Five reasons to choose StemRNA 3rd Gen reprogramming

[fa icon="calendar"] Mar 2, 2018 10:07:01 AM / by Anca Haralambie

RNA Reprogramming is used globally for the generation of integration-free induced pluripotent stem cells (iPSCs) by researchers in biotech, academia, research hospitals and government agencies. This reprogramming method is gaining more and more interest and core labs and biotech companies focusing on regenerative medicine and GMP compatibility are adopting this technology and make it their standard.

 

REPROCELL’s latest evolution in the Stemgent RNA reprogramming kit series combines a unique non-modified RNA and microRNA technology to generate induced pluripotent stem cells (iPSCs). This novel StemRNA 3rd Gen reprogramming kit provides stem cell researchers with a new level of simplicity, versatility, and time saving, enabling cellular reprogramming of human fibroblasts, cells derived from blood and now urine even on difficult to reprogram patient samples.

StemRNA-3G-kitStemgent® StemRNA™ 3rd Gen reprogramming technology — the most rapid, robust, and clinically- relevant integration-free method on the market. REPROCELL offers StemRNA-3rd Gen Reprogramming Kits and custom iPSC production services to clients in industry and academia with over 15 years’ experience as leaders in stem cell research. REPROCELL’s custom services provide iPSCs and differentiated functional cell types suitable for your own use in the commercialization of services and products as well as research purposes. The 3rd generation RNA technology is faster, more stable and footprint-free, allowing to deliver iPSCs in around half the time of its competitors.

 

 

Five Reasons to Choose StemRNA 3rd Gen Reprogramming

 

1  Requires No Screening of iPSCs

Non-viral, non-DNA, non-integrating cellular RNA reprogramming allows for the generation of clinically-relevant iPSC lines without the need to screen clones for vector retention (footprint-free) — this saves you time and money! [See data in ref. 1]

 

2  Generates High Quality iPSCs

StemRNA-reprogrammed iPSCs are robust in culture and display very low clone-to-clone variability with normal karyology.

In the Figure 1, reprogramming progression and iPSC characterization are shown on fibroblasts, UPCs and EPCs as performed by our scientist. The left figure illustrates the progressive speed and robustness of the reprogramming process using the StemRNA 3rd Gen technology. The right figure shows, pluripotency validation of generated iPSCs by marker expression and by in vitro and in vivo differentiation assays.

 

Figure 1 (below): StemRNA 3rd Gen reprogramming technology provides high-quality, fully pluripotent iPSCs starting from skin (fibroblasts), blood, or urine

Figure 1a: Reprogramming Timeline

iPSC-reprogramming-timeline

Reprogramming protocol:
• No conditioned medium
• No feeders
• No small molecules
• No screening required
• No cell passage or splitting
iPSC Colony Morphology and Pluripotency.
Primary somatic cells at Day 1 and morphology of emerging iPSC colonies are shown at progressively later stages. The pluripotency marker TRA-1-60 shows increasing levels of expression with iPSC maturity. Expanded iPSC clones (passaged cells) illustrate stable iPSC lines out beyond passage 8.

 

Figure 1b: Pluripotency

iPSC-defferentiation-1 Immunocytochemical Staining of Urine-derived iPSCs.
The morphology and expression of key pluripotency markers and DAPI are shown by immunostaining of UPC-RNA-iPSCs (passage 7) with various antibodies.

 

Figure 1c: Differentiation

iPSC-pluripotency

Validation of Tri-lineage Differentiation of Urine-derived iPSCs.
In vitro differentiation of UPC-RNA-iPSCs (passage 11) into early endoderm (AFP), neuronal cells (nestin) and cardiomyocytes (Troponin T) is shown by immunocytochemistry.

iPSC-defferentiation-2

Validation of Tri-Lineage Differentiation of EPC-derived iPSCs.
Histological analysis of teratoma resulting from the injection of EPC-RNA-iPSCs (passage 13) into the kidney capsule of immunocompromized mice.

In addition, StemRNA 3rd Gen-derived iPSCs readily convert to naïve stem cells under typical conditons for such conversions[3]

 

3  Yields Robust and Consistent Results

The combination of a unique microRNA cocktail with the use of non-modified RNAs increases the overall efficiency (Figure 2) and enables success for refractory or difficult-to-reprogram patient-derived samples across multiple different somatic cell types (Figure 1)

Figure 2 (below): Non-modified RNA leads to a greatly increased efficiency or iPSC generaton.  Human fibroblasts were either mock transfected, transfected with traditional modified RNA, or transfected with StemRNA 3rd Gen non-modified RNA. Colonies were stained for alkaline phosphatase and counted on day 19 after the start of transfections. Data from (2).

