluated in this study were heparin, HA, CSA (chondroitin-4-sulfate), CSB (dermatan sulfate), and CSC (chondroitin-6-sulfate). Cell cultures were conducted in 24-well culture plates, with all culture surfaces represented on each plate by three replicate wells. Control culture surfaces were of three types: untreated culture wells, wells coated with type I collagen, and chitosan-coated wells. To prepare the collagen-coated wells, 0.25 ml of a 1 mg/ml solution of collagen in 1 mM HCl was evaporated to dryness in each well and then equilibrated with HEPES buffered saline. Pure chitosan culture surfaces were prepared by covering the bottom of each well with 50 μl of sterile chitosan solution. The solution was then evaporated to dryness at room temperature in a biological laminar flow hood. This process produced a coating of dry chitosan acetate. The coated wells were then washed with 0.5 ml of 0.1 M NaOH to remove the acetate. This was followed by equilibration with 1.5 ml of 50 mM HEPES buffered saline (pH 7.4). GAG-chitosan complex surfaces were prepared by first coating wells with 50 μl of sterile chitosan solution and again evaporating to dryness. The coated wells were covered with 0.5 ml of sterile GAG solution and allowed to equilibrate for ∼6 h. This procedure resulted in the deposition of a GAG-chitosan complex membrane on the dish. The formed complex membranes were then washed four times with 1.5 ml of HEPES buffered saline. In the Results section, membranes formed by this procedure are designated “regular” membranes. Preparation of GAG-Depleted Complex Membranes Heparin-chitosan and CSB-chitosan complex membranes were prepared in 24-well plates as described above. The membranes were then washed six times with phosphate buffered saline (PBS) (pH 7.4). For each wash, 0.5 ml of PBS was added to each well and the plate was incubated at 37°C for 4 h, after which the solution was aspirated. Cells were seeded onto the membranes after the sixth wash. Procurement and Culture of CD34+ Cells Human umbilical cord blood was collected post-partum in accordance with a protocol approved by the Wayne State University Institutional Review Board. CD34+ cells were isolated and partially purified using an immunomagnetic separation kit (mini MACS, Miltenyi Biotec; Auburn, CA). In brief, anticoagulated cord blood (heparin, 1 mg/ml) was mixed with an equal volume of Iscove's modified Dulbecco's medium (IMDM) and layered onto Histopaque density medium (D1.077, Sigma Chemical Co.). After centrifugation at 1,500 rpm for 35 min, mononuclear cells were retrieved from the interphase band and washed three times with tissue culture medium. Immunomagnetic separation was then conducted following the manufacturer's instructions. An aliquot of the CD34-enriched cell suspension was taken for analysis prior to cell seeding. From a typical run, ∼1 million mononuclear cells were recovered from 20 ml of blood with CD34+ percentages ranging from 30% to 60%. The isolated cells were seeded into 24-well tissue culture plates containing the control and GAG complex surfaces. Seeding density was 15,000-30,000 cells per well with 0.5 ml of culture medium per well. The culture medium consisted of IMDM supplemented with 10% fetal calf serum, 1% bovine serum albumin (BSA), 5 × 10−5 M β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin B, 5 ng/ml stem cell factor (SCF), and 10 ng/ml interleukin 3 (IL-3). Half-volume medium changes were done at three-day intervals without cell removal. The total culture duration was six weeks. Culture with Soluble GAGs The effects of soluble heparin and CSB were evaluated in noncoated tissue culture wells using GAG-supplemented medium. The test GAGs were individually dissolved in culture medium at concentrations of 7 μg/ml or 160 μg/ml. Cells were seeded into noncoated wells using the GAG-supplemented medium, and cultures were conducted as described above. For these studies, the control condition consisted of cells in noncoated wells with the normal culture medium. Analysis of Cell Phenotype Culture plates were sacrificed after two, four, and six weeks of culture for cell counting and phenotype analysis. Nonadherent and weakly adherent cells were collected from individual wells by pipette rinsing with PBS. Cells were pelleted once and resuspended in 0.6 ml of PBS containing 1% bovine albumin. Staining was conducted at 4°C using a fluorescently labeled anti-CD34 antibody (HPCA-2-PE, Becton Dickinson; San Jose, CA). After 30 min of incubation, cells