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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.

SUBMITTER: Chou BK

PROVIDER: S-EPMC4367508 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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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.

Chou Bin-Kuan BK Gu Haihui H Gao Yongxing Y Dowey Sarah N SN Wang Ying Y Shi Jun J Li Yanxin Y Ye Zhaohui Z Cheng Tao T Cheng Linzhao L

Stem cells translational medicine 20150305 4

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 clinical ...[more]

PMID: 25742692

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Project description:BackgroundHuman-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability. To satisfy the sizable requirement for clinical applications of hiPSCs, large-scale, expansion-oriented, xeno-free, and cost-effective media are critical. Although several xeno-free media for hiPSCs have been generated over the past decades, few of them are suitable for scalable expansion of cultured hiPSCs because of their modest potential for proliferation and high cost.MethodsIn this study, we developed a xeno-free ON2/AscleStem PSC medium (ON2) and cultured 253G1 hiPSCs on different matrices, including iMatrix-511 and gelatin nanofiber (GNF) in ON2. Over 20 passages, we evaluated cell proliferation by doubling times; pluripotency by flow cytometry, immunofluorescence staining and qRT-PCR; and differentiation ability by three germ layer differentiation in vitro and teratoma formation in severe combined immunodeficiency mice, followed by histological analysis. In addition, we compared the maintenance effect of ON2 on hiPSCs with StemFit® AK02 (AK02N) and Essential 8™ (E8). Besides 253G1 hiPSCs, we cultivated different hiPSC lines, including Ff-l01 hiPSCs, ATCC® ACS-1020™ hiPSCs, and Down's syndrome patient-specific ATCC® ACS-1003™ hiPSCs in ON2.ResultsWe found that 253G1 hiPSCs in ON2 demonstrated normal morphology and karyotype and high self-renewal and differentiation abilities on the tested matrices for over 20 passages. Moreover, 253G1 hiPSCs kept on GNF showed higher growth and stemness, as verified by the shorter doubling time and higher expression levels of pluripotent markers. Compared to AK02N and E8 media, 253G1 hiPSCs grown in ON2 showed higher pluripotency, as demonstrated by the increased expression level of pluripotent factors. In addition, all hiPSC lines cultivated in ON2 were able to grow for at least 10 passages with compact clonal morphology and were positive for all detected pluripotent markers.ConclusionsOur xeno-free ON2 was compatible with various matrices and ideal for long-term expansion and maintenance of not only healthy-derived hiPSCs but also patient-specific hiPSCs. This highly efficient medium enabled the rapid expansion of hiPSCs in a reliable and cost-effective manner and could act as a promising tool for disease modeling and large-scale production for regenerative medicine in the future.

Project description:A prerequisite for the realization of human pluripotent stem cell (hPSC) therapies is the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable expansion and directed differentiation of hPSCs. Comparative analyses of commercially available microcarriers clearly show the need for developing synthetic substrates supporting the adhesion and growth of hPSCs in three-dimensional cultures under agitation-induced shear. Moreover, the low seeding efficiencies during microcarrier loading with hPSC clusters poses a significant process bottleneck. To that end, a novel protocol was developed increasing hPSC seeding efficiency from 30% to over 80% and substantially shortening the duration of microcarrier loading. Importantly, this method was combined with the engineering of polystyrene microcarriers by surface conjugation of a vitronectin-derived peptide, which was previously shown to support the growth of human embryonic stem cells. Cells proliferated on peptide-conjugated beads in static culture but widespread detachment was observed after exposure to stirring. This prompted additional treatment of the microcarriers with a synthetic polymer commonly used to enhance cell adhesion. hPSCs were successfully cultivated on these microcarriers in stirred suspension vessels for multiple consecutive passages with attachment efficiencies close to 40%. Cultured cells exhibited on average a 24-fold increase in concentration per 6-day passage, over 85% viability, and maintained a normal karyotype and the expression of pluripotency markers such as Nanog, Oct4, and SSEA4. When subjected to spontaneous differentiation in embryoid body cultures or directed differentiation to the three embryonic germ layers, the cells adopted respective fates displaying relevant markers. Lastly, engineered microcarriers were successfully utilized for the expansion and differentiation of hPSCs to mesoderm progeny in stirred suspension vessels. Hence, we demonstrate a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable, xeno-free propagation and differentiation of human stem cells intended for therapies.

Dataset Information

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.

Publications

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.

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