The initial finding that pluripotency could be induced in human somatic cells revolutionized the field of regenerative medicine, since patient-specific stem cells can now be generated to further examine the causes and mechanisms of various human diseases. Since the discovery of human iPSCs in 2007 1, various studies have focused on improving the reprogramming methods in order to increase the induction efficiency, as well as to further simplify the protocol. iPSCs are commonly generated from dermal fibroblasts. However, skin biopsies are required to isolate fibroblast cells, highlighting the necessity to identify an alternative source of cells for reprogramming that would involve less invasive surgical procedures for isolation.
Cord blood cells and peripheral blood mononuclear cells (PMNCs) are attractive sources for the generation of iPSCs due to the low invasiveness of their collection, as well as the abundance of blood banks for potential donors. iPSCs were first derived from human peripheral blood in 2009 2. CD34+ cells were mobilized from peripheral blood and subsequently transduced with retroviruses delivering OCT4, SOX2, KLF4, and MYC vectors. Although the reprogramming was successful, use of retroviral vectors requires genomic integration of transgenes that may increase the risk of tumor formation during clinical applications. Thus, recent studies have focused on generation of “integration-free” iPSCs. Yet, development of integration-free methods often means compromising the reprogramming efficiency. iPSC induction in CD34+ cells using non-integrating episomal plasmids resulted in ~0.03% reprogramming efficiency 3.
Recently, in Stem Cells, Yamanaka’s group reported a protocol that increased the efficiency of iPSC induction from CD34+ cord blood and peripheral blood 4. They previously identified an efficient combination of episomal plasmids for reprogramming of adult fibroblasts, termed the “Y4” combination, consisting of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and an shRNA for TP53 5. Transfection of CD34+ cells from human cord blood with the Y4 combination resulted in up to 0.1% reprogramming efficiency across two donors. iPSC induction efficiency of PMNCs isolated from peripheral blood with the Y4 mixture, on the other hand, was inconsistent across donors. To further increase the reproducibility of iPSC induction from multiple donors, the authors added a vector encoding EBNA1, which is required for episomal plasmid replication and should thereby increase expression of the episomal plasmids. Addition of the EBNA1 vector to the Y4 mixture resulted in 0.1% reprogramming efficiency in PMNCs across seven donors. Both, the CD34+- and PMNC-derived iPSCs were molecularly and functionally identical to hESCs.
In summary, Okita et al identified a new protocol allowing efficient generation of integration-free iPSCs from blood. Previous studies reported ~0.02%- 0.03% induction efficiency from peripheral blood 2 and isolated CD34+ cells 3. Here, the authors reported a reprogramming efficiency of ~0.06%, with a maximum of 0.1%. In addition, the new protocol could induce iPSCsfrom frozen PMNCs as efficiently as from freshly isolated cells. Thus, with the increasing number of potential donors available at cord blood banks, iPSCs can be now efficiently generated for use in autologous or allogeneic stem cell therapy.
1 Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872, doi:10.1016/j.cell.2007.11.019 (2007).
2 Loh, Y. H. et al. Generation of induced pluripotent stem cells from human blood. Blood 113, 5476-5479, doi:10.1182/blood-2009-02-204800 (2009).
3 Mack, A. A., Kroboth, S., Rajesh, D. & Wang, W. B. Generation of induced pluripotent stem cells from CD34+ cells across blood drawn from multiple donors with non-integrating episomal vectors. PLoS One 6, e27956, doi:10.1371/journal.pone.0027956 (2011).
4 Okita, K. et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 31, 458-466, doi:10.1002/stem.1293 (2013).
5 Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nat Methods 8, 409-412, doi:10.1038/nmeth.1591 (2011).