Generation of Induced Pluripotent Stem Cells from CD34+ Cells across Blood Drawn from Multiple Donors with Non-Integrating Episomal Vectors

Abstract

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.

An Efficient Nonviral Method to Generate Integration-Free Human-Induced Pluripotent Stem Cells from Cord Blood and Peripheral Blood Cells

“…Mack et al purified and expanded CD34 þ cells from PMNCs and carried out transduction with episomal vectors, which were similar to T1, but used L-MYC instead of C-MYC [17]. They estimated that the reprogramming efficiency was 0.03% on average and obtained approximately five iPSC colonies per 1 ml of blood.…”

“…The colonies were counted 16-25 days after plating, and the colonies similar to human ESCs were selected for further cultivation and evaluation. Generation of iPSC from CD34 þ rich population was performed as described previously with slight modification [17]. Briefly, purified CD34 þ cells from PMNC was expanded for 6 days in StemSpan SFEM medium (StemCell Technologies Inc, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 10 ng/ml IL-3, 100 ng/ml IL-6, and 300 ng/ml of Flt3 ligand, stem cell factor (SCF), and TPO.…”

Human iPSC-Derived Neural Crest Stem Cells Promote Tendon Repair in a Rat Patellar Tendon Window Defect Model

“…21,50,51 Thus, NCSCs represent a unique cell source for regenerative medicine. Based on recent progresses in safety 52 and efficiency, 53 iPSCs can be made at clinical-grade therapeutic cells for future applications. Therefore, multipotent NCSCs derived from iPSCs may be used as a valuable and unlimited cell source for tissue regeneration, including neural and mesenchymal tissues (tendon, ligament, etc.).…”

Good Cell Culture Practice for stem cells and stem-cell-derived models

“…A range of transcription factors has been used with Oct4 and Sox2 being most crucial and any combination of others like C-or L-Myc, Klf4, Lin-28 and Nanog. Delivery systems include Retro/Lentivirus, Sendai Virus, mRNA, piggybac, and episomal-based approaches (Yu et al, 2007(Yu et al, , 2009Shi et al, 2008;Mack et al, 2011;Woltjen et al, 2009;Stadtfeld et al, 2008). These methods may be performed with BVDV may cause slight changes in growth rate but this virus is non-cytopathic and microscopic changes in the culture will not be detected.…”