Stem Cells, Vol. 14, No. 1, 146-150,
January 1996
© 1996 AlphaMed Press
FLT3/FLK-2 (STK-1) Ligand Does Not Stimulate Human Megakaryopoiesis In Vitro
Mariusz Z. Ratajczaka,
Janina Ratajczaka,
John Forda,
Robert Kregenowa,
Wojtek Marlicza,
Alan M. Gewirtza,,b
a Department of Pathology and Laboratory Medicine, and
b Internal Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
Key Words. Hematopoiesis • Megakaryocytopoiesis • FLT3/FLK-2 (STK-1) receptor • FLT3/FLK-2 (STK-1) ligand • Growth factors • Antisense oligodeoxynucleotides
Dr. Alan M. Gewirtz, 513B-Stellar-Chance Laboratories, University of Pennsylvania School of Medicine, 422 Curie Blvd., Philadelphia, PA 19104, USA. e-mail: gewirtz{at}a1.mscf.upenn.edu
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Abstract
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It has not yet been determined if the FLT3/FLK-2 or STK-1 Ligand (STK-1 L)/FLT3/FLK-2 or STK-1 receptor (STK-1R) axis has the ability to regulate human megakaryopoiesis in vitro. To address this question, we exposed normal human CD34+ marrow mononuclear cells to recombinant human STK-1L alone, or in combination with other growth factors. Colony-forming unit-megakaryocytic/thrombocytes (CFU-Meg) and BFU-E-derived colonies were then enumerated, and effects on colony size and maturation noted. As assessed by these parameters, STK-1L had no demonstrable effect on megakaryocyte colony formation. Similarly, suppressing STK-1R expression with oligodeoxynucleotides also had no influence on CFU-Meg-derived colony formation. To begin to derive a physiologic explanation for these findings, we examined freshly isolated normal human megakaryocytes for the presence of STK-1L and STK-1R mRNA. In contrast to a growing number of growth factors and growth factor receptors which appear to be expressed by megakaryocytes, normal mature human megakaryocytes express neither STK-1R or STK-1L mRNA. Accordingly, our results led us to hypothesize that if STK-1/ STK-1L have any effects on megakaryocyte development in vitro, they are likely subtle and of uncertain physiologic significance.
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Introduction
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Receptors with intrinsic tyrosine kinase activity, such as KIT and FMS, and their respective ligands are known to play an important role in hematopoietic cell development [1]. A newer member of this family, known variously as FLT3/FLK-2 and STK-1R (stem cell tyrosine kinase receptor-1) in the murine and human systems, respectively [2, 3], has also been shown to influence hematopoietic cell development. Expression of STK-1R, in contrast to KIT and FMS, is restricted to CD34+ hematopoietic cells, and functional studies with antisense oligodeoxynucleotides (ODN) have suggested that the receptor may contribute to hematopoietic cell development in general, but most importantly to myelopoiesis at the early stem/progenitor cell level [3].
The ligand for FLT3/FLK-2 (STK-1) – STK-1L has also recently been cloned [4, 5]. In contrast to its receptor, its mRNA is expressed by a variety of cell types [4, 5]. STK-1L function in these various cell types is unclear, but when CD34+ hematopoietic cells are exposed to the recombinant form of this cytokine, thymidine incorporation is stimulated, suggesting an effect on cell proliferation [2]. In colony stimulating assays, STK-1L appears to have little or no effect on in vitro hematopoietic progenitor cell growth by itself. However, it costimulates myeloid and B lymphocyte progenitors when employed with kit ligand (KL), interleukin 3 (IL-3) and GM-CSF [4]. Interestingly, STK-1L does not appear to stimulate erythropoiesis either alone or in combination with erythropoietin (EPO) and KL [2, 4]. Recently however, the stimulatory effect of STK-1L in combination with other cytokines on human erythropoiesis has been reported [6].
Because the role of STK-1R/STK-1L axis in human megakaryocytopoiesis has not yet been evaluated, the goal of the present study was to determine if normal human megakaryocytes express STK-1 or its ligand, and if the ligand stimulates megakaryocytopoiesis in vitro.
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Materials and Methods
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Cells
Normal light-density bone marrow mononuclear cells (MNC) were obtained from five consenting normal human donors and depleted of adherent cells and T lymphocytes (A-T-MNC) as previously described [7–10]. CD34+ cells were enriched from the A-T-MNC using the Dynal CD34 progenitor cell selection system according to the manufacturer's protocol (Dynal, Oslo, Norway). Briefly, 4 x 107 A-T-MNC were suspended in 0.9 ml of incubation buffer (Ca++, Mg++ free phosphate-buffered saline [PBS]), 2% bovine serum albumin (BSA) to which was added 100 µl of M450 Dynabeads complexed with anti-CD34 antibodies. The cells were incubated for 30 min at 4°C on a rotating rack. Cell/bead rosettes were isolated using a myeloid progenitor cell magnet (Dynal, Oslo, Norway). The dynabeads were subsequently removed from the CD34 enriched cells using the protocol recommended by Dynal. The purity of CD34+ cells was >95% as assayed by fluorescence-activated cell sorting, and viability exceeded 95% as assessed by trypan blue exclusion.
