Research On The Biological Characteristics And In-Vivo Function Of Human Cord Blood-Derived Megakaryocytes Expanded In Vitro

OBJECTIVE: Hematopoietic stem cell (HSC) transplantation is now a well-established therapeutic modality for broadening spectrum of medical problems. Umbilical cord blood (CB) has emerged as an alternative source of HSC transplantation in recent years due to the lack of a human leukocyte antigen (HLA) -identical sibling donor in the majority of candidates. However, two major limitations hamper the widespread use of CB as a source of hematopoietic stem cells for marrow replacement. First, it contains a finite number of hematopoietic stem and progenitor cells which may be not enough for the hematopoietic rescue of large recipients undergoing transplantation, as well as pantients with diseases knouwn to be resistant to engraftment, such as Fanconi anemia, chronic myelogenous leukemia, and severe aplastic anemia. Second, in comparison with bone marrow (BM) or peripheral blood (PB), CB transplantation is characterized by a delayed engraftment and , in particular, a very slow platelet recovery. A strategy currently under investigation to reduce this prolonged period of thrombocytopemia is the simultaneous graft of CB stem cells and ex vivo-expanded HSC and /or megakaryocyte (Mk) progenitors. The CB contains a high number of primitive progenitor cells with gteater in vitro expansion ability and in vivo engraftment capability when compared with other sources. Furthermore, the reduced alloreactivity of cord blood cells contributes to the lower risk of graft-vs-host-disease (GVHD) in patients transplanted with CB when compared to those transplanted with allogeneic adult BM.Several growth factor combinations have been tested to identify culture conditions that can support a large expansion of primitive repopulating stem cells.CB CD34⁺ can be expanded in stroma-free cultures containing F1t-3 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), interleukin-3(IL-3) and so on. This cytokine combination amplifies CB progeitors and precursors of all hematopoietic lineages, without a concomitant loss but rather an increase, of the in vivo repopulating ability of primitive stem cells. Neverthless, it has not been established so far whether CB CD34⁺ cells, after several weeks of expansion in this culture system, still retain not only the same in vivo repopulation ability, but also the same proliferation and differentiation potential towards the Mk lineage as the unmanipulated CD34⁺ CB cells. Only a few studies have shown that primitive non-obese diabetic severe combined immunodeficient (NOD/SCID) mouse repopulating stem cells from CB can be expanding after in vitro culture.In CB transplants, the Mk lineage takes the longest time to engraft. However, to date, if only a few experimental studies have addressed the issue of the short-term engragtment ability of fresh CB stem cells, even fewer have addressed that of ex-vivo expanded stem cells. Using the NOD/SCID mouse model, the short-term repopulation ability and the differentiation and maturation potiential of human hematopoietic lineages in and ex vivo experimental model can be analyzed.In this study CD34⁺ cells were obtained by immunomagnetic beads methods and cultured in serum-free and stroma-free medium containing the following three different cytokine combinations: TPO+SCF+IL-3+IL-6, TPO+SCF+IL-3 and TPO+SCF. The cells of the 3^rd, 7^th, 10^th and 14^th day were collected and then the expression of cell surface molecules was examined by fluorescence microscope. Flow cytometer (FCM) was performed to examine cells apoptosis. The maturation evaluation of Mk ploidy and colony forming unit- megakaryocyte (CFU-Mk) were also carried. At last the change of their specific gene expression was tested by real time fluorescent quantitation PCR. The collecting cells were then transplanted into NOD/SCID mice.METHODS: 1. CD34⁺ cell purification and ex vivo expansion CB samples were obtained by draining the cord blood into sterile collection tubes containing the anticoagulant