Ex vivo expansion of hematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. In this study, we investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis on prolonged maintenance and expansion of cord blood CD34+ cells. Enriched CD34+ cells (1 x 105/mL, n=5) or mononuclear cells (1 x 106/mL, n=8) were cultured in X-VIVO-10 medium for 14, 21, 28 and 35 days without supplementary cytokine or medium changing. Our results showed that the presence of NTL (200 ng/mL) or FL-3 ligand (FL, 40 ng/mL) significantly preserved populations of early stem/progenitor cells (total CFU, BFU/CFU-E, CFU-GM, CFU-GEMM) in these cultures, compared with respective controls at various time points. In the ex vivo expansion study (n=16), the presence of stem cell factor (S, 50 ng/mL), thrombopoietin (T, 50 ng/mL), FL (F, 80 ng/mL) effectively expanded total nucleated cells (TNC) at day 8 (116 ± 20.2 fold) and day 12 (424 ± 68.8 fold), as well as all subsets of progenitor cells as demonstrated by flow cytometry and CFU assays. The presence of NTL (200 ng/mL) significantly increased TNC (148 ± 24.5 fold at day 8; 572 ± 91.9 fold at day 12; P < 0.01) and expansion of early progenitor cells (CD34+, CD34+CD38−, CFU-GEMM) and committed CFU of the myeloid (CFU-GM), erythroid (BFU/CFU-E) and the megakaryocytic lineage (CFU-MK) (P < 0.01 compared with respective TSF cultures). There was also slight but consistent increase of CD61+CD41+ cells in the presence of NTL (8.58 ± 2.14 x 105 vs. 7.30 ± 1.82 x 105 cells/mL, P < 0.001). Significantly, the increased expansion was not only contributed by the higher TNC, but also by the increase in the proportion of CD34+ cells, CD34+CD38− cells and the density of differential CFU. Six weeks after enriched CD34+ cells at day 0 or expanded cells at day 12 were infused into sub-lethally irradiated NOD/SCID mice, human CD45+ cells were detectable in the BM, spleen and PB of the mice. In the BM, there were engraftments of human hematopoietic cells of the early (CD34+), myeloid (CD33+, CD14+), B-lymphoid (CD19+) and megakaryocytic (CD61+) lineages. In animals that received day 12 expanded cells in the TSF + NTL group, there was a significant increase of human CD45+ cells in the BM (19.3% vs. 11.5%, P = 0.03, n = 15) when compared with those only exposed to TSF, and a trend of increased engraftment in their spleen (P = 0.07, n = 14). Comparison of the complete amino acid sequences of NTL and FRIL (a dicot mannose-binding lectin shown to preserve hematopoietic stem cells, PNAS, 96, 646–650, 1999) showed 10.2% identity and both peptides contain putative functional/structural sites such as those for N-myristoylation, casein kinase II phosphorylation, protein kinase C phosphorylation and N-glycosylation. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34+ cells for transplantation.
The use of combination of Chinese herbs as a treatment for thrombocytopenia has been reported to be effective and safe. We have reported that Angelica Polysaccharide (extracts of Angelica Sinensis) has promoting effect on blood stem cells and megakaryocytopoiesis (Yang et al, Blood, 2002, 100 (11); 53a). Sanqi, Radix Notoginseng, is the dried roots of Panax notoginseng (Burk.) F. H. Chen (Araliaceae). It has been used for treatment of trauma and bleeding due to internal and external injuries. Its main constituents are ginsenosides (a kind of saponins), as well as notoginsenosides (only rich in Notoginseng species). Although Sanqi is a well-known haemostatic drug, its effects and mechanisms on megakaryocyte/platelet production have not been studied. The objective of this study was to compare the effect of a purified notoginsenoside R1 (NR1) and thrombopoietin (TPO) on thrombopoiesis in irradiated mice. NR1 (2.5 mg) and TPO (0.25 ug) were dissolved in distilled water and given by intra-peritoneal injection daily for 14 days starting from the day after radiotherapy. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from NR1, TPO, and vehicle control groups on day 0, 7 and 14. On day 14, the mice were sacrificed and bone marrow cells were harvested for CFU-MK, CFU-GM, BFU-E and CFU-F (fibroblastoid) assays (n=5). Our results showed that NR1 enhanced the recovery of platelets, WBC, and RBC count. Moreover, NR1 also promoted the CFU-F (12 ± 0.7 vs 19 ± 0.38 colonies/2 x 106 cells, p=0.0034), CFU-MK (22 ± 1.9 vs 26 ± 3.8 colonies/2 x 105 cells, p=0.025), CFU-GM (26 ± 5.2 vs 37 ± 4.3 colonies/2 x 105 cells, p=0.002), and BFU-E (13 ± 2.9 vs 18 ± 1.9 colonies/2 x 105 cells, p=0.003) formation. Similar results were obtained in TPO-treated group. In in-vitro study, we further analyzed the effect of NR1 (0–50mM) on mouse CFU-MK formation using a plasma clot colony assay. The results showed that NR1 (20 mM) enhanced TPO (50 ng/ml)-induced CFU-MK formation (19 ± 2.2 vs 30 ± 6.8 colonies/2 x 105 cells, p=0.02, n=5). Furthermore, the effect of NR1 (5–50 mM) on the growth of bone marrow stromal cells was also investigated using CFU-F assay. NR1 (50mM) had a promoting effect on CFU-F growth (18 ± 3.7 vs 24 ± 1.8 colonies/2 x 106 cells, p=0.043, n=5). Our studies showed that NR1 enhances thrombopoiesis in vivo and the growth of bone marrow stromal cells as well as megakaryocytes in vitro. Therefore, we speculate that the thrombopoietic activity of NR1 may be mediated via promoting the progenitor of platelet, megakaryocytes, and bone marrow stromal cells. Although TPO has been used as an agent for the recovery of platelet production after the onset of thrombocytopenia, long-term clinical usage of TPO may induce potential side effects such as thrombosis. Here we reported that the effect of NR1 is comparable with TPO on the production of platelets in irradiated mice.