were washed and resuspended in PBS/BSA. Cells were counted and analyzed for CD34 expression using a flow cytometer (Becton Dickinson). GAG Quantification Assays GAGs in solutions and in membrane washes were quantified by an alcian blue-based colorimetric method [19]. A 30 μl volume of the sample or a GAG standard solution was transferred into a 96-well microassay plate and mixed with 240 μl of alcian blue dye solution (1.4 mg/ml in 0.05 M sodium acetate). After 10 min of equilibration, the absorbance of the solution at 480 nm was measured. The GAG concentration was determined from a standard curve prepared using solutions of the test GAG (0 to 30 μg/ml) in PBS. The GAG content of complex membranes was determined using a colorimetric method based on binding of toluidine blue [20, 21]. To assay bound GAG in complex membranes on a 24-well plate, the overlying solution was first aspirated and the membrane rinsed once with PBS. A 600 μl volume of toluidine blue solution (0.075 mg/ml in water) was then applied for 10 min. The decrease in absorbance of the dye solution at 630 nm was measured and correlated with GAG mass from a standard curve. To prepare the standard curve, 2 to 10 μg of the test GAG in 150 μl of water was mixed with 150 μl of 0.15 mg/ml toluidine blue solution. Next, 300 μl of cyclohexane was added, and the mixture was vortexed. The mixture was allowed to separate, and the organic layer with associated toluidine blue-GAG complex was removed by aspiration. The decrease in the absorbance of the aqueous layer at 630 nm was then measured. Heparin Desorption Kinetics To evaluate the kinetics of heparin desorption from complex membranes, 500 μl of PBS were applied to each of a series of heparin-chitosan membranes in 24-well plates. The plates were incubated at 37°C, and solutions were collected at time intervals up to 96 h. The heparin content of solutions and membranes was then measured as described above. Statistical Analysis Cell expansion on GAG-chitosan and other test surfaces was compared to expansion on the control plastic surface by applying Student's t-test to corresponding time points. Significance was tested at a probability level of 5%. Results Culture Membrane Formation and Morphology In solution, chitosan is a positively charged polymer and is capable of forming insoluble ionic complexes with negatively charged polymers such as the glycosaminoglycans. Similarly, dehydrated chitosan salts can be complexed in situ by rehydrating with an aqueous solution of a negatively charged polymer. We have previously used this ionic interaction to microencapsulate primary hepatocytes [15, 22]. Similar chitosan-GAG complexes have also been applied as aids to wound healing and tissue regeneration [23-25]. Membranes produced by the GAG-chitosan complex formation procedure appeared granular and textured under the phase contrast light microscope. Numerous ridges and convolutions could be clearly seen. In contrast, untreated chitosan surfaces were smooth and transparent. Complex membranes exhibited uniform staining with toluidine blue, indicating uniform GAG distribution. After six weeks of culture, toluidine blue staining was still significant, indicating that measurable quantities of GAG remained throughout the culture period. Cell Morphology and Adhesion While the significance of cell shape in these studies is uncertain, it is generally believed that the most primitive hematopoietic progenitors are poorly adherent and thus may be more likely to exhibit a spherical morphology. In light of this, we briefly report our morphological observations. In general, the vast majority of proliferating cells were nonadherent. In addition to individual cells, some formation of both detached and adherent cell aggregates was observed on the CSC-chitosan surfaces. The number and size of these aggregate colonies increased during the culture period (Fig. 1). Cells of a variety of shapes and sizes were also observed on the CSC-chitosan surfaces in the latter half of the culture period. In the case of noncoated polystyrene surfaces, multiple cell shapes and sizes were also observed, but few aggregates were seen up to week 6. On the heparin-chitosan surfaces, no colony formation or aggregation was seen, and all cells maintained their original spherical shape. On the chitosan and HA-chitosan surfaces, some aggregate formation was observed at the end of two weeks, but aggregate frequency subsequently declined. In the case of CSB, a small number of