Reprogramming Human Somatic Cells to Pluripotency Using RNA
by
Matthew Angel
M.S., Electrical Engineering and Computer Science, MIT, 2008
B.S.E., Electrical Engineering, Princeton University, 2003
Submitted to the Department of Electrical Engineering and Computer Science
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
at the
ARCHIVES
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
MASSACHUSETTS INSTTTE
S OF
TECHNOL0GY
MAR 29F
February 2012
© 2012 Massachusetts Institute of Technology
All rights reserved
JLBRARIES
Signature of Author
Department of Electri
gineering and Computer Science
October 17, 2011
Certified by
Mehmet F. Yanik
Associate Professor of Electrical Engineering and Computer Science
10
-. 0
-hesis Supervisor
Certified by
Ford Profess
glas A. Lauffenburger
& Head, Department of Biological Engineering
i Thesis Committee Chairman
Accepted by
,j,(f esie A. Kolodziejski
'
Professor of Electrical Engineering and Computer Science
Chair of the Committee on Graduate Students
Reprogramming Human Somatic Cells to Pluripotency Using RNA
by
Matthew Angel
Submitted to the Department of Electrical Engineering and Computer Science
on October 17, 2011 in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
Abstract
Somatic cells can be reprogrammed to a pluripotent stem-cell state by ectopic expression of
defined proteins. However, existing reprogramming methods take several weeks, suffer from low
efficiencies, and most use DNA-based vectors, which carry mutagenesis risks. Here, we describe
efficient and rapid reprogramming of human cells using RNA. Within two weeks, fibroblasts
from 7 adult patients, including 5 Parkinson's patients aged 53 to 85, formed colonies that
exhibited gene expression consistent with pluripotent stem cells. Established lines generated
teratomas in vivo, and differentiated into tyrosine hydroxylase-positive neurons in vitro. Genetic
analysis using array comparative genomic hybridization with an 8.9kb median probe spacing
demonstrated that RNA reprogramming can yield lines free of copy number variations. The very
high efficiency of this technique allowed us to reprogram single adult fibroblasts to pluripotency
with a 44% success rate (n = 9). Our results suggest that the efficiency and kinetics of
reprogramming methods need not be limited by a fundamental stochastic element as has been
suggested. Due to the high efficiency, speed, reliability, and integration-free nature of RNA
reprogramming, this technique will likely become the method of choice for generating diseaseand patient-specific pluripotent stem cells.
Thesis Supervisor: Mehmet F. Yanik
Title: Associate Professor of Electrical Engineering and Computer Science
Acknowledgements
The author is deeply indebted to C.B. Rohde and M.A. Scott for their thoughtful
suggestions, frequent insights, and unwavering support during the first few years of failed
experiments, and the last few months, when all of the pieces finally came together.
M.F. Yanik is to be thanked for creating the environment in which this work was done,
and for allowing the author to pursue the lengthy and, as he was often reminded, expensive
experimentation that eventually led to success.
Without the guidance of D.A. Lauffenburger, this thesis would very likely have never
been completed. He and D.M. Freeman, in addition to critical commentary, helped the author
face and overcome challenges that can arise when one works on a method with the potential for
immediate, broad utility.
S. Yamanaka and K. Okita were extremely kind in agreeing to replicate the
reprogramming protocol described in Chapter 6 using RNA supplied by the author. The author
has difficulty recalling another instance in which he has behaved so ambitiously as to send a tube
of RNA to the other side of the Earth, expecting to hear word of stem cells two weeks later.
Portions of this thesis are based on two of the author's papers, "Innate Immune
Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins",
published in PLoS ONE in July 2010, and "Reprogramming Human Somatic Cells to
Pluripotency Using RNA", submitted to Nature in April 2011, transferred to Nature
Biotechnology in August 2011, and currently under review at Nature Methods.
The author has been supported by a Hugh Hampton Young Memorial Fund Fellowship,
the MIT Biotechnology Training Program, which is funded by the National Institute of General
Medical Sciences of the National Institutes of Health, by research assistantships in the
Biophotonics, Bioscreening, and Nanomanipulation Group, later renamed the High-Throughput
Neurotechnology Group in the Research Laboratory of Electronics at MIT, and by research and
teaching assistantships in the Department of Electrical Engineering and Computer Science at
MIT.
Finally, the author would like to thank his parents, who routinely encourage him to value
scientific, professional, and personal integrity above all else.
Contents
1.
1.1 Somatic-Cell Reprogramming
2.
10
Introduction
10
. . . . . . . . . . . . . . . . . . . . . . . .. .
11
in vitro-Transcribed RNA is an Efficient Protein-Expression Vector
2.1 Design and Synthesis of Stable, Efficiently Translated in vitro-Transcribed
RN A
. . . . . . . . . . . . . . . . . . . . . . . . . ... . .
2.2 Expression of Reprogramming Proteins in Human Fibroblasts Using RNA
. .
14
.
14
.. .
2.3 Sum m ary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
3.
11
.. . . . . . . .
Innate-Immune Suppression Enables Frequent Transfection with RNA
17
Encoding Reprogramming Proteins
3.1 The Innate-Immune Response Triggered by Transfection of Human Fibroblasts
. . . .
w ith RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
3.2 A Combinatorial siRNA Screen for Suppressing Innate Immunity to Enable
Frequent RNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.
18
3.3 Sum m ary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
RNA Transfection Enables Sustained Expression of Functional Proteins
25
4.1 Sustained Expression of Functional Lin28 by Repeated RNA Transfection
-.
25
. . . . .
28
. . . . . . . . .
33
4.2 Expression of Functional Transcription Factors by RNA Transfection
4.3 Sum m ary . . . . . . . . . . . . . . . . . . . . . . . . .
. .
5.
Cap 1-Capped RNA Yields More Protein and is Less Immunogenic than
ARCA-Capped RNA
35
5.1 Expression of Proteins from in vitro-Transcribed RNA Containing the NonCanonical Nucleotides, pseudouridine and 5-methylcytidine
6.
. . . . . . . . . .
35
5.2 A Culture Medium for High-Efficiency RNA Transfection . . . . . . . . . . .
36
5.3 Immunogenicity of Cap 1-Capped and ARCA-Capped RNA . . . . . . . . . .
39
5.4 Sum mary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43
Reprogramming Human Fibroblasts to Pluripotency Using RNA
44
6.1 Early Morphological Changes During RNA Reprogramming Resemble
Mesenchymal-to-Epithelial Transition . . . . . . . . . . . . . . . . . . . . . .
45
6.2 Rapid and Efficient Formation of hES Cell-Like, Oct4/Sox2*/Nanog*/Tra- 181 + Colonies in Cultures of Primary Human Fibroblasts Transfected with RNA
Encoding Reprogramming Proteins
7.
. . . . . . . . . . . . . . . . . . . . . . .
45
6.3 c-Myc is Required for RNA Reprogramming . . . . . . . . . . . . . . . . . .
45
6.4 Reprogramming Single Human Fibroblasts to Pluripotency Using RNA
. . . .
52
6.5 Sum m ary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
56
Characterization of RNA-Reprogrammed Cell Lines
57
7.1 G ene Expression
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
57
7.2 Teratoma and Directed Differentiation . . . . . . . . . . . . . . . . . . . . . .
61
7.3 Short Tandem Repeat (STR) Analysis . . . . . . . . . . . . . . . . . . . . . .
61
7.4 Karyotype . . . . . . . . . . .
61
.
. . . . . . . . . . . . . . . . . . . . . . . .
7.5 Copy-Number Variation (CNV) Analysis by High-Resolution Array
Comparative Genomic Hybridization (aCGH)
8.