To date, there is no ideal treatment for thrombocytopenia. We have proposed a possible mechanism of serotonin (5-HT) on megakaryocyte (MK) differentiation and platelet formation (Yang et al, Blood, 2003 suppl). The root of polygonum multiflorum thunb (Heshouwu) is an important ingredient of many commonly used prescriptions in Chinese medicine for promoting blood production. Polygonum multiflorum extracts (PME) also inhibit monoamine oxidase (MAO) and increase the level of 5-HT. Therefore, we hypothesize that polygonum multiflorum may have a promoting effect on thrombopoiesis via inhibition of monoamine oxidase (MAO) to increase 5-HT levels. The objective of this study was to investigate the hematopoietic role of PME in irradiated mice. PME (125 mg/kg/day) and TPO (12.5 ug/kg/day) were given by intra-peritoneal injection daily for 21 days starting from the day after irradiation (4 Gy). Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from PME, TPO, and vehicle control groups on day 0, 7, 14 and 21. On day 21, the mice were sacrificed and bone marrow cells were harvested for CFU-MK, CFU-GM, BFU-E, CFU-GEMM and CFU-F (fibroblastoid) assays (n=8). We also investigated the in vitro effect of PME on CFU-F formation. Our results showed that PME enhanced the recovery of platelets, WBC, and RBC counts. Moreover, PME also promoted CFU-MK (30 ± 8 vs 15 ± 3 colonies/2 x 105 cells, p<0.01), CFU-GM (38 ± 7 vs 28 ± 7 colonies/2 x 105 cells, p<0.05), BFU-E (19 ± 3 vs 12 ± 4 colonies/2 x 105 cells, p<0.05), and CFU-F formation (36 ± 11 vs 23 ± 7 colonies/2 x 106 cells, p<0.01). Similar results were obtained in TPO-treated group. In in-vitro study, we further analyzed the effect of PME (0–500 ug/ml) on mouse CFU-F formation. The results showed that PME at 100–500 ug/ml significantly enhanced CFU-F formation (p<0.05, n=6). Our studies showed that PME enhances thrombopoiesis in vivo and the growth of bone marrow stromal cells in vitro. Therefore, we speculate that the thrombopoietic activity of PME may be mediated via promoting the bone marrow stromal cells. Although TPO has been effective as an agent for the recovery of platelet production after the onset of thrombocytopenia, long-term clinical usage of TPO may induce potential side effects such as thrombosis. Here we reported that the effect of PME is comparable with that of TPO on hematopoiesis and the production of platelets.
