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.