Expansion of CD34
+cells from human umbilical cord blood
by FL and/or TPO gene transfected human marrow stromal
cell lines
ZHANG Yi (
Æ
Ê
) , TANG Peixian (Tang Pei-hsien
O
),
JIN Ying (
\
) , LI Xiusen (
'.
) , ZHANG Shuangxi (
Æ
) ,
WU Ying (
Q
ç
) & MAO Ning (
Á
)
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China Correspondence should be addressed to Zhang Yi (email: [email protected])
Received December 1, 2000
-897,.9 To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were trans-fected into human stromal cell line HFCL. To establish gene transtrans-fected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 52.1)-fold, total progenitor cells (CFC) by (74.5 5.2)-fold and CD34+ cells by 15.7-fold. Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 5.5)-fold and (57.0 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34+ cells can be extensively expanded ex vivo by using gene transfected stromal cells along with cytokines.
Keywords: ex vivo expansion, CD34+ cells, stromal cell line, Flt3 ligand, thrombopoietin.
Human umbilical cord blood (CB) has been established as an important source of hemato-poietic stem/ progenitor cells (CD34+ cells). To date, over 3000 CB transplantations have been performed for children with malignant and nonmalignant diseases. However, there are potential limitations to the widespread use of CB; there may be enough hematopoietic stem cells to recon-stitute children, but the ability to engraft an adult from CB may require ex vivo manipulations. Many studies showed that the combination of thrombopoietin (TPO) and Flt3 ligand (FL) was able to support expansion of human CD34+ cells. On the other hand, contact with stromal cells was reported to preserve human CD34+ cells quality during ex vivo manipulation[1, 2]. This study was designed to determine the extensive amplification of CB CD34+ cells by FL and/or TPO gene transfected human bone marrow stromal cell lines.
1 Materials and methods
1.1 Materials
1.1.1 Human stromal cell line and umbilical cord blood samples. The human stromal cell line HFCL was a kind gift from Prof. Jiang Xueying (Institute of Hematopoiesis, Academy of Chinese Medical Sciences, Tianjin, China). Stromal cell lines HDF (gene transferred FL), HLT (gene transferred TPO) and HFT (gene transferred FL and TPO) were established by Dr. Zhang Yi[3].
CB samples were obtained from Peking Union Medical College (PUMC) Hospital, China. Immunomagnetic isolation system and CD34 progenitor cell isolation reagents were purchased from Miltenyi Biotech (Germany).
1.1.2 Human cytokines and antibodies. Recombinant human stem cell factor (rhSCF), recom-binant human Flt3 ligand (rhFL), recomrecom-binant human thrombopoietin (rhTPO), recomrecom-binant hu-man granulocyte colony-stimulating factor (rhG-CSF), recombinant huhu-man granulocyte-macro- phage colony-stimulating factor (rhGM-CSF), recombinant human interleukine-3 (rhIL-3), and recombinant human erythropoietin (rhEPO) were purchased from Preptech (UK).
The monoclonal antibodies of CD34-PE and CD38-FITC were products from Pharmingen (USA).
1.1.3 Reagents. BSA, methylcellulose, 2-mercaptoethanol, L-glutamine, and hydrocortisone were produced by Sigma (USA). Cell medium and horse serum were from Gibco (USA). LTC-IC medium (Myelocult H5100) was from Stem Cell (Canada).
1.2 Methods
1.2.1 Cord blood collection and CD34+ cells separation. CB samples were obtained from nor-mal full-term deliveries after obtaining fornor-mal parental consent according to guidelines established by the PUMC Hospital. CB samples were collected by drainage of blood into sterile polypropyl-ene bags containing preservative-free sodium heparin. CB samples were preserved and transported at 0 4 . CD34+ cells were isolated in 4 h. CB mononuclear cells were isolated by density cen-trifugation over Ficloo-Hypaque (density 1.077 g/cm3).
CD34+ cells were immunomagnetically enriched from CB samples by utilizing MACS CD34 Progenitor Cell Isolation Kit according to the manufacturer’s instructions[4]. The efficiency of the purification was verified by flow cytometry counterstaining with CD34 monoclonal antibodies and CD38 monoclonal antibodies.
