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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 1  |  Page : 233-239

Fast recovery of platelet production in NOD/SCID mice after transplantation with ex vivo expansion of megakaryocyte from cord blood CD34+ cells


1 Department of Pediatrics, The Second Hospital of Shandong University, Jinan, Shandong Province, China
2 Department of Pediatrics, Qilu Hospital of Shandong University, Jinan, Shandong Province, China

Date of Web Publication8-Mar-2018

Correspondence Address:
Prof. Xiuli Ju
Department of Pediatrics, Qilu Hospital of Shandong University, 107# Wenhua Xi Road, Jinan, Shandong Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.193893

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 > Abstract 

Background: Cord blood transplantation (CBT) can be a life-saving procedure in the treatment of a broad variety of disorders, including hematologic, immune, and genetic diseases. However, delayed platelet recovery hinders the application of CBT.
Purpose: The aim of this study was to determine the optimal combination of cytokines to amplify megakaryocyte (Mk).
Methods: CB CD34+ cells were obtained by immunomagnetic isolation and amplified under four different cytokine combinations. CD34+ cells of the group with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (FL), and interleukin-6 (IL-6) were collected on days 0, 3, 7, 10, and 14. Immunophenotype was analyzed by flow cytometry (FCM). Polyploidic Mk cultured cells were collected on days 7 and 14 for colony-forming unit-Mk assay. The NOD/SCID mice were injected with expanded CD34+ cells, and the peripheral blood (PB) and bone marrow (BM) were tested on 3, 7, and 14 days.
Results: The group with TPO, SCF, FL, and IL-6 reached the maximal total expansion fold and Mk population at day 7, which was slightly reduced later. After transplantation into NOD/SCID mice with expanded CD34+ cells, the human CD41+ cells were detected in mice PB on day 3 and in BM on day 7, then disappeared after 14 days. The expressing of activated platelet CD 42b+/CD62P+ increased gradually after transplantation.
Conclusion: Platelets can recover rapidly in vivo by means of expanded CD34+ cells with various cytokines. In our system, a group of TPO, SCF, FL, and IL-6 represents the best cytokine combination for expansion of Mk progenitor cells from CB CD34+ cells.

Keywords: Expansion, hematopoietic stem cells, megakaryocyte, platelet recovery, transplantation


How to cite this article:
Wang H, Ge W, Zhuang Y, Fu J, Li D, Ju X. Fast recovery of platelet production in NOD/SCID mice after transplantation with ex vivo expansion of megakaryocyte from cord blood CD34+ cells. J Can Res Ther 2018;14:233-9

How to cite this URL:
Wang H, Ge W, Zhuang Y, Fu J, Li D, Ju X. Fast recovery of platelet production in NOD/SCID mice after transplantation with ex vivo expansion of megakaryocyte from cord blood CD34+ cells. J Can Res Ther [serial online] 2018 [cited 2019 Nov 12];14:233-9. Available from: http://www.cancerjournal.net/text.asp?2018/14/1/233/193893


 > Introduction Top


Hematopoietic stem cells (HSCs) transplantation has become an important means to treat pediatric patients with malignant disease. Umbilical cord blood (CB) has emerged as an alternative source of HSCs in recent years because of the lack of a human leukocyte antigen (HLA)-identical sibling donor in the majority of candidates.[1],[2] However, two major concerns limit the widespread use of CB as a source of HSCs for marrow replacement. First, CB contains a finite number of HSCs and progenitor cells, which may be insufficient for the hematopoietic rescue of large recipients undergoing transplantation, as well as patients with diseases known to be resistant to engraftment, such as Fanconi anemia, chronic myelogenous leukemia, and severe aplastic anemia.[3] Second, in comparison with bone marrow (BM) or peripheral blood (PB), CB transplantation (CBT) is characterized by a delayed engraftment and, in particular, a very slow platelet recovery.[4],[5],[6] To reduce this prolonged thrombocytopenia, an effective strategy is to transplant both CB stem cells and ex vivo-expanded HSCs simultaneously and/or megakaryocyte (Mk) progenitors. CB contains a high number of primitive progenitor cells in vitro with a greater expansion ability and in vivo engraftment capability compared with other sources.[7] Furthermore, the reduced alloreactivity of CB cells contributes to the lower risk of graft-versus-host disease than those transplanted with allogeneic adult BM.[8]