. . . . . . . . . . . . . . . . .
66
7.6 Sum m ary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
66
Conclusions
69
8.1 Proposed Directions for Future Research
. . . . . . . . . . . . . . . . . . . .
70
Appendices
A
73
Methods
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
73
A.1.1 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. 1.2 in vitro Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . .
73
A. 1Chapters 2-4
73
74
A.1.3 RNA Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. 1.4 Quantitative RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . .
74
A. 1.5 siRNA-Mediated Knockdown . . . . . . . . . . . . . . . . . . . . . . .
74
A. 1.6 Immunocytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . .
74
A.1.7 Western Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
75
A.2 Chapters 5-7
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .
. .
75
A.2.1 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
75
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
75
A.2.3 Autologous Feeder Preparation . . . . . . . . . . . . . . . . . . . . . . .
76
A.2.4 RNA Reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . .
76
. . . . . . . . . . . . . . . . . . . . .
77
A.2.2 RNA Synthesis
A.2.5 Single-Cell RNA Reprogramming
.. . . . . .
77
A.2.7 Immunocytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . ..
77
A.2.6 qRT-PCR
. . . . . . . . . . . . . . . . . . . . . . . . .. .
. . . . . . . . . . . . . . . . . . .
78
A.2.9 Karyotyping
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
A.2.10 Microarray
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
A.2.l1 aCGH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
. . . . . . . . . . . . . . . . . . . . . . . . . .
78
A.2.8 Short Tandem Repeat (STR) Analysis
A.2.12 Teratoma
A.2.13 Directed Differentiation
Bibliography
83
List of Figures
2.1
Design of an in vitro-transcription template for the synthesis of stable, efficiently
translatable RN A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
2.2
In vitro-transcribed RNA encoding reprogramming proteins . . . . . . . . . . . .
13
2.3
RNA transfection yields ES-cell-level expression of reprogramming proteins in
primary human fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
2.4
RNA transfection yields correctly localized protein . . . . . . . . . . . . . . . . .
16
3.1
Upregulation of innate immune genes following long-RNA transfection . . . . . .
20
3.2
Repeated long-RNA transfection causes cell death in human fibroblasts . . . . . .
21
3.3
Innate immune suppression enables frequent long-RNA transfection . . . . . . . .
22
3.4
Repeated, combined expression of reprogramming proteins in primary human
fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
4.1
Repeated RNA transfection yields sustained, high-level expression of Lin28 . . . .
26
4.2
Lin28 expressed using RNA is functional, and modulates let7 miRNA expression
27
4.3
RNA transfection yields functional MyoDI protein that modulate downstream
targ ets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4
RNA transfection yields functional Sox2 and Nanog that modulate a downstream
target, H m ga2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5
31
Transfection with RNA encoding Sox2 overcomes upregulation of Hmga2 induced
by co-transfection with a let7 inhibitor . . . . . . . . . . . . . . . . . . . . . . . .
5.1
29
32
Substituting 5-methylcytidine for cytidine decreases translation of pseudouridinecontaining RN A
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
37
5.2
RNA reprogramming medium enables high-efficiency RNA transfection
. . . . .
38
5.3
Cap 1-capped RNA is less toxic than ARCA-capped RNA . . . . . . . . . . . . .
41
5.4
Cap 1-capped RNA is less immunogenic than ARCA capped RNA, and B 18R
effectively suppresses the innate immune response caused by frequent RNA
transfection
6.1
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cells undergo morphological changes resembling MET within 4 days of RNA
reprogram m ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2
42
46
Both RNA reprogramming medium and B18R are required for RNA
reprogramming using feeders
. . . . . . . . . . . . . . . . . . . . . . . . . . ..
47
6.3
RNA reprogramming of adult human cells is rapid
. . . . . . . . . . . . . . . . .
48
6.4
RNA reprogramming of adult human cells is efficient . . . . . . . . . . . . . . . .
49
6.5
RNA reprogramming efficiently generates Nanog-positive colonies by day 14 . . .
50
6.6
Effect of Myc variants on RNA reprogramming efficiency . . . . . . . . . . . . .
51
6.7
Temporal requirements of c-Myc expression during RNA reprogramming . . . . .
53
6.8
Plating single fibroblasts for single-cell RNA reprogramming
. . . . . . . . . . .
54
6.9
Reprogramming single cells using RNA . . . . . . . . . . . . . . . . . . . . . . .
55
7.1
Cells reprogrammed using RNA express pluripotency markers . . . . . . . . . . .
58
7.2
Cells reprogrammed using RNA exhibit gene expression consistent with the
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
59
7.3
Cells reprogrammed using RNA exhibit ES cell-like gene expression patterns . . .
60
7.4
Parkinson's patient-derived RNA-reprogrammed cells forn teratomas in vivo . . .
62
7.5
Parkinson's patient-derived RNA-reprogrammed cells differentiate into
pluripotent stem -cell state
dopaminergic neurons in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . .
63
7.6
STR analysis of RNA-reprogrammed iPS cell lines . . . . . . . . . . . . . . . . .
64
7.7
G-banded karyotypes of seventeen RNA-reprogrammed cell lines, including two
lines derived from single, Parkinson's patient-derived fibroblasts . . . . . . . . . .
7.8
65
RNA reprogramming can generate pluripotent stem cells free of copy number
variations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
67
List of Tables
3.1
siRNA concentrations for screening
. . . . . . . ...
..............
5.1
RNA Reprogramming Medium
. . . . . . . . . . .
..............
7.1
Summary of iPS cell-line characterization experiments
..............
A.1
in vitro-transcription template synthesis . . . . . . ..
..............
A.2
RNA synthesis
. . . . . . . . . . . . . . . . . . . ..
..............
A.3
Primers for RT-PCR of endogenous transcripts
. . .
..............
A.4
Antibodies . . . . . . . . . . ..............
..............
Chapter 1
Introduction
1.1 Somatic-Cell Reprogramming
Somatic cells can be reprogrammed to a pluripotent stem-cell state by maintaining expression of
specific combinations of proteins through several rounds of cell division'-7. Although methods
for reprogramming human somatic cells using non-viral DNA vectors have been reported" , the
low efficiencies and mutation risks of these methods will likely limit their therapeutic potential.
Reprogramming by direct protein transduction has also been demonstrated'
n", however
reprogramming human cells using recombinant proteins is currently an extremely inefficient
process. Expressing reprogramming proteins by repeated transfection with protein-encoding
RNA could avoid the limitations of both DNA- and protein-based reprogramming techniques.
However, transfection with long, in vitro-transcribed RNA triggers a potent innate immune
response in human cells, even when the RNA is capped and polyadenylated to mimic eukaryotic
mRNA. To address this problem, we explored methods of suppressing innate immunity to
enable frequent transfection with protein-encoding RNA.
Chapter 2
in vitro-Transcribed RNA is an
Efficient Protein Expression Vector
2.1 Design and Synthesis of Stable, Efficiently Translated in
vitro-Transcribed RNA
The T7 bacteriophage RNA polymerase is widely used to synthesize large quantities of RNA in
vitro from a DNA template. Requirements of T7-based in vitro transcription include the
presence of a double-stranded region containing the T7 promoter sequence,
"taatacgactcactataggg", wherein the last base in this sequence is the first nucleotide incorporated
by the polymerase into the RNA transcript. We constructed linear, double-stranded templates
based on our previously described design' 2 (Fig. 2.1) containing the T7 promoter at the 5'-end,
and encoding a variety of proteins previously shown to have reprogramming activity, including
Oct4, Sox2, Klf4, c-Myc, Utfl, Nanog, Lin28, MyoD1, and Aicda. The protein-coding
sequences were surrounded by the human
p-globin (Hbb)
5'- and 3'-untranslated regions (UTRs),
which contain sequence elements that confer stability to mRNA molecules"-"'. In addition, a
strong Kozak consensus sequence was included between the 5'-UTR and the protein coding
sequence to promote efficient translation of the encoded protein. We synthesized capped,
polyadenylated transcripts using these templates and appropriate RNA-polymerase, capping, and
poly(A)-polymerase enzymes (Fig. 2.2 and Appendix A).
