[Effect of human umbilical cord mesenchymal stem cells on the CD34+ cells transplantation in NOD/SCID mice].

OBJECTIVE: To study the effect of human umbilical blood (UB) mesenchymal stem cells (MSC) on the CD34(+) cells transplantation in NOD/SCID Mice. METHODS: Umbilical blood CD34(+) cells (3.5 x 10(5) cells) alone or combined with umbilical cord MSC cells were transplanted into NOD/SCID mice that had been irradiated with (137)Cs (3.0 Gy) before transplantation. Changes in peripheral blood cells within 6 post-transplantation weeks were detected. The mice were sacrificed 6 weeks after transplantation. The human hematopoietic cells (hCD45(+)) and multi-lineage engraftment cells (CD3/CD19, CD33, CD14, CD61, and CD235a) in NOD/SCID recipients bone marrow, spleen, and peripheral blood were analyzed by flow cytometry. RESULTS: In the 3rd post-transplantation week, white blood cells (WBC), platelets (PLT), and red blood cells (RBC) began to increase in both two groups. In the 6th post-transplantation week, WBC and PLT counts in CD34(+) + MSC group reached peak levels and were significantly higher than CD34(+) alone group (P < 0.05), while RBC level was not significantly different between these two groups P > 0.05). hCD45(+) cell levels in bone marrow and peripheral blood were (42.66 +/- 2.57) % and (4.74 +/- 1.02) % in CD34(+) + MSC group, which were significantly higher than those in CD34(+) alone group [(25.27 +/- 1.67) % and (1.19 +/- 0.54) %, respectively, P = 0.006]. Also in the 6th post-transplantation week, the proportions of CD19(+), CD33(+), CD14(+), CD61(+), and CD235a(+) in CD34(+) + MSC group were significantly higher than those in CD34(+) alone group (P < 0.05), while the proportion of CD3(+) T lymphocyte in CD34(+) + MSC group was significantly lower than that in CD34(+) alone group (P = 0.003). The amplification of CD19(+) B lymphocyte was significantly higher than other blood cell lineages (P < 0.05). CONCLUSION: The co-transplantation of MSC cells and CD34(+) cells can promote hematopoietic stem cell transplantation and hematopoietic recovery in vivo.

[Effect of mesenchymal stem cells on multiple myeloma cells growth and inhibition of bortezomib induced cell apoptosis].

OBJECTIVE: To investigate the role of mesenchymal stem cells (BMSCs) in multiple myeloma (MM) bone marrow (BM) microenrivonment and their effect on myeloma cells survival and bortezomib induced apoptosis. METHODS: BMSCs were derived from BM of untreated myeloma patients (MM-BMSCs) and healthy donors (HD-BMSCs), respectively. The phenotype, proliferation time and cytokine secretion of MM-BMSCs were detected and compared with HD-BMSCs. Then BMSCs were co-cultured with myeloma cell line NCI-H929 and bortezomib in vitro. The NCI-H929 cells proliferation and bortezomib induced cell apoptosis were investigated. RESULTS: MM-BMSCs and HD-BMSCs were isolated successfully. The phenotype of MM-BMSCs was similar to that of HD-BMSCs. Expressions of CD73, CD105, CD44 and CD29 were positive, but those of CD31, CD34, CD45 and HLA-DR (< 1%) negative. The proliferation time of MM-BMSCs was longer than that of HD-BMSCs (82 h vs 62 h, P < 0.05). Moreover, over-expressions of IL-6 and VEGF in MM-BMSCs culture supernatant were detected as compared with that in HD-BMSCs [(188.8 ± 9.4) pg/ml vs (115.0 ± 15.1) pg/ml and (1497.2 ± 39.7) pg/ml vs (1329.0 ± 21.1) pg/ml, respectively]. MM- BMSCs supported survival of the myeloma cells NCI-H929 and protected them from bortezomib induced cell apoptosis. CONCLUSIONS: MM-BMSCs is benefit for myeloma cells proliferation and against cell apoptosis induced by bortezomib. Over-expression of IL-6 and VEGF maybe play a critical role in these effects.

[Study of influence of umbilical cord mesenchymal stem cells on CD34+ cells in vivo homing in NOD/SCID].

