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a Department of Oncological Sciences, University of Torino Medical School,
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b Division of Clinical Oncology, Institute for Cancer Research and Treatment, Candiolo, Torino, Italy;8 {+ [& M8 z0 n5 Q& e
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c The Pediatric Department, University of Torino Medical School, Torino, Italy% K$ {9 c6 ^ k3 e* g
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Key Words. Cord blood ? CD34 cell expansion ? NOD/SCID ? Megakaryocyte engraftment# Q/ Q1 |# @5 m. W9 F) }' |
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Wanda Piacibello, M.D., University of Torino Medical School, Department of Oncological Sciences, Institute for Cancer Research and Treatment, Laboratory of Clinical Oncology, Prov. 142, 10060 Candiolo, Torino, Italy. Telephone: 39-011-9933349; Fax: 39-011-9933522; e-mail: wanda.piacibello@ircc.it/ l* x" k3 s i3 G. f/ H
7 l6 s5 r6 F) f/ j. [* h# VABSTRACT
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' m5 S D" S$ vThe numbers of hematopoietic progenitors and stem cells in cord blood (CB) may be enough to support engraftment in children, but their ex-vivo expansion might be required to successfully engraft an adult. Moreover, a long-lasting severe post-transplant thrombocytopenia is often observed even in pediatric patients .2 M) U1 U/ z% `. O. A; U Y4 U
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Therefore, two important aspects of the biology of ex-vivo expanded cells relate to cultured cells: either maintaining their self-renewal capacity and multilineage differentiation potential, or improving their short-term engraftment ability when transplanted into myeloablated recipients. Several growth factor combinations have been tested to identify suitable culture conditions to induce expansion of primitive stem cells (SCs). So far, only a few studies have shown that primitive non-obese diabetic severe combined immunodeficient (NOD/SCID) mouse repopulating stem cells from CB can be expanded (a few or several-fold) after in vitro culture .
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( \! Q- b2 ^& V, ~In CB transplants, the megakaryocyte (Mk) lineage takes the longest time to engraft. However, to date, if only a few experimental studies have addressed the issue of the short-term engraftment ability of fresh CB SCs, even fewer have addressed that of ex-vivo expanded SCs . Using the NOD/SCID mouse model, the short-term as well as the long-term repopulating ability and the differentiation and maturation potential of human hematopoietic lineages in an in vivo experimental model can be analyzed .
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1 h& b! J* ]+ X- ^, [+ j3 oThus, by means of this in vivo model we set up experiments to evaluate the Mk lineage reconstitution ability and functional platelet release by baseline CB CD34 cells (b34 ) and CB CD34 cells reisolated after a 4-week expansion (4w34 ) in the presence of Flt-3 ligand (FL), thrombopoietin (TPO), and stem cell factor (SCF).
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* H4 U+ V2 ~! m$ m2 X% sMATERIALS AND METHODS
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: Z! p+ S1 L' IMegakaryocyte Reconstitution in NOD/SCID Mice by Baseline and Expanded CD34 Cells (b34 and 4w34 )9 ~% U# Y" ~ n1 t$ F