11
T7 Promoter
4
CDS
yNhel
TAATACGACTCACTATAGGGACATT
a a
Y
HBB 3'-UTR
CAAACAGCTAGCCACCATG e e ACCGGTGCTCGC e e e CATTGC
HBB5'-UTR
A
Kozak
AgelA
Figure 2.1. Design of an in vitro-transcription template for the synthesis of stable,
efficiently translatable RNA. The transcribed strand of an Hbb-UTR-stabilized in vitrotranscription template encoding an arbitrary protein is shown. The long arrow indicates the first
transcribed base, and short arrows indicate restriction-enzyme cleavage sites, which are used for
template assembly.
Oct4
2kb
1.5kb
-
Sox2
KIf4
c-Myc
Utf1
Nanog
Lin28
MyoD1
Aicda
WO elN
som-
1kb
0.5kb
Poly(A)-*
+
-
+
-
+
-
+
-
+
-
+
-
+
Figure 2.2. In vitro-transcribed RNA encoding reprogramming proteins. Transcripts are
shown both before and after enzymatic addition of a poly(A) tail. Samples were resolved on a
denaturing formaldehyde-agarose gel.
2.2 Expression of Reprogramming Proteins in Human
Fibroblasts Using RNA
Electroporating cells with 1pjg of each RNA (in a 50ptL total volume, see Appendix A) resulted
in protein expression at or above ES-cell levels within 6-12 hours for Oct4, Sox2, Nanog, Lin28,
and MyoDl (Fig. 2.3). We previously showed that the intracellular half-life of RNA of this
design is approximately 9-12 hours, and appears largely independent of the protein-coding
sequence 12. Interestingly, several of the factors (Oct4, Nanog, and MyoD 1) showed a dramatic
reduction in expression 12-24 hours after transfection, while other factors (Sox2 and Lin28)
remained at high levels for more than 24 hours, suggesting that in this system, the duration of
protein expression is likely determined primarily by the stability of the protein.
Immunostaining showed the correct sub-cellular localization of all factors (Fig. 2.4).
Notably, unlike direct protein transduction11 , RNA transfection resulted in strict nuclear
localization of transcription factors, suggesting that RNA transfection yields a greater fraction of
intact protein than does direct protein transduction.
2.3 Summary
RNA transfection is a simple and powerful method for expressing proteins in cells. Here we
have shown that this method can be used to express reprogramming proteins in primary human
fibroblasts at levels comparable to, and in some cases far exceeding, the levels of these proteins
endogenously expressed in hES cells. Furthermore, the strict nuclear localization of transcription
factors expressed using RNA suggests that the majority of the expressed protein is intact.
H9
RNA (pg)
0 0.1 0.2 0.5 1 2
H9
Oct4
Hours after transfection
6 12 18 24 30 36 48
WI'W
P-actin
Sox2
-actin
S-
Nanog
P-actin ow
Lin28
1-actin
00
4ISM
Am*
Rh30
0 0.1 0.2 0.5 1
we
ese
MyoD1 gi
@-actin -w
2
es mme
e
n
Rh30
m
2 4 6
8 10 12
saro
M a 00
es
0-4V090
-..
Figure 2.3. RNA transfection yields ES-cell-level expression of reprogramming proteins in
primary human fibroblasts. Western blots showing expression levels and lifetimes of Oct4,
Sox2, Nanog, Lin28, and MyoDI proteins in MRC-5 human fetal lung fibroblasts transfected
with protein-encoding RNA, relative to levels in hES (H9) and rhabdomyosarcoma (Rh30) cells.
p-actin
was used as a loading control. Left panels: The amount of RNA per 50pjL
electroporation volume was varied as indicated. Cells were lysed 6 hours after transfection.
Right panels: Cells were transfected with 1p[g of RNA, and lysed at the indicated times.
+
--..
Figure 2.4. RNA transfection yields correctly localized protein. Expression and sub-cellular
localization of Oct4, Sox2, Klf4, Utfi, Nanog, Lin28, and MyoD1 proteins following RNA
transfection. Cells were fixed and stained 6-12 hours after transfection. For each protein,
identical camera settings and exposure times were used for the RNA-transfected and mocktransfected samples.
Chapter 3
Innate-Immune Suppression Enables
Frequent Transfection with RNA
Encoding Reprogramming Proteins
The mechanisms by which cells distinguish endogenous RNA from the exogenous RNA
produced during viral infection are the subject of ongoing investigation and debate
17-23
In
humans, exogenous RNA is a pathogen-associated molecular pattern (PAMP) for which toll-like
receptor 3,7/8 (Tlr3,7/8)2 4 -27 , and members of the Rigi receptor family' 8 are pattern-recognition
receptors (PRRs). Once activated, these PRRs initiate cascades of intracellular signaling that
result in upregulation of PRRs, hypersensitizing cells to subsequent exposure to exogenous
RNA. PRR activation also results in the production of type I interferons, which hypersensitize
nearby cells. In addition, long, exogenous RNA molecules bind and activate Eif2ak2, blocking
translation of both exogenous and endogenous RNA 2 8 , 2 9 . Although the innate immune response
to exogenous RNA is initiated and regulated by intra- and extracellular signaling networks
containing a great deal of redundancy, RNA viruses have evolved methods of disrupting these
pathways by destroying or inhibiting specific immune-related proteins to enable persistent
infection3 0 . We hypothesized that mimicking viral immunoinhibition by co-transfecting cells
with an siRNA cocktail designed to directly knock down expression of immune-related proteins
could desensitize cells to exogenous RNA, and thus enable repeated long-RNA transfection.
3.1 The Innate-Immune Response Triggered by
Transfection of Human Fibroblasts with RNA
Primary human fibroblasts transfected with in vitro-transcribed RNA encoding Lin28 quickly
upregulated many genes involved in the immune response to viral RNA including Ifnbl, Tlr3,
Rarres3,Eij2ak2, Stat],2, Ifit1,2,3,5, OasI,2,3,L, and Isg2O (Fig. 3.1). Performing a second
transfection after 48 hours resulted in significant cell death (Fig. 3.2).
3.2 A Combinatorial siRNA Screen for Suppressing Innate
Immunity to Enable Frequent RNA Transfection
We previously showed that combined, siRNA-mediated knockdown of immune-related proteins
could rescue primary human fibroblasts from the cell death caused by frequent transfection with
protein-encoding in vitro-transcribed RNA12. However, effective combinations all included
siRNA targeting p53, suggesting incomplete immune suppression. To identify more effective
immunosuppressive siRNA mixtures, we conducted a two-stage combinatorial siRNA screen
starting from a list of known viral targets (Fig. 3.3). In both stages, fibroblasts were
electroporated with siRNA three times at 48-hour intervals (Table 3.1).
Table 3.1. siRNA concentrations for screening.
