| Hematopoietic stem cells (HSCs) are generally defined as cells having the self-renewing potential and the capacity to give rise to differentiated cells of all hematopoietic lineages. Hematopoietic stem cell transplantation (HSCT) is performed for complete healing of hematologic disorders and as a supportive therapy after high-dose chemotherapy against malignant diseases. HSCs can be collected from peripheral blood (PB), bone marrow (BM), and umbilical cord blood (UCB). Human UCB is thought to be an attractive alternative to BM or growth factor mobilized PB as a source of hematopoietic progenitors, because UCB contains a high number of primitive hematopoietic cells. The frequency of graft-versus-host disease (GVHD), which is a severe side effect of HSCT in patients, is reduced among patients receiving transplants from UCB. However, the total number of UCB HSCs harvested from one donor's UCB may be enough hematopoietic stem cells to reconstitute children, but it is not sufficient for HSCT in an adult patient. To solve this problem, it is necessary to expand the HSCs from UCB in vitro. In the present study, we attempted to introduce the thrombopoietin (THPO) and Flt-3 ligand (FL) extramembranous domain gene into human bone marrow-derived mesenchymal stem cells (hMSCs) and to assess the supportive effects of the transduced hMSCs on ex vivo expansion of HSCs from UCB in synergy with extra cytokines.hMSCs, derived from mesodermal cells, have actively self-proliferative and multi-differentiative capacities into various cell types. hMSCs were isolated successfully from human bone marrow and their growth characteristics were also investigated in this report. The hMSCs were adherent and fibroblast-like, and maintained similar morphology over serial passages. The time of primary cultures was approximately 14d. Analysis of growth kinetics indicated that growth curve of the hMSCs was classical, showing "S" type, and the hMSCs demonstrated a strong proliferative capacity after passaging. The time of each passage culture among the passage-2 to passage-20 hMSCs was about 6d, whereas among the passage-20 to passage-30 hMSCs, the time of each passage culture was about 9d. In addition, population doubling time (PDT) varied from 30h to 59h during the 200-days' culture period. Importantly, PDT inceaesed slightly from 30h to 40h during the 125-days' culture period, however, among the later 75d, PDT inceaesed sharply from 40h to 59h. Cell cycle status analysis by measuring DNA content, revealedthat 81.5% of the hMSCs were in Go/G| phase, showing classical characteristics of stem cells. Flow cytometric analysis indicated that the hMSCs were universally positive for CD 166, CD29 and CD44, and negative for CD45, CD34 and HLA-DR.THPO and FL was two early-acting cytokines during hematopoiesis. THPO and FL extramembranous domain gene were cloned successfully from human fetal hepatocyte RNA with abundant THPO and FL mRNAs using reverse transcription-polymerase chain rection (RT-PCR) technique. Sequence analysis demonstrated that both THPO and FL extramembranous domain gene were consistent with the sequences from Genebank data, except that signal peptide sequence of THPO gene produced a base pair mutation. In order to introduce THPO and FL extramembranous domain gene into the hMSCs, Bicistronic retroviral vector pLXPFIT was constructed by internal ribosome entry site (IRES). Next, the hMSCs was transfected with pLXPFIT retroviral vector using a combined method (prestimulation+centrifugation+ multi-cycle transfection). RT-PCR and western blotting analysis demonstrated that THPO and FL extramembranous domain mRNAs and proteins levels in the transduced hMSCs were significantly higher than that in the primary hMSCs. And enzyme linked immunosorbent assay (ELISA) showed that secretive expressions of THPO and FL extramembranous domain proteins in the transduced hMSCs were stable and effective during 8-passages' culture period. In addition, flow cytometric analysis indicated that EGFP fluorescence expression in the EGFP-transduced hMSCs did not decreased during 12-passages' culture period. These results demonstrated that THPO and FL extramembranous domain genes had been transduced successfully into the hMSCs and also, THPO and FL extramembranous domain proteins had been effectively expressed.Next, the supportive effects of the transduced hMSCs on ex vivo expansion of primitive hematopoietic cells from UCB were investigated. The results showed that the outputs of total number of nucleated cells, CD34+ cells, CFU-Cs and CFU-GEMM in the transduced hMSCs coculture system were 910-, 121-, 62- and 90-fold respectively during the first serum-containing culture period (2wk), which were significantly higher than that in other control groups. Although the outputs of total number of nucleated cells, CD34+ cells and CFU-Cs during the first period (2wk) under serum-free condition decreased slightly, expansion of CFU-GEMM in both serum-containing and serum-free system had the similar results. In order to determine whether the transduced hMSCs could support long-term ex vivo expansion of cobblestone area-forming cell(CAFC) from UCB, a second culture period (6wk) hematopoietic expansion experiment was performed. The results showed that the number of hematopoietic cells (HPCs) in the coculture with the primary hMSCs decreased through 6 weeks. The HPCs expansion on TPO/FL-transduced hMSCs showed a tendency similar to that on primary hMSCs up to 4 weeks. However, as TPO/FL-transduced hMSCs were able to survive longer than the primary hMSCs (up to 6wk), the number of HPCs expanded on TPO/FL-transduced hMSCs was significantly higher than that on primary hMSCs during the fourth, fifth, and sixth weeks.Finally, the effects of the transduced hMSCs on ex vivo expansion of more primitive HSCs from UCB were investigated. Ex vivo expansion of long-term culture-initiating cells (LTC-ICs) and SCID-repopulating cells (SRCs) from UCB was determined. Firstly, the LTC-ICs frequency of HPCs from different culture system was determined by limiting dilution assay. The results demonstrated that the outputs of LTC-ICs in the transduced hMSCs during 2-weeks' and 4-weeks' culture period increased by 10- and 25-fold respectively, significantly higher than that in other culture system. Secondly, SRC assay was performed to confirm whether SRCs from HPCs had been expanded. The results demonstrated that the percentage of human CD45+ cells in the BM mononuclear cells of NOD/SCID mice transplanted with HPCs from the transduced hMSCs system was 54-60%, suggesting that SRC had been expanded effectively. Finally, the multilineage surface markers of these human CD45+ cells were determined by flow cytometric analysis. The results showed that glycophorin A, CD33, CD14, CD41, CD34 and CD19 markers were positive, suggesting that SRC from the HPCs produced by the transduced hMSCs coculture sysyem had differentiated into most of the blood cells.In conclusions, a novel THPO/FL-transduced hMSCs had been developed successfully, and these cells could effectively support ex vivo expansion of CFU-GEMM, LTC-ICs and SRCs (more primitive HSCs) from UCB CD34+ cells in synergy with extra cytokines. The THPO/FL-transduced hMSCs coculture system here we had developed potentially has many important applications in the field of clinical cord blood HSCT.
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