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Megakaryocyte Cultures
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Colony-forming unit-megakaryocytic/thrombocytes (CFU-Meg) and BFU-Meg colonies were cultured from CD34+ cells as described [8, 9]. Briefly, 2 x 104 CD34+ MNC were suspended in 0.4 ml Iscove's modified Dulbecco's medium (IMDM) and mixed with 1 ml Iscove's Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Grand Island, NY) supplemented with CaCl2, BSA, 0.6 ml heat-inactivated horse serum (Hyclone, Logan, UT), and 0.2 ml fresh frozen bovine plasma (GIBCO, Gaithersburg, MD). CFU-Meg colony growth was stimulated with EPO (5 U/ml), IL-3 (20 U/ml), and IL-6 (40 U/ml) or with thrombopoietin (TPO) (50 ng/ml). BFU-Meg colony growth was stimulated with EPO (5 U/ml), IL-3 (20 U/ml), IL-6 (40 U/ml), GM-CSF (5ng/ml), IL-1
(100 pg/ml) and KL (100 ng/ml). In some cases, recombinant STK-1L (Genzyme, Cambridge, MA) was also added to cultures, either alone, or in combination with the other growth factors employed. After 11 days culture (CFU-Meg) or 21 days culture (BFU-Meg) in a fully humidified CO2 incubator at 37°C, colonies were enumerated by immunofluorescence with an antiplatelet glycoprotein IIb/IIIa monoclonal antibody (Immunotech, Marseille, France) as we have previously reported [8, 9].
Antisense ODN Treatment Protocol
CD34+ MNC were suspended in Iscove's DMEM supplemented with 2% heat inactivated bovine calf serum (Hyclone), washed once by centrifugation, and then resuspended in polypropylene tubes in the same medium (104 cells/0.4 ml). Unmodified (natural backbone) ODN corresponding to codons 1-6 of the human STK-1R cDNA sequence [3] were added to the cultures at an initial concentration of 100 µg/ml. 16 h later, an additional 50 µg/ml of the same sequence were added for a total concentration of 150 µg/ml; ~0.007 µM. ODN sequences employed were as follows: STK-1 Sense (S) ODN-5'-CGA GGC GGC ATC CGA GGG-3'; STK-1 Antisense (AS) ODN-5'-CCC TCG GAT GCC GCC TCG-3'; and Scrambled (Scr) ODN- 5'-CTC CGG CTG AGG CCC TCC-3'. After a total ODN exposure time of 24 h, the unwashed cells were plated in plasma clot cultures.
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mRNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
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For RT-PCR-directed analysis of STK-1 mRNA expression, total cellular mRNA was extracted from ODN-treated MNC in suspension culture, or from freshly isolated normal human megakaryocytes using a Quick-Pre mRNA purification KITTM (Pharmacia, Piscataway, NJ). In the case of ODN-treated CD34+ cells, mRNA was isolated from 104 cells after 72 h exposure. In the case of normal human megakaryocytes, mRNA was isolated from 100 morphologically recognizable cells isolated using a micromanipulator from an elutriation enriched preparation [11]. Final mRNA pellets were washed with 75% ethanol and suspended in 10 µl of distilled water, autoclaved x 3.
Reverse transcription of mRNA was carried out as follows: 4 µl of the original 10 µl sample was heated to 65°C for 10 min and then cooled on ice for 3 min. 100 U of Moloney murine leukemia reverse transcriptase (GIBCO BRL, Gaithersburg, MD), 50 ng of random primers (Boehringer Mannheim, Indianapolis, IN), 40 U of RNAzin (Promega, Madison, WI) and nucleoside triphosphates (dNTPs) (50 mM each) were added to the tube and incubated for 1 h at 37°C. Specific oligo primer pairs employed were as follows: STK-1 sense: 5'-CTC GTT GTT TTT TCT GCA ATG-3' (nts 97-117); STK-1 antisense: 5'-GAT GGA AGC AGA TAC ATC CAC-3' (nts 324-304) amplified product-228 bp [3]; STK-1L sense: 5'-AAC AAC CTA TCT CCT CCT GCT-3' (nts 30-50); STK-1L antisense: 5'-GGC ACA TTT GGT GAC AAA GTG-3' (nts 337-317) [4].
Amplification of ß-actin was performed in all mRNA samples to validate both the integrity of the mRNA employed and the PCR reaction [8, 9].
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Southern Analysis of RT-PCR Products
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10 µl of amplified product were electrophoresed on a 4% agarose gel and transferred to a nylon filter. Filters were prehybridized and then probed with an ODN 32P-end labeled with T4 polynucleotide kinase (Promega, Madison, WI) specific for the cDNA of interest. For STK-1: CTT CCC CAC TGA TGA ATC ATT GTT (nt 181-205) [3], and for STK-1L: CAC GGT GAC TGG GTA ATC TTG AAG CAG GTA [4] (nt 150-180). Hybridization was detected by autoradiography as described previously [3].
