Cytokine-Dependent Proliferation of Human CD34 Progenitor Cells in the Absence - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:11 |只看该作者 |倒序浏览 |打印

作者：Henriette M. Goselinka, Pieter S. Hiemstrab, Peter van Noortb, Rene M.Y. Bargea, Roel Willemzea, J.H. Frederik Falkenburga作者单位：a Laboratory of Experimental Hematology, Department of Hematology,b Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands





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      【摘要】
   In this study, we demonstrate that the synthesis and release of serine proteinases by hematopoietic cells affects the in vitro proliferation of hematopoietic progenitor cells (HPCs) in response to proteins, including hematopoietic growth factors (HGFs), transferrin, insulin, and albumin in serum-free cultures. In serum-free cultures, bone marrow mononuclear cells or the CD34¨C progeny of the CD34  cells were shown to release the serine proteinases human neutrophil elastase (HNE), cathepsin G (Cath G), and proteinase 3 (Pr3). In the absence of serum, we showed that HNE, Cath G, and Pr3 rapidly and dose-dependently degraded HGF and other proteins present in the medium, resulting in decreased proliferation of HPCs. Addition of the serine proteinase inhibitors 1¨Cproteinase inhibitor (1-PI) or the secretory leukocyte proteinase inhibitor (SLPI), but not leupeptin, aprotinin, or AEBSF (4--benzenesulfonylfluoride hydrochloride), could completely prevent the degradation of proteins relevant to the growth of hematopoietic cells. Thus, the addition of serine proteinase inhibitors like 1-PI or SLPI may be critical for the expansion of CD34  cells or gene transfer into CD34  cells or other hematopoietic cells in vitro using serum-free media under good manufacturing practice conditions.
      【关键词】 Hematopoietic progenitor cells Serum-free culture Hematopoietic growth factor Serine proteinase2 I/ I# m1 R6 z# [
   INTRODUCTION" X9 l" J7 ?. e. B* @' n5 \# N
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Serum-free media are increasingly used for in vitro studies of hematopoietic cells and for expansion of stem cells, dendritic cells, or other hematopoietic cells for clinical use . Plasma or serum contains proteinase inhibitors that can neutralize these proteinases. We hypothesized that bone marrow accessory cells and also the progeny of the CD34  cells during in vitro maturation synthesize and release proteinases that in the absence of plasma or serum may degrade proteins in the culture media, thereby influencing the proliferation and differentiation of HPCs in response to HGF.