CB CD34+ can be expanded in stoma-free cultures containing thrombopoietin (TPO), Flt-3 ligand (FL), stem cell factor (SCF), interleukin-6 (IL-6), and other cytokines. This cytokine combination amplifies CB progenitors and precursors of all hematopoietic lineages, without a concomitant loss but rather an increase.[9],[10],[11] Much effort has been devoted to study cytokine cocktails to support the expansion of Mk progenitors ex vivo.[12] Nevertheless, whether CB CD34+ cells, after several weeks of expansion with cytokines combination, can retain not only the same in vivo repopulation ability but also the same proliferation and differentiation potential toward the Mk lineage as the unmanipulated CB CD34+ cells remain unclear. Only a few studies have shown that platelet can recover with expanded CB CD34+ cells by NOD/SCID (nonobese diabetic severe combined immunodeficient) mouse model.[13]

In CBT, engraftment of the Mk lineage shows the longest delay. However, to date, only a few experimental studies have addressed the issue of the short-term engraftment ability of fresh CB stem cells, and even fewer have addressed that of ex vivo-expanded stem cells.[14],[15] Using the NOD/SCID mouse model, the short-term repopulation ability and the differentiation and maturation potential of human hematopoietic lineages in ex vivo experimental model can be analyzed.[16]

In this study, CD34+ cells were obtained by immunomagnetic beads methods and cultured in serum-free and stroma-free medium containing the following four different cytokine combinations: TPO + SCF + FL + IL-6, TPO + SCF + FL, TPO + SCF, and TPO. The cells were collected on days 3, 7, 10, and 14, and then the expression of cell surface molecules was examined. Mk ploidy and colony-forming unit-Mk (CFU-Mk) formation were also evaluated. Finally, the collected cells were then transplanted into NOD/SCID mice.


 > Methods Top


CD34 + cell purification and expansion

Human CB (n = 12) cells were obtained after obtaining informed consent from full-term deliveries in the Department of Obstetrics in Qilu Hospital of Shandong University. The use of CB was approved by the Ethics Committee of Qilu Hospital of Shandong University. Mononuclear cells (MNCs) were isolated from CB using Ficoll (density 1.077 ± 0.001 g/ml, TBD, Tianjin, China), washed twice, and resuspended with phosphate buffer saline (PBS). The samples were washed with MACS buffer (pH 7.2) containing 0.5% bovine serum albumin (BSA) and 2 mmol/L ethylenediaminetetraacetic acid. The CD34+ cells were isolated using immunomagnetic columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The procedure was performed twice to obtain a high purity of CD34+ cells. CD34+ cell purity was analyzed using FCM (Guava easyCyte 8HT, EMD Millipore, Billerica, MA, USA) and Guava InCyte software (version 2.8, EMD Millipore, Billerica, MA, USA).

The CB CD34+ cells were plated in 24-well plates using StemSpan™ SFEM serum-free medium (Stem Cell Technologies, Vancouver, Canada) at 5 × 105 cells/mL, and the medium was refreshed each week. Cultures were incubated with different cytokines at 37°C in a humidified atmosphere with 5% CO2 for 14 days. The experiment was divided into four groups: (1) TPO + SCF + FL + IL-6 (all from RD, USA); (2) TPO + SCF + FL; (3) TPO + SCF; and (4) TPO. The final concentration of cytokines was 50 μg/L TPO, 100 μg/L SCF, 100 μg/L Flt3-L, and 100 μg/L IL-6. Total cells were collected and counted on days 0, 3, 7, 10, and 14.