OBJECTIVE: To investigate the effect and the potential mechanism of umbilical cord (UC) derived mesenchymal stem cells (MSCs) on umbilical cord blood (UCB) derived CD34+ cells in vivo homing in xenotransplanted NOD/SCID mice model. METHODS: CD34+ cells and MSCs were derived from fresh UCB and UC, respectively. CD34+ cells (5 x 10(5) per mice) and MSC cells (5 x 10(6) per mice) were co-transplanted into irradiated NOD/SCID mice intravenously. CD34+ cells (5 x 10(5) per mice) alone were transplanted into the mice as control group. CD34+ cells home in bone marrow and spleen of recipient mice were detected 20 hours after transplant by FACS and RT-PCR, and the homing efficiencies were calculated. The effect of MSCs on CD34+ cells chemotactic function was investigated after co-cultured UCB CD34+ cells with UC MSCs in vitro. After 4 and 7 days coculture, the homing related adhesion molecules (the CD49e, CD31, CD62L, CD11a) expressed on CD34+ cells were detected by FACS. RESULTS: 1) The homing efficiencies in bone marrow in experimental and control group were (7.2 +/- 1.1)% and (5.4 +/- 0.9)%, respectively (P < 0.05). 2) Human GAPDH gene was detected in bone marrow in experimental group and in spleen in both groups. 3) The migration efficiency of CD34+ cells was significantly higher in experimental group (35.7 +/- 5.8)% than in control group (3.5 +/- 0.6)% (P < 0.05). 4) The expression of CD49e, CD31, CD62L on CD34+ cells kept higher level in MSCs cocultured group than in CD34+ cells alone group. CONCLUSIONS: MSCs can efficiently increase homing of CD34+ cells to bone marrow and spleen in vivo by keeping a high level of homing adhesion molecules expression and improving migration efficiency of UCB CD34+ cells.

[Differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood and bone marrow].

The purpose of this study was to explore the differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood (CB) and bone marrow (BM). Mononuclear cells (MNCs) were obtained from CB or BM by Ficoll-Hypaque density gradient separation. CD34+ cells were purified by magnetic cell sorting (MACS). The selected CD34+ cells were seeded in serum-free conditions stimulated with thrombopoietin (TPO), TPO+interleukin 11 (IL-11), or TPO+IL11+heparin for 14 days. Amplification product (CD34+, CD41a+, and CD34+ CD41a+ cells) immunophenotypes, megakaryocyte apoptosis rates and the DNA content were measured by fluorescence-activated cell sorting (FACS). The colony-forming units of granulocytes and monocytes (CFU-GM), burst-forming units of erythrocytes (BFU-E), and colony-forming units of megakaryocytes (CFU-Mk) were also evaluated by the colony-forming units (CFU) assay. The results indicated that CD34+ cells derived from CB showed higher expansion ability of total cell counts, CD41a+ and CD34+ CD41a+ cells than those derived from BM for all days 14 of culture (p<0.05, respectively). There were no significant differences in CFU-GM, BFU-E, and total CFU-Mk counts between CB and BM-derived CD34+ cells on day 0 (p>0.05, respectively), but CB-derived CFU-Mk seemed mainly large colonies, and the number of large colonies was higher than that from BM (p<0.05) on day 0. There were no significant differences in expansion ability of CFU-GM between CB and BM-derived cells on days 7, 10, and 14 of culture (p > 0.05, respectively), but the expansion ability of BFU-E and CFU-Mk derived from CB cells was higher than that from BM (p<0.05, respectively). There were no significant differences in apoptosis rates of megakaryocyte from two source cells for days 14 of culture. Megakaryocytes derived from CB mostly showed the 2N DNA content (>90%) for days 14 of culture, while those cells derived from BM showed the increased DNA content, and 4N, 8N or more ploidy cells gradually increased with prolonging of culture time. It is concluded that CB-derived CD34+ cells have a greater proliferation potential than that derived from BM, which is therefore proven to be a better cell source for megakaryocyte progenitor expansion in vitro.

[The role of stromal cell-derived factor and its receptor-CXCR4 in G-CSF-induced hematopoietic stem cell mobilization].