1.2.2 Expansion groups. Two culture systems were used in this study. Stromal cell-free culture system constituted 4 groups according to different cytokines, i.e. SCF+IL-3; SCF+IL-3+FL; SCF+IL-3+TPO; SCF+IL-3+FL+TPO. Coculture system of gene transferred stromal cell lines was divided into 4 groups according to different stromal cell lines, i.e. HFCL+SCF+IL-3;
HDF+SCF+IL-3; HLT+SCF+IL-3; HFT+SCF+IL-3.
1.2.3 Ex vivo expansion cultures of CD34+ cells. All cultures were maintained in IMDM with 12.5%HS, 12.5%BSA, 2 mmol/L L-glutamine, 10−4 mol 2-mercaptoethanol, 10−6 mol hydrocorti-sone and various combinations of recombinant human cytokines including SCF at 50 ng/mL, IL-3 at 10 ng/mL, FL at 10 ng/mL and TPO at 10 ng/mL.
CD34+ cells (1×104 cells/mL) were cocultured with or without a confluent layer of the stro-mal cell lines in quadruplicate flat-bottomed 24-well plates in 1 mL of medium. The wells were grown at 37 at 5% CO2. Cultures were fed at weekly intervals by removing half of the medium
and replacing it with fresh complete medium. Stromal cell line layers were changed twice a week in each microwell. The total number of hematopoietic cells, CD34+ cells and hematopoietic pro-genitor cells was analyzed.
1.2.4 Clonal hematopoietic progenitor cells culture. Aliquots from initial CB samples or cul-tured cells were incubated in methylcellulose media at concentrations of 1×103 cells/mL for puri-fied CD34+ cells and 5×103 100×103 cells/mL for cultured cells in 24-well plates. One milliliter of culture mixture contained 1.3% methylcellulose, IMDM, 30% HS, 1% deionized fraction V BSA, 10−4 mol/L 2-mercaptoethanol, 2 mmol/L L-glutamine, and various growth factors. The op-timum concentration of each growth factor was as follows: SCF 10 ng/mL, IL-3 10 ng/mL, GM-CSF 10 ng/mL, G-CSF 10 ng/mL and EPO 4 U/mL. The cells were incubated at 37 under a humidified atmosphere of 5% CO2 for 14 days, and then colony scoring was performed for
CFU-GM and BFU-E under an inverted microscope. The positive wells were defined as contain-ing at least one colony of 50 or more cells.
1.2.5 LTC-IC assay. Bone marrow stromal cells were irradiated with 10 15 Gy 60Coand plated in 24-well tissue plates to form pre-established stromal layers for the long-term cultures according to Dexter’s method. After 4 weeks expansion cells were seeded on top of the irradiated bone marrow stroma in culture LTBMC-medium (Myelocult H5100 supplemented with 10−6 mol/L hydrocortisone), for another 4 weeks at 33 with weekly changes of half the medium. Every week, non-adherent cells were removed, combined with the corresponding trypsinized ad-herent cells, washed and assayed for cell colony-forming units (CFC) in methylcellulose medium. Colonies were scored 2 weeks later. The frequency of wells in which there were no clonogenic progenitors was determined according to the number of the initial input population, and frequency of LTC-IC was calculated. The total number of LTC-IC initially present was scored 5 8 weeks later.
1.2.6 Statistics. Results of experimental points from different experiments were reported as the standard error of the mean. The significance of differences between mean values was determined by using the two-tailed Student’s t-test.
2 Results
2.1 CB CD34+ cells separation and purification
CD34+ cells were enriched significantly by using the MACS system. Flow cytometric analy-sis showed that the purities of CD34+ and CD34+CD38− cells are 95.0% and 10.0% respectively (fig. 1).
Fig. 1. Purity of isolated CD34+ cells from human cord blood analyzed by flow cytometry.
2.2 Evaluation of supportive effects on ex vivo expansion of human CD34+ cells on the total number of expansion cells, CFC, CFU-GM and BFU-E in the coculture systems of transgenic stromal cell lines
In 4 weeks, the effects of different cytokines and transgenic stromal cell lines were first as-sessed on ex vivo expansion of human CD34+ cells in two culture systems by 8 different groups. In the stroma-free cytokine-containing culture, cells were grown at the edge of plates at first and then moved to the center of plates. However, in stromal coculture, cells were grown on the top of the stromal cell lines. In the first week, the number of total nucleated cells of the different culture systems increased. In weeks 2, 3, 4, the number of total nucleated cells in coculture system was significantly increased compared with that in stroma-free culture system (P 0.05). The most po-tent combination of SCF+IL-3+FL+TPO increased the total nucleated cells by (893.3 52.1)-fold in week 4 (table 1).