aggregates was observed in the latter half of the culture. Figure 1. Open in new tabDownload slide Cord blood CD34+ cells on GAG-chitosan membranes. Phase contrast micrographs. A: CSB, week 4. B: heparin, week 4. C: attached colonies on CSC, week 4. D: CSB-depleted membranes, week 6. Note the adherent cell population. Figure 1. Open in new tabDownload slide Cord blood CD34+ cells on GAG-chitosan membranes. Phase contrast micrographs. A: CSB, week 4. B: heparin, week 4. C: attached colonies on CSC, week 4. D: CSB-depleted membranes, week 6. Note the adherent cell population. With the exception of the aggregate colonies described above, cell attachment to the GAG-chitosan membranes was negligible for most of the culture period. On the more highly textured heparin-chitosan and CSB-chitosan membranes, many cells appeared to be wedged in membrane niches and valleys, but these could be easily detached with gentle washing. Growth of a fully spread, strongly adherent cell population was observed in the noncoated and collagen-coated wells during weeks 3 to 6 of culture. To examine the growth contributions of the adherent cells, two- and four-week-old plates, from which the nonadherent and weakly adherent cells had been removed by washing, were supplied with fresh medium for an additional two weeks. No significant cell growth was observed, indicating that the critical proliferative cell population was either nonadherent or only weakly adherent. Cell Proliferation Most surfaces appeared to support large increases in total cell number (Fig. 2). With the exception of pure chitosan and CSA-chitosan membranes, all produced greater than a 25-fold increase in total cells after six weeks of culture. Cell numbers on chitosan membranes appeared to plateau at about a 15-fold increase. Interestingly, CSA-chitosan membranes repeatedly showed an inhibitory effect on cell proliferation and survival, usually resulting in complete cell death by the end of week 2. In contrast, cells on CSC-chitosan consistently exhibited the highest total growth with approximately a 45-fold increase. Figure 2. Open in new tabDownload slide Effect of GAG-chitosan surfaces on total cell proliferation. Right and left y-axes display total cell number and expansion ratio respectively. The expansion ratio was obtained by dividing the total cell number at any time point by the number of cells initially seeded. Data points and error bars represent the combined mean and standard deviation from two experimental runs, with a minimum of three replicate culture wells per run. Lower error bars have been omitted for clarity. Asterisks indicate statistically significant differences with respect to control. Figure 2. Open in new tabDownload slide Effect of GAG-chitosan surfaces on total cell proliferation. Right and left y-axes display total cell number and expansion ratio respectively. The expansion ratio was obtained by dividing the total cell number at any time point by the number of cells initially seeded. Data points and error bars represent the combined mean and standard deviation from two experimental runs, with a minimum of three replicate culture wells per run. Lower error bars have been omitted for clarity. Asterisks indicate statistically significant differences with respect to control. In contrast to the observed increases in total cells, only heparin-chitosan and CSB-chitosan membranes consistently exhibited significant expansion of the CD34+ population, as shown in Figure 3. In the case of heparin-chitosan, proliferation peaked at week 4 with a 35-fold increase of the CD34+ population compared with 25-fold expansion with CSB-chitosan. The other surfaces generally exhibited CD34+ cell increases of less than 10-fold. While actual expansion numbers varied considerably from one isolation to the next, these two surfaces always exhibited superior growth of the CD34+ cell fraction. In general, CSB membranes tended to produce their maximum growth of CD34+ cells within the first two weeks of culture, whereas heparin membranes tended to peak at week 4. It should be noted that the apparent declines in CD34+ cell numbers after week 4 probably represent transitions to more differentiated stages and the associated loss of CD34 expression. With the exception of heparin and CSB membranes, all surfaces exhibited negligible CD34+ content after six weeks. These results suggest that heparin and CSB interacted with the progenitor cells in such a manner as to either preferentially enhance the proliferation