In this study, we analyzed the synthesis and release of serine proteinases by bone marrow accessory cells, purified CD34  cells, and their progeny in serum-free cultures and their influence on the proliferation of HPCs in response to HGF. We investigated the substrates of the serine proteinases in these HPC cultures and the specific serine proteinase inhibitors that could neutralize the suppression of HGF-induced growth of HPCs. We demonstrate that in serum-free media, bone marrow accessory cells degrade HGF and other proteins, including human serum albumin (HSA), through the production and release of various serine proteinases. Addition of specific serine proteinase inhibitors to these cultures completely prevented this degradation.
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MATERIALS AND METHODS
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Enzymes, Cytokines, Antibodies, Inhibitors, and Specific Neutralizers& V( P( Q1 I: U% ?
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Human neutrophil elastase (HNE), cathepsin G (Cath G), and the nonenzymatic neutrophil defensins (human neutrophil peptides . Neutrophil proteinase 3 (Pr3) was a generous gift from Dr. M.R. Daha (Leiden University Medical Center, Leiden, The Netherlands). Urokinase (Medacinase) was provided by Medac GmbH (Hamburg, Germany, http://www.medac.de). Human recombinant stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) were provided by Amgen (Thousand Oaks, CA, http://www.amgen.com). Human interleukin-3 (IL-3), GM-CSF, and IL-6 were gifts from Novartis International (Basel, Switzerland, http://www.novartis.com), and erythropoietin (EPO) was provided by Cilag (Janssen-Cilag, Herentals, Belgium, http://www.janssen-cilag.be). The polyclonal rabbit antibodies against HNE, Cath G, and Pr3 were raised in the author¡¯s laboratory (P.S.H.) and provided by Calbiochem (catalog no. 219358; San Diego, http://www.emdbiosciences.com) and Dr. Daha, respectively. The purified Immunoglobulin G (IgG) fractions from these rabbit anti-sera showed a high titer (1:1,000) against its specific proteinase. 1¨Cproteinase inhibitor (1-PI), purified from human plasma, was obtained from SERVA (Inglesheim, Germany, http://www.serva.de). Human recombinant secretory leukocyte proteinase inhibitor (SLPI) was a generous gift from Dr. R.C. Thompson (Synergen Inc., Boulder, CO, http://www.synergen.com). Leupeptin, aprotinin (purified from bovine lung), and 4-(2-Aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF) were purchased from Boehringer Mannheim (Mannheim, Germany, http://www.boehringer.com).
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Cell Preparations
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After informed consent, bone marrow samples were obtained from healthy donors. The cells were separated over Ficoll isopaque, and the interphase cells were depleted of mature monocytes and T lymphocytes as described previously . These bone marrow mononuclear cells (BMMNCs) were cryo-preserved in liquid nitrogen until use. Purified CD34  cells and CD34¨C cells were obtained from thawed BMMNCs, using Dynabeads M-450 (Dynal Biotech, Oslo, Norway, http://www.dynal.no) according to the manufacturer¡¯s instructions. Beads containing antibodies were removed from the CD34  cells using a sheep polyclonal antibody anti-CD34 Fab IgG (DE-TACHaBEAD CD34; Dynal Biotech). FACS (fluorescence-activated cell sorter) analysis showed that the purified CD34  fractions contained >90% CD34  cells. Eosin dye exclusion of the unseparated BMMNCs and the CD34¨C and CD34  populations (n = 3) resulted in viability percentages of 88% ¡À 7%, 91% ¡À 6%, and 100% ¡À 0% (mean value ¡À SD), respectively.
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Cell Cultures to Analyze Synthesis and Release of Serine Proteinases
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To analyze the synthesis and release of serine proteinases by BMMNCs or subpopulations, cultures were performed in serum-free medium in roundbottomed 96-well microtiter plates (Costar, Corning Incorporated, Corning, NY, http://www.corning.com) in aliquots of 0.15 ml culture medium per well, containing 250,000 viable BMMNCs per ml, 250,000 viable CD34¨C cells per ml, or 30,000 viable CD34  cells per ml. The culture medium consisted of Iscove¡¯s modified Dulbecco¡¯s medium (BioWhittaker, Verviers, Belgium, http://www.cambrex.com) supplemented with 0.6% (wt/vol) purified clinical-grade HSA (Central Laboratory of the Blood Transfusion Services, Amsterdam, The Netherlands, http://www.sanguin.com), 20 µg/ml cholesterol (Sigma, St. Louis, http://www.sigmaaldrich.com), 10 µg/ml insulin (Sigma), 5 x10¨C5 M ß-mercaptoethanol, 200 µg/ml human transferrin APO-F (apolipoprotein F precursor; Boehringer Mannheim) saturated with FeCl3 ¡¤ 6H2O, to which a mixture of HGF was added, including SCF (at a final concentration of 50 ng/ml), IL-3 (50 ng/ml), GM-CSF (10 ng/ml), G-CSF (10 ng/ml), IL-6 (1 ng/ml), and EPO (2 IU/ml). After various time intervals up to 14 days of culture, aliquots of cell suspensions were centrifuged (10 minutes, 350g) and supernatants were harvested. To analyze the intracellular protein content, cell pellets were lysed for 1 hour at 20¡ãC in phosphate-buffered saline containing 0.1% (vol/vol) Triton-X 100 (T-6878; Sigma). All samples were analyzed in serial dilutions (l:5¨C1:50) using the sandwich-type enzyme-linked immunosorbent assay (ELISA) to determine total content of HNE and Cath G )¨C0.1% H2O2 as substrate. The reaction was stopped by addition of 50 µl oxalic acid 2%, and absorbance was read at 415 nm. The lower limit of detection of the assay was less than 1 nM Pr3.8 S& X% j' m7 ?7 S* R
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To visualize possible degradation of cytokines by factors released by BMMNCs, culture medium containing 100 µg/ml transferrin, 0.05% (wt/vol) HSA, and 200 µg/ml G-CSF or GM-CSF was incubated in the presence or absence of 500,000 BMMNCs per ml. At day 0, 3, 6, 9, or 14 of incubation, 100 µl culture medium was harvested and centrifuged (10 minutes, 350g). Quantities of 5 µl and 20 µl of each supernatant were loaded under reducing conditions onto a 15% (wt/vol) SDS-PAGE and run simultaneously with molecular mass markers. As positive controls for the degradation of G-CSF or GM-CSF by serine proteinases, 1 µg of each cytokine was incubated for 0¨C24 hours with 18 µM HNE in a final volume of 10 µl. The gels were stained with Coomassie Blue to visualize the proteins.4 O+ n6 x% M7 z- _3 h) z7 G9 A0 i