StemRNA-3G-fig-2

 

4  Uses Safe and Clinically Relevant Methods

The non-modified mRNA in the StemRNA 3rd Gen method yields high reprogramming efficiencies of between 1-4% of primary cells, depending on the cell type, with iPSCs ready to be clonally expanded by day 10-14.

 

Figure 3 (below): Time line for fibroblast reprogramming with StemRNA 3rd Gen technology

 

StemRNA-3G-fig-3

5  Safe and clinically compatible

The non-modified RNAs of our StemRNA 3rd Gen technology are synthesized using a GMP-compatible manufacturing process.  The reprogramming protocol and the iPSC maintenance medium (NutriStem® hPSC XF Medium) are chemically defined, xeno-free and serum-free.  NutriStem is produced under cGMP compliance, with an FDA drug master file available.  The iPSCs are cryopreserved in ReproCryo™ DMSO-free medium.

 


 

Table 1: StemRNA 3rd Gen Technology Produces The Highest Quality iPSCs Available

 

Comparison of Current Non-Integrative Reprogramming Technologies1
Reprogramming technology Sendai
ThermoFisher
Episome
ThermoFisher
StemRNA
1st & 2nd Gen
REPROCELL
(Stemgent2)
StemRNA
3rd Gen
REPROCELL
(Stemgent2)
Non-integrative ?  
No vector retention × ×  
DNA-free/ Virus-free ×  ×    
No screening required × ×
Normal karyology and stability of iPSC ++3 +3  +++3 +++
Reprogramming efficiency ++3 
0.08%
+3
0.01%
++3 
1%
++
2-4% 
Reprograms refractory patient lines  × ×     
Time to usable iPSCs 8-10 weeks
(Passage 10+)
10-12 weeks
(Passage 10+)
5-6 weeks
(Passage 4-5)
4 weeks
(Passage 4-5)

1Data based on fibroblast reprogramming. 2Stemgent Inc. was acquired by REPROCELL Inc. (Japan) in 2014. 3Based on ref. 1.

 


 

REPROCELL_labs_worldwide

REPROCELL has reprogramming technical experts and stem cell laboratories on three continents 

 

Download our posters to find out more

ISSCR2015-StemRNA-NM-mobile

ISSCR2015: A novel 4 transfection protocol for deriving iPS cell lines from human blood-derived endothelial progenitor cells (EPCs) and adult human dermal fibroblasts using a cocktail of non-modified reprogramming and immune-evasion mRNAs

ISSCR2016-StemRNA-NM_poster

ISSCR2016: Use of non-modified RNAs for the derivation of clinically-relevant iPS cell lines from human blood, urine and skin cells using GMP-compliant reagents

 

References

  1. Schlaeger TM; Daheron L; Brickler TR; Entwisle S; Chan K; Cianci A; DeVine A; Ettenger A; Fitzgerald K; Godfresy M; Dupta D; McPherson J; Malwadkar P; Gupta M; Bell B; Doi A; Jung N; Li X; Zon LI; Rubin LL; Feinberg AP; Meissner A; Cowan CA; Daley GQ. A comparison of non-integrating reprogramming methods. Nature Biotechnology 33:58 (2015)
     
  2. Poleganov MA; Eminli S; Beissert T; Herz S; Moon JI; Goldmann J; Beyer A; Heck R; Burkhart I; Barea Roldan D; Türeci Ö; Yi K; Hamilton B; Sahin U. Efficient reprogramming of human fibroblasts and blood-derived endothelial progenitor cells using non modified RNA for reprogramming and immune evasion. Human Gene Therapy 26:751 (2015)
     
  3. Liu X; Nefzger CM; Rossello FH; Chen J; Knaupp AS; Firas J; Ford E; Pflueger J; Paynter JM; Chy HS; O'Brien CM; Huang C; Mishra K; Hodgson-Garms M; Jansz N; Williams SM; Blewitt ME; Nilsson SK; Schittenhelm RL; Laslett AL; Lister R; Polo JM. Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming. Nature Methods 14:1055 (2017)

 

Topics: iPSC Reprogramming, Drug Discovery, Stem Cells

Anca Haralambie

Written by Anca Haralambie

Business Development and Distributor Sales Representative, REPROCELL