of CD34+ cells or to inhibit transitions to more differentiated phenotypes. Figure 3. Open in new tabDownload slide Effect of GAG-chitosan surfaces on proliferation of CD34+ cells. Right and left y-axes display CD34+ cell number and expansion ratio, respectively. The expansion ratio was obtained by dividing the number of CD34+ cells at any time point by the number of CD34+ cells initially seeded. Data points and error bars represent the combined mean and standard deviation from two experimental runs, with a minimum of three replicate culture wells per run. Lower error bars have been omitted for clarity. Asterisks indicate statistically significant differences with respect to control. Figure 3. Open in new tabDownload slide Effect of GAG-chitosan surfaces on proliferation of CD34+ cells. Right and left y-axes display CD34+ cell number and expansion ratio, respectively. The expansion ratio was obtained by dividing the number of CD34+ cells at any time point by the number of CD34+ cells initially seeded. Data points and error bars represent the combined mean and standard deviation from two experimental runs, with a minimum of three replicate culture wells per run. Lower error bars have been omitted for clarity. Asterisks indicate statistically significant differences with respect to control. CD34+ Cell Percentages and Enrichment Figure 4 illustrates the trends in CD34+ cell percentages observed in culture. Although high levels of overall cell proliferation were observed on many surfaces (Fig. 2), only heparin and CSB membranes typically showed significant enrichment of the culture with CD34+ cells, and both maintained CD34+ percentages above the initial values for at least four weeks of culture. On these two surfaces, CD34+ content generally exceeded 60%, and it went as high as 100% on some heparin membranes (data not shown). In contrast, the other membranes and controls never exceeded the initial seeding value and usually exhibited steady declines. Figure 4. Open in new tabDownload slide Effect of GAG surfaces on CD34+ cell percentages. Data points are the mean and standard deviation from two experimental runs, with a minimum of three replicate culture wells per run. Asterisks indicate statistically significant differences with respect to control. Figure 4. Open in new tabDownload slide Effect of GAG surfaces on CD34+ cell percentages. Data points are the mean and standard deviation from two experimental runs, with a minimum of three replicate culture wells per run. Asterisks indicate statistically significant differences with respect to control. GAG Desorption Studies In the culture studies, GAG immobilization was accomplished by forming an ionic complex membrane. The amount of sulfated GAG which could be initially immobilized per well appeared to be independent of GAG type (Table 1). Since the ionic environment changed drastically going from membrane formation conditions to cell culture conditions, partial dissociation of the complexes during culture was considered likely. So in order to better understand the role of soluble, desorbed GAG in the observed growth phenomena, the desorption kinetics were studied. Experiments were conducted to evaluate the effect of medium changes on the GAG content of both the membrane and the medium. Conditions were chosen to mimic those of actual cell cultures. Heparin was used as the test GAG for this study. The results of these experiments are summarized in Figure 5. With each 50% medium change, a quantity of heparin desorbs from the membrane. After 24 days, approximately 10% of the heparin initially complexed remains on the chitosan membrane. The magnitude of this residual fraction appeared unaffected by the frequency of medium changes (data not shown). The presence of stable bound heparin after 28 days was confirmed with toluidine blue staining and by the observation that GAG-complex membranes were still insoluble in 0.1 M acetic acid after four weeks of culture, whereas pure chitosan membranes dissolved easily. The kinetics of heparin desorption between medium changes was also examined (Fig. 6). The desorption followed a hyperbolic curve which reached an equilibrium state after ∼5 h. While the initial desorption removed about 50% of the membrane heparin, subsequent medium changes removed approximately 20% of the remaining heparin. The results of these studies indicate that the GAG content of both membrane and culture medium declined with each medium change over the course of a culture. Thus