OBJECTIVE: To explore the role of stromal cell-derived factor (SDF-1) and its specific receptor CXCR4 in the G-CSF-induced hematopoietic stem/progenitor cells (HSPCs) mobilization in human healthy donor. METHODS: The changes of SDF-1/CXCR4 in bone marrow (BM) and peripheral blood (PB) of healthy donors during G-CSF-induced mobilization were detected by enzyme-linked immunosorbent assay (ELISA), immunohistological staining and flow cytometry. SDF-1 neutralizing antibody wes injected into BALB/c mice to further test its effect on mobilization. RESULTS: SDF-1 concentration in mobilized BM (mBM), steady state BM (ssBM) and PB were(7.23 +/- 0.66) microg/L, (5.43 +/- 0.35) microg/L and (5.42 +/- 0.52) microg/L, respectively. SDF-1 protein levels were decreased in the BM (P < 0.05) after 5-day G-CSF injection, and its concentration gradient between BM and PB disappeared (P > 0.05). Significant up-regulation of CXCR4 expression was observed on mBM CD34 cells in healthy donors. The rate of CXCR4 expression on CD34 cells in ssBM, mBM and mobilized PB were (40.98 +/- 21.56)%, (65.80 +/- 24.68)% and (27.54 +/- 26.03)%, respectively. Comparing with that in ssBM and mBM, CXCR4 expression on mobilized PB CD34+ cells were significantly decreased (P < 0.05). Inhibition of SDF-1 signal by blocking monoclonal antibodies significantly reduced G-CSF-induced mobilization in BALB/c mice. This resulted in significant decrease of white blood cell count and progenitors mobilized into peripheral circulation. CONCLUSION: G-CSF induces HSPCs mobilization by decreasing bone marrow SDF-1 and down-regulating CXCR4 expression on HSPCs.

[Effects of stromal cell-derived factor 1 and platelet factor 4 on the adhesion characteristics and chemotactic function of ex vivo expanded umbilical cord blood CD34+ cells].

To investigate the effects of stromal cell-derived factor 1 (SDF-1) and platelet factor 4 (PF4) on the homing-related function of expanded ex vivo umbilical cord blood CD34(+) cells, purified cord blood CD34(+) cells were cultured in serum-free medium containing a HGF combination of FL + SCF + TPO (FST) with either 100 ng/ml SDF-1 alone, 100 ng/ml PF4 alone, or both of these 2 cytokines. The expansion rate of CD34(+) cells, colony formation, homing-related functions including expression of homing-related adhesion molecules of expanded CD34(+) cell, adhesion activity and chemotactic function of the re-selected expanded CD34(+) cells were evaluated at different time points. The results showed that expansion rate of CD34(+) cells and expansion multiple of CFU in SDF-1 groups were higher than those in control. The expression of CD49e on the expanded CD34(+) cells was remarkable up-regulated, in contrast, expression of CXCR-4 on the expanded CD34(+) cells was remarkable down-regulated in SDF-1 groups. The expression of CD49e, CD54 and CXCR-4 on the expanded CD34(+) cells were remarkably up-regulated in the PF4 groups. In all the SDF-1 group, PF4 group and SDF-1 plus PF4 group, the ability of expanded CD34(+) cells adhering to fibronectin layer were higher than those in the control on day 10. Spontaneous migration rate of expanded CD34(+) cells in SDF-1 groups were higher than those in control, while SDF-1-induced migration rate were lower than those in control on day 10. SDF-1-induced migration rate in PF4 groups were higher than those in control on day 10. Spontaneous and SDF-1-induced migration rate of expanded CD34(+) cells in the SDF-1 plus PF4 groups were higher than those in control on day 10. It is concluded that, SDF-1 and PF4 can up-regulate expression of adhesion molecules on expanded CD34(+) cells, and retain the adherent and migration ability of expanded CD34(+) cells, which is helpful for the homing of expanded CD34(+) cells. In short, SDF-1 and PF4 are helpful for the homing-related function of the expanded UCB HSPC.

[Comparison of expanding dendritic cells derived from cord blood and mobilized peripheral blood by two-step culture method].

