Efficient Generation of Integration-Free iPS Cells from Human Adult Peripheral Blood Using BCL-XL Together with Yamanaka Factors

Abstract

The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source—peripheral blood—has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34+ cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3−/CD19− cells or T/B cell-depleted MNCs for 4–6 days led to the generation of 20–30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

“…The third, MBX (modified vector expressing BCL-XL), expresses BCL-XL alone. The configuration of these vectors is similar to the three vectors previously published [22]. We also made a different episomal vector, called GBX (modified vector expressing GFP and BCL-XL), expressing green fluorescent protein (GFP) and human BCL-XL, linked by a P2A self-cleavage peptide, using a piggyBac (PB)-based plasmid (provided by Dr. H. Bai) in the laboratory of Dr. Z. Wang of Johns Hopkins University.…”

“…First, we deleted and replaced the DNA fragment (from NruI to NheI, ∼2.7 kb) containing the hygromycin selection gene controlled by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) promoter and polyadenylation DNA sequences and the cytomegalovirus (CMV) promoter, with a short linker restoring the NruI and NheI sites. Next, we inserted the spleen focus-forming virus (SFFV) long terminal repeat promoter/enhancer (∼400 bp), which showed better gene expression than the CMV promoter in human hematopoietic cells [20,22,24]. The DNA fragment of the SFFV promoter/enhancer was synthesized by GenScript (Piscataway, NJ, http://www.genscript.com), based on the DNA sequence of the pSFFV-neo-BCL-XL expression vector [24].…”

“…Since 2011, several publications have demonstrated that episomal vectors are capable of reprogramming human blood MNCs to integration-free iPS cells [16][17][18][19][20][21][22]. We, and several other groups, have focused on using either hematopoietic progenitors (expressing the CD34 surface antigen) or myeloid-erythroid cells, both lacking V(D)J rearrangements such as found in committed T and B cells.…”

Positive Correlation Between the Efficiency of Induced Pluripotent Stem Cells and the Development Rate of Nuclear Transfer Embryos When the Same Porcine Embryonic Fibroblast Lines Are Used As Donor Cells

“…In this study, we found that the efficiencies of iPSC induction were different among different PEF donors. Generally, iPSC induction efficiency is determined by the percentage of the marker gene expression in cells if there is a reporter system in the donor cells, such as the Oct4 promoter driving the enhanced green fluorescent protein (EGFP) system, or determined by the numbers of stable iPSC lines derived from the donor cells (Su et al, 2013;Xue et al, 2013;Zhao et al, 2009). The number of AP-positive colonies is not considered an ideal index for the efficiency of iPSC induction, because AP-positive colonies cannot represent the full reprogramming status; it is only an early reprogramming marker .…”

End of Inevitability: Programming and Reprogramming

“…Although the four factors used originally by Yamanaka's group (Oct4, Klf4, Sox2 and c-Myc, otherwise designated OKSM) [2,7] remain commonly used, there are intense efforts to complement the "Yamanaka factors" with other factors to increase efficiency of the repogramming process, including factors as diverse as, e.g. bcl-xl [8] and E-cadherin [9].…”