the effects of exposure to a specific GAG moiety would also be expected to vary with time. We should note that heparin has the highest molecular charge density of the GAGs examined. It is therefore likely to exhibit the highest binding strength of any GAG in a GAG-chitosan complex. This higher binding strength would translate into a reduced desorption rate, thus suggesting that the desorption rates for the other sulfated GAGs could reasonably be expected to be higher. This argument may not hold for hyaluronate, since the high molecular weight of this molecule (∼1,000,000) may play a much more significant role in determining desorption kinetics. Table 1: Quantity of GAG initially bound per well. Approximately 1 mg of chitosan was deposited per well. Each well had an area of 1.9 cm2. Data are mean and standard deviation from six wells. GAG . Initial amount (μg/cm2) . Heparin 110.60 ± 4.97 CSA 99.45 ± 5.55 CSB 102.31 ± 5.36 CSC 115.25 ± 5.48 GAG . Initial amount (μg/cm2) . Heparin 110.60 ± 4.97 CSA 99.45 ± 5.55 CSB 102.31 ± 5.36 CSC 115.25 ± 5.48 Open in new tab Table 1: Quantity of GAG initially bound per well. Approximately 1 mg of chitosan was deposited per well. Each well had an area of 1.9 cm2. Data are mean and standard deviation from six wells. GAG . Initial amount (μg/cm2) . Heparin 110.60 ± 4.97 CSA 99.45 ± 5.55 CSB 102.31 ± 5.36 CSC 115.25 ± 5.48 GAG . Initial amount (μg/cm2) . Heparin 110.60 ± 4.97 CSA 99.45 ± 5.55 CSB 102.31 ± 5.36 CSC 115.25 ± 5.48 Open in new tab Figure 5. Open in new tabDownload slide Effect of culture medium changes on bound and soluble heparin levels. A: heparin content of the solution. B: heparin content of the membrane. Data points are the mean and standard deviation of four replicate samples. Figure 5. Open in new tabDownload slide Effect of culture medium changes on bound and soluble heparin levels. A: heparin content of the solution. B: heparin content of the membrane. Data points are the mean and standard deviation of four replicate samples. Figure 6. Open in new tabDownload slide Fractional heparin content of heparin-chitosan membrane versus time after addition of fresh culture medium. Values are normalized to the heparin quantity initially immobilized. Figure 6. Open in new tabDownload slide Fractional heparin content of heparin-chitosan membrane versus time after addition of fresh culture medium. Values are normalized to the heparin quantity initially immobilized. Effects of Soluble and Bound GAG Levels on Cell Proliferation The heparin desorption studies confirmed the transient nature of GAG levels in cultures on the “regular” membranes. Since the precise nature of a GAG effect might be expected to vary with either solution concentration or surface density, we conducted additional cultures with the goal of clarifying the concentration effects of GAGs in both membranes and solutions. Heparin and CSB were chosen for these studies because of their overall superior effect on CD34+ cell growth. Alongside “regular” membranes prepared with four brief buffer washes, cultures were conducted on GAG-depleted membranes, where the membrane was subjected to extensive equilibrium washing to desorb GAG. The GAG level at cell seeding for these membranes was estimated to be characteristic of weeks 4 to 6 in the regular membrane cultures. For heparin, this represented a membrane content of <10 μg/cm2 at cell seeding, compared with >100 μg/cm2 for regular membranes. The effects of soluble GAG in the absence of a membrane were also examined using concentrations characteristic of the early and late phases of a regular membrane culture (i.e., 160 and 7 μg/ml). The results of these studies are presented in Figures 7 and 8. Figure 7. Open in new tabDownload slide Effects of regular membranes, GAG-depleted membranes, and soluble GAGs on total cell proliferation. Right and left y-axes display total cell number and expansion ratio, respectively. The expansion ratio was obtained by dividing the total cell number at any time point by the number of cells initially seeded. Data points and error bars represent the mean and standard deviation from a representative culture, with a minimum of three replicate culture wells. Lower error bars have been omitted for clarity. Figure 7. Open in new tabDownload slide Effects of regular membranes, GAG-depleted membranes, and soluble GAGs on total cell proliferation. Right and left y-axes display total cell number and expansion ratio, respectively. The expansion ratio was obtained by