Table 1 The increase of the total nucleated number in ex vivo expansion of cord blood Culture time /d Groups 7 14 21 28 SCF+IL-3 26.0 1.0 152.7 2.1 331.3 25.3 624.0 42.3 SCF+IL-3+FL 37.8 1.0 188.0 9.0 376.7 18.6 710.7 88.8 SCF+IL-3+TPO 33.3 2.1 176.7 7.1 367.3 26.4 694.7 58.3 SCF+IL-3+FL+TPO 52.0 6.6 215.0 7.8 428.0 25.0 762.7 72.6 SCF+IL-3+HFCL 31.8 1.9 166.4 2.6 340.7 57.1 638.7 17.2 SCF+IL-3+HDF 46.8 6.8 191.6 8.6 394.0 37.5 796.0 52.5 SCF+IL-3+HLT 43.0 1.0 183.7 21.2 390.7 28.1 764.0 62.9 SCF+IL-3+HFT 64.2 3.4 228.3 6.7 474.7 16.2 893.3 52.1
The total expansion effects of CFC (colonies of CFU-GM and BFU-E) were also studied in the 8 groups. After 4 weeks of culture, the number of CFC was remarkably increased in different groups. Maximal expansion of CFC was observed during the first two weeks of culture. SCF+IL-3+HFT manifested the most potent combination, with the total progenitor cells increased by (74.5 5.2)-fold, while in another combination of SCF+IL-3+FL+TPO the increment was by (60.8 5.8)-fold. In the other 6 groups, the expansion effects of CFC in transgenic coculture sys-tems manifested higher increment of
CFC than in stroma-free culture systems (fig. 2).
To determine whether cells gener-ated the CFU-GM and BFU-E, the ex-pansion cells were incubated in me-thylcellulose media. As a result, slight increase for CFU-GM and BFU-E in transgenic coculture systems was noted for the first week. Interestingly, the number of CFU and BFU-E was sig-nificantly increased in culture systems in the second week, and an increase by (78.1 5.5)-fold and (57.0 19.7)-fold
in CFU-GM and BFU-E was obtained on day 14 of culture in the presence of SCF+ IL-3+HFT(SI+HFT). The expansion efficiency for committed progenitor including CFU- GM and BFU-E and the volume of clonal cells were gradually decreased following expansion time (data not shown). These data demonstrated that it is possible to promote the expansion of hematopoietic progenitor cells in vitro using transgenic stromal cell lines with several other cytokines.
2.3 Effect of gene transfected stromal cell lines on the expansion of cord blood CD34+ cells CD34+ cells derived from a single delivery were cultured in different culture systems, in-cluding stroma-free liquid culture system and gene transfected stromal cell lines coculture system. During culture on day 7, 14 and 28, aliquots of harvested cells were labeled with PE-conjugated CD34 and FITC-conjugated CD38 antibodies; the presence of CD34+ cells and CD34+CD38− cells was analyzed by flow cytometry. CD34+ cells were significantly increased by 15.7- and 12.1-fold on day 7 of culture in the presence of SCF+IL-3+HFT(SI+HFT) and SCF+IL-3+FL+TPO (SI+FT). On day 14, the expansion of CD34+ cells was decreased by 3.9- and 2.7-fold. Until day 28, the expansion fold of CD34+ cells was only 0.8 and 0.4. Otherwise, on day 14 and 28, CD34+CD38− cells were expanded 28- and 16-fold, 5.3- and 1.5-fold respectively. These results indicate that the combination with FL and TPO could effectively support rapid expansion of primitive progenitor cells, but the mechanism of hematopoietic-supportive remains unknown (table 2).