Immunophenotyping analysis

Totally, 5 × 105 CD34+ cells were collected on days 0, 3, 7, 10, and 14. After washing twice with PBS, the cells were labeled using the following monoclonal antibodies: mouse antihuman CD34-FITC, CD42b-PE, CD41a-PE, CD61-FITC, CD49d-PE, CD49e-FITC, CD11a-FITC, CD54-PE, and mouse IgG1-FITC, IgG2-PE (BD, Pharmingen, USA). Cells in PBS at a volume of 100 with 10 μL of monoclonal antibody were incubated at 25°C for 30 min. After washing with PBS, the cells were resuspended in 100 μL of PBS and detected by FCM (Guava easyCyte 8HT, EMD Millipore, Billerica, MA, USA), and data were analyzed by Guava InCyte software (Version 2.8, EMD Millipore, Billerica, MA, USA).

Megakaryocyte ploidy

Polyploidic Mk cultured cells were collected on days 7 and 14, washed thrice, and resuspended in PBS with 10% BSA. The cells were then incubated with CD41-PE for 30 min at 4°C before fixation in 70% ethanol for 20 min. For DNA staining, the cells were incubated with ribonuclease (RNase, 50 mg/L) and propidium iodide (50 mg/L) for 1 h at 4°C. Mk polyploidy of CD41+ cells was analyzed by FCM.

CFU-Mk assay: Totally, 1 × 104 CD34+ cells were seeded into 24-well plates for semisolid culture (MethoCult GF H4434, Stem Cell Technologies, Vancouver, Canada) with different cytokines for 2 weeks. The experiment was divided into four groups as described before. The total number of colonies was scored for the CFU-Mk assay using a microscope.

Transplantation into NOD / SCID mice

NOD/SCID mice were purchased from the Institute of Experimental Animal of Chinese Academy of Medical Sciences (Shanghai, China) and raised under sterile conditions. The Ethics Committee of Animal Experimentation from our hospital approved the procedures. Six-week-old female mice (weighing about 18 ± 0.6 g each) were treated with 350 cGy of total-body sublethal irradiation from a 137 Cs source 4 h to 6 h before transplantation. We selected the experiment group with the highest expansion fold and cell maturity on day 7 to evaluate the function of expanded cells in vivo. 24 mice per group were injected with 1 × 106 (0.5 ml) CD34+ cells expanded after 7 days via the tail vein for each animal, nonexpanded CD34+ cells were set as control 1, and normal saline of the same volume was set as control 2. Mice were tested on 0, 3, 7, and 14 days after transplantation; six mice were sacrificed for each time point.

Detection of human megakaryocyte in peripheral blood and bone marrow of NOD/SCID mice

Mice were sacrificed on 0, 3, 7, and 14 days after transplantation. We first obtained the PB from the heart of the mouse. Then, BM was flushed from femora and tibiae using Iscove's modified Dulbecco's medium. MNCs were isolated from BM or PB using Ficoll (Density 1.088 ± 0.001 g/ml, TBD), washed twice, and resuspended in PBS. Then, MNCs were labeled using flowing monoclonal antibodies: mouse antihuman CD41a-PE and mouse IgG-PE (BD, Pharmingen). Cells in PBS at a volume of 100 with 10 μL of monoclonal antibody were incubated for 30 min at room temperature (RT). The cells were resuspended in 100 μL of PBS after washing with PBS and immediately analyzed by FCM.

Isolation and activation of platelet in peripheral blood of NOD/SCIDmice

PB from NOD/SCID mice was drawn into ACD tubes (BD Vacutainer), and platelet-rich plasma was separated by centrifugation at 200 ×g for 15 min at RT with no brake. The platelet-rich plasma was collected, and platelets were washed twice using citrate buffer at 440 ×g for 10 min at RT. Platelets were counted and resuspended to a final concentration of 1 × 108 cells/ml in Tyrode's buffer. Agonists used to activate the platelets using adenosine diphosphate (ADP, Sigma) at 20 μM for 30 min at 37°C. The platelets were then washed once with Tyrode/PGE/Hep buffer (to prevent further platelet activation and aggregation) by centrifuging at 2000 ×g for 10 min at RT. To assess platelet activation, expression of CD62P (using PE-mouse antihuman CD62P) was detected on CD42b+ (FITC-mouse IgG anti-human CD42b) platelets. Finally, platelets were washed and immediately analyzed by FCM.