dividing the total cell number at any time point by the number of cells initially seeded. Data points and error bars represent the mean and standard deviation from a representative culture, with a minimum of three replicate culture wells. Lower error bars have been omitted for clarity. Figure 8. Open in new tabDownload slide Effect of regular membranes, GAG-depleted membranes, and soluble GAGs on CD34+ cell proliferation. Right and left y-axes display CD34+ cell number and expansion ratio, respectively. The expansion ratio was obtained by dividing the number of CD34+ cells at any time point by the number of CD34+ cells initially seeded. Data points and error bars represent the mean and standard deviation from a representative culture, with a minimum of three replicate culture wells. Lower error bars have been omitted for clarity. Figure 8. Open in new tabDownload slide Effect of regular membranes, GAG-depleted membranes, and soluble GAGs on CD34+ cell proliferation. Right and left y-axes display CD34+ cell number and expansion ratio, respectively. The expansion ratio was obtained by dividing the number of CD34+ cells at any time point by the number of CD34+ cells initially seeded. Data points and error bars represent the mean and standard deviation from a representative culture, with a minimum of three replicate culture wells. Lower error bars have been omitted for clarity. All conditions, with the exception of 160 μg/ml heparin, supported cell growth. Figure 7 shows that 160 μg/ml heparin in solution completely inhibited cell proliferation. In fact, microscopic examination confirmed that this concentration was toxic, and all seeded cells died. Of the remaining heparin conditions, the regular membranes exhibited the lowest level of proliferation (25-fold increase at week 4), and the depleted membrane exhibited approximately a 50-fold increase at week 4. Similar total cell results were obtained with the CSB conditions, with the notable difference that 160 μg/ml CSB in solution supported cell proliferation well. The CD34+ cell results indicated that the regular membranes produce the highest proliferation for heparin, with a peak of 40-fold at four weeks. The heparin-depleted membranes and 7 μg/ml heparin in solution conditions showed similar profiles with continuously increasing CD34+ cell numbers up to week 6. In contrast, the 160 μg/ml CSB condition exhibited the highest CD34+ cell proliferation for this GAG with a 40-fold increase at week 2. CD34+ cell percentage data (not shown) indicated that in addition to regular heparin and CSB membranes, 160 μg/ml CSB also produced significant CD34+ cell enrichment. While most proliferating cells were nonadherent in the soluble GAG conditions, we noted significant proliferation of a heterogeneous adherent cell population in these membrane-free cultures. This population was apparent with both CSB concentrations as well as the low heparin case. While adherent cells were also observed on the depleted membranes, their numbers were significantly lower. Taken together, these results indicate that soluble GAGs contribute to the proliferative effects of the GAG-chitosan membranes. The effects are strongly concentration-dependent, and optimal level is dependent on the particular GAG. The results also suggest that the highly transient nature of the GAG levels above regular membranes may play a major role in the maintenance of and enrichment with CD34+ cells seen with these two GAGs. Discussion The difficulties associated with generating large quantities of hematopoietic stem cells (HSCs) severely limit the widespread use of allogeneic HSC transplantation as an effective treatment for hematologic and malignant disease. In addition, the success rate of hematopoietic transplant therapies could be significantly increased if large quantities of transplant material with a high incidence of HSCs could be reliably generated. In most hematopoietic expansion schemes, expansion and lineage differentiation proceed in parallel, and HSC expansion per se is either limited or nonexistent. This limitation is exacerbated by the fact that the mechanism by which stem cells are induced to undergo either self-renewal or lineage differentiation is poorly understood. The difficulty of controlling HSC growth in vitro can be partly attributed to the many complex interactions existing in most experimental systems and points to the need for alternate systems by which factors that are believed to play a role in HSC renewal can be