Fig. 2. The increase of committed progenitors derived from CD34+ cellsafter 2 weeks of culture in different groups (SI=SCF+
Table 2 The increase of CD34+ cells and CD34+CD38- cells after 4 weeks of culture in vitro SCF+IL-3+FL+TPO SCF+IL-3+HFT Culture time/d CD34+/CD34+CD38−(1 104) CD34+/CD34+CD38−(1 104) 0 0 0 7 12.1/− 15.7/− 14 2.7/16 3.9/28 28 0.4/1.5 0.8/5.3
2.4 Effect of gene transfected stromal cell lines on the support of long-term culture initiating cells (LTC-IC)
Long-term culture initiating cells (LTC-IC) are identified as a subset of extremely primitive hematopoietic progenitor cells for which it needs as long as 5 weeks to perform colonies in culture. Our experiments showed that over a 4-week culture period in 8 different groups, non-adherent cells and adherent cells were mixed and then seeded on top of the irradiated bone marrow stroma in culture LTBMC-medium for 4 weeks at 33 with weekly changes of half the medium. The output of LTC-IC was assayed weekly. The results demonstrated that generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, i.e. SCF+IL-3 +FL+TPO and SCF+IL-3+HFT; the total numbers of CFU-GM in two combinations were 3.0 0.0 and 4.5 2.1; and there was no significant difference between these two groups statistically. Since HFT was a transgenic stromal cell line which FL and TPO genes were transfected into hu-man stromal cell line HFCL, these studies suggest that the combination with FL and TPO could effectively support primitive hematopoiesis.
3 Discussion
It is well documented that human umbilical cord blood (CB) is a rich source of stem and progenitor cells, which can be used as an alternative to bone marrow for reconstituting hemato-poiesis in the allograft setting. The successful cell dose for CB transplantations is around (3 5) ×105/kg, which restricts the clinical application in adult patients. The study on the expansion of CD34+ cells in CB seems extremely important. According to the recent reports, in the bone mar-row, a complex interplay of cell-cell adhesion in contact with hematopoietic cells and stromal cells constitutes the basic regulation of hematopoiesis[5 7]. Bone marrow stromal cells are the major factor in hematopoietic environment, and the self-renewal, proliferation, differentiation, migration and apoptosis of hematopoietic stem and progenitor cells are tightly regulated by stromal cells[8, 9]. Aiuti et al.’s studies[10] showed induced expansion of CD34+ progenitor/stem cells during the first 4 weeks of coculture and that this CD34+ population was maintained for up to 10 weeks in vitro. Kawada et al.[11] found that in the coculture system the stromal cell line dramatically supports the rapid expansion of cryopreserved cord blood CD34+CD38- cells in synergy with TPO/FL. Within 7 days of culture, a 100-fold increase in CD34+CD38- cells was obtained. CFC, CFU-GEMM and LTC-IC were enhanced by 10- to 30-fold, 10- to 20-fold and 25- fold, respectively. In this study
we assessed the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, and we draw the following conclusions: CD34+ cells were enriched significantly using the MACS system. In week 4, the total number of nucleated cells and number of CD34+ cells and CFC were expanded in different culture systems including SCF, IL-3, FL, TPO and transgenic stromal cell lines. The number of total nucleated cells and CFC (colonies of CFU-GM and BFU-E) in transgenic stromal coculture system was significantly increased compared to that in stroma-free culture system, and the number of total nucleated cells increased by (893.3 52.1)-fold. The maximal expansion of CFC was observed during the first two weeks of culture. SCF+IL-3+HFT manifested the most potent combination, with the number of total progenitor cells (CFC) increased by (74.5 5.2)-fold. The expansion effects of CFC in HFT group could be more significantly increased than those in other culture groups (P 0.05). The expansion effi-ciency for CFC was gradually decreased following expansion time. The ability of hematopoietic cells to generate the CFU-GM and BFU-E was different in different culture systems. Slight in-crease for CFU-GM and BFU-E in transgenic coculture systems was noted. Interestingly, the number of CFU-GM and BFU-E was significantly increased in culture systems in the second week. SCF+IL-3+HFT manifested the most potent combination. The expansion effects of CD34+ cells and CD34+CD38− cells indicated that SCF+IL-3+HFT was identified as the most po-tent combination, CD34+ cells were significantly increased by 15.7-fold on day 7 of culture, and then CD34+ cells were gradually decreased. However, on day 14 and 28, CD34+CD38− cells were expanded 28-fold and 5.3-fold. These results indicate that the transgenic stromal cell lines could effectively support rapid expansion of CD34+ cells in synergy with FL/TPO. Furthermore, CD34+CD38- cells were maintained for about 4 weeks during culture, but the mechanism of he-matopoietic-supportive remains unknown. Our conclusion indicates that cord blood CD34+ cells and the proliferation of CFU-GM and BFU-E can be extensively expanded ex vivo by using gene transfected stromal cells along with cytokines. The expansion effects of CD34+ cells in different culture systems were different by stromal cell lines and cytokines. A simple and effective method for ex vivo expansion of primitive hematopoietic progenitor cells required suitable cytokines and stromal cell.
