Preliminary Study Of Ex Vivo Amplification Of Human Cord Blood Hematopoietic Stem/Progenitor Cells

Umbilical cord blood is one good alternative source of hematopoietic stem/progenitor cells (HSPCs) for clinical treatment. However, the number of the HSPCs obtained from one donor of cord blood is only sufficient for child patients which limited the application of cord blood transplantation. To treat the teenager and adult patients, the ex vivo amplification of cord blood HSPCs before transplantation is needed. Currently, the conventional way to amplify the HSPCs ex vivo is to directly culture the suspending HSPCs in Petri-dishes, which is dependent on the high dose of combined cytokines (CKs). However, the cells amplified in such a 2 dimensional system can not keep long-term hematopoiesis. It is urgent to develop new methods to amplify HSPCs ex vivo.In the first part of this research, we studied a new alginate 3 dimensional (3D) culture system to amplify human cord blood HSPCs. The HSPCs live in the bone marrow microenvironment. The interaction between HSPCs, HSPCs with stromal cells and the extracellular matrices is one of the factors keeping the self-renewal capacity of the HSPCs. A 3D culture system is designed to mimic such a microenvironment. In our research, we encapsulate the cord blood mononuclear cells (CBMCs) in alginate 3D static and rotating culture system, and compared to the conventional 2D system. The data showed that under low concentration of CKs, the cell number and CD34+ cells both decreased in 2D system, however, increased significantly in 3D static culture system. Similar results were achieved in the 3D rotating system under much lower CKs. The CFU-G/GM increased in the 3D systems. The amplified cells achieved greater engraftment in after being transplanted to NOD/SCID mice.In the second part of the research, we studied the effect of the pl8ink4c down-regulation to the HSPCs. The proliferation and differentiation of cell are restrictly regulated by cell cycle.3 major types of the molecules, cyclins, CDKs and CKIs. are involved in the cell cycle regulation. P18ink4c is one of the CKIs. which stop the cell at GO phase by inhibiting the activity of cyclin D-CDK4/6 complex at early G1 phase. Multiple organmegaly and tissue hyperplasia were seen in pl8ink4c-/-mice. Mice bone marrow transplantation model show the enhanced self-renewal capacity of p18ink4c -/- bone marrow cells. In our research, we designed an optimal siRNA sequence of the human pl8ink4c gene, constructed the p18-siRNA expressed DNA plasmid and ADV vector. We transfected the human cord blood purified CD34+ cells with synthesized siRNA, p18ink4c-siRNA-plasmid and ADV-pl8ink4c-siRNA. The results show that the p18ink4c expression of human cord blood CD34+ cells was down-regulated, cell number increased and the loss of CD34+ cells declined to some extent. However, the efficiency of the transfection was too low to meet the clinical application.In our research of these two parts, we achieved or partially achieved the amplification of human cord blood HSPCs by changing the culture system or knock-down the CKI, respectively. The data of 3D alginate culture system provide a promising result which was not reported before. However, the low efficiency of currently available techniques in human HSPCs transfection limits the amplification effect of down-regulation of p18ink4c.