Efficient generation of iPS Cells from Adult
Human Fibroblasts with Five Factors SelfReplicating RNA (5F srRNA) Containing Oct4,
Sox2, Klf4, Glis1 and cMyc
Naohisa Yoshioka1,2, Min Lu2, Vi Chu2 and Steven F Dowdy1
1University of California, San Diego, La Jolla, CA, USA
2Cellular Assay, Biological Reagents & Kits, R&D, MilliporeSigma, Temecula, CA, USA
Results
Introduction
The generation of induced Pluripotent Stem Cells (iPSCs) has the potential
to develop regenerative medicine therapies to treat diseases. We
previously devised a RNA-based approach to generate iPSCs that uses a
polycistronic synthetic, self-replicating RNA (srRNA) that simultaneously
expresses four reprogramming factors, including OCT4, KLF4, SOX2, and
GLIS1 or c-MYC (OKS-iG or OKS-iM). We observed in some adult human
fibroblasts that the four factor (4F) srRNAs, especially OKS-iM, resulted in
low yield of iPSCs. To overcome this limitation, we engineered a five
factors (5F) srRNA that included OCT4, KLF4, SOX2, GLIS1 and c-MYC (5F
srRNA). The 5F srRNA efficiently generated iPSCs from adult human
fibroblasts, including a donor from cardiomyopathy patient. Interestingly,
5F srRNA induced LIN28, which was originally used for human iPSC
generation with OCT4, SOX2 and NANOG, and frequently used for
increasing iPSC generation with Yamanaka Factors (OCT4, SOX2, KLF4 and
cMYC). We also observed that 5F srRNA accelerated reprogramming by a
week compared to 4F srRNAs. In summary, the 5F srRNA has a
significantly greater potential for iPSC generation from difficult and slow
growing adult human cells from patient’s cells and for cell-based therapy
applications.
1: Induction of LIN28A is observed in 5F srRNA transfected cells
A
B
3: Summary of iPSC generation with srRNAs
C
(A) Expression of reprogramming
factors from srRNAs in BJ cells. (B)
TaqMan PCR analysis of Lin28A
and Nanog. Cells were cultured in
Ad-DMEM/10%FBS or ES medium
containing bFGF. (C) Western blot
analysis of Lin28A and Nanog.
2: iPS cell generation by 4F, 5F and 6F srRNAs
A: iPSC generation with neonatal fibroblasts (BJ cells)
Methods
Self-replicative RNA (srRNA) system for iPSC generation
A
Schematic of VEE-RF self-replicative RNA
B: iPSC generation from human adult fibroblasts
FB#31/55yrs/m
B
FB#32/54yrs/f
FB#33/24yrs/m (Cardiomyopathy)
C
RNA synthesis
RNA transfection & iPSC generation
C: Teratoma formation
D: srRNA degradation in
iPSC clones
+B18R
*All experiments were performed with 6-well plate, and number of AP+
colonies were calculated by starting well.
-B18R
4: 5F-iPS cell generation with iPSC booster
1: Marker: 6, 4, 3, 2, 1.5, 1, 0.5, 0.2 kb
2: OKSiG RNA, T7 transcribed
3: OKSiG RNA, Capped & Poly A tailed
4: 5F RNA, T7 transcribed
5: 5F RNA, Capped & Poly A tailed
(A) Scheme of VEE-iPSC reprogramming factors RNA. Reprogramming factors were cloned after nonstructural proteins (nsP1,2,3,4) with puromycin selection marker. (B) VEE-srRNAs were synthesized
with T7 RNA polymerase, and then enzymatically added 5’ cap and poly A tail. (C) Scheme of iPSCs
generation with VEE-srRNAs to get integration-free iPSCs.
Summary
Discussion
Comparison of Methods for iPSC generation
-5F-srRNA induced LIN28A independent of ES medium (bFGF), while
Nanog expression was ES medium dependent.
-5F-srRNA is now available form MilliporeSigma (SCR703, Human OKSGcMyc Simplicon RNA)
Retrovirus
VEE RNA + B18R,
Sendai virus,
Transgene expression
-Human iPS Reprogramming Boost Supplement (SCM088, MilliporeSigma)
was dramatically increased the efficiency of iPSC generation.
Human iPS Reprogramming
Boost Supplement
(#SCM088, MilliporeSigma)
was treated during 5F-iPSC
generation with BJ cells. AP
staining on day 21.
(A) Comparison of iPSC generation with 4F-iM, 4F-iG, 5F and 6F srRNAs in BJ cells. iPSC
colonies were stained with Alkaline Phosphatase, Tra-1-60 and SSEA4. (B) Comparison of iPSC
generation with 4F-iM, 4F-iG and 5F srRNAs in Adult human fibroblasts. (C) Teratoma formation
of 5F-iPSC clones from BJ, FB#31 and FB#33. (D) iPSC clones were picked up and cultured in
the absence of B18R. The srRNA become undetectable in 5 passaging of cells.
-We improved the 5F-srRNA for human iPSC generation.
-5F-srRNA worked very efficiently for adult human fibroblasts.
- Booster
+ Booster
Retrovirus
O/S/K/M or Glis1
stable
-integrative
VEE Replicon
O/S/K/M and/or Glis1
stable
-need B18R protein
-limit to transfectable cells
Sendai virus
O/S/K/M
mRNA
O/S/K/M and Lin28
Plus culture condition, etc
transient
-everyday transfection
-efficiency of patient cells?
Plasmid
O/S/K/M and Lin28, Nanog,
Plus p53KD or SV40LT, etc
transient
-high risk of integration
-effect of KD of p53 pathway?
- B18R
Stable Sendai virus
mRNA transfection
Plasmid (episomal),
1
10
day (s)
20
Integration
30
stable/transient
-laborious to get good quality of virus
-take time to become virus free
40
Reference: Yoshioka N, et. al. (2013) Efficient Generation of human iPSCs by a synthetic self-replicative
RNA. Cell Stem Cell 13: 246-254.
Acknowledgments: We thank NC Chi and DC Deacon for FB#31, #32, #33 cells, LS Goldstein for HFB
cells, ARI Muotri and NM Varki for teratoma analysis, RH Quintanilla and U Lakshmipathy for TaqMan
probes for srRNA and reagents.
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