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To evaluate short-term Mk-engraftment, 2.5 x 105 b34 cells were injected into cohorts of sublethally irradiated NOD/SCID mice that were sacrificed 1, 2, and 3 weeks after inoculation. BM cells of the sacrificed animals were obtained from both femurs and tibias and assessed for the presence of human hematopoietic cells. Human cells in the murine BM were quantified for myeloid, lymphoid, erythroid, and Mk lineage antigen expression. Fluorescence-activated cell sorter (FACS) analysis of the BM of the sacrificed animals showed that at week 1 post-transplant levels of human CD45 cells were low but clearly detectable (3.7 ± 2.8). Within the human cells, the most represented were the CD34 and the CD19 subpopulations (Table 1). Erythroid cells, identified by CD71 and GpA expression were also present. Surprisingly, no cells belonging to the Mk lineage could be found. At week 2 post injection, the levels of human CD45 cells were quite similar. At week 3, human cell engraftment further increased. Only at this time point the Mk subpopulation, although at very low percentages, became detectable (Table 1).$ J) j/ t1 \& a) [
" `- ~: I. \1 f4 m& @+ WTable 1. Short-term engraftment of NOD/SCID mice transplanted with fresh (b34 ) or ex vivo expanded (4w34 ) cells! L2 C# y5 x+ P: t4 p
, E$ |1 A, w- s1 H3 i" dTo evaluate the short-term Mk-engraftment capacity of more expanded primitive cells, in five different experiments, 2.5 x 105 CD34 cells were cultured in triplicate stroma-free liquid cultures in the presence of FL, SCF, and TPO as described . After 4 weeks of expansion they yielded a mean of 48.5 ± 2.4 x 106 total cells that included 1.9 ± 0.43 x 106 CD34 cells. Following immunoselection, only 2.5 x 105 CD34 (4w34 ) cells were injected in each mouse. In these mice the levels of human engraftment at week 1 were similar to those found in b34 -transplanted mice and increased with time. Here the growth of some CFU-Mk colonies could be achieved. CFU-Mk number increased at weeks 2 and 3 (Table 1). By contrast, in mice injected with b34 cells, no CFU-Mk colonies could be found at week 1. Only at week 3 were there as many Mk colonies as those found much earlier (week 1) in 4w34 transplanted mice.9 [+ \$ h/ V2 K$ @$ `- s: `3 S" |
( Q5 M5 X3 O$ P$ n! g8 XLong-term Mk-engraftment was evaluated in NOD/SCID mice sacrificed 6–8 weeks after inoculation. Table 2 shows the mean engraftment level of ten mice injected with 2.5 x 105 b34 cells. Flow cytometry analysis showed that the human cells belonged to all hematopoietic lineages; cells of the Mk lineage were found in all mice. CFU-Mk colonies were detected in plasma clot cultures seeded with the BM cells of the transplanted animals (Table 2).8 ]% h. O" H- o& l# s/ N
7 u4 ]. h g9 R2 I# ^Table 2. Six- to 8-week engraftment of NOD/SCID mice transplanted with b34 and 4w34 CB cells7 M. \/ c6 n" A/ i I9 l7 D
5 v- M1 W! z$ B4 i2 J# c# ]The results of injection of 2.5 x 105 4w34 cells are reported in Table 2. All the mice, after 6–8 weeks from the injection, were successfully engrafted (10/10). The mean engraftment level of the ten mice injected with this cell dose was 15 ± 5%. FACS analysis showed that human cells belonged to all hematopoietic lineages (not shown).
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Moreover, five mice were transplanted with all of the CD34 cells generated by initial 2.5 x 105 CD34 cells during a 4-week expansion. At 6–8 weeks post-transplant the BM engraftment levels were very high (79 ± 11.4) and the Mk population was well-represented (3.6 ± 0.4% of the total BM).