Statistical analysis

All data are presented as mean ± standard error mean of separate experiments. SPSS (version 14.0; SPSS Inc., Chicago, IL, USA) was used for statistical analysis. They were compared via ANOVA followed by a Student's t-test. Differences between values were considered statistically significant at P < 0.05.


 > Results Top


CD34+ cell purification

The median CD34+ purity was 93 ± 3.4% after immunomagnetic bead selection for all 12 samples. The average number of CD34+ was 1.18 ± 0.38 × 106.

Expansion of megakaryocyte

Results showed that the maximum expansion of the Mk population in the four experiments occurred at day 7, which was slightly reduced later [Figure 1]. Group 1 (TPO with SCF, FL, and IL-6) reached the highest total cell quantities. In the presence of TPO, SCF, FL, and IL-6, the proportion of CD34−/CD41+ phenotype of Mk reached 6.99 ± 2.08, 40.49 ± 7.35, 36.70 ± 6.90, and 30.65 ± 4.94-fold in total cell numbers on days 3, 7, 10, and 14, respectively. Expanded by Group 2 (TPO, SCF, and FL), the CD34−/CD41+ cells reached 6.43 ± 0.99, 36.76 ± 7.24, 31.48 ± 5.58, and 27.04 ± 5.25-fold on days 3, 7, 10, and 14, respectively. In the absence of FL and IL-6 (Group 3), the CD34−/CD41 + cells reached 5.01 ± 1.54, 28.10 ± 4.23, 25.15 ± 3.03, and 20.96 ± 2.81-fold, respectively, on days 3, 7, 10, and 14. By contrast, TPO only (Group 4) induced the lowest CD34−/CD41+ cells, only reaching 4.13 ± 1.12, 22.06 ± 2.71, 18.20 ± 2.98, and 15.79 ± 3.21-fold, respectively, on days 3, 7, 10, and 14. The CD34+/CD41+ cells that were expanded by four factors reached maximum total cell quantities compared with other experiments on day 14, however, had no significant statistical significance. The proportion of CD41+, CD42b+, and CD61+ cells phenotype of progenitors of Mk lineage continuously increased with induction by cytokineon days 3, 7, 10, and 14. The TPO with SCF, FL, and IL-6 experiment reached higher expanded fold and cell maturity at day 14.
Figure 1: Megakaryocyte immunophenotype in four groups with different cytokine combinations (n = 12). (a) CD34−/CD41+, (b) CD34+/CD41+, (c) CD41+, (d) CD61+, and (e) CD42b+ cell amplification effects. *The cell amplification effect of the thrombopoietin + stem cell factor + FL + interleukin-6 combination was better than the effects of other experiment groups (P < 0.05). On day 14, the total number of the cell subsets was significantly higher than that on day 10 (P < 0.05)

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[Figure 2] shows the surface adhesion molecules of Mk in the presence of different cytokine combinations. The adhesion molecules which can help HSC adhesion and homing play an important role in the megakaryocyte development TPO with SCF, FL, and IL-6 induced a significant and large production of CD49d, CD11a, and CD49e compared with the other experiments. The expression of adhesion molecules increased at each experiment induced by cytokine on days 3, 7, 10, and 14. By contrast, the expression of CD54 decreased at each experiment.
Figure 2: Surface adhesion molecules of megakaryocyte in the presence of different cytokine combinations (n = 12). Percentages of (a) VLA-4-positive cells, (b) VLA-5-positive cells, and (c) LFA-positive cells peaked on day 7 in all groups and then began to decline. (d) The percentage of ICAM-1-positive cells decreased with time (P < 0.05). *The percentage of VLA-4 (CD49d)-positive cells was markedly higher than that of the other adhesion molecules on day 14 (P < 0.05)

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Detection of colony-forming unit-megakaryocyte

