Cellular reprogramming of human peripheral blood cells

Abstract

Breakthroughs in cell fate conversion have made it possible to generate large quantities of patient-specific cells for regenerative medicine. Due to multiple advantages of peripheral blood cells over fibroblasts from skin biopsy, the use of blood mononuclear cells (MNCs) instead of skin fibroblasts will expedite reprogramming research and broaden the application of reprogramming technology. This review discusses current progress and challenges of generating induced pluripotent stem cells (iPSCs) from peripheral blood MNCs and of in vitro and in vivo conversion of blood cells into cells of therapeutic value, such as mesenchymal stem cells, neural cells and hepatocytes. An optimized design of lentiviral vectors is necessary to achieve high reprogramming efficiency of peripheral blood cells. More recently, non-integrating vectors such as Sendai virus and episomal vectors have been successfully employed in generating integration-free iPSCs and somatic stem cells.

Keywords: Cell fate conversion; Hematopoietic cells; Induced pluripotent stem cells; Peripheral blood; Reprogramming.

Figure 1

Different lentiviral vector design leads to differential transgene expression levels A. The SFFV promoter is stronger than other commonly-used promoters in human hematopoietic cells. Human cord blood CD34⁺ cells were transduced with four GFP-expressing lentiviral vectors at the same multiplicity of infection (MOI). Expression of GFP was analyzed by flow cytometry at 3 days after transduction. The backbone of the lentiviral vector was described in detail previously . SFFV, long-terminal repeat from spleen focus-forming virus; CAG, CMV immediate-early enhancer/chicken β-actin promoter; EF1, elongation factor 1α; PGK, phosphoglycerate kinase 1; GFP, green fluorescent protein. B. Linking multiple genes with 2A is a better strategy than dual promoters for the expression of multiple genes. The expression of reprogramming factors OCT4, SOX2 and KLF4 was driven by the SFFV promoter. 293T cells were transduced with four lentiviral vectors at the same MOI. Expression of OCT4 was analyzed by intracellular staining and flow cytometry at 3 days after transduction. The linkage of multiple genes with 2A leads to decreased expression of each one (Lenti SFFV-OCT4 vs. Lenti SFFV-OCT4-2A-SOX2 vs. Lenti SFFV-OCT4-2A-SOX-2A-KLF4). The use of dual promoters considerably decreases the transgene expression compared to the vector design of a single promoter with 2A linkers (Lenti SFFV-OCT4-2A-SOX2-2A-KLF4 vs. Lenti SFFV-OCT4-2A-SOX2-PGK-KLF4). The data shown here are representative of three independent experiments with similar results.