Inhibition of HPC Proliferation as Determined in Colony-Forming Unit Assays8 [5 G- T& _" z3 [! x2 a& l

Semi-solid colony assays were performed using culture medium supplemented with methylcellulose (Methocel 4,000 cps; Fluka, Freiburg, Germany, http://www.sigma-aldrich.com) at a final concentration of 1.1% (wt/vol), SCF (50 ng/ml), IL-3 (50 ng/ml), GM-CSF (10 ng/ml), G-CSF (10 ng/ml), IL-6 (1 ng/ml), and EPO (2 IU/ml). Cultures were assayed in sixfold in flatbottomed 96-well microtiter plates (Greiner, Alphen a/d Rijn, The Netherlands, http://www.greinerbiooneinc.com) in aliquots of 0.1 ml per well, containing either 103 BMMNCs or 150 CD34  cells. To determine a possible effect of proteinases produced by BMMNCs on HPC proliferation, BMMNCs were cultured in the presence or absence of a serine proteinase inhibitor (1-PI, SLPI, aprotinin, AEBSF, or leupeptin) at molar concentrations of 1¨C1,000 nM. Purified CD34  cells were cultured in the absence or presence of increasing concentrations of the serine proteinases HNE, Cath G, Pr3, or urokinase, or of the nonenzymatic peptides HNP1¨C3. In some experiments, the cultures were preincubated for 2 hours with 1-PI to neutralize the serine proteinases before addition of the CD34  cells. In all experiments, a control culture was performed using culture medium supplemented with 10% (vol/vol) human serum (blood type AB positive), which was derived from healthy blood donors, pre-screened for allo antibodies, and inactivated for 30 minutes at 56¡ãC. After 12 days of incubation in a humidified atmosphere of 5% CO2 at 37¡ãC; granulocyte-macrophage colony-forming units (CFU-GM) defined as aggregates of more than 50 cells and burst-forming units erythroid colonies (BFU-E) defined as hemoglobinized bursts were counted. Colony growth was expressed as percentage growth of the control cultures in the presence of 10% human AB serum.! V  m5 ]6 R. y; A3 V  x0 f* o

Statistical Analysis

Data are expressed as mean ¡À SD, or the paired Student¡¯s t-test was used to analyze the significant difference of results.
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RESULTS

Production and Release of Serine Proteinases by BMMNC, CD34¨C, or CD34  Bone Marrow Cells

The synthesis of serine proteinases and their release into the medium was studied at the start and after various time intervals from initiation of the culture of bone marrow cells in the presence of HGF. Unfractionated BMMNCs showed a stable viability of 88% ¡À 7% up to day 6 of culture, followed by an increase of nonviable cells. In Table 1, the presence of HNE, Pr3, and Cath G is shown in cultures of 3 x 105 unseparated BMMNCs or after separation of BMMNCs into CD34¨C and CD34  populations from day 0 of incubation to day 14.. k7 L7 x# K8 f2 ?, o  j7 |" b