The production of CFU-Mk by induced CD34+ cells was evaluated [Table 1]. The maximum production of CFU-Mk was obtained with CD34+ cells induced by TPO, SCF, FL, and IL-6 on day 10 (a 40-fold increase of the initial number of CFU-Mk). This finding confirmed the presence of progenitors of Mk lineage.
Table 1: Fold expansion of colony-forming unit-megakaryocyte with different cytokine combinations (n=12)

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Detection of polyploidic megakaryocyte

[Table 2] shows the production of polyploidic Mk induced by different cytokine combinations. Only 2N and 4N DNA contents of Mk were produced on day 7; no ≥8N ploidy was detected. In the presence of TPO, SCF, FL, and IL-6, ≥8N ploidy was observed on day 14, and 2N and 4N ploidies were decreased, which indicated the maturity of Mk.
Table 2: Effects of different cytokine combinations on megakaryocyte DNA content (n=12)

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Megakaryocyte and platelet production in bone marrow and peripheral blood of NOD/SCID mice

Based on the ex vivo experiment, we selected 5.67 × 105 CD34+ cells expanded by Group 1 (TPO, SCF, FL, and IL-6) and Group 2 (TPO, SCF, and FL) for 7 days. When induced by Group 1, the total number of cells reached 2.25 × 107; the number of CD34+ cells induced by Group 2 was 1.97 × 107. After sublethal irradiation, expanded cells were injected via tail vein. The human CD41 + cells (CD41+% Hu) could be detected in most experiments except for the control 2 from day 3 after transplantation [Table 3]. When induced by TPO, SCF, FL, and IL-6, human CD41+% was detected on day 3 in PB and on day 7 in BM, both peaked on day 14. When induced by TPO, SCF, and FL or nonexpanded CD34+ cells, human CD41+% Hu in PB or BM peaked at day 7 and decreased thereafter. Human CD41+% in PB in the control 2 was detected only on day 7.
Table 3: Expressing the human CD41+ cells in nonobese diabetic/severe combined immunodeficient mice peripheral blood and bone marrow (n=24 for each group)

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The functionality of human platelet in NOD/SCID mice peripheral blood

We detected human platelet that activated by ADP (weak agonist). Thrombin induces granule secretion resulting in CD62P expression on the platelet membrane. The expressing of platelet CD42b+/CD62P+ increase gradually after transplantation [Figure 3].
Figure 3: Expressing of CD42b+/CD62P + platelet after activated by adenosine diphosphate. *The percentage of CD42b+/CD62P + platelet was the highest than that of the other experiment group on day 14 (P < 0.05). On day 14, the expressing of CD42b+/CD62P + platelet was significantly higher than that on day 7 (P < 0.05)

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 > Discussion Top


CD34+ cells can be expanded ex vivo in a stroma- and serum-free system.[12],[16] Several growth factor combinations have been tested to identify culture conditions that can support a large expansion of primitive repopulating stem cells. The previous studies showed that TPO is sufficient to expand CD34+ cells with short- and long-term PLT production potential.[17] Lazzari et al.[12] investigated the effect of different cytokine cocktails, including IL-6, IL-11, Flt3-L, and TPO, combined with serum or serum-free medium, on the ex vivo expansion of CD34+ cells from CB. Their final results support the possibility of maintaining long-term expansion of CB HSCs in a simple stroma- and serum-free system. Cortin et al.[11] used a multistep statistical strategy to quantify individual and interactive effects of cytokines on megakaryopoiesis using a large-scale screening of 13 cytokines. They found that TPO, SCF, IL-6, IL-9, and Flt3-L could stimulate Mk maturation; by contrast, erythropoietin and IL-8 are inhibitors of Mk maturation. Pineault et al.[18] showed that the presence of TPO and low concentrations of Flt3-L, SCF, IL-3, IL-6, and IL-11 stimulate the expansion of Mk progenitors. Several studies have used cytokine cocktails to support the expansion of Mk progenitors ex vivo.[9],[10],[11] In the preliminary experiments, we found that several cytokines could induce umbilical CB differentiation into granulocyte but not Mk. In the present study, we selected TPO, SCF, FL, and IL-6 for Mk expansion. The final concentration of cytokines was 50 μg/L TPO, 100 μg/L SCF, 100 μg/L Flt3-L, and 100 μg/L IL-6. In the preliminary experiments, we test lower concentrations of IL-6 (50 μg/L). We find high concentration is better than lower concentration of IL-6.