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Statistical Analysis
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Arithmetic means and standard deviations were assessed using Instat 1.14 (GraphPad 1993, San Diego, CA) run on a Macintosh LCII computer. Experiments were performed on cells obtained from five healthy donors. Cells at each point were cultured in quadruplicate.
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Results and Discussion
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STK-1L alone failed to stimulate the formation of human megakaryocyte colonies when added to cultures of human CD34+ MNC (data not shown). When added in combination with IL-3 (20 U/ml), IL-6 (40 U/ml) and EPO (5 U/ml) (Fig. 1
) or TPO (50 ng/ml) (Table 1) at doses of 25 or 100 ng/ml, it did not further stimulate CFU-Meg cloning efficiency or proliferative capacity as evidenced by number or size of the colonies. Further, morphologic examination of the colonies failed to suggest that STK-1L had any effect on megakaryocyte maturation. Similarly, when STK-1L was added in combination with EPO (5 U/ml), IL-3 (20 U/ml), IL-6 (40 U/ml), GM-CSF (5 ng/ml), IL-1 (100 pg/ml) and KL (100 ng/ml), it did not influence BFU-Meg colony formation (Table 2).
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Fig. 1. Effect of STK-1L on CFU-Meg colony formation. CD34+ marrow cells (1 x 104/ml) were cloned in the presence of IL-3, IL-6 and EPO alone or with these same growth factors plus 25 or 100 ng/ml of recombinant human STK-1L. The number of colonies are frequencies/1 x 104 cells plated.
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Table 1. Effect of STK-1L on CFU-Meg colony formation. CD34+ marrow cells (1 x 104/ml) were cloned in the presence of TPO alone or with TPO plus 25 or 100 ng/ml of recombinant human STK-1La
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Table 2. Effect of STK-1L on BFU-Meg colony formation. CD34+ marrow cells (1 x 104/ml) were cloned in the presence of [IL-3, IL-6, EPO, GM-CSF, IL-1, KL] alone or with these same growth factors plus 25 or 100 ng/ml of recombinant human STK-1L
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It is possible that only minute amounts of STK-1L might promote maximal stimulation of megakaryocyte colony formation and that such amounts might already be present in the serum used to supplement the cultures. If this were true, adding more STK-1L might not lead to further augmentation of colony formation. Conversely, inhibiting the expression of STK-1R would be expected to either diminish the numbers of colonies formed or perturb their development. To assess this possibility, we examined CFU-Meg growth from CD34+ MNC which had been exposed to STK-1R S, AS or Scr ODN. In comparison to an untreated control, none of the ODN sequences effected megakaryocyte colony formation (Fig. 2A). Figure 2B demonstrates that the STK-1R AS ODN was able to specifically eliminate the target mRNA at the 72-h time point. Accordingly, transient loss of the STK-1R expression did not adversely affect CFU-Meg growth.
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Fig. 2A. Effect of STK-1R ODN on CFU-Meg colony formation. CD34+ marrow cells (1 x 104/ml) were exposed to S, AS, or Scr ODN as detailed in the text, and then cloned in the presence of IL-3, IL-6 and EPO. After 11 days in culture, CFU-Meg were enumerated. Colonies formed by untreated control cells (C) are also shown. The number of colonies are frequencies/1 x 104 cells plated.
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Fig. 2B. Effect of STK-1R targeted ODN on mRNA levels in normal human CD34+ marrow cells. Total RNA was extracted from untreated cells (C), or cells exposed to the ODN as indicated in the text. RNA was reverse transcribed and then amplified by PCR. Southern blot analysis of STK-1R and ß-actin cDNA amplified from untreated control (C) cells, or cells exposed to S, A, or Scr ODN is shown in the upper and lower panels, respectively.
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Finally, we also examined freshly isolated, morphologically recognizable normal human bone marrow megakaryocytes for STK-1R and STK-1L mRNA expression. Using cells that were essentially 100% pure and a sensitive RT-PCR assay, we were able to detect ß-actin mRNA but could not detect either STK-1R or STK-1L mRNA (data not shown). As expected however, STK-1R and STK-1L mRNA were detected without difficulty from, respectively, CD34+ cells and stromal cells prepared from the same sample.
These results are of interest from several points of view. First, though the number of cytokines which affect in vitro megakaryopoiesis has grown in recent years [12, 13] and now includes recombinant TPO [14], our results suggest that STK-1L will not be added to this group. Second, some cytokines support the growth of certain lineages, not by promoting proliferation, but rather by inhibiting cells from undergoing apoptosis. Recent studies from our laboratory suggest that STK-1L may be among such growth factors [7] but not, apparently, for cells of the megakaryocyte lineage. These results are in accord with those which have found that STK-1L has no demonstrable effect on in vitro erythropoiesis at the level of the BFU-E or CFU-E [2, 4]. Finally, these results add, albeit indirectly, to a growing body of data which suggest that megakaryocytes and erythrocytes share a common progenitor which derives from the pluripotent hematopoietic stem cell at a point divergent from the offspring which populate the myeloid progenitor cell compartment.
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Footnotes
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Provisionally accepted October 25, 1995.
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References
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Received September 21, 1995;
accepted for publication November 9, 1995.
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Abstract
Introduction