To compare the expansion efficiency and function of dendritic cells derived from CB-CD34+ cells and MPB-CD34+ cells by using two-step culture method, enriched CB-CD34+ cells or MPB-CD34+ cells with immunoadsorption were primarily cultured in the presence of FL, SCF, TPO, GM-CSF for 10 days, and then further cultured with a combination of GM-CSF, IL-4, TNF-alpha, CD40Ab and PGE2 to induce DC. The DC phenotypes were detected by flow cytometry, the expansion efficiency and cell function were evaluated by mix-lymphocyte reaction (MLR), IL-12 level was detected by using ELISA and the chemotactic function mediated by secondary lymphoid tissue chemokine (SLC) was determined with Transwell plate. The results indicated that after 10 days of expansion, there were no significant difference in the percentage of CD14+CD1a- cells between CB and MPB [(40.48 +/- 16.85)% vs (28.07 +/- 23.19)%, P > 0.05], but the expansion of total cells in CB was higher than that in MPB (388.88 +/- 84.63-fold vs 79.67 +/- 10.32-fold, P < 0.01), so the yield of CD14+CD1a- cells from CB was significantly higher than that from MPB too (189.42 +/- 25.02-fold vs 28.74 +/- 23.27-fold, P < 0.01). The percentage of CD83+ DCs cultured with CD40Ab/PGE2 derived from CB were higher than those cultured with TNF-alpha derived from MPB respectively [(34.52 +/- 11.22)% vs (3.70 +/- 2.27)% and (36.69 +/- 13.36)% vs (7.34 +/- 3.364)% respectively, P < 0.01]. In the same circumstance, the yield of CD83+ DCs derived from CB was much more than that from MPB (198.72 +/- 117.53 times vs 33.95 +/- 6.19 times, P < 0.01). There were no difference in stimulating capacity, IL-12 secretion and migration capacity between DCs derived from CB and MPB. It is concluded that DCs induced from CB-CD34+ cells by two-step culture possess similar functions with that from MPB-CD34+ cells, but the yield of DCs from CB CD34+ cells is much more than that from MPB CD34+ cells.

[The effect of matrix metalloproteinase-9 in granulocyte colony stimulation factor-induced stem cell mobilization].

OBJECTIVE: To investigate the effects of matrix metalloproteinase-9 (MMP-9) on granulocyte colony stimulation factor (G-CSF)-induced hematopoietic stem/progenitor cell (HSPC) mobilization in healthy donors of hematopoietic stem cells. METHODS: Peripheral blood (PB) samples and bone marrow (BM) blood samples were collected from 12 healthy donors of hematopoietic stem cell before and 5 days after G-CSF-induced mobilization. CD34(+) cells were isolated and purified. ELISA was used to detect the protein expression of MMP-9 in the peripheral blood and BM blood of the healthy donors. The protein expression of MMP-9 in the BM blood was detected by ELISA and immunohistochemistry, and the stromal cell-derived factor-1 (SDF-1) level in the BM blood was detected by ELISA. The mRNA expression of MMP-9 in the BM blood samples was detected by RT-PCR. HT1080 cells rich in MMP-9 were cultured. CD34(+) cells were co-cultured with the supernatant of HT1080 cell culture fluid. CD34(+) cells cultured in Iscove's modified Dulbecco's medium were used as control group. Fluorescence-activated cell sorter was used to detect the CXCR4 expression on the surface of the CD34(+) cells. In the transwell experiment CD34(+) cells were divided into 4 groups: control group, o-phenanthroline (MMP-9 chemical inhibitor, MPI) group, HT1080 sup group, and HT1080 + MPI group to be co-cultured with buffer, o-phenanthroline, supernatant of culture fluid of HT1080 cells, or supernatant of culture fluid of HT1080 cells Flow cytometry was used to calculate the cell migration capacity. RESULTS: The MMP-9 level of BM and PB of the healthy donors 5 days after G-CSF mobilization were 278 ng/ml +/- 34 ng/ml and 392 ng/ml +/- 284 ng/ml respectively, both significantly higher than those before G-CSF mobilization (42 ng/ml +/- 17 ng/ml and 27 ng/ml +/- 12 ng/ml respectively (P < 0.01 and P < 0.05). Western blotting showed that the SDF-1 level in the supernatant 5 days after G-CSF mobilization was 5.9 ng/ml +/- 1.0 ng/ml, significantly lower than that before G-CSF mobilization (7.2 ng/ml +/- 0.7 ng/ml, P < 0.05). The CXCR4 levels of the CD34(+) cell from both PB and BM blood were up-regulated after co-culture with the supernatant of HT1080 cells (both P < 0.05). The migration capacity of CD34(+) cells cultured in the supernatant of HT1080 cells was increased significantly (P < 0.05), however, this effect could be inhibited by MIP (P < 0.05). The PB WBC numbers of the G-CSF group and G-CSF + MPI group were 14.9 x 10(6)/L +/- 4.3 x 10(6)/L and 12.3 x 10(6)/L +/- 1.2 x 10(6)/L respectively, the PB WBC numbers of the G-CSF + MPI group was significantly lower than that of the G-CSF group (P < 0.05), however, significantly higher than that of the negative control group (6.8 x 10(6)/L +/- 2.5 x 10(6)/L, P < 0.05). The CFU of the G-CSF group was (84 +/- 10) U/2 x 10(5) MNC, significantly higher than that of the G-CSF + MPI group, (69 +/- 3) U/2 x 10(5) MNC (P < 0.05). The BM MNC number of the G-CSF group was 12.7 x 10(6)/L +/- 0.7 x 10(6)/L, not significantly different from that of the G-CSF + MPI groups (13.1 x 10(6)/L +/- 1.3 x 10(6)/L; P > 0.05). CONCLUSION: MMP-9 probably facilitates HSPC mobilization by degrading SDF-1, up-regulating CXCR4 expression on the CD34(+) cells, and increasing the migration ability of CD34(+) cells.