Table 1. Serine proteinase levels in cultures of unseparated bone marrow mononuclear cells (BMMNCs) and in purified CD34¨C and CD34  subpopulations in response to hematopoietic growth factors5 _8 p$ _. L6 w( a

At day 0, HNE, Pr3, and Cath G were demonstrated to be intracellularly present in the unseparated BMMNCs. Cell lysis resulted in release of 5.7 ¡À 2.1 nM, 4.0 ¡À 2.7 nM, and 1.9 ¡À 1.4 nM protein, respectively. Cell-sorting experiments illustrated that at initiation of the culture, HNE, Pr3, and Cath G were present in the CD34¨C, but not in the CD34 , cell population. During culture, the intracellular content of HNE, Pr3, and Cath G in CD34¨C cells decreased. In contrast, the progeny of the CD34  cell population showed a rapid intracellular increase of HNE, Pr3, and Cath G from day 3 of culture.0 m/ h4 r% r8 h5 q  {9 H: y) W) J

The first day, the culture supernatants of 3 x 105 BMMNCs per ml contained 4.9 ¡À 3.4 nM HNE, 7.8 ¡À 5.5 nM Pr3, and 0.83 ¡À 0.31 nM Cath G. Cell sorting of BMMNCs into CD34  and CD34¨C cells revealed that release of HNE, Pr3, and Cath G into the medium was initially caused by the CD34¨C cell population. During culture, the release of HNE and Cath G, but not Pr3, by the CD34¨C cells decreased. In contrast, from day 3 of culture, an increase of HNE, Cath G, and Pr3 was found in the medium of the progeny of the CD34  population, which at day 6 had differentiated into mainly CD33  and CD13  cells (data not shown). As demonstrated in Figure 1, the increase of HNE, Pr3, and Cath G in the culture medium of CD34  cells after day 6 coincided with an increase of the numbers of nonviable cells in the population (Fig. 1), corresponding with a decrease in proliferation of CD34  cells in response to HGF.
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Figure 1. Expansion of purified CD34  cells in response to hematopoietic growth factor (HGF). Thirty thousand CD34  cells were cultured in serum-free medium in the presence of HGF and analyzed for total cell numbers and viability by eosin dye exclusion (n = 3). After 6 days of culture, the nonviable cell numbers increased. p = .01 at day 9 as compared with the nonviable cell numbers at day 6.
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Inhibition of HGF-Induced HPC Growth by Serine Proteinases0 f2 P* }8 a2 R6 h8 _0 G4 Z

HNE, Cath G, as well as Pr3 were analyzed for their ability to decrease HGF-induced proliferation of highly purified CD34  cells in the absence of serum. Addition of 0.5¨C2.5 nM of HNE or Cath G and 5¨C10 nM of Pr3 resulted in a more than 80% reduction of CFU-GM and BFU-E growth (Fig. 2A). In contrast, addition of the human serine proteinase urokinase or the non-enzymatic human HNP1¨C3 did not suppress HGF-induced HPC growth. A 2-hour preincubation of the proteinases with a neutralizing concentration of 1-PI before addition to the culture medium completely reversed the effect induced by HNE, Cath G, or Pr3 (Fig. 2B), illustrating the inhibitory activity of the serine proteinases HNE, Cath G, or Pr3 on the HGF-induced proliferation of CD34  cells.( F3 ^) ~1 i9 x) M/ p
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Figure 2. Effect of serine proteinases on hematopoietic progenitor cell growth of CD34  cells. One hundred fifty CD34  cells were cultured in semi-solid serum-free medium containing hematopoietic growth factors in the absence or presence of 1¨C20 nM HNE, Pr3, Cath G, HNP1¨C3, or urokinase (n = 3). Colony growth is expressed as percentage growth of the control culture in the presence of 10% serum blood type AB. The absolute colony numbers in the control culture were 10 ¡À 3 CFU-GM and 13 ¡À 2 BFU-E per 150 CD34  cells (mean value ¡À SD). HNE, Cath G, or Pr3 dose-dependently reduced CFU-GM and BFU-E growth up to 80% (A). ap  .005 at 2.5 nM HNE or Cath G and bp .005 at 5 nM Pr3 as compared with the cultures in the absence of HNE, Cath G, or Pr3. (B): The reduction of colony growth was completely abolished after neutralization with 2.5-fold nM 1¨Cproteinase inhibitor. Abbreviations: Cath G, cathepsin g; HNE, human neutrophil elastase; HNP1¨C3, human neutrophil proteins 1¨C3; Pr3, proteinase 3.6 v' U* \5 y" [# z

