Derivation Technologies

The HMGU human iPSC Core Facility utilizes established and optimized protocols for generating human iPS cells, relying primarily on methods that do not involve introduction of constructs into the genome, including episomsal plasmids and modified mRNAs (mmRNAs). Minimizing genetic alternations is important when considering clinical application of iPS cells, and for disease modeling purposes. The Core Facility is currently one of few labs around the world that provides for free a panel of human iPS cell lines reprogrammed by mmRNAs, and offers mmRNA reprogramming of adult human somatic cells as a service. The Core Facility produces human iPS cell lines by reprogramming several types of somatic cells, including fibroblasts, peripheral blood mononuclear cells (PBMCs), and lymphoblastoid cell lines. The Core Facility processes established cell cultures for reprogramming, and also generates primary cell cultures for reprogramming from biopsies and donated blood specimens.

Episome reprogramming

We offer two options for integration free episome reprogramming:

The Epi5 method is intended for peripheral blood mononuclear cell (PBMC) reprogramming with very low probability for plasmid integration. The protocol includes an option for CD34+ population enrichment (consisting of blood progenitors and stem cells), a step that often improves reprogramming efficiently.  The cell are transfected (by nucleofection) with a total of five episome vectors harboring an oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) backbone, individually expressing the reprogramming factors, POU5F1 (OCT4), LIN28A, NANOG, KLF4, CMYC and SOX2. The protocol also includes a phase of transient expression of p53 shRNAs (short inhibitory RNAs) that improve the reprogramming efficiency (Okita et al. 2011). For samples that are difficult to reprogram we boost reprogramming by including an episomal vector carrying an additional expression cassette of CMYC, LIN28A and NANOG (Okita et al. 2011).

The 4in1 method is intended for integration-free reprogramming of human fibroblasts. It is performed via nucleofection of a single vector harboring polycistronic expression cassette of reprogramming factors, POU5F1 (OCT4), KLF4, SOX2 and CMYC cloned in a minicircle plasmid backbone. The protocol also includes a phase of transient expression of p53 shRNAs (short inhibitory RNAs) which improves the reprogramming efficiency (Diecke et al. 2015).

 

 

mRNA

The mmRNA is currently the ultimate method  for reprogramming with minimal risk for genetic alternations. The RNA cocktail consists of synthetic mRNAs prepared in vitro and modified with 3’ and 5’ UTR elements that increase their stability. We have optimized the mmRNA reprogramming protocol using POU5F1 (OCT4), LIN28A, KLF4, CMYC and SOX2 for human and primate skin fibroblasts. The reprogramming schedule includes 14 transfection rounds performed daily, and additional 20 days of culturing for generating stable clones iPS cell clones.