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

Abstract

Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ‡10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. STEM CELLS

Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience

“…Indeed, several existing cell repositories have already banked hundreds of fibroblast lines from diseased and healthy donors. Several other somatic cell types have been successfully reprogrammed into iPSCs and used for disease modelling with varying efficiency, including keratinocytes 138 , dental pulp cells 139,140 , several blood cell types 141–143 and exfoliated renal epithelial cells present in urine 144 (TABLE 1). Each has advantages and disadvantages; for example, blood is easy to obtain, but the efficiency of reprogramming is rather low.…”

Re‐entering the pluripotent state from blood lineage: promises and pitfalls of blood reprogramming

“…Vectorbased nonintegrating strategies make use of Sendai-viral vectors or episomal vectors. Several groups have reported successful attempts in transfecting different types of blood cells with oriP-EBNA1-based episomal vectors encoding reprogramming factors and SV40-T antigen to derive iPSCs [11,18,19,[26][27][28]. It has been reported by different groups that both aPB and CB cells could be reprogrammed by a temperature-sensitive Sendai virus expressing OSKM factors [6,14].…”

“…To further enhance the clinical applicability, various research groups have established clinically compliant protocols for blood reprogramming [27,40]. Clinically, blood collection and storage techniques are developed and routinely practised.…”

“…To date, episomal vector-based blood reprogramming has been first established by Chou et al [25]. Several groups have reported successful attempts in transfecting different types of blood cells with oriP-EBNA1-based episomal vectors encoding reprogramming factors and SV40-T antigen to derive iPSCs [11,18,19,[26][27][28].…”

Looking into the future: Using induced pluripotent stem cells to build two and three dimensional ocular tissue for cell therapy and disease modeling

“…The same is not true for iPS cell derivation and differentiation into RPE. There have been a number of protocols developed for the generation of transgene, virus, proto-oncogene, and xeno-free iPS cells (Chou et al, 2015; Goh et al, 2013; Okita et al, 2008). Similarly, protocols have been published to generate RPE under xeno-free conditions from pluripotent stem cells (Pennington et al, 2015; Sridhar et al, 2013).…”