Stage 1:
Target
Ifnbl
Tlr3
Rarres3
Eif2ak2
Statl
Stat2
Tp53
Cdknla
Final Concentration/nM
200
200
200
200
200
200
800
200
Stage 2:
Target
I+E
I+E+T3
I+E+S2
I+E+T3+S2
I+E+T3+S 1+S2
I+E+T3+S2+TP+C
I+E+T3+S1+S2+TP+C+R
Final Concentration/nM
200+200
200+200+200
200+200+200
200+200+200+200
200+200+200+100+200
200+200+200+200+800+200
200+200+200+100+200+800+200+100
Total Concentration/nM
400
600
600
800
900
1800
2000
RNA encoding Lin28 was included in the second and third transfections. Cells were counted 24
hours after the last transfection to assess viability. In the first stage, we transfected cells with
siRNA targeting a single gene from the following list: Ifnb], T/r3, Rarres3,Eif2ak2, Stat],
Stat2, Tp53, and Cdknla. The highest viability was observed in cultures transfected with siRNA
targeting either Ifnbl or Ei/2ak2. In the second stage, we co-transfected cells with siRNA
mixtures, all including siRNA targeting both Ifnbl and Eif2ak2. Combined knockdown of Ifnb]
and Ei/2ak2 resulted in a significant increase in cell survival compared to cells transfected with
protein-encoding RNA only (p = 0.03), while adding siRNA targeting Stat2 resulted in complete
rescue of the cells (p < 0.005), which continued to proliferate at a rate comparable to the mocktransfected control.
Using this technique, we were able to transfect fibroblasts every 24 hours with RNA
encoding Oct4, Sox2, Klf4, and Utfl, a combination of factors capable of reprogramming human
fibroblasts to a pluripotent stem-cell state3 ' (Fig. 3.4). Many transfected cells expressed high
levels of all four factors, and many mitotic cells were observed.
C
<
Mock
E Long RNA
10000
1000
-
"0~
LA
0
C'
w&
(3eI4
100-
10T
_
I T,-I IiIi~ i~1~-i
I
I
T _
I
I
I
I
I
I
CDW
_j WQ<Q<
TI
-z
z
DfO
Figure 3.1. Upregulation of innate immune genes following long-RNA transfection. MRC-5
fibroblasts were transfected with 0.4ptg of RNA per well of a 24-well plate using a lipid-based
transfection reagent (see Appendix A). Expression of innate immune genes was measured by
quantitative RT-PCR 24 hours after transfection. Gapdh was used as a loading control. Error
bars indicate the standard deviation of replicate samples.
700
2 "dTransfection
600
SMock
E
LI,
400
-
1 st
Transfection
300
C
G)
a200
£
*-Long RNA
U)
u loo
3
Days
Figure 3.2. Repeated long-RNA transfection causes cell death in human fibroblasts. MRC5 fibroblasts were electroporated twice with 0.5pg/50pL of Lin28-encoding RNA at 48-hour
intervals. Samples of cells transfected with RNA (black circles) and mock-transfected cells
(gray squares) were trypsinized and counted at the indicated times. Data points and error bars
indicate the mean and standard error of two independent experiments. Data points are connected
for clarity.
0.29
0.45
0.06
0.05
0.47
SEMW
0.07
0.08
0.07
0.07
0.08
Stat1 (Si)
Stat2 (S2)
0.19
0.21
0.07
0.07
Tp53 (TP)
0.20
0.07
-
Cdknla (C)
l+E
0.34
0.65
0.07
0.09
Ic:
siRNA Mixture
No siRNA
lfnbl (1)
Tlr3 (T3)
Rarres3 (R)
Eif2ak2 (E)
I+E+T3
l+E+S2
I+E+T3+S2
I+E+T3+S1+S2
l+E+T3+S2+TP+C
I+E+T3+S1+S2+TP+C+R
IViability
0.67
0.95
0.98
0.95
1.04
0.84
0.09
0.11
0.09
0.11
0.11
0.09
I0
iO
13
ILe+
-
*
----
*
**
-u-I
**
**
0.
0
0.2
0.4
0.6
0.8
*I
1
Viability
Figure 3.3. Innate immune suppression enables frequent long-RNA transfection. Combined
knockdown of Ifnb1, Eif2ak2, and Stat2 rescues cells from the innate immune response triggered
by frequent long-RNA transfection. MRC-5 fibroblasts were transfected as in Fig. 3.2, but with
the indicated siRNA on day 0, and 0.5tg of Lin28-encoding RNA and additional siRNA on days
2 and 4 (Table Si). Samples of cells were trypsinized and counted 24 hours after the second
long-RNA transfection (day 5). Values indicate cell count relative to mock-transfected cells.
tStandard error of replicate samples (n = 4). *p < 0.05, **p < 0.005.
Lfl
6
Ln
+
Figure 3.4. Repeated, combined expression of reprogramming proteins in primary human
fibroblasts. Frequent transfection of primary human fibroblasts with a mixture of RNA
encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utfl yields sustained, ES cell-level
expression. Cells were reverse transfected with an immunosuppressive siRNA cocktail
(Lipofectamine RNAiMAX, Invitrogen), and then transfected with protein-encoding RNA
(0.1 pg of RNA per factor per well) using a lipid-based transfection reagent every day for three
days (see Appendix A). Cells were fixed and stained 8 hours after the last transfection. For each
protein, identical camera settings and exposure times were used for the mock-transfected, RNAtransfected, and hES-cell samples. Bottom row: Cells expressing reprogramming proteins
undergo mitosis. Utfl localized to chromosomes in mitotic cells (arrow).
3.3 Summary
Generating autologous pluripotent stem cells for therapeutic applications will require the
development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitrotranscribed, protein-encoding RNA is a straightforward method of directly expressing high levels
of reprogramming proteins without genetic modification. However, long-RNA transfection
triggers a potent innate immune response characterized by growth inhibition and the production
of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes
cell death.
RNA viruses have evolved methods of disrupting innate immune signaling by destroying
or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral
targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize
cells to exogenous RNA. We show that combined knockdown of interferon-p (Ifnb]), EiJ2ak2,
and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA
transfection. Using this technique, we were able to transfect primary human fibroblasts every 24
hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utfl.
Our results demonstrate that suppressing innate immunity enables frequent transfection
with protein-encoding RNA. This technique represents a versatile tool for investigating
expression dynamics and protein interactions by enabling precise control over levels and timing
of protein expression. Our finding also opens the door for the development of reprogramming
and directed-differentiation methods based on RNA transfection.
Chapter 4
RNA Transfection Enables Sustained
Expression of Functional Proteins
4.1 Sustained Expression of Functional Lin28 by Repeated
RNA Transfection
To determine whether RNA transfection could sustain high-level expression of biologically
active protein through multiple rounds of cell division, we repeatedly transfected fibroblasts with
siRNA targeting Ifnbl, Eif2ak2, Stat2, and Tlr3, and RNA encoding Lin28. Lin28 is a
cytoplasmic, RNA-binding protein that is highly expressed both in embryonic stem cells, where
it regulates cell growth32 , and in several cancers where it interferes with the maturation of
members of the let7 family of miRNAs3 3 -
,
which regulate Hmga2 and downstream targets such
as Snail that promote metastasis and invasion 3- . We transfected fibroblasts five times at 48hour intervals with Lin28-encoding RNA, and measured the levels of Lin28 protein and let7a
miRNA (Figs. 4.1-4.2). Lin28 protein was detected as early as six hours after the first
transfection, remained highly expressed for two to three days, and was detected up to five days
after each transfection. The level of let7a miRNA began to decrease two days after the first
transfection, and in cells transfected only once, reached approximately 50% of the level in mocktransfected cells (p = 0.02), while in repeatedly transfected cells the level of let7a continued to
decrease, reaching approximately 10% of the level in mock-transfected cells one day after the
25
H9
Lin28
1-actin
Mock 6h 12h 18h 1d
:s
2d 3d
,
4d
5d
76d
7d
Lin28
1S
Tran
sfectiont
Lin
p-actin8d
8d
d
9d
tLin28
2"' Transfectionto-actin **i
6W*
tLin
P-actin 1
SV
28
3 rd Transfection
0
EA
**
Lin28
4th TransfectionI
1
12d 13d
ii
-actin geKW 0
0"
eseO
5th Transfectiontl
Figure 4.1. Repeated RNA transfection yields sustained, high-level expression of Lin28.
