Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood

Abstract

Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2–3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-015-9586-8) contains supplementary material, which is available to authorized users.

Can cord blood banks transform into induced pluripotent stem cell banks?

“…These are described in our recent blood reprograming work [30]. In brief, using a density test, we found that re-programing could be achieved with 50 mL of peripheral blood samples, which corresponds to 25,000e30,000 mononuclear cells using a gene integrationefree Sendai virus vector.…”

Introductory Chapter: Update on Mesenchymal and Induced Pluripotent Stem Cells

“…The capacity of human iPSCs to retain patient-specific genomic, transcriptomic, proteomic, metabolomic, and other visualized big data information makes it possible to extend their applications beyond disease modelling into the field of personalized medicine which encompasses the adoption of novel prevention and treatment strategies based on individual variability [65]. The emergence of modern iPSC technology, with the capacity of these stem cells to undergo unlimited self-renewal and differentiation into any type of cell, has a great potential to advance translational applications including stem cell therapies and the generation of large-scale collections of cell lines for research purposes [67]. Recently, genomic editing technologies have been applied to correct the mutations in disease-specific iPSCs to create gene-corrected iPSCs that can be utilized in autologous stem cell-based therapies [64].…”

Blood Derived Induced Pluripotent Stem Cells (iPSCs): Benefits, Challenges and the Road Ahead

“…These iPSCs were, in turn, successfully differentiated to GABAergic neurons [18]. Moreover, recent studies demonstrate the successful derivation of iPSCs from PBMCs collected from Alzheimer disease (AD) and Parkinson disease (PD) patients [20-25]. Undeniably, blood is becoming a routinely used source for iPSC generation.…”

“…Furthermore, in our experience, reprogramming success is not adversely affected when performed on PBMCs extracted from aged patients, in clear contrast with dermal fibroblasts extracted from elderly patients. In support of this convenient source of iPSCs we, as well as other laboratories, have successfully demonstrated the feasibility of using this accessible resource for cell reprogramming and modeling diseases like ASD, AD and PD [3,20-25]. …”