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Title: Design of artificial microenvironment for cord blood CD34+ cells expansion ex vivo
Authors: ZHANG YUE
Keywords: Hematopoietic stem cell, Mesenchymal stem cells, 3D Cell Culture, Surface modification, Fibronectin
Issue Date: 4-Apr-2006
Citation: ZHANG YUE (2006-04-04). Design of artificial microenvironment for cord blood CD34+ cells expansion ex vivo. ScholarBank@NUS Repository.
Abstract: Allogeneic transplantation of CB is limited by insufficient number of HSC as well as PHPC. An efficient ex vivo expansion of CB can overcome this limitation. An ideal ex vivo expansion condition should mimic the in vivo hematopoietic microenvironment, which can efficiently expand the cell number while still maintaining the a??stemnessa?? of the cells.MSC, as non hematopoietic, well-characterized skeletal and connective tissue progenitor cells within the bone marrow stroma, have been investigated as support cells for the culture of HSC/PHPC. They are attractive for the rich environmental signals that they provide and their immunologic compatibility and supporting effect in transplantation. In this study we clearly demonstrated the benefit of MSC inclusion in HSC expansion ex vivo. We also compared the contact and non-contact cultures of the two cell types; the cell-cell direct interaction was necessary for the efficient expansion of HSC.HSC-MSC co-cultures have only been performed in 2D configuration. We postulate that a 3D culture environment that resembles the natural in vivo hematopoietic compartment would be more conducive for regulating HSC expansion. The result showed that the direct contact between MSC and HSC in 3D cultures led to statistically-significant higher expansion of CB CD34+ cells compared to 2D co-cultures: 891- versus 545-fold increase in total cells, 96- versus 48-fold increase of CD34+ cells, and 230- versus 150-fold increase in CFC. NOD/SCID mouse engraftment assays also indicated a high success rate of hematopoiesis reconstruction with these expanded cells. This enhanced expansion result was probably due to the ECM secreted by MSC, especially FN, which facilitated the interaction with the seeded CD34+ cells.Based on this finding, we hypothesize that the immobilization of ECM protein FN onto the surface of culture systems might represent another stroma free expansion strategy. A key problem in FN immobilization is the change of bioactivity during the immobilization process. By comparing the FN bioactivity after two common immobilization pathways: conjugation and adsorption, we found that the adsorption method maintained a normal conformation of FN, leading to less change of its bioactivity in the access of RGD domains and cell adhesion ability. The covalent conjugation process, on the other hand, induced FN unfolding and fibrillogenesis ex vivo, which blocked the access of active RGD domains and suppressed fibroblast cells adhesion. This bioactivity variance on the level of immobilization methods may provide us a way to control the function of the immobilized protein, which can be helpful for the construction of an artificial niche.
Appears in Collections:Master's Theses (Open)

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