Introduction STEM CELLS 1994;12:

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1 Growth Factor Stimulation of Cryopreserved CD34 Bone Marrow Cells Intended for Transplant: An In Vitro Study to Determine Optimal Timing of Exposure to Early Acting Cytokines Mariusz Z. Ratajczak, Janina Ratajczak, David A. Kregenow; Alan M. Gewirtza.b Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA; bdepartment of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA Key Words. Bone marrow transplantation Cryopreservation CD34 cells IL-1p IL-3 KL Abstract. Stimulating CD34 hematopoietic cells with growth factors prior to transplantation may decrease the duration of post-transplant aplasia. The optimal time in which to deliver this stimulus remains unclear. We therefore sought to determine if progenitor cell cloning efficiency, as reflected by colony forming units-granulocytemacrophage (CFU-GM) colony formation, differed when growth factor stimulation was carried out prior to freezing or after thawing. To address this issue, CD34 marrow cells were suspended in Iscove s medium with 20% bovine calf serum. They were then stimulated with kit ligand (KL), interleukin-3 (IL-3), and interleukin-lp (IL-1p) for 36 h either prior to cryopreservation or immediately after thawing. We observed that cells stimulated prior to freezing formed more CFU-GM colonies than cells stimulated after thawing. We also found that CD34 cells stimulated with growth factors either before or after freezing gave rise to more CFU-GM colonies than thawed cells plated without prestimulation. Thus, the cloning efficiency of stored marrow, as reflected by CFU-GM colony formation, may be effectively enhanced by pretreating cells with hematopoietic growth factors. Our data further suggest that the optimal time for this treatment is before cryopreservation as opposed to immediately after thawing. Based on these results, we hypothesize that stimulation of CD34+ hematopoietic cells prior to cryopreservation may simplify cell processing, enhance subsequent engraftment, Correspondence: Dr. M. Z. Ratajczak, Department of Pathology and Laboratory Medicine, 230 John Morgan Bldg., University of Pennsylvania, 36th St. & Hamilton Walk, Philadelphia, PA 19104, USA. Received April 8, 1994; provisionally accepted May 7, 1994; accepted for publication August 15, OAlphaMed Press /94/$5.00/0 and bring about significant cost savings by decreasing the amounts of recombinant growth factors administered to patients in order to facilitate marrow recovery. Introduction Marrow aplasia remains a significant cause of morbidity and mortality in the post-transplant period [ 11. Committed progenitors are responsible for the first wave of hematopoietic reconstitution after transplant since they proliferate and differentiate in the marrow before the true stem cells. Therefore, increasing the number and growth potential of committed progenitors would likely be a useful strategy for decreasing the duration of post-transplant marrow aplasia [2,3]. This is especially true since initial concerns that progenitor cell expansion might occur at the expense of the stem cell compartment appear to be unfounded [4,5]. Administration of recombinant human hematopoietic growth factors along with a marrow graft is widely used to ameliorate post-transplant aplasia. Though clearly effective, systemic treatment of bone marrow transplant recipients with hematopoietic growth factors is extremely expensive. An alternative strategy would be to stimulate marrow cells in suspension prior to transplantation [6]. The obvious advantage of this approach is that much less growth factor is required to stimulate cells in a small volume ex vivo than after they have been transplanted into a recipient. In addition, cytokines which might be useful for expansion but are poorly tolerated in vivo may also be used. Indeed, early reports suggest that STEM CELLS 1994;12:

2 600 Optimizing the Proliferative Capacity of Cryopreserved Marrow this approach is worthwhile. For example, stimulation of human marrow cells with interleukin 3 (IL-3) and granulocyte-macrophage colonystimulating factor (GM-CSF) in vitro in liquid cultures for three days prior to transplantation resulted in statistically significant improvements in marrow engraftment and transplant-related mortality [6]. We have also reported that growth factor stimulation is effective for cryopreserved marrow cells [7]. CD34' cells stimulated before freezing with kit ligand (KL), IL-3, and IL-lP gave rise to twice as many myeloid and erythroid colonies after thawing when compared with unstimulated controls. Knowing that it is desirable to transplant cryopreserved hematopoietic cells as soon as possible after thawing, we wondered if stimulation prior to cryopreservation would be as