Generation of induced pluripotent stem cells from CD34+ cells across blood drawn from multiple donors with non-integrating episomal vectors

Mack AA, Kroboth S, Rajesh D, Wang WB
Source: PLoS ONE
Publication Date: (2011)
Issue: 6 (11): e27956
Research Area:
Stem Cells
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
PBMC, human
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b
The CD34 nucleofection kit and device (Lonza; Allendale, NJ USA) were used for transfections. For CD34+ cells, 3.5 mg of each plasmid in Combination Set 1 and 3 ug of each plasmid for Combination Set 2 except for the L-myc containing plasmid where 2 mg was transfected using program U-08. Conditions for PBMC reprogramming relied on 1exp106 cells per transfection, program T-16, and DNA
The methodology to create induced pluripotent stem cells (iPSCs) affords the opportunity to generate cells specific to the individual providing the host tissue. However, existing methods of reprogramming as well as the types of source tissue have significant limitations that preclude the ability to generate iPSCs in a scalable manner from a readily available tissue source. We present the first study whereby iPSCs are derived in parallel from multiple donors using episomal, non-integrating, oriP/EBNA1-based plasmids from freshly drawn blood. Specifically, successful reprogramming was demonstrated from a single vial of blood or less using cells expressing the early lineage marker CD34 as well as from unpurified peripheral blood mononuclear cells. From these experiments, we also show that proliferation and cell identity play a role in the number of iPSCs per input cell number. Resulting iPSCs were further characterized and deemed free of transfected DNA, integrated transgene DNA, and lack detectable gene rearrangements such as those within the immunoglobulin heavy chain and T cell receptor loci of more differentiated cell types. Furthermore, additional improvements were made to incorporate completely defined media and matrices in an effort to facilitate a scalable transition for the production of clinic-grade iPSCs