[The expression and clinical significance of early differentiation antigens in acute leukemia].

OBJECTIVE: To evaluate the expression and clinical significance of early differentiation antigens of hematopoietic cells CD(34), CD(90) and CD(133) in acute leukemia (AL). METHODS: The expression of CD(34), CD(90) and CD(133) on leukemic blasts in 76 AL patients was detected with three-color direct flow-cytometry and CD(133) mRNA was detected with hemi-quantitative reverse transcriptive polymerase chain reaction (RT-PCR). RESULTS: (1) CD(34) and CD(133) expression in AL patients was significantly higher than that in controls (46.37% vs 0.47%, 21.93% vs 0.29%, P < 0.01), but there was no obvious difference of CD(90) expression between them (0.51% vs 0.25%, P > 0.05). The expression of CD(133) antigen was highly correlated with CD(133) mRNA expression in both of the normal control donors and AL patients (r = 0.932, P < 0.01). (2) The positive rates of CD(34), CD(90) and CD(133) in all AL patients were 63.2%, 7.9% and 42.1%, respectively. The expression of CD(90) in ALL was higher than that in AML (P < 0.05). Positive expression of CD(133) in AML-M(4) was significantly higher than that in other AML subtypes (P < 0.01). The positive rate of CD(34) in B-ALL was much higher than that in T-ALL (P < 0.05). (3) CD(133) expression in AML was significantly correlated with the expression of CD(34) and HLA-DR (P < 0.01). (4) The expression of CD(34), CD(90) and CD(133) was not associated with the clinical prognostic factors such as cytogenetic or molecular aberrations, initial peripheral blood WBC counts, lactate dehydrogenase level, multiple drug resistant expression and age. (5) There was a trend toward lower completely remission (CR) rate and overall survival rate in CD(34), CD(90) and CD(133) positive cases, but only CD(34)(+)/CD(133)(+) cases had significant lower CR rate than negative ones (P < 0.05). CONCLUSIONS: It is indicated AL has significantly higher CD(34) and CD(133) expression as compared with the normal controls. CD(133)/CD(34) co-expression might provide adverse prognostic stratification of acute leukemia.

[The expression of CD133 in acute leukemia and its clinical significance].

OBJECTIVE: To evaluate the expression of CD133 and its clinical significance in acute leukemia (AL) patients. METHODS: The expression of CD133 and CD133 mRNA in leukemic blasts from 76 AL patients were detected by three-color flow-cytometry and hemi-quantitative RT-PCR respectively. RESULTS: (1) CD133 mRNA expression was highly correlated with CD133 expression in both of normal donors and AL patients groups. The expression of CD133 in AL patients was significantly higher than that in control group (P < 0.01). (2) The positive rates of CD133 and CD133 mRNA in AL group were 42.1% (32/76) and 46.1% (35/76) respectively. There was no significant difference in CD133 expression between AML-M(3) and normal control, AML and ALL, as well as T-ALL and B-ALL. The expression of CD133 in AML-M(4) were significantly higher than those in other AML subtypes (81.8% vs 43.7% and 81.8% vs 46.9% at CD133 and CD133 mRNA level, respectively, P < 0.01). (3) The expression of CD133 in AML was significantly correlated with the expression of CD34 and HLA-DR (P < 0.001). (4) The expression of CD133 had no relationship with the clinical prognostic factors such as cytogenetic or molecular aberrations, WBC counts, LDH, mdr1 expression and age. (5) There was a trend toward lower CR rate in CD133(+) cases, but only CD34/CD133(+) double positive cases had significant lower CR rate than that of negative ones (44.4% vs 71.4%, P < 0.05). CONCLUSIONS: AL had significantly higher CD133 expression compared to normal control. The detection of CD133 expression might help to identify AL type and predict therapeutic outcomes. Co-expression of CD133/CD34 might convey adverse prognosis of AL.