effective as stimulation post-thawing. If so, cell handling might be greatly simplified. The goal of the present study was to address this question. We found to our surprise that CD34' marrow cells stimulated prior to freezing formed more colony forming unit-granulocyte-macrophage (CFU-GM) colonies than cells stimulated after thawing. Materials and Methods Cells Normal light-density bone marrow mononuclear cells (MNC) were obtained from six consenting normal human donors and depleted of adherent cells and T lymphocytes (A-T-MNC) as described [8]. CD34+ cells were enriched from the A-T-MNC population by incubation with anti-hpca-1 murine monoclonal antibody (Becton Dickinson, Mountainview, CA) and subsequent immunoselection of antibody-labeled cells with magnetic beads according to the manufacturer's protocol (Dynal, Oslo, Norway). Briefly, 2 x lo7 A-T-MNC were incubated in 1.5 ml Eppendorf tubes for one h in one ml of Iscove's modified Dulbecco's minimal essential medium (IMDM) (GIBCO, Grand Island, NY) supplemented with 5% bovine calf serum (BCS) (Hyclone, Logan, UT) with 50 ml of anti-cd34 antibodies (anti-hpci) at 4 C on a rotating rack. The cells were then washed three times by centrifugation in IMDM + 5% BCS and again resuspended in one ml of IMDM + 5% BCS. Polystyrene beads (150 ml) (Dynal) covered with sheep antimouse antibodies were added, and the cells were incubated for 45 min at 4 C on a rotating rack. Next, the cells were transferred to five ml polystyrene plastic tubes and collected for two min using a magnetic rack (Dynal). The magnetic separation was repeated three times. After each separation, the medium and cells not attached to the tube walls were removed by gentle aspiration. After the third separation, the cells were resuspended in LMDM + 10% BCS. Final purity of CD34' cells collected in this manner was -80%. Cell viability was assayed using the trypan blue exclusion test (>95% viable), and cell number was counted using a hemocytometer. Stimulation of CD34'A-T-MNC The isolated CD34' cells were resuspended in IMDM + 20% BCS and placed in 4 ml polypropylene plastic tubes in 0.5 ml aliquots containing 2 x lo4 CD34' cells. The cells were then either stimulated with cytokines as described below and cryopreserved, or cryopreserved and then stimulated in the same manner after thawing. Control samples were either: a) plated without being frozen or exposed to cytokines, orb) exposed to the same combination of cytokines on ice, immediately frozen, thawed after four weeks, and plated after being washed of freezing medium. The cells were stimulated with KL (100 ng/ml), IL-3 (20 Ulml), and IL-lP (100 pg/ml) for 36 h in suspension in a fully humidified incubator at 37 C and 5% CO, (Fisher Scientific, Pittsburgh, PA). Cryopreservation of CD34+ A-T-MNC The CD34' MNC suspension (0.5 ml aliquots) was transferred from polypropylene tubes to two ml cryotubes (Corning Glassworks, Corning, NY). The volume of the freezing medium was increased to one ml and the concentration of BCS and dimethylsulfoxide (DMSO) (Sigma, St. Louis, MO) was adjusted to 20% and lo%, respectively. The tubes and the freezing medium were kept on ice. The samples were immediately transferred to a mechanical freezer at -80 C (Fisher Scientific). Four weeks later, all the samples were thawed and plated in semisolid methylcellulose cultures. Thawing of CD34' A-T-MNC After storage for four weeks, the stored cryotubes were placed in a 37 C water bath

3 Ratajczak/Ratajczak/Kregenow/Gewirtz 60 1 for two min. One ml of the cell suspension from each tube was transferred to a 50 ml plastic Corning tube containing 30 ml of ice-cold IMDM supplemented with 20% BCS. After incubating for 10 min on ice, the tubes were centrifuged, and the sediment was resuspended in fresh culture medium. The viability of the thawed cells was assayed using the 0.5% trypan blue exclusion test. The fraction of dead cells unable to exclude dye was calculated per 100 cells examined. Cell Cultures To culture granulocyte-macrophage colonies formed by CFU-GM, 2 x lo4 CD34+A-T-MNC were washed from the medium in which the cells were stimulated or cryopreserved and suspended in 0.4 ml IMDM, mixed with 1.8 ml of HCC-4230 methylcellulose medium (Terry Fox Laboratories, Vancouver, Canada) supplemented with L-glutamine (0.125 mm) and plated in duplicate 35-mm Petri dishes, 1.O ml/dish. CFU-GM colonies were stimulated with GM-CSF (5 ng/ml) t IL-3 (20 U/ml). Only human recombinant growth factors (generous gift of Genetics Institute, Cambridge, MA) were used in these experiments. Cultures were grown in a fully humidified incubator at 37 C and 5% COz (Fisher Scientific). CFU-GM colonies (more than 50 cells/colony) were counted on day 11 with an inverted microscope using standard morphological