After ex vivo expansion of CD34+ cells in short-term culture, we assessed whether the CD34+ cells could be reconstituted in vivo in long-term culture. Many studies identified the content of long-term initiating cells (LTC-IC) able to support reconstitution long-term hematopoiesis in
vivo[12, 13]. LTC-IC was a population of primitive progenitor cells in vitro in long-term culture. Assessment of characteristics and content by LTC-IC can provide important information concern-ing transplantation. Graft failure can be predicted by testconcern-ing the ability of stem cells to produce LTC-IC in long-term stroma-supported culture. In our studies, the expansion of hematopoietic cells in long-term culture by week 4 was seeded on the 60Co irradiated human stromal cell layers, then cultured for the next 4 weeks. The results showed that LTC-IC was found in two
combina-tions, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT. The conclusion suggests that since HFT was a transgenic stromal cell line with FL and TPO genes transfected into human stromal cell line HFCL, the combination of FL and TPO could effectively support primitive hematopoiesis.
Acknowledgements We are grateful to Drs. Pan Lingya, Tong Ying and Zu Fuli for providing human cord blood. This work was supported by the National High Technology Program of China (Grant No. BH-030501).
References
1. Luens, K. M., Travis, M. A., Chen, B. P. et al., Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+Thy+Lin- cells with preserved ability to engraft
SCID-hu bone, Blood, 1998, 91: 1206—1215.
2. Piacibello, W., Sanavio, F., Garetto, L. et al., Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood, Blood, 1997, 89(8)L 2644—2653.
3. Zhang Yi, Tang Peixian, Li Xiusen et al., Expression of human FL ligand and thrombopoietin genes in a bone marrow stromal cell line by internal ribosome entry site (IRES) sequence, Chin. J. Hematol. (in Chinese), 1999, 20(12): 624 627. 4. Zhou, S., Mao, N., Zhao, M. et al., Effect of recombinant FLT3 ligand (rhFL) on in vitro expansion of human cord blood
CD34+ cells, Natl. Med. J. China, 1998, 78: 37—39.
5. Dexter, T. M., Allen, T. D., Lajtha, L. G. et al., Conditions controlling the proliferation of hematopoietic stem cells in vitro, J. Cell Physiol., 1997, 91: 335—344.
6. Petzer, A. L., Hogge, D. E., Lansdorp, P. M. et al., Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium, Pro. Natl. Acad. Sci. USA, 1996, 93: 1470—1474.
7. Sirchia, G., Rebulla, P., Placental/umbilical cord blood transplantation, Haematologica, 1999, 84: 738—747.
8. Wineman, J., Moore, K., Lemischka, I. et al., Functional heterogeneity of the hematopoietic microenvironment: Rare stromal elements maintain long-term repopulating stem cells, Blood, 1996, 87: 4082—4090.
9. Szilvassy, S. J., Weller, K. P., Lin, W. et al., Leukemia inhibitory factor upregulates cytokine expression by a murine stro-mal cell line enabling the maintenance of highly enriched competitive repopulating stem cells, Blood, 1996, 87: 4618—4628.
10. Aiuti, A., Fredrich, C., Sieff, C. A. et al., Identification of distinct elements of the stromal microenvironment that control human hematopoietic stem/progenitor cell growth and differentiation, Exp. Hematol., 1998, 26: 143—157.
11. Kawada, H., Ando, K., Tsuji, T. et al., Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system, Exp. Hematol., 1999, 27: 904—915.
12. Alcorn, M. J., Holyoake, T. L., Richmond, L. et al., CD34+ cells isolated from cryopreserved peripheral-blood progenitor
cells can be expanded ex vivo and used for transplantation with little or no toxicity, J. Clin. Oncol., 1996, 14: 1839—1847. 13. Van Hennik, P. B., Breems, D. A., Withagen, C. M. et al., Graft-failure can be predicted by testing the ability of stem cells
