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Extracellular matrix-dependent generation of integration- and xeno-free iPS cells using a modified mRNA transfection method

ABSTRACT: In this study, we sought to examine whether an extracellular matrix (ECM)-based xeno-free culture system that we recently established could be used together with a microRNA-enhanced mRNA reprogramming method for the generation of clinically safe iPS cells. The notable features of this method are (1) the use of a xeno-free/feeder-free culture system for the generation and expansion of iPS cells rather than the conventional labor-intensive culture systems using human feeder cells or human feeder-conditioned medium and (2) the enhancement of mRNA-mediated reprogramming via the delivery of microRNAs. Strikingly, we observed the early appearance of iPS cell colonies (~11 days), substantial reprogramming efficiency (~0.2-0.3%), and a high percentage of ESC-like colonies among the total colonies (~87.5%), indicating enhanced kinetics and reprogramming efficiency. Therefore, the combined method established in this study provides a valuable platform for the generation and expansion of clinically safe (i.e., integration- and xeno-free) iPS cells, facilitating immune-matched cell therapy in the near future.

ORGANISM(S): Homo sapiens

PROVIDER: GSE68035 | GEO | 2016/04/21

SECONDARY ACCESSION(S): PRJNA281595

REPOSITORIES: GEO

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Gene expression profiles in human iPS cells cultured by different four methods

Project description:Hepatocyte-like cells differentiated from human iPS cells are expected to be utilized in pharmaceutical research and regenerative medicine. Recently, various culture methods for human iPS cell maintenance have been developed. However, it is not well known whether human iPS cell maintenance method affects hepatic differentiation potency. In this study, we cultured human iPS cells using four maintenance methods: ReproStem medium with feeder cells (mouse embryonic fibroblasts), AK02N medium with iMatrix-511 (E8 fragments of laminin511), Essential 8 medium with Vitronectin N (N-terminal domain of vitronectin), TeSR-E8 medium with Vitronectin XF (xeno-free vitronectin). Then, these human iPS cells were differentiated into the hepatocyte-like cells. Interestingly, the gene expression levels of definitive endoderm markers in the definitive endoderm cells generated from human iPS cells cultured with ReproStem or TeSR-E8 medium were higher than those in other groups. The gene expression level of foregut marker, HHEX, in the definitive endoderm cells generated from human iPS cells cultured with ReproStem medium was higher than that in other groups. Consistently, the expression levels of hepatocyte markers, albumin and urea secretion capacity, and CYP3A4 activity in the hepatocyte-like cells generated from human iPS cells cultured with ReproStem medium were higher than those in other groups. Our data indicated that the most suitable human iPS cell maintenance method for efficient hepatic differentiation was the on-feeder method which uses mouse embryonic fibroblasts, but not feeder-free methods. In conclusion, human iPS cell maintenance method largely affects hepatic differentiation potency.

2018-09-25 | GSE119698 | GEO

Expression data of induced pluripotent stem cell

Project description:We present a robust serum-free system for the rapid and efficient reprogramming of mouse somatic cells by Oct4, Sox2 and Klf4. The elimination of fetal bovine serum and oncogene c-Myc allowed reprogramming cells to be detected as early as Day 2 and reached greater than 10% of the population at Day 7 post retroviral transduction. The resulting iPS colonies were isolated with high efficiency to establish pluripotent cell lines. Based on this method, we further developed iPS-SF1 as a dedicated reprogramming medium for chemical screening and mechanistic investigations. Comparasion of iPS cell lines derived from serum-free culture condition, MEF cells cultured in serum and serum-free condition and ES cell lines was shown.

2009-03-18 | GSE15267 | GEO

DNA microarrays of three shRNA control iPS clones (Ctrl 2,3,4) and shECAD iPS clones (shECAD 4,8,9)

Project description:E-cadherin upregulation is an early event of reprogramming of fibroblasts to induce pluripotent stem cells (iPS). Knocking down of E-cadherin by shRNA impairs iPS generation, though some colonies with great morphorlogical difference to shRNA control colonies remain. To illustrate the molecular and functional difference between shECAD iPS clones and shRNA control iPS clones, three respective iPS clones (shECAD 4,8,9 and Ctrl 2,3,4) were derived and DNA microarrays were run to analyze the transcriptional profile of these clones. OG2 MEFs were infected with Sox2, Klf4, Oct4 and c-Myc (SKOM) plus either Luciferase shRNA (shLUC) or E-cadherin shRNA (shECAD) retrovirus. At Day 6 post infection cells were split onto feeder cells. Several colonies from SKOM+shLuc and SKOM+shECAD were picked out at Day 14 post infection respectively and three cell lines were established, namely Ctrl 2,3,4 for SKOM+shLuc iPS and shECAD 4,8,9 for SKOM+shECAD iPS. All clones were maintained on feeder cells in mESC medium. RNA were extracted from these six cell lines and DNA microarrays were run to analyze the transcriptional profile.