criteria. Each culture was plated in quadruplicate. Statistical Analysis Arithmetic means and standard deviations were calculated on a MacIntosh LCIl using Instat 1.14 (GraphPad Software, San Diego, CA) software. The data were subjected to statistical analysis using the Student's t-test for unpaired samples. Statistical significance was defined as p < Table I. Viability of unstimulated non-cryopreserved (A), unstimulated cryopreserved (B), and cryopreserved bone marrow CD34' cells stimulated either before (C) or after (D) freezing Viability (%*SD) 100*0 83*6 82*9 89*11 control cells, -84% of the CFU were recovered using either approach. CFU-GM colony formation by variously manipulated cells is shown in Figure 1. In accord with our initial experiments [7], CD34+ cells that were cultured for 36 h in the presence of early-acting cytokines before cryopreservation formed more CFU-GM colonies than cells frozen without growth factor stimulation (p = ). In addition, CD34+ cells that were prestimulated with growth factors prior to freezing began to proliferate earlier and gave rise to larger colonies than CD34' cells which were frozen without growth factor prestimulation (data not shown). CD34' cells. stimulated in suspension with early-acting cytokines immediately after thawing also manifested significantly enhanced cloning efficiency (p = 0.O004) and formed larger,000 1 T * Results We initially sought to determine the temporal effect of growth factor stimulation on subsequent viability of cryopreserved CD34+ marrow cells (Table I). We found that the viability of treated marrow cells was unchanged by stimulation with growth factors either prior to, or after freezing at -80 C (p = 0.13). We also found that when compared with unfrozen A B C D Fig. 1. CFU-GM colony formation by 2 X lo4 bone marrow CD34' cells; A) Unstimulated noncryopreserved cells; B) Unstimulated cryopreserved cells; C) Cells stimulated for 36 h before cryopreservation; D) Cells stimulated for 36 h immediately after thawing. *p < 0.05 in comparison with D.

4 602 Optimizing the Proliferative Capacity of Cryopreserved Marrow colonies than unstimulated cryopreserved cells. However, the proliferative potential of the CD34 cells stimulated before cryopreservation appears to exceed that of cells which were stimulated after thawing. The difference observed was relatively small (-25%) but was statistically significant (p = 0.045). Therefore, in a direct comparison, stimulation of hematopoietic cells prior to cryopreservation was at least equivalent to, and possibly better than, stimulation of cells post-thawing. Discussion The early-acting cytokines employed in this study, namely KL, IL-3 and IL- 1 p, are important for the survival and growth of bone marrow progenitor cells. They inhibit apoptosis [9] and have already been successfully used for ex vivo expansion of noncryopreserved human and murine hematopoietic progenitors [2, 4, 5, 101. Nevertheless, these proteins are quite expensive and add significantly to the cost of the transplant procedure. Making use of these materials in a more efficient manner would be very desirable. Examining ways in which this could be done was a major objective of this study. Our initial studies in this area suggested that exposing hematopoietic cells to growth factors prior to freezing enhanced their survival and subsequent cloning efficiency in comparison with cells frozen without such stimulation 171. However, the optimal time of this stimulation was not addressed. We were particularly interested in knowing whether stimulation before freezing would be comparable to stimulation after thawing [6] in terms of progenitor cell cloning efficiency. If stimulation could be effectively accomplished prior to cryopreservation, and if this strategy ultimately proved beneficial in the setting of clinical marrow transplantation, this form of marrow pretreatment might be easier to accomplish than stimulation of marrow cells post-thawing but prior to transplantation. The results presented in this paper suggest that prestimulation of marrow before, as opposed to after, cryopreservation may be optimal. Several caveats apply to this conclusion, however. First, we report here only effects on CFU-GM cloning efficiency. It might be argued that our conclusions are tenuous without demonstrating similar enhanced cloning efficiency on less mature progenitors. It is our impression however, that CFU-GM behavior in culture parallels that observed for less mature CFU when these are directly compared [ 11, 121. Nevertheless, our predictions regarding the utility of this strategy do assume, with some support from the literature [ 1 11, that the proliferative potential of CD34+ cells, as assessed by the cloning efficiency of CFU-GM, correlates positively with bone