MRC-5 fibroblasts pre-transfected with a cocktail of siRNAs targeting Ifnbl, Eif2ak2, Stat2, and
Tlr3 were transfected five times with 0.5ptg of Lin28-encoding RNA and additional siRNA at 48hour intervals. Cells were lysed at the indicated times, and the amount of Lin28 protein was
analyzed by western blot.
p-actin was used as a loading control.
A
B
1.2
1.2
*
I
0)_ 0.8
T
0.6
0.4
"
10.
(3 0.8
|0.6
0.4
'
0.2
-
0.2
>
0
0
0
2
4
6
8
Days
10
12
0
1
2
3
4
Days
5
6
Figure 4.2. Lin28 expressed using RNA is functional, and modulates let7 miRNA
expression. Sustained expression of Lin28 downregulates its target, mature let7 miRNA.
Transfections were conducted as in Fig. 4.1. A. Data points indicate mature let7a levels in cells
transfected once (circles), twice (squares), three times (diamonds), four times (triangles), or five
times (crosses), relative to the level in mock-transfected cells. A solid smoothed line connects
data points corresponding to cells transfected once, and a dashed smoothed line connects data
points corresponding to cells transfected five times (dark symbols). U47 RNA was used as a
loading control. Error bars indicate the standard error of replicate samples. B. let7a
downregulation is Lin28-specific. Cells were transfected as in (A), but with MyoD1-encoding
RNA. Error bars indicate the standard error of replicate samples.
fifth transfection (p = 0.004) (Fig. 4.2A). Regardless of the number of transfections, let7a
expression returned to normal levels approximately four days after the final transfection. The
level of let7a miRNA in cells repeatedly transfected with MyoD 1-encoding RNA remained
within 70% of the level in mock-transfected cells, suggesting that the decrease in let7a
expression following transfection with Lin28-encoding RNA was not a non-specific effect of
long-RNA transfection (Fig. 4.2B). The observed decrease in the level of mature let7a miRNA
in cells transfected five times over the course of ten days with Lin28-encoding RNA indicates
both that each transfection was efficient, and that the translated protein was active.
4.2 Expression of Functional Transcription Factors by
RNA Transfection
We next used RNA transfection to express other known reprogramming proteins. Many of these
proteins are transcription factors, which, unlike Lin28, must translocate to the nucleus and
interact with genomic DNA to exert their function. We electroporated fibroblasts with RNA
encoding the skeletal-muscle master gene MyoD1, and detected a high level of MyoDl protein
six hours after transfection (Fig. 4.3A). Two targets of MyoDI that are normally silenced in
fibroblasts, M-cadherin (Cdh15) and desmin (Des), were detected as early as six hours after
transfection, and their expression peaked after 12-24 hours (Fig. 4.3B). Interestingly, expression
of Cdhl5 and Des in cells treated with the demethylating agent 5-aza-dC showed similar
dynamics, but reached a peak level ten times higher than in the untreated cells, suggesting that
MyoD1 -induced activation of Cdh15 and Des in fibroblasts is inhibited by genomic methylation.
Because of the high transfection efficiency and activity of proteins expressed by longRNA transfection, we hypothesized that this technique could be used to investigate early targets
of reprogramming factors in somatic cells. Genes encoding pluripotent-stem-cell master
regulators such as Oct4 and Nanog are highly methylated in somatic cells, and as a result
transient expression of proteins that promote transcription of these genes (Oct4, Sox2, and
Nanog, for example) does not immediately cause their expression. Instead, somatic cell
Rh30
Hours after
transfection
6 12 24 48
B
0-
AZA-
AZA+
C1 .5
MyoD1
10
1
0
U.'
L=0
5
MyoD1
0
1-actin
12
.0
------ 0
24
36
A
1
O-actin
m
MyoD1
*41..
0-actin
-
0
12
24
36
48
15
.5
MyoD1
0
48
10
0
--
5
.5
LU0
0
0
0
0
.-------12
24
48
36
Hours after transfection
0
0
12
24
36 48
Hours after transfection
Figure 4.3. RNA transfection yields functional MyoD1 protein that modulate downstream
targets. A. Expression of MyoD 1 protein in fibroblasts. Fibroblasts cultured for three days with
or without 2.5 pM 5-aza-dC (AZA) were electroporated with 1pgg/50pL of MyoDl-encoding
RNA. Cells were lysed at the indicated times, and the amount of MyoD 1 protein in each sample
was analyzed by western blot. B. Expression of MyoDI in fibroblasts activates its normally
silent targets, Cdh15 and Des in a methylation-dependent manner. Cells were transfected as in
(A), and expression of CdhJ5 and Des was measured by RT-PCR at the indicated times (squares,
mock-transfected cells; circles, RNA-transfected cells).
reprogramming may first require downregulation of somatic-cell genes, together with
upregulation of ES-cell genes that are not completely silenced in somatic cells. One such gene,
Hmga2, encodes a small chromatin-associated protein that cooperates with other factors to
40
regulate gene expression. Hmga2 is highly expressed in embryonic stem cells , young neural
stem cells 41 , and many human cancers, and is moderately expressed in various adult tissues
including fibroblasts. Overexpressing Hmga2 induces pituitary tumours in mice by binding to
and inhibiting retinoblastoma protein 4 2 , a tumour suppressor. Hmga2-induced pituitary
43
adenomas exhibit >5-fold downregulation of Sox2 compared with normal pituitary tissue . We
hypothesized that a reciprocal relationship might exist as a mechanism by which Sox2-expressing
stem cells regulate Hmga2 expression. We transfected fibroblasts with RNA encoding Oct4,
Sox2, Nanog, Lin28 or MyoDI, and measured expression of Hmga2 after 24 hours (Fig. 4.4).
As expected, cells transfected with RNA encoding Lin28 (which downregulates let7 miRNA,
which itself downregulates HInga2) showed slight overexpression of Hmga2 (p = 0.03), while
the level of Hmga2 mRNA in cells transfected with RNA encoding Nanog was approximately
3.5 times that in mock-transfected cells (p = 0.002), and expression in cells transfected with
RNA encoding Sox2 was approximately 0.5 times that in mock-transfected cells (p = 0.002).
The high level of Hinga2 expression in ES cells, combined with the upregulation of Hmga2
observed in fibroblasts transfected with RNA encoding Nanog suggests that Hmga2 may be an
early downstream target of Nanog in fibroblasts during reprogramming.
To determine whether downregulation of Hinga2by Sox2 is sufficient to counteract
Hmga2 upregulation caused by inhibition of let7, we co-transfected cells with a let7-miRNA
inhibitor and RNA encoding Sox2, and measured Hmga2 expression after 24 hours (Fig. 4.5).
While cells transfected with only the let7 inhibitor showed approximately 3-fold upregulation of
Hmga2, those transfected with both the inhibitor and Sox2-encoding RNA expressed Hmga2 at
the same level as mock-transfected cells, suggesting that an ES-cell level of Sox2 is sufficient to
replace let7-mediated downregulation of Hnga2. The competing roles of Sox2, Nanog, and
Lin28 in the regulation of Hmga2 highlight the complex interactions between these factors that
likely take place during reprogramming.
4
0
a)
.
