Publications (7)24.56 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: A method to produce clinically useful platelets in vitro would help overcome the frequent shortages, donor deferrals, disease transmission, and alloimmunization with volunteer donor-derived platelets. Using CD34 positively selected cord blood cells, we investigated ways to increase platelet quality and yield in a three-dimensional modular perfusion bioreactor system. We found a two- to threefold increase in platelet numbers produced only when the early phases of the culture process were carried out at 5% oxygen, versus when 20% oxygen was used throughout the culture period (p<0.05), and much more than when 5% oxygen was used throughout. When the medium was routed through the cell-scaffold construct, versus when it flowed under and over the construct, or just intermittent feeding was used, the number of platelets increased two- to threefold (p<0.05), and enhanced collagen-induced aggregation. The 5% oxygen early in the culture process mimics the marrow adjacent to the bone where early progenitors proliferate. Flow through the cell-scaffold construct creates shear forces that mimic the flow in central venous sinuses of the marrow and enhances platelet production from proplatelets. The use of altered oxygen levels and cross flow enhanced platelet numbers and quality, and will contribute to eventual in vitro platelet production for clinical use.
- [Show abstract] [Hide abstract] ABSTRACT: In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis; however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating TpoR and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine.
- [Show abstract] [Hide abstract] ABSTRACT: Methods producing human platelets using growth on plastic, on feeder layers, or in suspension have been described. We hypothesized that growth of hematopoietic progenitors in a three-dimensional (3D) scaffold would enhance platelet production sans feeder layer. We grew CD34 positively selected human cord blood cells in surgical-grade woven polyester fabric or purpose-built hydrogel scaffolds using a cocktail of cytokines. We found production of functional platelets over 10 days with two-dimensional (2D), 24 days with 3D scaffolds in wells, and more than 32 days in a single-pass 3D perfusion bioreactor system. Platelet numbers produced daily were higher in 3D than 2D, and much higher in the 3D perfusion bioreactor system. Platelet output increased in hydrogel scaffolds coated with thrombopoietin and/or fibronectin, although this effect was largely obviated with markedly increased production caused by changes in added cytokines. In response to thrombin, the platelets produced aggregated and displayed increased surface CD62 and CD63. Use of 3D scaffolds, especially in a bioreactor-maintained milieu, may allow construction of devices for clinical platelet production without cellular feeder layers.
- [Show abstract] [Hide abstract] ABSTRACT: The ACE System is a potent biological engineering system consisting of a mammalian artificial chromosome engineered with multiple site-specific integration sites, expression-optimized shuttle vectors to specifically transfer genes, and a proprietary integrase to catalyze specific incorporation of a payload onto the ACE. ACEs are promising gene delivery vehicles for gene-based cell therapies as they are stably maintained, non-integrating, autonomously replicating, and are easily isolated to high purities by flow sorting. We published the first reports of the transfer and stable transgene expression of a mammalian artificial chromosome into hMSCs and hHSCs (Stem Cells 22:324–33, 2004; Exp Hematol 33:1470–1476, 2005). We will update our progress, including an hMSCs enrichment strategy resulting in stable EPO transgene expression, more than 50 days post EPO-ACE transfer, at levels of 100–200 IU/cell/day.We also report the first successful ACE transfer into human embryonic stem cells (hESCs). We quantified the delivery of IdUrd- labeled ACEs to hESCs, via flow cytometry at 24–48 hours post- transfection, a screening technique that utilizes a FITC-conjugated anti-BrdUrd B44 clone antibody that binds to the IdUrd-DNA adduct. We detected IdUrd-labeled ACEs in 13% of the cells 24–48 hours post-transfer. Gene expression studies are underway. The combination of ACEs and the multipotency of adult & embryonic stem cells represent a unique approach for the study of stem cell fate/biology and for the development of novel gene-based cell therapies.
- [Show abstract] [Hide abstract] ABSTRACT: The transfer of mammalian artificial chromosomes (MACs) to hematopoietic stem and progenitor cells (HSPCs) presents a promising new strategy for ex vivo gene therapy that alleviates numerous concerns surrounding viral transduction along with a unique platform for the systematic study of stem cell biology and fate. Here we report the transfer of a satellite DNA-based artificial chromosome (an ACE), made in mouse cells, into human cord blood hematopoietic cells. A GFP-Zeo-ACE encoding the genes for humanized Renilla green fluorescence protein (hrGFP) and zeomycin resistance (zeo) was transferred into CD34 positively selected cord blood cells using cationic reagents. Post ACE transfer, CFU-GM-derived colonies were generated in methylcellulose in the presence or absence of bleomycin. Bleomycin-resistant cells expressed GFP and contained intact autonomous ACEs, as demonstrated by fluorescent in situ hybridization. Moreover, when the cells from these plates were replated in methylcellulose, we observed secondary bleomycin-resistant CFU-GM-derived colonies, demonstrating stable chromosome retention and transgene function in a CFU-GM progenitor. To our knowledge this is the first report demonstrating the transfer of a mammalian artificial chromosome and the stable expression of an encoded transgene in human hematopoietic cells.
The Ohio State University
Columbus, Ohio, United States
- Department of Pathology