Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

ArticleinJournal of Visualized Experiments 68(68) · November 2012with32 Reads
DOI: 10.3791/4327 · Source: PubMed
Abstract
Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)(1-4). Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies(5). We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors(6). These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs(7-9). In one study, a Tet inducible version of the STEMCCA vector was employed(9), which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies. Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described(10,11) in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.
    • "Additionally, the delivering strategies for inducing iPSCs have been improved. The retroviral or lentiviral vectors were routinely used to integrate the reprogramming genes into the host genome to induce iPSCs [30, 57] . Viral delivery system are efficient and reproducible in reprogramming , however, the random integration of transgenes into genome increases the risk of tumor formation and may cause mortality in chimeric and progeny mice derived from iPSCs [58] . "
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    • "For human reprogramming experiments, BJ fibroblasts were first infected with control or SUMO2 pGIPZ shRNA (V3LHS_388696 and V3LHS412780; GE Dharmacon) viruses. Two days later, cells were infected with the human pHAGE-STEMCCA virus (Sommer et al., 2012) to initiate reprogramming. Cells were replated in fibroblast medium (DMEM and 10% FBS) and cultured for 4 days. "
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    • "pluripotent stem cells were generated as described previously [59, 60]. Briefly, 4 mL of human peripheral blood was collected into a BD Vacutainer CPT Cell Preparation Tube and centrifuged to produce a buffy coat containing peripheral blood mononuclear cells (PBMCs). "
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