citrate-phosphate dextrose with written informed consent. Mononuclear cells (MNC) were isolated from CB using Percol density centrifugation within 4-6 hours. The CD34⁺ fraction was isolated with superparamagnetic microbead selection using a high-gradient magnetic field and a miniMACS column. Five hundred thousand CD34⁺ cells were seeded in serum-free medium with the following cytokine combinations: 1) TPO + SCF + IL-3 + IL-6; 2) TPO + SCF + IL-3; 3) TPO + SCF.2. Megakaryocyte characterization1) Megakaryocyte immunophenotype The cells of the 3^rd, 7^th, 10^th and 14^th day were collected, the expression of Mk and adherence facter cells surface molecules CD42b, CD41a., CD61 and CD49d, CD49e, CD 11 a, CD54 was examined by fluorescence microscope.2) Megakaryocyte apoptotic Cultured cells were treated with Annexin V/FITC and propidium iodide (PI), then analyzed on a FACS machine.3) Colony forming unit- Megakaryocyte (CFU-Mk) assay Cultured cells were seeded in human methylcellulose medium at 37℃in a fully humidified atmosphere at 5% CO₂. Colony scoring was performed by microscope.4) Megakaryocyte ploidy Culture cells were incubated with propidium iodide to stain the DNA in a solution containing RNase, and analyzed CD41 on a FACS machine.5) Megakaryocyte specific gene expression The cDNA of cultured cells were synthezed by using RevertAid^TM First Strand cDNA Synthesis kit, and the specific gene expression was detected by real time fluorescent quantitation PCR.3. In vivo function1) Injection of cells in NOD/SCID mice 16 NOD/SCID mice were separated in 4 groups and handled under sterile conditions and maintained in cage microislators. Sublethally irradiated (350 cGy of total body irradiation )6- to 8-weeks old mice were injected through the tail vein with cells as following: First group, cultured 7-10 days cells induced by TPO+SCF+IL-3+IL-6 combinations; Second group, cultured 7-10 days cells induced by TPO+SCF combinations; Third group, unexpanded CD34⁺ cells; Forth group, the same volum normal sodium.2) Analysis of mice BM The bone marrow cells were obtained from mice femoral bone and tibia bone. Fluorescence microscope was used to analyzed the levels of human cells in the BM of the mice.3) Human platelet detection in NOD/SCID mouse peripheral blood Platelet appearancing in murine PB after injection was also assessed by Fluorescence microscope.RESULTS: 1. In the first part of this work we studied the effect of various cytokine combination on the differentiation of CD34⁺.The results demonstrated that CB CD34⁺ cells produced the largest amount of CD41⁺, CD42b⁺ and CD61⁺ cells after 10 and 14 days of cultured in the present of TPO + SCF + IL-3 + IL-6, TPO + SCF + IL-3 and TPO + SCF. Maxmum production of less mature progenitors (CD34⁺CD41⁺ cells) was obtained at a much earlier time point (day 7) with TPO + SCF + IL-3 + IL-6. The expression of CD49d⁺, CD11a⁺ and CD49e⁺ cells reached high level at day 14, however CD54⁺ cells declined with the time of culture. Ploidy analysis shows that TPO + SCF + IL-3 + IL-6 indued formation of 16N and 32N cells of CD34⁺. Maximum production of Mk obtained with TPO + SCF + IL-3 + IL-6 combinations. It was confirmed by the analysis Mk apoptosis a, CFU-Mk assay and real time fluorescent quantitation PCR.2. The expanded Mks can be engrafted the NOD/SCID mice bone marrow after 1 weeks , the expression of CD45⁺ cells was (5.47±1.76)%,(6.63±0.57)% respectively. The percentage of CD41⁺ cells was (1.70±0.17)%,(2.21±0.31) % respectively. Human platelet in peripheral blood of the mice can be detected after 3 days, and reached the highest level after 7 and 14 days respectively.CONCLUSION: CD34⁺ cells of unbilical cord blood have the potential ability to differentiate into mature Mks ex vivo, maximum production of mature Mks were obtained at 7-10 day in TPO + SCF + IL-3 + IL-6 combinations. By means of serial transplants we found that ex-vivo expanded cells are capable of sustaining long-time Mk engragment. Short-time engraftment of ex-vivo expanded cells seems even more efficient than unmanipulated cells.