[Changes of the migration ability of the cord blood CD(34)(+) cells during short-term ex vivo expansion].

OBJECTIVE: To study the effect of ex vivo expansion on the migration ability and the CXCR4 expression of umbilical cord blood (CB) hematopoietic stem/progenitor cells (HSPC). METHODS: CD(34)(+) cells isolated from fresh CB samples were cultured in a serum-free and stroma-free culture system. On day 7, 10 and 14, CD(34)(+) cells were re-selected from the expanded cells, and the expression of CXCR4 and the transmigration ability of these CD(34)(+) cells were evaluated respectively and compared with those of the precultured fresh CD(34)(+) cells. RESULTS: (1) SDF-1 induced a higher migration percentage of fresh or expanded CB CD(34)(+) cells than that of uninduced ones. (2) Both of the uninduced and SDF-1-induced migrations were slightly reduced in the first week and then much more reduced in the second week expansion (P < 0.05). (3) The number of the CD(34)(+)CXCR4(+) cells were significantly increased during the culture period, but there was a downtrend of CXCR4 expression on CD(34)(+) subset; the expression levels on day 10 and 14 were lower than that on day 0. CONCLUSIONS: The expanded HSPC would sustain the chemotactic activity during one-week-culture, but with further extended culture time their intrinsic homing potential would be partly impaired.

[Preliminary study on extensive amplification of human dendritic cells differentiated from cord blood CD34+ progenitor cells by two-step culture].

OBJECTIVE: To Explore a two-step culture system to generate a large number of dendritic cells (DC) differentiated from cord blood (CB) CD(34)(+) cells. METHODS: Enriched CB CD(34)(+) cells with immunoadsorption were primarily cultured in the presence of stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (Tpo) and interleukin-3 (IL-3) for 7 (group I), 10 (group II) or 14 days (group III) respectively, and then further cultured with GM-CSF, IL-4 and TNF-alpha for 5 - 8 days to induce DC. The expansion and cell function were evaluated by flow cytometry (FCM) and mix-lymphocyte reaction (MLR), and detection of IL-12 in the supernatant by using ELISA. RESULTS: The total nucleated cells with 53.39 +/- 20.59-, 307.17 +/- 119.59- and 1117.25 +/- 335.49-folds expansion could be respectively obtained after 7 - 14 days of expansion culture. After DC induction, CD(1a)(+) cells were 21.40 +/- 16.70-, 143.2 +/- 60.35- and 150.8 +/- 42.16-fold increase as compared to the initial nucleated cells. Comparing with that in group I, the CD(1a)(+) cells were much more in groups II and III; but there was no difference between the latter two groups (P > 0.05). The cultured cells in the three groups showed almost the same allo-stimulatory capability and IL-12 excretion when the second culture duration maintained 8 days, while the capability and excretion were greatly decreased when the duration shortened to 5 days (P < 0.05). CONCLUSION: A plenty of functionally mature DC could be obtained from the CD(34)(+) cells in the two-step culture system of 7 - 10 days HSC expansion followed by 8 days DC induction.

Short-term ex vivo expansion sustains the homing-related properties of umbilical cord blood hematopoietic stem and progenitor cells.