Substrates of Enzymatic Activity of Serine Proteinases
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To analyze the kinetics of the effect of serine proteinases present in the medium, culture medium with or without HGF was incubated with HNE for 2 hours. HNE was then neutralized or not neutralized by a second incubation with 1-PI for 2 hours before highly purified CD34  cells were added (Table 2). Addition of HNE to HGF in the culture medium completely abrogated colony formation of CFU-GM and BFU-E. Addition of HNE to HGF for 2 hours followed by neutralization of HNE with 1-PI for another 2 hours already resulted in a 68% reduction of CFU-GM and 15% of BFU-E colonies, indicating a rapid effect of HNE. If HGF was added after neutralization of HNE by 1- PI, normal HPC growth was observed, illustrating that degradation products from the proteins were not responsible for the reduced HPC growth (Table 2).
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Table 2. Sequential inactivation of HGF by HNE and the inhibitory effect of 1-PI& ?# h* q5 t% I6 c' G0 t

To analyze whether the inhibitory effect was induced by rapid removal of cell surface receptors from the cell membrane by the proteinases, HNE was first incubated for 2 hours with the CD34  cells prior to addition of 1-PI for another 2 hours followed by addition of the HGF. This resulted in only 5% ¡À 4% and 25% ¡À 11% reductions of CFU-GM and BFU-E colony formation, respectively (n = 2, data not shown), illustrating that although a direct effect on surface receptors may be present, this was likely not to be the main mechanism of proteinase-induced growth inhibition of HPCs in the presence of HGF." S/ a: S' b7 U5 N
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To illustrate degradation of HGF and other proteins in the medium by HNE, culture medium containing G-CSF, IL-3, or GM-CSF was incubated with HNE for 2 hours, and the proteins were analyzed on SDS-PAGE. Within 2 hours of incubation, 100 µg/ml of G-CSF, IL-3, and GM-CSF were strongly degraded into small fragments in the presence of 18 µM HNE (Fig. 3), illustrating the rapid destruction of proteins in the medium by proteinases.

Figure 3. Degradation of hematopoietic growth factors by HNE. To visualize the proteins, serum-free medium containing high concentrations of G-CSF, IL-3, or GM-CSF (100 µg/ml) and 0.6% HSA (wt/vol) was incubated in the absence or presence of 18 µM HNE for 2 hours at 37¡ãC. The medium was analyzed on SDS-PAGE, and the proteins were stained with Coomassie Blue. Within 2 hours, 100 µg/ml of G-CSF, IL-3, and GM-CSF was strongly degraded into small fragments by HNE. Abbreviations: HNE, human neutrophil elastase; HSA, human serum albumin; IL, interleukin; M, molecular mass marker.  z- }$ c; Q' [4 T; o' V( t

To analyze whether a similar HGF degradation was observed in serum-free cultures of BMMNCs, 200 µg/ml of GM-CSF or G-CSF was incubated in the presence or absence of 5 x 105 BMMNCs per ml (Fig. 4). In the absence of BMMNCs, no degradation of proteins was detected during the culture periods. In contrast, BMMNCs strongly degraded GM-CSF and G-CSF within 24 hours of culture. These observations illustrate that in serum-free medium cultures of BMMNCs, HGFs present in the medium were degraded by proteinases produced by the BMMNCs.