marrow engraftment. Second, the cocktail of growth factors we employed may not be optimal [ 121. Similarly, the amount of time we exposed the cells to growth factors may also be suboptimal. In this regard, we found that extending prestimulation beyond 36 h resulted in a decrease in the formation of large CFU-GM colonies with more than 100 cells and an increase in cluster formation. Accordingly, while exposing cells to growth factors for periods of less than 36 h may be acceptable, exposure periods in excess of 36 h appear to be detrimental. One could reasonably expect then that our results might be even better if the stimulation we provided is optimized. Third, the experimental model system utilized for this work employs certain techniques which, at the moment, are not standard for clinical bone marrow transplantation. We used human CD34 cells, but this marrow cell fraction is being used more frequently for transplantation [ll]. In addition, cryopreserved cells were frozen and stored in a mechanical freezer at -80 C. We and others have previously shown that this is an inexpensive and convenient method for long-term storage purposes [7, 11, 13-18]. Finally, we appreciate that the true utility of the modification to cell handling we propose can only be determined in the setting of clinical bone marrow transplantation. Short of this, studying the ability of cells treated in this manner to rescue marrow ablated SCID mice may help to answer this question. Such studies are now ongoing in our laboratory. References 1 Armitage JO. Bone marrow transplantation. N Engl J Med 1994;330: To LB. Is our current strategy in manipulating hemopoiesis in autologous transplantation correct? Stem Cells 1993;11: Zucali JS. Stem cell manipulation for better treatment outcome. Cancer Res Ther Control 1993;3:

5 Ratajczak/Ratajczak/Kregenow/Gewirtz Muench M, Firpo MT, Moore MAS. Bone marrow transplantation with interleukin- l plus kit-ligand ex vivo expanded bone marrow accelerates hematopoietic reconstitution in mice without the loss of stem cell lineage and proliferative potential. Blood 1993;81: Williams DA. Ex vivo expansion of hematopoietic stem and progenitor cells-robbing Peter to pay Paul? Blood 1993;81: Naparstek E, Hardan Y, Ben-Shahar M, Nagler A, Or R, Mumcuoglu M, Weiss L, Samuel S, Slavin S. Enhanced marrow recovery by short preincubation of marrow allografts with human recombinant interleukin-3 and granulocyte macrophage colony-stimulating factor. Blood 1992;80: Ratajczak MZ, Ratajczak J, Kregenow DA, Kuczynski WI, Skorski T, Gewirtz AM. Cytokine stimulation of CD34' bone marrow cells prior to cryopreservation enhances their post-thawing proliferative potential. Folia Histochem Cytobiol 1994;3: Ratajczak MZ, Luger SM, DeRiel K, Abrahm J, Calabretta B, Gewirtz AM. Role of the kit protooncogene in normal and malignant human hematopoiesis. Proc Natl Acad Sci USA 1992;89: Brandt JE, Bhalla K, Hoffman R. Effects of interleukin-3 and c-kit ligand on the survival of various classes of human hematopoietic progenitor cells. Blood 1994;83: McAlister IB, Teepe M, Gillis S, Williams DE. Ex vivo expansion of peripheral blood progenitor cells with recombinant cytokines. Exp Hematol 1992;20: Ciavarella D. Hematopoietic stem cell processing and storage. Biotechnology 1991 ;19: Brugger W, Mocklin W, Heimfeld S, Berenson RJ, Mertelsmann R, Kanz L. Ex vivo expansion of enriched peripheral blood CD34' progenitor cells by stem cell factor, interleukin-lj3, IL-6, IL-3, interferon-y, and erythropoietin. Blood 1993;8 1 : Ahmed T, Wuest D, Ciavarella D, Ayello J, Feldman EJ, Biguzzi S, Gulati S, Hussain F, Mittelman A, Asceanso J, Arlin AA. Marrow storage techniques: a clinical comparison of refrigeration versus cryopreservation. Acta Haematol 1991 ;85: Clark J, Pati A, McCarthy D. Successful cryopreservation of human bone marrow does not require a controlled-rate freezer. Bone Marrow Transplant 1991;7: Jackson J, Kloster T, Lisbeth W, Damon M, Rehder B, Schmechel B, Ward B, Kessinger A. Peripheral blood-derived stem cells can be successfully cryopreserved without using controlled-rate freezing. Prog Clin Biol Res 1992;333: Ratajczak MZ, Kuczynski WI, Ratajczak J, Light B, Luger S, Gewirtz AM. A simple efficient method for cryopreserving bone marrow cells in a -8O'C mechanical freezer. Blood 1993;82(suppl 1):652a. 17 Spitzer G, Verma DS, Fisher R, Zander A, Vellekop L, Litam J, McCredie K, Dicke KA. The myeloid progenitor cell-its value in predicting hematopoietic recovery after autologous bone marrow transplantation. Blood 1980;55: Stiff JP, Koester AR, Weidner MK, Dvorak K, Fisher RI. Autologous bone marrow transplantation using unfractioned cells cryopreserved in dimethylsulfoxide and hydroxyethyl starch without controlled rate freezing. Blood 1987;70: