The Time Effect Of PCNA-ASODN On Ex Vivo Expansion And Differentiation Of Cord Blood CD34～+ Hematopoietic Precursor Cells

Backgroud and Objective: Human cord blood is a rich source of hematopotic stem/progenitorcells. It has been proved to be a valuable alternative source of hematopoietic precursor cellsbesides bone marrow and peripheral blood. Till now cord blood transplantation has been widelyused in treating adolescent and adult haematological malignancies and marrow failure syndromes.However, one of the main limiting factors for extensive clinical use of cord blood allogeneictransplantation in adult and adolescent is the lower infused nucleated cell and CD34⁺ cell dosethat can be collected from umbilical cord blood (UCB). Ex vivo expansion of UCBhematopoietic precursor cells is developing very fast, might help to overcome this limitation.Hematopoietic precursor cells from cord blood have a large proliferative capacity on thestimulation of cytokines, but also become more mature and lose self-renew activity, and itsability of the long-term hematopoietic reconstitution has been affected. Therefore, how to expandthe number of hematopoietic precursor cells while remaining its ability of self-renew andmultilineage differentiation potential as more as possible has become a current research hot spot.In recent years, many studies have been carried out establishing a combination cytokines ormodeling bone marrow hematopoietic microenvironment in vitro that could allow ex vivoexpansion of the more primitive hematopoietic precursor cells, but despite this concerted effort,no definitive agreement on a common expansion protocol has so far been reached. In contrast tothe large studies on the protocol of cytokines combination, less information is known about cellcycle regulation of hematopoietic precursor cells. Not only regulated by cytokines,hematopoietic cells proliferation and differentiation is also closely associated with cell cycleregulators. Proliferation cell nuclear antigen(PCNA) is an important cell cycle regulatory proteinand the key factor driving cells into S-phase from G₁-phase, and also plays an important role inregulating haematopoiesis. In order to fred the best time-point of transfecting PCNA antisenseoligodeoxynucleotide (PCNA-ASODN), investigate the correct opportunity that interfere in thecell cycle regulation when hematopoietie precursor cells has been expanded in vitro, andimprove the the effect of ex vivo expansion of hematopoietic precursor cells, we study the timeeffect of PCNA-ASODN on ex vivo expansion and differentiation of cord blood CD34⁺hematopoietic precursor cells by transfecting PCNA-ASODN at different time point.Methods: CD34⁺ cells were purified from fresh cord blood by immunomagnetic beads, then wereincubated in liquid culture system with growth factors combination(SCF+IL-6+IL-3+Tpo+ FL). Cells were divided into 4 groups:â‘ control group: without PCNA-ASODN;â‘¡experimental group 1: transfected PCNA-ASODN at 48h;â‘¢experimental group 2: transfectedPCNA-ASODN at 72h;â‘£experimental group 4: transfected PCNA-ASODN at 96h. All groupsare changed half of medium after 3 d, and each experimental group is carded out the cationicliposome mediated transfection of PCNA-ASODN according to the preset time point. AfterCD34⁺ cells incubated 7 days, we count the number of nuclear cells; the percentage of differentkinds of hematopoietic cells and cell cycle were measured by flow cytometry; PCNA proteinlevel in cells was detected by immunocytochemistry.Results: Compared to the control group, PCNA were lowly expressed in three experimentalgroups, and the down-regulation of PCNA protein level at experimental group 3 was the mostobvious; The nuclear cells and CD34⁺ cells of all group were expanded obviously, and the foldsexpanded of the nuclear cells at the experimental group were lower than that of the controlgroup, but the folds expanded of the CD34⁺ CD38^- cells at the experiential group were higherthan that of the control group, especially the experimental group 2, reached (55.1±4.2) folds;Both the percentage of CD34⁺ cells and the percentage of CD34⁺ CD38^- cells at the experimentalgroup were higher than that of the control group, and both that of the experimental goup 1 werethe highest among all group, (42.6±2.0)% and (82.4±2.4) % respectively; In addition, theproportion of G₀/G₁ phase cells was (92.6±3.5)% before incubated. After incubated, thenumbers of S/G₂/M phase cells increased greatly, but that of the experimental group were lowerthan that of the control group. The proportion of the S/G₂/M phase cells at experimental group 3was (38.8±2.7)%, which was the least in all groups.Conclusion: During the ex vivo expansion of cord blood CD34⁺ cells, low-concentrationPCNA-ASODN was transfected by cationic liposome at different time point. The outcome showthat the regulating effects of PCNA-ASODN had the time effect on the ex vivo expansion ofcord blood CD34⁺ cells. PCNA-ASOND could decrease PCNA expression effectively, and afterincubated 7 days, the proportion of G₀/G₁ phase cells, the percentage of CD34⁺ cells and thepercentage of CD34⁺ CD38^- cells were different at different group. Consequently, our studydemonstrates that PCNA-ASODN could postpone the cell differentiation of cord bloodhematopoietic precursor cells and improve the expanded effect of primitive hematopoietic cells.However, in order to obtain the optimal effects of ex vivo expansion on cord bloodhematolopoietic precursor cells, the interference in the cell cycle regulatorys should select thecorrect condition.