CU
L
Oct4
Sox2 Nanog Lin28 MyoD1
Figure 4.4. RNA transfection yields functional Sox2 and Nanog that modulate a
downstream target, Hmga2. Regulation of Hinga2 expression by reprogramming proteins.
Hmga2 expression in fibroblasts transfected with RNA encoding the indicated protein was
measured by RT-PCR 24 hours after transfection. Values are given relative to mock-transfected
cells. Gapdh was used as a loading control. *p < 0.005.
31
C
*
0
T
II
~1~
(.
C1
1
Mock
Sox2
Lin28
MyoDI
Figure 4.5. Transfection with RNA encoding Sox2 overcomes upregulation of Hmga2
induced by co-transfection with a let7 inhibitor. Hmga2 expression in fibroblasts cotransfected with RNA encoding reprogramming proteins and a let7a inhibitor was measured by
RT-PCR, 24 hours after transfection. Values are given relative to mock-transfected cells that
received neither long RNA nor the let7a inhibitor. Gapdh was used as a loading control. *p
0.004.
4.3 Summary
RNA transfection is a versatile tool for investigating expression dynamics and protein
interactions. In addition, the ability to maintain high-level expression of defined proteins in
human cells for many days without genetic manipulation highlights the potential importance of
RNA transfection in the development of reprogramming methods for therapeutic applications.
Although techniques for in vitro synthesis of large quantities of capped, polyadenylated RNA
have been available for some time44'45, as have a variety of delivery techniques including
electroporation and lipid-mediated transfection 1 3,46, the potent immune response triggered by
RNA transfection has largely limited its use to studies of immunity, and has prevented the
development of RNA-based reprogramming methods.
Here we have shown that combined knockdown of Iffnb1, Eif2ak2, and Stat2 rescues
human fibroblasts from the innate immune response triggered by frequent transfection with
protein-encoding RNA, and enables sustained, high-level expression of active proteins.
Interestingly, while we previously found that p53 knockdown alone increased the rate of
recovery of cells transfected with long RNA' 2 , we now demonstrate that combined knockdown
of Ifnb], Eif2ak2, and Stat2 is sufficient to allow frequent transfection with protein-encoding
RNA, eliminating the need for p53 knockdown, which may facilitate the use of RNA transfection
in therapeutic applications as p53 is crucial for the maintenance of genomic integrity.
RNA transfection enables precise control over the timing and level of expression of
encoded proteins. We used this characteristic to investigate the regulation of downstream targets
of the cytoplasmic RNA-binding protein Lin28, and the transcription factor and skeletal-muscle
master regulator MyoDl 1. In addition, we used RNA transfection to search for early targets of
reprogramming factors in fibroblasts, and we provide evidence that the pluripotent-stem-cell
master genes and reprogramming factors Sox2 and Nanog are novel upstream effectors of the
proto-oncogene Hmga2.
Finally, while we have shown that siRNA-mediated immunosuppression alone is
sufficient to enable frequent RNA transfection, the use of small-molecule immunosuppressants
(for example, glucocorticoids such as cortisone 47 or dexamethasone), and/or protein
4
immunosuppressants such as B 18R, a vaccinia-virus-encoded soluble type I interferon receptor '
either alone or in combination with siRNA may increase the quantity of RNA that can be
delivered to cells and the frequency of transfection, two parameters that will likely be critical in
49,
the design of efficient RNA-based reprogramming methods. The discovery that innate immune
suppression enables frequent RNA transfection thus provides a clear path toward the
development of culture and transfection protocols for RNA-based reprogramming.
Chapter 5
Cap 1-Capped RNA Yields More
Protein and is Less Immunogenic than
ARCA-Capped RNA
The mRNA of higher eukaryotes is methylated at the 2'-O of the second nucleotide. RNA
containing this modification, which together with the 7-methylguanosine cap forms the Cap I
structure, is translated more efficiently by eukaryotic cells than RNA containing a standard cap50 .
In addition, the Cap 1 structure can be generated enzymatically, resulting in near 100% capping
efficiency 1, while co-transcriptional incorporation of synthetic cap analogues results in a
mixture of capped and uncapped RNA 5 2,s3. Also, Cap 1-capped RNA evades detection by
receptors of the innate immune system.54 '
We therefore reasoned that incorporating the Cap 1
structure into in vitro-transcribed RNA could result in reduced induction of innate immunity
during repeated transfection.
5.1 Expression of Proteins from in vitro-Transcribed RNA
Containing the Non-Canonical Nucleotides, pseudouridine
and 5-methylcytidine
We synthesized capped, tailed RNA encoding Oct4 and substituted pseudouridine (')triphosphate, 5-methylcytidine (5mC)-triphosphate or both T-triphosphate and 5mC-triphosphate
protocol1 2,5 6
for UTP and CTP in the in vitro-transcription reaction. We followed our published
to generate RNA containing either the Cap 0 structure or the Cap 1 structure, which has recently
been shown to reduce the immunogenicity of RNA by inhibiting restriction by members of the
IFIT family of pathogen recognition receptors54 . We plated fibroblasts in 6-well plates at a
density of 1x10 5 cells/well. The following day, we transfected the cells with 0.5ug/well of Oct4encoding RNA. The culture medium was replaced 4 hours after transfection, and the plates were
fixed and stained for Oct4 protein 12 hours after transfection (Fig. 5.1).
RNA based on the design that we previously described 12,56 (unmodified, Cap 1) yielded many
cells with brightly stained nuclei. Incorporating T increased the amount of translated protein by
approximately 4 fold, while incorporating 5mC yielded a negligible increase. These results
agree with the results presented by Kariko, et al. that T increases protein translation from in
57
vitro-transcribed RNA, and that the effect of 5mC-incorporation is more modest . Incorporating
both T and 5mC decreased the amount of protein translation relative to unmodified RNA by
roughly 2 fold. We obtained nearly identical results from independent batches of RNA encoding
GFP and mCherry, and using 5mC-triphosphate obtained from two different vendors. We also
obtained similar results using RNA synthesized with the Cap 0 structure, although with every
nucleotide combination, protein translation was significantly reduced when compared to the
corresponding Cap 1-capped RNA.
5.2 A Culture Medium for High-Efficiency RNA
Transfection
We previously reported that human fibroblasts transfected with in vitro-transcribed RNA
encoding reprogramming factors can translate correctly localized, functional protein5 6 . However,
although the viability of fibroblasts cultured in standard hES cell-culture medium is high, we
found that the efficiency of RNA transfection in this medium is low (Fig. 5.2). To solve this
A
Unmodified
4W
i+
SmC (Trilink) SmC (Trilink)
qj+
5mC (BA)
B
B
_____________________________________________
Cap
0
28
o~
C
.2
Unmodified
W
mC (Trilink)
W +5mC
W + 5mC
(Trilink)
(IBA)
Figure 5.1. Substituting 5-methylcytidine for cytidine decreases translation of
pseudouridine-containing RNA. A. MRC-5 fibroblasts were transfected with Oct4-encoding
RNA containing complete substitution with pseudouridine ('T) and/or 5-methyleytidine (5mC)
and either the Cap 0 or Cap I structure. Cells were fixed and stained 12 hours after transfection.
