Episomal iPSC Reprogramming Kit: #RF202

Product Description

The human episomal iPSC reprogramming kit from ALSTEM is one of the best choices for producing footprint-free iPSCs, providing a source of iPSCs for all stages of your pluripotent stem cell research. Optimized by the ALSTEM team, this episomal kit has proven to be successful in inducing pluripotency in a number of different somatic cell types.

Features

  • Generate transgene-free, virus-free, and footprint-free iPS cells
  • Episomal kit for human induced pluripotent stem cell (iPSC) generation
  • Safe for all stages of your research from basic research to pre-clinical research
  • Flexible in source cell selection
  • Enhanced reprogramming efficiency by puromycin selection
  • Optimized for feeder-free reprogramming
  • Addition of small molecules are not required 

Optimize Reprogramming with Puromycin Selection


Figure 2. (A) A typical image of the human dermal fibroblasts transfected with RFP using Neon Transfection Devices (24 hours after electroporation). ~ 30% of fibroblasts are RFP positive. (B) After puromycin selection, more than 90% of fibroblasts are expressing RFP (~5 days after puromycin selection).

Figure 1. Characterization of iPSCs derived from human dermal fibroblasts using ALSTEM episomal vectors. Bright field image of hiPSC colonies (top left), immunostaining of hiPSC colonies expressing ESC specific markers OCT4, SSEA-3, and TRA-1-81.
Product Specifications
Product Name Human iPS Cell Reprogramming Episomal Kit
Catalog # RF202
Component
hiPSC Reprogramming Episomal Vectors (cat.no. RF202_1)
RFP Control Vector (cat.no. RF202_2):
Each kit contains sufficient material for 10 reprogramming experiments
Shipping Ambient temperature
Storage and Stability Store at -20 °C upon receipt. This product is stable up to 6 months when stored as directed.
Quality Control Each lot of Human iPS Cell Reprogramming Episomal Kit is tested to ensure human fibroblasts can be reprogrammed to iPSCs.
Restricted Use For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Protocol Download
 
  • Overview
  • Procedure

Description

Induced pluripotent stem cells (iPSCs) are genetically reprogrammed from adult cells, which are similar to natural pluripotent stem cells, such as embryonic stem cells (ESCs). iPSCs exhibit a pluripotent stem cell-like state, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, teratoma formation, and potency and differentiability. While these artificially generated cells are not known to exist in the human body, they show qualities remarkably similar to those of embryonic stem cells. Therefore, iPSCs are an invaluable resource for drug discovery, cell therapy, and basic research.

Human iPSCs were first generated in 2007 through retrovirus- or lentivirus-mediated gene transduction. However, retroviral or lentiviral vectors require integration into host chromosomes to express reprogramming genes. Integration-free human iPSCs have been generated using several methods, including adenovirus, Sendai virus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. The reprogramming efficiency of these integration-free methods is impractically low in most cases. Direct delivery of proteins or RNA is labor-intensive, requiring repeated delivery of the reprogramming factors. Modifying Sendai virus vectors or preparing synthesized mRNAs are technically demanding.

The Human iPS Cell Reprogramming Episomal Kit is an optimized mixture of multiple vectors that can reprogram somatic cells to iPSCs without integration. The episomal vectors have the oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) backbone that delivers the reprogramming factors as well as puromycin resistant gene. This system has been successfully demonstrated in reprogramming of fibroblasts, as well as other adult cells, to iPSCS. High expression of transgenes due to oriP/EBNA-1 mediated nuclear import and retention of vector DNA allows iPSC derivation in a single transfection. The reprogramming efficiency is further enhanced by puromycin selection. In addition, silencing of the viral promoter which drives EBNA-1 expression and the loss of the episomes at a rate of ~5% per cell cycle due to defects in vector synthesis and partitioning allows the removal of episomal vectors from the iPSCs without any additional manipulation.

For optimal reprogramming with the Human iPS Cell Reprogramming Episomal Kit, culture the fibroblasts in Fibroblast Medium until the day of transfection. After transfection, allow the cells to recover in Fibroblast Medium for 24 hours, and then add puromycin to remove the untransfected cells. Reseed the cells on feeders about 5 days post transfection, and switch the culture medium to hESC culture medium next day. This reprogramming protocol is very simple and the effective. PD0325901, CHIR99021, A-83-01, hLIF, or HA-100 is not required for reprogramming.

 

Create Your Own Footprint-free iPS Cells

Human iPS Cell Reprogramming Episomal Kit is a cost-effective plasimd mix of multiple episomal vectors which encode four reprogramming factors. Other reprogramming methods, such as lentivirus and retrovirus, contain transgenes that can integrate into the host genome, potentially disrupting the genome or causing unpredictable results. This episomal mix is able to efficiently generate transgene-free, virus-free and footprint-free induced pluripotent stem cells (iPSCs) in both feeder and feeder-free conditions. Optimized by ALSTEM team, this episomal mix has proven to be successful in inducing pluripotency for a number of different somatic cell types. 

These episomal vectors are introduced into the cell by electroporation. As oriP/EBNA1 vectors, they contain all the reprogramming factors and replicate extrachromosomally only once per cell cycle. At this replication rate, the episomes are lost at a rate of approximately 5% per cell generation.

Generate iPSC Lines from a Wide Variety of Somatic Cell Types

iPS cells have been generated by episomal vectors from a range of somatic cells including fibroblasts, bone marrow mononuclear cells, PBMCs, lymphoblast B cells, and various disease-type fibroblasts and PBMCs. Each kit provides enough material for 10 reprogramming experiments.