What alternative methods exist for reprogramming adult cells into iPS cells, excluding methods that introduce foreign genetic material into the host cells? If there are any, what are the major limitations of these methods?
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Pluripotent stem cell (PSC) culture
Company: Life Technologies
Job Title: Customer Training Manager
We currently offer several reprogramming products that are considered non-integrating, for a complete list of reprogramming options available, visit lifetechnologies.com/reprogramming.
CytoTune –iPS Sendai Reprogramming Kits – http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/sendai-virus-reprogramming.html?cid=fl-cytotune
The CytoTune™-iPS Reprogramming Systems use vectors based on replication-incompetent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune™ Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint) and divide out over time.
The CytoTune™ –iPS systems work with both fibroblast and blood samples in feeder and feeder free conditions. There are multiple publications that reference other cell types that have been successfully reprogrammed. Learn more at this link – http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/sendai-virus-reprogramming/cytotune-publications.html
We also have two different Episomal reprogramming systems:
Epi5™ Episomal iPSC Reprogramming Kit – http://www.lifetechnologies.com/order/catalog/product/A15960?ICID=search-product
The Epi5™ Reprogramming Vectors contain an optimized mixture of three episomal vectors with an oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) backbone for delivering the reprogramming genes, Oct4, Sox2, Lin28, L-Myc, and Klf4. High transfection efficiency due to oriP/EBNA-1 mediated nuclear import and retention of vector DNA allows iPSC derivation in a single transfection (Yu et al.,2011). In addition, silencing of the viral promoter driving 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 (Nanbo et al., 2007).
We have also shown that we can use Lipofectamine® 3000 (https://www.lifetechnologies.com/order/catalog/product/L3000015?ICID=search-product) for reprogramming with the Epi5™ kit and get excellent efficiency with fibroblast reprogramming.
Episomal iPSC Reprogramming Kit – http://www.lifetechnologies.com/order/catalog/product/A14703?ICID=cvc-reprogramming-c1t1
The Episomal iPSC Reprogramming Vectors are an optimized mixture of three vectors that can reprogram somatic cells to iPSCs without integration. The three episomal vectors have the oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) backbone that delivers the reprogramming genes, Oct4, Sox2, Nanog, Lin28, L-Myc, Klf4, and SV40LT. This system has successfully demonstrated reprogramming of fibroblasts, CD34+ cells, and Peripheral Blood Mononuclear Cells (PBMCs). High transfection efficiency due to oriP/EBNA-1 mediated nuclear import and retention of vector DNA allows iPSC derivation in a single transfection.2 In addition, silencing of the viral promoter driving 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.
Current limitations to episomal systems are the need to use electroporation systems to enable the vectors to enter the cell. Keep in mind, the ability to use Lipofectamine 3000 with the Epi5™ reprogramming system does ameliorate some of this issue.