SDF-1 is the ligand to the chemokine receptor CXCR-4. A small synthetic peptide agonist of SDF-1 (CTCE-0214) has been shown to expand human cord blood hematopoietic stem and progenitor cells. In this study, we investigated whether a brief exposure of expanded cord blood hematopoietic cells to CTCE-0214 can improve engraftment of the cells into NOD/SCID mice. Published in vivo studies demonstrated that the administration of CTCE-0214 to transplanted NOD/SCID mice mobilized human colony forming cells (CFC) and enhanced human thrombopoiesis (Exp Hematol 32, 300, 2004). Our earlier study showed that CTCE-0214 added to single factors of thrombopoietin (TPO), stem cell factor (SCF), or Flt-3 ligand (F3L) synergistically increased the survival of enriched cord blood CD34+ cells (Blood 102, 960a, 2003). In this study, we further investigated the effects of CTCE-0214 on the ex vivo expansion of CD34+ cells to multi-lineage progenitors and the homing and engraftment capacity of expanded human progenitor cells after a short in vitro exposure to the peptide prior to infusion into NOD/SCID mice. Enriched CD34+ cells (MACS) derived from cord blood were cultured for 8 days in serum-free medium QBSF-60 containing TPO (50 ng/ml), SCF (50 ng/nl) and F3L (80 ng/ml) (TSF), with or without CTCE-0214 (0.01 ng/ml) (TSF+CTCE-0214) added at day 4. Progenitor cells expanded for 8 days in the absence of CTCE-0214 were pulsed with the peptide (100 ng/ml) for 4 hours (TSFpCTCE-0214). Results are summarized in Table. CTCE-0214 significantly (N=30, p≤0.05, paired t-test) increased the fold expansion of total nucleated cells (TNC), CD34+ cells, CD34+CD38- cells, CFU-GM, CFU-E, and CFU-MK (total CFC). Expanded progenitor cells (with and without CTCE-0214) were then infused into irradiated NOD/SCID mice. After 6 weeks, enhanced engraftments of human CD45+ cells (p≤0.05, N=21) were demonstrated in the bone marrow (BM) of mice that received cells cultured in TSF+CTCE-0214. Interestingly, a short pulse of cells expanded in TSF to CTCE-0214 for 4 hours also significantly increased the NOD/SCID engraftment (N=18), although no major changes to the in vitro read-out parameters were observed. The mechanism could be associated with the increased homing capacity of progenitor cells after pulsing with CTCE-0214. In conclusion, our results showed that CTCE-0214 enhances the proliferation of early progenitor cells in culture and exposure to the peptide can enhance the engraftment potential of expanded cells in NOD/SCID mice. The SDF-1 peptide agonist could be developed for application to hematopoietic stem cell transplantation and ex vivo expansion. NOD/SCID Engraftment of Expanded Cord Blood Stem Cells TSF TSF+CTCE-0214 TSFpCTCE-0214 *Fold expansion (mean±SE); **% human CD45+ cells in BM of mice TNC* 84.6±10.4 123.5±15.3 88.5±11.2 CD34+* 8.5±1.3 14.1±2.1 9.6±1.6 CD34+CD38−* 24.6±4.8 48.7±8.6 27.5±5.3 Total CFC* 46.9±6.5 87.9±10.7 50.6±6.4 NOD/SCID** 2.8±0.9 6.7±2.5 8.3±4.0
Despite progress in the development of effective treatments against T-cell acute lymphoblastic leukemia (T-ALL), about 20% of patients still exhibit poor response to the current chemotherapeutic regimens and the cause of treatment failure in these patients remains largely unknown. In this study, we aimed at finding mechanisms that drive T-ALL cells resistant to chemotherapeutic agents. By screening etoposide sensitivity of a panel of T-ALL cell lines using DNA content and PARP cleavage as apoptosis markers, we identified an apoptosis-resistant cell line, Sup-T1. Western blot analysis and caspase activity assay showed that Sup-T1 cells were deficient in etoposide-induced activation of caspase-3 and caspase-9. In addition, mitochondrial cytochrome c release was not evident in etoposide-treated Sup-T1 cells. However, addition of exogenous cytochrome c in cell-free apoptosis reactions induced prominent caspase-3 activation, indicating that the chemoresistance observed in Sup-T1 cells was due to its insusceptibility to the drug-induced mitochondrial alterations. Analysis of the basal expression of the Bcl-2 family proteins revealed that the levels of Bcl-2 was higher in Sup-T1 cells, while Bax and BimEL levels were lower, when compared to etoposide-sensitive T-ALL cell lines. Gene silencing using antisense oligonucleotide to Bcl-2 and overexpression of Bax did not resensitize cells to etoposide-induced apoptosis. On the contrary, transient transfection of BimEL into Sup-T1 cells significantly restored etoposide sensitivity. Further experiments revealed that the lack of BimEL expression in Sup-T1 cells was due to the rapid degradation of newly-synthesized BimEL by the proteosomal pathway, as treatment of Sup-T1 cells with a proteosome inhibitor significantly restored the protein level of BimEL. Moreover, treatment with proteosome inhibitor resulted in mobility shift of BimEL, which was sensitive to phosphatase digestion. Furthermore, treatment of Sup-T1 cells with JNK inhibitor resulted in accumulation of BimEL, and pretreatment with JNK inhibitor restored sensitivity of Sup-T1 cells to etoposide-induced apoptosis, indicating that constitutive activation of the JNK pathway in Sup-T1 cells was responsible for promoting BimEL phosphorylation, and this may serve as a signal targeting BimEL to the proteosome for degradation. Altogether, our findings provide the first evidence that JNK activation correlates inversely with BimEL level by promoting its phosphorylation and degradation. This, in turn, reduces the sensitivity of T-ALL cells to chemotherapeutic agents.
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