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The presence of human CFU-Mk in the BM of mice was evaluated. Overall, 1,900 ± 302, 2,200 ± 159 and 8,259 ± 1,102 human Mk colonies were detected in mice transplanted with b34 and the two concentrations of 4w34 cells, respectively.3 {6 F- }; ~0 O% u4 S; F" M
O0 q5 M0 @) I l2 v+ e/ F* OTo evaluate the effective long-term Mk-engraftment of expanded cells, the unseparated BM cells of three primary mice, harvested 6 weeks after injection of 2.5 x 105 4w34 cells, were transplanted in three secondary and subsequently in three tertiary sublethally irradiated recipients. In these experiments of serial transplants, mice were successfully engrafted, and the Mk population was well-represented (Fig. 1).8 e, E- x* j- G# ]
& C( w+ ^, f: Z; l+ @; XFigure 1. Human megakaryocyte engraftment in serial transplant NOD/SCID mice. FACS profile of marrow cells from a representative NOD/SCID mouse that 6 weeks earlier was transplanted with 2.5 x 105 4wCD34 (CD34 cells immunoselected after 4 weeks of expansion in presence of FL, SCF, and TPO). The BM of this primary mouse was injected into a secondary sublethally irradiated NOD/SCID mouse sacrificed 6 weeks after transplantation; the BM of this mouse was injected in a tertiary mouse also sacrificed 6 weeks after transplantation. Human CD45 cells represented 22% of the BM cells of the primary mouse, 7% of the secondary recipient that had received 25 x 106 unseparated BM cells of the primary mouse, and 2.5% of the tertiary mouse that received 30 x 106 total BM cells of the secondary recipient. FACS analyses of human CD41 expression in the BM of primary, secondary, and tertiary mice were performed on total BM: the percentages of CD41 cells were respectively: 1.3%, 0.6%, and 0.1%. The bottom panels represent the analysis of the CD41 population within the CD45 cell gate in each of the three recipients.9 R: q2 J2 q. f" W, V# z
5 R, e3 F) ]) t; b% e( j# KPlatelet Production in NOD/SCID Mice* l6 c& Y" K5 b2 D# W3 r/ V; }
2 P! u# Y1 R. A9 r5 j- |) IThe appearance of HuPlts in the mouse PB after CB injection was monitored from week 1 by FACS analyses. After total body irradiation murine platelets decreased from a mean of 1.45 ± 0.3 x 1012/l to a mean of 0.45 ± 0.1 x 1012/l at week 1 and increased to a mean of 0.8 ± 0.2 x 1012/l at week 3.
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HuPlts were detected by staining PB cells with an anti-CD41 mAb against HuPlts surface GP IIb/IIIa. After transplantation of b34 cells, a maximum of 0.3% HuPlts was detected only at week 3 (Fig. 2). At week 4 HuPlt count was a mean of 4.5%; the percentage of the HuPlts was similar at 6–8 weeks after transplant in some transplanted mice. By contrast in the PB of mice injected with 2.5 x 105 4w34 cells, 0.5% of HuPlts were seen as early as week 1, even if the human CD41 cells in the murine BM were below the FACS detection limit (: i8 y" H! \$ ]+ y# w! _
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Figure 2. Human platelet appearance. Kinetics of HuPlt appearance in the PB of NOD/SCID mice injected with 2.5 x 105 baseline CD34 cells, 2.5 x 105 CD34 cells immunoselected from 4-week expanded cultures and with all the CD34 progeny of 2.5 x 105 initial CD34 cells expanded for 4 weeks. Results show the mean ± SD of the percentage of HuPlts detected by FACS analysis in the murine PB at the indicated time points after transplantation (4 mice per experimental point, 3 separate experiments).
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" S/ H; L. O; ZFigure 3. Flow cytometric analysis of human platelets in peripheral blood of NOD/SCID mice. A) PB samples (from a representative untransplanted mouse, a normal human donor, and a mouse injected 3 weeks previously with 2.5 x 105 4w34 cells) were labeled with a mAb against human CD41a. Analysis was performed within the platelet population gate based upon forward and side scatter. B) Flow cytometric analysis of HuPlts from a mouse transplanted 3 weeks previously with 2.5 x 105 4w34 cells before and after thrombin stimulation. On the y-axis PE-anti-human CD41a, on the x-axis FITC-anti-human CD62P. CD62P is expressed only on activated platelets. FACS analysis is carried out within the gate of HuPlts.
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HuPlt activation in response to in vitro challenge with thrombin was tested. Thrombin induces granule secretion resulting in CD62P expression on the platelet membrane. After thrombin stimulation, the expression of CD62P on human CD41 platelets was increased (Fig. 3B).
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DISCUSSION
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& L* L/ l& K1 g- N" e4 SSupport for this work was provided by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy and from the Ministero dell’Universit角 e della Ricerca Scientifica e Tecnologica (MURST), Rome to W.P. and to M.A., and from CNR (Progetto Finalizzato Oncologia). The authors wish to thank Mrs. L. Ramini for invaluable secretarial assistance.
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