Identical camera settings and exposure times were used to capture each image. Two random
fields are shown for each sample. B. Relative protein translation was determined by analyzing
the images shown in (A). A background threshold was determined by taking the maximum pixel
intensity outside a cell nucleus, and subtracting that value from all of the pixels before
calculating the mean pixel intensity. The same threshold was used for all of the images. Error
bars indicate the standard error of intensity from the two random fields.
a
hES-cell medium
RNA reprogramming medium
b
60
50
840
30
20
10
a. 0
0
6
-
12 18 24 30 36 42 48
Hours after transfection
hES-cell medium
RNA reprogramming medium
1mm
Figure 5.2. RNA reprogramming medium enables high-efficiency RNA transfection. CCD1124Sk adult dermal fibroblasts were plated at 200,000 cells/well of a 6-well plate and were
cultured either in standard hES cell-culture medium (DMEM/F 12 + 20% serum replacer
(Invitrogen) + IX NEAA + 2mM L-glutamine + 50uM p-mercaptoethanol + 20ng/mL bFGF) +
200ng/mL B 18R or in RNA reprogramming medium (with 200ng/mL B 18R). A single RNA
transfection was performed as described in Appendix A. Cells were fixed and stained for Oct4 0,
6, 12, 24, and 48 hours after transfection. a, A representative image of cells fixed 12 hours after
transfection showing many more cells expressing Oct4 when cultured in RNA reprogramming
medium. b, Cells translate approximately 30 times more protein when cultured in RNA
reprogramming medium vs. standard hES-cell medium. Images of random 4mm x 4mm fields
were thresholded to remove the extra-nuclear background, and the average pixel intensity was
determined. Error bars: s.e.m. (n=4).
problem, we used a culture medium (Table 5.1) that enables high-efficiency RNA transfection,
which we called "RNA reprogramming medium".
RNA reprogramming medium consists of a simple buffered mixture of amino acids,
vitamins, lipids, and other supplements. The only proteins are insulin, transferrin, bFGF, and
human serum albumin. Other than ascorbic acid58 , the medium contains no small molecules that
are known to enhance reprogramming. Fibroblasts transfected with RNA encoding a mixture of
reprogramming factors, and cultured in RNA reprogramming medium, translated approximately
30 times more Oct4 protein after 12 hours than fibroblasts cultured in standard hES cell-culture
medium, but transfected under otherwise identical conditions (Fig. 5.2). We thus used RNA
reprogramming medium for all subsequent RNA transfection experiments.
5.3 Immunogenicity of Cap 1-Capped and ARCA-Capped
RNA
Adult human dermal fibroblasts transfected daily with ARCA-capped RNA suffered massive cell
death after only three transfections (Fig. 5.3). In stark contrast, cells transfected under identical
conditions with Cap 1-capped RNA exhibited high viability. Samples of RNA were extracted
each day and analyzed for expression of innate immune-related genes (Fig. 5.4). Cells
transfected with ARCA-capped RNA upregulated interferon-p (IFNB 1), toll-like receptor 3
(TLR3), retinoic acid receptor responder (tazarotene induced) 3 (RARRES3, a.k.a. RIG-I),
IFITI, IFIT2, IFIT3, OAS 1, OAS2, and OASL. Four of these genes, IFIT2, IFIT3, OAS2, and
OASL, were >1 00-fold upregulated after only two transfections. These four genes were less
upregulated in cells transfected with Cap 1-capped RNA (4,6,7, and 12-fold, respectively),
suggesting that Cap I-capped RNA is less immunogenic than ARCA-capped RNA, despite
phosphatase treatment of the ARCA-capped RNA to remove the 5'-triphosphate from the
uncapped fraction 59. Supplementation of the culture medium with B 18R, a vaccinia virusencoded decoy receptor for Type I interferons4 8 ' 49, dramatically reduced the upregulation of
innate immune-related genes in cells transfected with either ARCA-capped or Cap 1-capped
RNA (Fig. 5.4). However, even in the presence of B 18R, three of the genes that we measured,
39
Table 5.1. RNA Reprogramming Medium.
2
Amino Acids
mg/L
D-alpha-tocopherol acetate
glycine
18.75
Inorganic Salts
mg/L
calcium chloride
116.6
L-alanine
L-alanyl-L-glutamine
L-arginine hydrochloride
L-asparagine
L-aspartic acid
4.45
542
147.5
7.5
6.65
cupric sulfate
ferric nitrate
ferric sulfate
magnesium chloride
0.0013
0.05
0.417
L-cysteine hydrochloride
L-cystine dihydrochloride
17.56
magnesium sulfate
28.64
48.84
31.29
potassium chloride
311.8
L-glutamic acid
L-histidine hydrochloride
7.35
31.48
sodium bicarbonate
2438
L-isoleucine
54.47
L-leucine
L-lysine hydrochloride
59.05
sodium chloride
sodium phosphate dibasic
sodium phosphate monobasic
L-methionine
91.25
17.24
zinc sulfate
Other
L-phenylalanine
35.48
HEPES
L-proline
17.25
D-glucose
3151
L-serine
26.25
2.39
L-threonine
53.45
hypoxanthine
linoleic acid
L-tryptophan
L-tyrosine disodium salt dihydrate
9.02
55.79
L-valine
Vitamins
52.85
MR/L
biotin
choline chloride
0.0035
8.98
lipoic acid
cholesterol
cod liver oil fatty acids
(methyl esters)
Tween-80
Pluronic F-68
D-calcium pantothenate
folic acid
2.24
2.65
phenol red
putrescine dihydrochloride
niacinamide
pyridoxal hydrochloride
2.02
sodium pyruvate
6999.5
71.02
62.5
0.432
mg/L
15mM
0.042
0.105
4.5
10
25
0.1%
8.1
0.081
55
pyridoxine hydrochloride
2 thymidine
2.031 insulin
0.365
5
riboflavin
0.219
transferrin
5
selenius acid
thiamine hydrochloride
vitamin B12
2.17
0.68
i-inositol
ascorbic -aci d-2-phosphate
12.6 bFGF
1 B18R
human serum albumin
.005
0.5%
0.02
0.2
ARCA -B18R
ARCA +BIR
Can 1 +B18R
0
CU
CU
0
CU
0
O')
I
I
Figure 5.3. Cap 1-capped RNA is less toxic than ARCA-capped RNA. CCD- 1124Sk adult
dermal fibroblasts were plated at 50,000 cells/well of a 6-well plate, and were transfected under
identical conditions with identical quantities of either ARCA-capped or Cap 1-capped RNA as
described in Appendix A.
RARRES3
TLR3
IFNBI
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Days of transfection
Days of transfection
IFIT2
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IFITI
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Days of transfection
Days of transfection
Days of transfection
OASI
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C 101
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041
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+
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Days of transfection
ARCA-capped RNA without B18R
Cap 1-capped RNA without B18R
+
-
2
-
ARCA-capped RNA +200ng/mL B18R
Cap 1-capped RNA +200ng/mL BI8R
Figure 5.4. Cap 1-capped RNA is less immunogenic than ARCA capped RNA, and B18R
effectively suppresses the innate immune response caused by frequent RNA transfection.
RNA was extracted from the cells shown in Fig. 5.3, and gene expression was measured by RTPCR. An asterisk ("*") indicates that due to the excessive cell death in wells transfected with
ARCA-capped RNA without B I8R, the quantity of RNA extracted from these samples on day 4
and day 5 was insufficient for analysis. Error bars: s.d. (n = 2 replicate samples).
IFIT2, IFIT3, and OASL, were still upregulated in cells transfected with ARCA-capped RNA
(23, 11, and 14-fold, respectively), compared to cells transfected with Cap 1-capped RNA (2.2,
2.0, and 1.0-fold, respectively), showing that under these conditions, B 18R was not able to
completely suppress the immune response to ARCA-capped RNA.
5.4 Summary
Here, we present a culture medium for high-efficiency RNA transfection. Fibroblasts cultured
and transfected in this medium expressed 30-times more Oct4 protein when transfected with
Oct4-encoding RNA, than when cultured in standard hES cell-culture medium, and transfected
under identical conditions.