studied individually. This study illustrates an in vitro system designed to present individual GAGs in both soluble and immobilized form. Hence, it provides a mechanism by which the role of GAGs in hematopoietic control may be studied without the confounding factors associated with a stromal cell layer. CD34-enriched cord blood cells were cultured on defined ECM polysaccharide membranes, and their proliferative activity was evaluated. Our results show that it is possible not only to expand cell numbers greatly but also to simultaneously enrich a culture in CD34+ cells using this system. The data presented are from a total of three cord blood culture runs. However, in several previously conducted experimental runs, the trends of heparin and CSB superiority are consistent and reproducible. Those additional runs are not incorporated, because they did not include all the GAGs presented here and some were of shorter duration. Since we wished to evaluate the differences between GAGs, we have presented only those runs in which all the GAGs were studied simultaneously. A number of interesting observations were made with regard to the relative effects of various GAGs on cell proliferation. First, the difference in response to CSA (chondroitin-4-sulfate) and CSC (chondroitin-6-sulfate) was striking given that the two GAG species differ only in the location of the sulfate group on the disaccharide repeating unit. CSC consistently exhibited the highest total cell proliferation of any GAG, although it performed poorly with regard to maintenance of the CD34+ population. In contrast, the apparent toxicity of CSA to these cells suggests that the effect may have been mediated by interactions involving highly specific recognition of CSA. Since CSA is not known to be inherently toxic to any cell type, the possibility of an apoptotic mechanism may bear further investigation. The superiority of CSB in CD34 maintenance also points to the existence of highly specific interactions. CSB differs from CSA and CSC by the location of the sulfate group and the presence of L-iduronic acid as a constituent monosaccharide [26]. Thus, it is likely that unique binding characteristics with cytokines and/or receptors may be key to its effect. In comparing heparin and CSB, we noted that although CSB generally reached its maximum CD34+ expansion before heparin (week 2 versus week 4), there appeared to be no correlation between the proliferation rate and the CD34+ percentage with either GAG. The observed total cell expansion was similar to that reported for the expansion of CD34+ cells in stroma-free, cytokine-based (specifically, IL-3 and SCF) systems [27-29]. The results of De Bruyn et al. [27] indicated that the expansion of CD34+ cells peaked at day 14, in all combinations of SCF, IL-3, IL-6, GM-CSF, and anti-transforming growth factor-β. After day 14, the population declined. In our experiments, expansion of the CD34+ population was observed with certain GAGs up to four weeks. Some cultures on heparin surfaces maintained high percentages of CD34+ cells with expansion even up to six weeks. These conditions, specifically those incorporating heparin and CSB, were able to produce a selective expansion of the more primitive progenitors while apparently inhibiting extensive differentiation. In spite of the fact that CD34 expression was maintained on heparin and CSB surfaces, the precise functional characteristics of the cells expanded in our cultures have not yet been determined. Gupta et al. [9] reported that heparin did not support maintenance of the LTC-ICs. Our results show that for heparin, the CD34+ culture outcomes ranged from minimal proliferation to extensive proliferation to full toxicity as a function of the heparin levels. This concentration effect is supported by reports that variations in GAG levels may play a significant role in the regulation of hematopoiesis [30, 31]. Notably, the activities of IL-3 and GM-CSF were shown to increase as the concentration of heparan sulfate increased [30]. Activity reached a maximum with increasing GAG concentration followed by a decline in activity with further increase in the GAG level. Our heparin desorption studies indicated that while the complex membranes were synthesized in such a manner as to ensure that the cell contact surface was comprised primarily of the GAG component, most of the GAG leached into solution over the course of the culture. This suggests that the observed effects may in fact be due mainly to soluble