Figure 4. Degradation of hematopoietic growth factors by BMMNCs. To be able to visualize the proteins, serum-free medium containing high concentrations of GM-CSF (A) or G-CSF (B) (200 µg/ml) and 0.6% human serum albumin wt/vol) was incubated in the absence or presence of 5 x 105 BMMNCs for 14 days or 6 days, respectively. At several intervals, the medium was analyzed on SDS-PAGE and the proteins were stained with Coomassie Blue. In the absence of BMMNCs, no degradation of GM-CSF and G-CSF was observed during the culture periods. In the presence of 5 x 105 BMMNCs, 200 µg/ml GM-CSF (A) and 200 µg/ml G-CSF (B) were strongly degraded within 24 hours of culture. Abbreviations: BMMNC, bone marrow mononuclear cell; M, molecular mass marker.

Inhibition of Proteinase Activity by Specific Serine Proteinase Inhibitors

To analyze whether the proteinases produced by accessory CD34¨C cells could be effectively neutralized by various serine proteinase inhibitors, BMMNCs were cultured in the absence or presence of serine proteinase inhibitors at molar concentrations of 1¨C1,000 nM. In the absence of inhibitors, only 25% ¡À 1% CFU-GM growth and 10% ¡À 10% BFU-E growth was observed as compared with cultures in the presence of control medium. Addition of 1 nM or higher of 1-PI or SLPI, but not of aprotinin, AEBSF, or leupeptin, completely restored HPC growth (Fig. 5), illustrating specificity of the serine proteinase inhibitors.0 p0 v5 f7 ?! P8 z: Y8 L9 R
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Figure 5. Effect of serine proteinase inhibitors on HGF-induced HPC growth from BMMNCs. One thousand BMMNCs were cultured in semi-solid serum-free medium containing HGF in the absence or presence of 1¨C1,000 nM 1-PI, aprotinin, AEBSF, leupeptin, or SLPI (n = 2). Colony growth is expressed as percentage growth of the control culture in the presence of 10% AB-serum. The absolute colony numbers in the control culture were 27 ¡À 14 CFU-GM and 21 ¡À 9 BFU-E per 103 BMMNCs. In the absence of inhibitors, only 25% ¡À 1% CFU-GM and 10% ¡À 10% BFU-E growth was observed (mean values ¡À SD). One nM 1-PI or 10 nM SLPI was sufficient to restore the HPC growth of 103 BMMNCs as compared with the control culture. One to 1,000 nM Aprotinin, AEBSF, or leupeptin could not restore the HPC growth of BMMNCs. ap = .06 and .05 at 1 nM 1-PI for CFU-GM and BFU-E, respectively; bp = .08 and .03 at 10 nM SLPI for CFU-GM and at 1 nM SLPI for BFU-E, respectively, as compared with the cultures in the absence of serine proteinase inhibitors. Abbreviations: AEBSF, 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride; 1-PI, 1¨Cproteinase inhibitor; BMMNC, bone marrow mononuclear cell; HGF, hematopoietic growth factor; HPC, hematopoietic progenitor cell; SLPI, secretory leukocyte proteinase inhibitor.2 u0 I8 @5 \( M2 p' J