BACKGROUND AND OBJECTIVES: The homing of stem cells to the bone marrow microenvironment following transplantation is a specific movement eventually leading to the stem cells lodging in specialized niches of hematopoiesis. The present study was designed to develop an ex vivo expansion system capable of preserving the homing potential of hematopoietic stem/progenitor cells (HSPC). DESIGN AND METHODS: Umbilical cord blood (UCB) CD34+ cells were expanded in QBSF-60 serum-free medium with a simple early-acting combination of cytokines and were re-selected from the expanded products at different time points. The homing-related characteristics and expansion rate of CD34+ cells were simultaneously examined. RESULTS: It was observed that the number of HSPC increased significantly under our expansion protocol. The expression of CD49d, CD44, CD11a and CD49e on expanded CD34+ cells increased or remained at the same levels as those on freshly isolated CD34+ UCB cells, while the expression of CD54 on expanded CD34+ cells was lower during the second week of culture than at the start. The spontaneous and SDF-1-induced adhesion of CD34+ cells was increased during the first 10 days of culture, with the adhesion rates reaching peak levels (62.8 12.8% and 90.5 11.7% for spontaneous and induced adhesion, respectively) on day 10. Neither spontaneous nor SDF-1-induced migration had changed significantly by day 7. INTERPRETATION AND CONCLUSIONS: These data demonstrate that, although ex vivo expansion may alter cell properties, our one-week expansion protocol can preserve most of the homing-related characteristics and activities of UCB HSPC.

[The effects of the short-term ex vivo expansion on the adhesion characteristics and chemotactic function of expanded ex vivo CD34(+) cells].

OBJECTIVE: To study the effect of ex vivo expansion on the adhesion activities and chemotactic function of umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPCs). METHODS: CD34(+) cells isolated from fresh UCB samples were cultured in serum-free and stroma-free culture system. After 7, 10 and 14 days' culture, CD34(+) cells were re-selected from the expanded products. Stromal cell- derived factor-1 (SDF-1) 100 ng/ml was added into the experimental CD34(+) cells and the absorbance at 570 nm of all groups was examined. 20 micro g/ml fibronectin (Fn) was added and the spontaneous adhesion between CD34(+) and FN was detected by MTT method. The homing-related functions including expression of homing-related adhesion molecules (CAM), adhesion activity and chemotactic function of the re-selected CD34(+) cells were evaluated and compared with those of the initial fresh CD34(+) cells. RESULTS: (1) The expression of CD49d, CD44, CD11a and CD49e on expanded CD34(+) cells increased or sustained the same levels as those of the fresh isolated UCB CD34(+) cells, while the expression of CD62L, CD54 and CD31 on expanded CD34(+) cells declined during the culture. (2) The spontaneous adhesion between CD34(+) and FN and SDF-1-induced adhesion continuously increased in the course of the first 10-day culture. The spontaneous adhesion rate and SDF-1-induced adhesion rate on day 0, day 7 and day 10 were 28% and 63%, 60% and 70%, 63% and 90% respectively. (3) The migration efficiency of re-selected CD34(+) cells on day 7 was almost the same compared to that of fresh CD34(+) cells. CONCLUSION: The expanded HSPCs sustain most of the homing-related characteristics and activities during one-week culture while extended culture may partly impair their intrinsic homing potential.

[Studies on the homing-related adhesion activities of UCB HSPC in short-term ex vivo expansion].

OBJECTIVE: To study the effect of ex vivo expansion on the adhesion activities of umbilical cord blood hematopoietic stem and progenitor cells (HSPC). METHODS: Fresh UCB CD(34)(+) cells were cultured in a serum and stroma-free culture system. At day 7, day 10 and day 14, CD(34)(+) cells were re-selected from the expanded products. The expression of adhesion molecules (CAMs) such as VLA-4, VLA-5, LFA-1, ICAM-1, HCAM, L-selectin and PECAM-1, and the adhesion activity of the expanded CD(34)(+) cells were evaluated and compared with those of precultured fresh CD(34)(+) cells. RESULTS: (1) The CD(34)(+) cells expressing homing-related CAMs were increased (from 15-fold increase for CD(34)(+) CD(54)(+) subset to 72-fold increase for CD(34)(+) CD(49e)(+) subset at day 14). (2) The expressions of CD(49d), CD(44), CD(11a) and CD(49e) on the expanded CD(34)(+) cells were increased or sustained the same levels as those on fresh UCB CD(34)(+) cells, while the expression of CD(62L), CD(54) and CD(31) on expanded CD(34)(+) cells declined with the cultivating. (3) Spontaneous adhesion and SDF-1-induced adhesion tended to be increased in the course of the first 10 day's culture. CONCLUSIONS: The culture system used in this study could substantially support the expansion of HSPCs expressing the above CAMs, and the expanded HSPCs would sustain their intrinsic adhesion potentials.