GAG interactions as opposed to interactions with surface-bound molecules. In addition, the desorption experiments showed that solution concentrations fall by an order of magnitude during culture. Thus, the relative importance of surface-bound versus soluble GAG may change with time, leading to a greater role for that immobilized on the surface at later times in culture. The mechanism by which GAGs may alter the proliferative disposition of an HSC-enriched population in our system is not yet clear. Clearly, the ability of GAG species to bind and enhance the activities of some hematopoietic growth factors is recognized and can be expected to play a role. By the same token, GAGs may shield, sequester, or otherwise reduce the availability of other growth factors. For any given growth factor, these GAG-mediated effects on HSCs can reasonably be expected to vary with the GAG concentration. In light of these possibilities, additional studies are clearly needed to identify concentration thresholds and the precise nature of the molecular interactions involved. Likewise, the relative roles of immobilized and soluble GAG must be further clarified. Our observations on the effects of soluble GAGs and GAG-depleted membranes suggest a number of potential mechanisms by which selective proliferation of and enrichment with CD34+ cells may have occurred in our system. The toxicity of high heparin levels suggests that the high solution concentration during the early stages of culture on regular membranes may have selectively killed or inhibited the proliferation of committed progenitors. Furthermore, the lower levels found later in culture may have provided appropriate enhancement of early progenitor growth. The GAG-chitosan membrane may also have had an effect separate to the growth factor activating ability of the GAG component. Prior work in our laboratory has shown that GAG-chitosan membranes limit the attachment and inhibit proliferation of a number of cell types, including fibroblasts, smooth muscle cells, and endothelial cells. Thus, it is likely that the surface would have limited attachment and spreading of contaminating stromal components and more differentiated cells in our CD34+ preparation. Limited cell spreading is often accompanied by reduced function. Since such cells are known to secrete differentiation-triggering soluble factors, reduced function may have correlated with a reduced differentiation signal, thus allowing proportionately greater self-renewal of CD34+ cells. This concept is supported by the observation that the reduction in GAG content later in cultures coincided with the appearance of an adherent population. In keeping with this idea, reductions in CD34+ cell content between weeks 4 and 6 may have been partially caused by an increasing differentiation-inducing signal from the growing adherent population. Finally, it has recently been shown that heparin is capable of activating a fibroblast growth factor receptor [32]. While this receptor may not be of major significance for HSC proliferation, the fact raises the possibility that similar direct GAG effects may occur in this system at some optimal concentration. It should be noted that since expression of the CD33 and CD38 antigens was not evaluated in our expanded cell population, the precise nature of the CD34+ cells at four weeks is not known. It is possible that the expanded population may represent mostly early progenitors already committed to a differentiation track, as opposed to true, uncommitted “stem cells.” In conclusion, this culture system has demonstrated the ability to produce simultaneous proliferation and CD34+ cell enrichment of a partially purified cord blood population by controlling the nature and levels of GAG moieties to which the cells are exposed. Acknowledgements The authors wish to acknowledge the advice and editorial assistance of Dr. James Eliason of the Karmanos Cancer Institute. We also wish to acknowledge Maureen Hoosang, R.N. and staff of the Hutzel Hospital, Labor and Delivery Center for assistance in the collection of cord blood samples. 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TI - Maintenance of CD34 Expression During Proliferation of CD34+ Cord Blood Cells on Glycosaminoglycan Surfaces
JF - Stem Cells
DO - 10.1002/stem.170295
DA - 1999-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/maintenance-of-cd34-expression-during-proliferation-of-cd34-cord-blood-KvW2u3NivB
SP - 295
EP - 305
VL - 17
IS - 5
DP - DeepDyve
ER -