DISCUSSION  U& S( d: A) R0 f/ d. F
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In vitro growth of human hematopoietic stem cells or progenitor cells in serum-free media is dependent on the presence of several proteins, including HGF, insulin, transferrin, and albumin . The CD34¨C cells at initiation of the culture and the progeny of CD34  cells during the in-vitro culture produced and released significant concentrations of the serine proteinases HNE, Cath G, and Pr3 into the culture medium. At the start of cultures of purified CD34  cells, no HNE, Cath G, or Pr3 was detected in the supernatant, resulting in initial normal proliferation of the cells. However, after 6 days of culture, when the CD34  cells had matured, the CD34¨C progeny released high concentrations of proteinases. Although, as expected, this resulted in normal numbers of CFU-GM and BFU-E colonies as measured at day 14 of cultures of purified CD34  cells, the colony size was smaller. We observed in liquid cultures of purified CD34  cells (viability > 99%) an exponential increase of viable cell growth up to day 6 followed by a decrease of expansion of viable cells and an increase of nonviable cells, while at the same time the release of serine proteinases in the supernatant increased. Therefore, after day 6, the increased concentration of serine proteinases was due to an increased production by the progeny of CD34  cells as well as an additional release into the medium caused by cell death during culture.. p5 O) e# ^/ c9 C  R! z
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These results explain the observation by others that in expansion studies of CD34  cells under serum-free conditions, the need for HGF was found to be at least 10-fold higher than in the presence of serum . This is probably not due to a spontaneous decay of HGF, because we demonstrated major degradation of HGF within 24 hours in the presence of BMMNCs but no degradation of proteins during a 14-day culture in the absence of BMMNCs. Depending on the purity of the CD34  population at day 0 and the expansion ratio of the cells, the need for HGF in serum-free cultures will increase when the numbers of CD34¨C cells increase. Exogenous addition of HNE, Cath G, or Pr3 to the cultures suppressed the HGF-induced growth of purified CD34  cells dose-dependently at concentrations as low as 1 nM. When HNE, Cath G, and Pr3 were neutralized for 2 hours with 1-PI before addition to the culture medium, normal colony growth was observed. Neutralization of the serine proteinases released by the BMMNCs was shown to be specific for certain serine proteinase inhibitors. Whereas addition of the serine proteinase inhibitors 1-PI or SLPI completely neutralized the serine proteinases, aprotinin, leupeptin, or AEBSF, which are commonly used in purification techniques to prevent degradation of proteins and peptides, could not prevent degradation of HGF by the serine proteinases produced by BMMNCs, indicating a specificity of serine proteinase inhibitors for different classes of serine proteinases. In normal serum, the concentration of 1-PI is 25 µM, resulting in a concentration of 2.5 µM 1-PI in culture medium containing 10% serum, which is a saturating concentration capable of inhibiting all serine proteinases released.

Other investigators  showed a direct toxic effect of HNE, Cath G, or Pr3 on endothelial cells and hepatocytes that could be inhibited by 1-PI. We demonstrated only a minor reduction of colony formation after short-term inactivation of cell membrane receptors by HNE before addition of 1-PI, indicating that rapid cell membrane receptor destruction was not the main mechanism responsible for the impaired proliferation of CD34  cells, although it may likely contribute to the overall inhibitory effect of proteinases on HGF-induced proliferation.4 k: `& L+ B/ w6 s1 t
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CONCLUSION

These results show the impact of serine proteinases produced by the progeny of CD34  cells on the cytokine-induced proliferation of progenitor cells or stem cells in serum-free cultures. These findings may have major implications for in vitro expansion of hematopoietic cells, in particular CD34  cells under serum-free conditions for clinical use under GMP conditions. The serine proteinases that are released in these cultures (HNE, Cath G, and Pr3) have preferential amino acid cleavage sites, like Ala, Val, Leu, and Phe , that frequently occur in HGF, other cytokines, other relevant proteins like albumin, as well as cell-bound proteins, including cytokine receptors. Consequently, the serum-free cultures will contain significant concentrations of undefined degraded products of these proteins. Although increasing the concentration of cytokines and other proteins in the cultures may compensate for the loss of the essential proteins relevant to in vitro expansions and genetic manipulations of CD34  or other hematopoietic cells, the presence of undefined protein fragments is undesirable for production of cellular products under GMP conditions. Frequent refreshment of the medium or reselecting the CD34  cells during culture may diminish the concentration of proteinases, but these multiple manipulations are likely to affect the cell expansion. Addition of defined serine proteinase inhibitors to serum-free cultures of these hematopoietic cells can prevent the degradation of relevant proteins present on the cell surface and in the medium, and will allow a more reliable culture of cellular products under GMP conditions for clinical use. Further research is required to investigate whether serine proteinase inhibitors might also influence the survival and growth of more primitive hematopoietic stem cells.

ACKNOWLEDGMENTS; x. G. f3 h: e  I* J/ `
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This study was supported by a grant from the J.A. Cohen Institute for Radiopathology and Radiation Protection.: ]; _' V" V+ o9 ]$ B# n

DISCLOSURES
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The authors indicate no potential conflicts of interest.- o' C$ v0 c' k+ r! U; `* O
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