Feeder-free derivation of human-induced pluripotent stem cells with synthetic messenger RNA

A cell and somatic cell technology, applied in the direction of genetically modified cells, cell culture active agents, artificially induced pluripotent cells, etc., can solve the problem of low efficiency of pluripotent cells

Active Publication Date: 2015-04-15
ALLELE BIOTECH & PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] A major difficulty in producing induced pluripotent stem cells (iPSCs)...

Method used

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  • Feeder-free derivation of human-induced pluripotent stem cells with synthetic messenger RNA
  • Feeder-free derivation of human-induced pluripotent stem cells with synthetic messenger RNA
  • Feeder-free derivation of human-induced pluripotent stem cells with synthetic messenger RNA

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Embodiment 1-I

[0075] The generation of embodiment 1-IVT template

[0076] Plasmid constructs for generating templates for in vitro transcription (IVT) of linear PCR products were constructed using ligation-independent cloning (LIC). We first constructed parental plasmids (pIVT) incorporating 5' and 3' untranslated regions (UTRs) flanked by open reading frame (ORF) inserts designed to accept encoding proteins of interest the insertion site. The sequences flanking the ORF were as described in Warren et al., Cell Stem Cell, 2010, comprising a low secondary structure leader sequence and a strong Kozak site (5'UTR) and murine α-globin 3'UTR. A linearized version of the pIVT vector with a 5' overhang was generated by re-annealing of two PCR products amplified from the plasmid using tailed primers. ORF PCR products with complementary overhangs were generated by a similar procedure, combined with vector PCR products, and transformed into DH5α bacteria by heat shock to clone gene-specific constr...

Embodiment 2

[0077] Example 2 – Generation of mRNA Mixtures

[0078] The mRNA synthesis process is outlined in Figure 5. Synthetic mRNA was generated using a 4:1 ratio of ARCA cap analog to GTP in the IVT reaction to generate a high percentage of capped transcripts. Complete replacement of CTP by 5m-CTP and UTP by pseudo-UTP in a nucleotide triphosphate (NTP) mixture was used to reduce the immunogenicity of the RNA product. Cap analogs and modified NTPs were purchased from Trilink Biotechnologies. Prepare a 2.5x NTP mix (ARCA:ATP:5m-CTP:GTP:pseudo-UTP of 15:15:3.75:3.75:3.75 mM) to replace the standard NTP supplied with the MEGAscript T7 kit (Ambion) used to perform the IVT reaction . Each 40 µL IVT reaction contained 16 µL of NTP mix, 4 µL of 10x T7 buffer, 16 µL of DNA template, and 4 µL of T7 enzyme. Reactions were incubated at 37°C for 4-6 hours and then treated with 2 μL of TURBO DNase for an additional 15 minutes at 37°C before purification on MEGAclear (Ambion) spin columns el...

Embodiment 3

[0079] Example 3 - Cells and media

[0080] Reprogrammed target cells include BJ neonatal fibroblasts (ATCC), HDF-f fetal fibroblasts, HDF-n neonatal fibroblasts and HDF-a adult fibroblasts (ScienCell) and XFF xenobiotic-free neonatal Fibroblasts (Millipore). For BJ, HDF and XFF cells, expansion cultures were in BJ Medium (DMEM+10% FBS), ScienCell Fibroblast Medium and FibroGRO Xeno-free Human Fibroblast Expansion Medium (Millipore) conduct. The feeder cells used were 3001G irradiated neonatal human foreskin fibroblasts (GlobalStem) and FibroGRO mitomycin C-inactivated xeno-free human neonatal fibroblasts (Millipore). Cell passaging steps associated with xeno-free feeder-based and feeder-free reprogramming assays were performed using TrypLE Select (Gibco), an animal product-free cell dissociation reagent.

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Abstract

The present disclosure relates generally to novel methods and compositions for using engineered reprogramming factor(s) for the creation of induced pluripotent stem cells (iPSCs) through a kinetically controlled process. Specifically, this disclosure relates to establishing combinations of reprogramming factors, including fusions between conventional reprogramming factors with transactivation domains, optimized for reprogramming various types of cells. More specifically, the exemplary methods disclosed herein can be used for creating induced pluripotent stem cells from various mammalian cell types, including human fibroblasts. Exemplary methods of feeder-free derivation of human induced pluripotent stem cells using synthetic messenger RNA are also disclosed.

Description

[0001] related application [0002] This application claims the benefit of priority to US Provisional Application USSN 61 / 646,292, filed May 13, 2012, the contents of which are incorporated herein in their entirety. field of invention [0003] The present disclosure generally relates to novel methods and compositions for generating induced pluripotent stem cells (iPSCs) through a kinetically controlled process using engineered reprogramming factors. In particular, the disclosure relates to the creation of combinations of reprogramming factors optimized for reprogramming various different types of cells, including fusions between conventional reprogramming factors and transactivation domains. More specifically, the exemplary methods disclosed herein can be used to generate induced pluripotent stem cells from a variety of different mammalian cell types, including human fibroblasts. Exemplary methods for the feeder-free derivation of human induced pluripotent stem cells using sy...

Claims

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Application Information

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IPC IPC(8): C12N5/10
CPCC12N2320/30C12N2501/603C12N2501/608C12N5/0696C12N2501/605C12N2310/14C12N2510/00C12N2501/602C12N2506/1307C12N15/111A61P43/00C12N2501/606
Inventor 王继武鲁吉·瓦兰倪毓辉
Owner ALLELE BIOTECH & PHARMA
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