Mk is the only polyploid cell in the human; the generation of platelet needs at least 95% Mk of 16–32 ploidy. In the different stages of development, Mk expresses different immune markers. The immune markers of Mk include CD34, CD41, and HLA-DR, as well as the periodically expressed markers PF4, CD61, and CD42b.[19] CD41 is increasingly expressed during Mk differentiation, and lower expression of CD41 means that the Mk cell population is immature.[20] Ploidy analysis is used to evaluate the degree of maturation of CD41+ cell produced in vitro. Adhesion molecules play important roles in HSC/progenitor cells adhesion and homing. In this study, we tested four adhesion molecules CD49d (VLA-4), CD49e (VLA-5), CD11a (LFA-1), and CD54 (ICAM-1). These four adhesion molecules can play an important role in the megakaryocyte development.[21],[22],[23] Similar to the previous studies,[24],[25] our data indicated that CB CD34+ cell-derived Mk cells gradually matured after day 7. According to our FACS results, the TPO, SCF, FL, and IL-6 combination reached relatively high expanded fold and cell maturity on day 14. Adhesion molecule tests showed the maximum efficiency of Mk progenitor cells was reached on day 7. CFU-Mk results and Mk polyploid analysis both support that the function of Mk may occur after 7 days of culture.

The NOD/SCID mouse transplantation model is widely used to study human HSCs because of T and B cell deficiency, NK cell hypofunction, macrophage function deficiency, and no hemolytic complement.[13],[26] NOD/SCID mice had received 250, 350, or 400 cGy irradiation before CB mononuclear or CD34+ cells transplantation. After 6 weeks of transplantation, 250 and 350 cGy irradiation groups showed lower death rate than the 400 cGy irradiation group; however, the implantation rate was similar. This result demonstrated the development of the NOD/SCID mouse model and techniques to assess human engraftment and analyze the effects of short-term cytokine exposure on the long-term repopulating capacity of CB stem cells.[14],[15] We selected the experiment group of higher expanded fold and cell maturity at day 7 to evaluate the function of expanded cells in vivo. In the preliminary experiments, we have tested the expression of megakaryocyte after 14 days of transplantation into mouse; found this period transplantation effect is poor. Our results showed that human platelets were recovered at 7 days in PB and BM injected with expanded or nonexpanded cells; in particular, injection with expanded cells exhibited faster recovery than injection with nonexpanded cells. PB platelets of the injected mice recovered significantly in the TPO, SCF, FL, and IL-6 treatment. The expressing of activated platelet CD42b+/CD62P+ increase gradually after transplantation. The results indicated that IL-6 is an effective cofactor for TPO, SCF, and FL. In conclusion, by means of expanded CD34+ cells with various cytokines, short-term engraftment by ex vivo-expanded cells seems more efficient than by nonexpanded cells. The combination of TPO, SCF, FL, and IL-6 might be the best cytokine combination for expansion of Mk progenitor cells from CB CD34+ cells.


 > Conclusion Top


Combination of TPO+SCF+FL+IL-6 can amplify megakaryocytes from CB CD34+ cells and accelerate platelet recovery in NOD/SCID mice.

Financial support and sponsorship

The present study was supported by the Grants of Major State Basic Research Development Program (2012CB966504); Shandong Province Natural Science Foundation (2014GSF118131andJinan201403010), and Basic Scientific Fund of Shandong University (2014QLKY02and2014QY003-11).

Conflicts of interest

There are no conflicts of interest.

 
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Salles II, Thijs T, Brunaud C, De Meyer SF, Thys J, Vanhoorelbeke K, et al. Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34(+) cells are functionally active in an ex vivo flow model of thrombosis. Blood 2009;114:5044-51.  Back to cited text no. 26
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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