2010-06-08 | E-GEOD-21062 | biostudies-arrayexpress

Transcription profi;ing of mouse induced pluripotent stem cells vs MEF cells to investigate culturing conditions

2009-03-28 | E-GEOD-15267 | biostudies-arrayexpress

Xeno-free and Chemically Defined Human System for Culturing Human Epidermal Keratinocytes

Project description:Developing an effective xeno-free and defined system for human keratinocyte culture has been one of the fundamental measures in current cellular therapy. For keratinocytes, in particular, such system will not only fulfill clinical safety and quality criteria, it will allow for the management of less severe burns and other skin injuries such as chronic wounds. To date, the expansion of autologous keratinocytes in the clinical settings still relies on 3T3 feeder co-culture system. Here we report a completely xeno-free and defined culture system by using human recombinant laminins as feeder replacement. We have thoroughly characterized the cells cultured in this new system both in vitro and in vivo. We found that laminin-511/-521, and the unanticipated laminin-411/-421, but not -332, -111, -121, -211, or LN-221 support adult human skin keratinocytes and could maintain their self-renewal properties comparable to the 3T3 system. We believe our new culture method should facilitate broader use of cultured epithelial cell products for today’s regenerative medicine.

2018-11-06 | GSE109645 | GEO

In vitro modeling of human germ cell development using pluripotent stem cells

Project description:In this study, we developed a feeder-, serum-, and animal product-free culture condition, and successfully induced high percentage of SLCs from human PSCs. Genome-wide expression analyses demonstrated a significant upregulation of germ cell specific genes in SLCs derived from PSCs compared to in vivo isolated human CD90+ SSCs. And furthermore, under this optimized feeder-free condition SLCs have went through meiosis and formed putative round spermatids. Our data demonstrated a robust feeder-, serum- and xeno-free protocol to induce differentiation of PSCs to SLCs from which putative haploid spermatids may develop.

2018-12-24 | GSE108496 | GEO

Reliability of 3D Cortical Spheroids Generation from Human Pluripotent Stem Cells

Project description:Here we assess the reliability of human cortical spheroid differentiation cultured in xeno-free, feeder-free conditions. We find high reliability in differentiation across multiple hiPSC lines as well as across experiments, and we anticipate that this directed differentiation approach will be useful for large scale generation of brain-region specific spheroids and disease modeling.

2018-11-06 | GSE120700 | GEO

Effect of Aid deletion on the global gene expression iPS cells

Project description:It has been shown that DNA demethylation has a pivotal role in the generation of induced pluripotent stem (iPS) cells. However, the underlying mechanism is still unclear. Previous reports indicated that activation-induced cytidine deaminase (Aid) is involved in DNA demethylation in several developmental processes and cell fusion-mediated reprogramming. Based on the reports, we hypothesized that Aid may be involved in DNA demethylation during the iPS cell generation. In this study, we examined the function of Aid in iPS cell generation using Aid knockout (Aid-/-) mice expressing a GFP reporter under the control of a pluripotent stem cell marker, Nanog. By the introduction of Oct3/4, Sox2, Klf4 and c-Myc, Nanog-GFP positive iPS cells could be generated from the fibroblasts and primary B cells of Aid-/- mice. The Aid-/- iPS cells showed normal proliferation and gave rise to chimeras, indicating their capacity for self-renewal and pluripotency. Microarray analysis demonstrated that the global gene expression of Aid-/- iPS cells was similar to that of Aid+/+ iPS cells. Aid+/+ and Aid-/- iPS colonies were generated from Aid+/+ and Aid-/- MEFs and picked up mechanically. The clones were passaged four times on feeder cells and two times on gelatin-coated dishes to exclude the contamination of feeder cells. Subsequently, the RNA was isolated. Six Aid+/+ iPS cell clones and six Aid-/- iPS cell clones were compared by microarray.

2014-04-10 | GSE51955 | GEO

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

Project description:Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue-culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with Inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI-supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications.

2016-06-02 | GSE82103 | GEO

Effect of Aid deletion on the global gene expression iPS cells

2014-04-10 | E-GEOD-51955 | biostudies-arrayexpress

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