Furthermore, to test the ability of 5-methyleytidine incorporation to increase protein
translation from pseudouridine-containing RNA, we synthesized Oct4-encoding RNA containing
combinations of these nucleotides. We transfected fibroblasts with these RNAs and measured the
expression of Oct4 protein by immunocytochemistry. We show that incorporation of
pseudouridine increases protein translation from in vitro-transcribed RNA, in agreement with
previous results 57. However, we find that adding 5-methylcytidine to pseudouridine-containing
RNA decreases protein translation to a level comparable to or less than that of unmodified RNA
in fibroblasts. Additionally, we show that our previously published RNA design, which
incorporates the Cap 1 structure, yields increased protein translation compared to RNA
containing the standard Cap 0 cap in both modified as well as unmodified RNA.
Finally, we demonstrate that Cap 1-capped RNA is less immunogenic than ARCAcapped RNA, and that primary human fibroblasts cannot tolerate frequent transfection with
ARCA-capped RNA, except in the presence of an immunosuppressant, and even then, only with
low viability. In contrast, cells exhibit high viability after frequent transfection with Cap Icapped RNA, either with or without an immunosuppressant.
Chapter 6
Reprogramming Human Fibroblasts
to Pluripotency Using RNA
Somatic cells can be reprogrammed to a pluripotent stem-cell state by ectopic expression of
defined proteins' 5 . However, existing reprogramming techniques take several weeks, suffer from
extremely low efficiencies, and use DNA-based vectors, which carry mutagenesis risks.
RNA transfection is a powerful method for expressing high levels of proteins both in vitro
and in vivo that avoids the integration risks of DNA-based vectors. However, transfection with
long, in vitro-transcribed RNA molecules triggers a potent innate immune response that causes
cell death. We previously demonstrated that suppressing the innate immune response of cells to
2
exogenous RNA enables frequent transfection with RNA encoding reprogramming proteins. '
56.
A recent publication reported low-efficiency (~1%) reprogramming of neonatal fibroblasts using
RNA containing a cap analogue (ARCA)5 9 . In Chapter 5, we showed that ARCA-capped RNA is
highly immunogenic, and that frequent transfection with this RNA results in excessive cell death,
even in the presence of a potent immunosuppressant. Here, we demonstrate that repeated
transfection with Cap 1-capped RNA encoding reprogramming factors results in efficient, rapid,
and reliable reprogramming of adult human fibroblasts to pluripotency.
6.1 Early Morphological Changes During RNA
Reprogramming Resemble Mesenchymal-to-Epithelial
Transition
Using RNA reprogramming medium, neonatal and adult human fibroblasts transfected daily with
Cap 1-capped RNA encoding Oct4, Sox2, Klf4, a stabilized c-Myc mutant (T58A) 60 , and Lin28
underwent extensive morphological changes resembling mesenchymal-to-epithelial transition
within 4 days (Fig. 6.1). No morphological changes were observed using either standard hES
cell-culture medium with B 18R or RNA reprogramming medium without B 18R (Fig. 6.2).
6.2 Rapid and Efficient Formation of hES Cell-Like,
Oct4+/Sox2+/Nanog+/Tra-1-81+ Colonies in Culture of
Primary Human Fibroblasts Transfected with RNA
Encoding Reprogramming Proteins
During the two weeks after the first transfection, hundreds of large, hES-cell-like colonies
emerged, which by day 14 could be picked for expansion (Fig. 6.3-6.4). More than 80% of these
colonies also expressed Nanog (Fig. 6.5), a highly specific marker of pluripotent stem cells.
6.3 c-Myc is Required for RNA Reprogramming
Myc variants have recently been shown to reprogram cells with varying efficiency6 1 . We tested
the ability of RNA encoding wild-type c-Myc-2 and c-Myc-2 (T58A) to reprogram adult
fibroblasts. Transfection with either wild-type c-Myc-2 or c-Myc-2 (T58A) resulted in many
Tra-1-81-positive colonies by day 14, while no morphological changes were observed when Myc
was omitted, and the cultures quickly deteriorated (Fig. 6.6). We next examined the temporal
requirements of c-Myc expression during RNA reprogramming. In the case of viral
reprogramming, maintaining expression of c-Myc for 3-5 days is sufficient to reprogram cells 62.
We found that expressing c-Myc for between 2-6 days using RNA was sufficient to generate
45
a
b
Figure 6.1. Cells undergo morphological changes resembling MET within 4 days of RNA
reprogramming. BJ fibroblasts were either mock transfected (a) or transfected with RNA as
described in Appendix A (b), and were imaged on day 4.
a
b
C
6.2. Both RNA reprogramming medium and B18R are required for RNA reprogramming
using feeders. CCD- 1124Sk adult dermal fibroblasts were reprogrammed using RNA as
described in Appendix A using standard hES cell-culture medium + 200ng/pL B 18R (a), RNA
reprogramming medium without B 18R (b) or RNA reprogramming medium + 200ng/pL B 18R
(c). Cells are shown on day 5 of reprogramming.
Day 14 (isolation)
Day 15 (BJ-iPS-1)
Figure 6.3. RNA reprogramming of adult human cells is rapid. BJ fibroblasts undergoing
RNA reprogramming. 50,000 cells were plated on mitotically inactivated feeders on day -1. Cells
were transfected every day for 12 days (day 0 to day 11) with RNA, with a single passage (1:20)
on day 6. The colony shown was picked on day 14 to establish the BJ-iPS-1 line.
Tr2.1 .1
0
C
1cm
-0
1cm
Figure 6.4. RNA reprogramming of adult human cells is efficient. Pluripotency-marker
expression of BJ and CCD-1I124Sk adult fibroblasts undergoing RNA reprogramming. Cells
were transfected as in Fig 6.3, and stained on day 14. For the adult cells, 10,000 cells were plated
on day -1, and the passage was performed on day 5 with a split ratio of 1:50. Colony counts are
shown in the corner of each image.
Feeders
Cells
RNA
+
+
+
+
+
+
-+
b
c Hoechst
Tra-1-81
Nanog
Figure 6.5. RNA reprogramming efficiently generates Nanog-positive colonies by day 14.
CCD- 1124Sk adult dermal fibroblasts were reprogrammed using RNA as described in Appendix
A and were stained for Tra- 1-81 (a) and Nanog (b) on day 14. Colony counts are shown in the
corner of each image. c, Higher magnification images of two representative colonies.
....
..
........
..........
..
-..........
.....
......
..
..
..
c-Myc-2 (T58A)
(1:50)
1cm
c-Myc-2 (wt)
(1:20)
Without Myc
(1:1)
Figure 6.6. Effect of Myc variants on RNA reprogramming efficiency. Parkinson's patientderived fibroblasts (AG20442) were reprogrammed using RNA encoding the indicated Myc
variant. Cells were passaged on day 5 using the indicated split ratio. A total of 12 transfections
were performed, and the cells were stained for Tra-1-81 on day 14. Colony counts are shown in
the corner of each image.
Tra- 1-81-positive colonies, supporting the conclusion that expression of c-Myc is necessary only
during the early stages of reprogramming (Fig. 6.7).
6.4 Reprogramming Single Human Fibroblasts to
Pluripotency Using RNA
The extremely high efficiency that we achieved in these experiments led us to determine whether
this method could be used to reprogram single cells. We isolated a single adult fibroblast (Fig.
6.8), and began transfections the following day (day 0). A total of 9 transfections were
performed, and by day 11 a small colony of compact, high-phase cells became visible (Fig. 6.9).
The resulting colony was picked on day 14. We made nine attempts to reprogram a single cell,
five using CCD-1 124Sk adult dermal fibroblasts, and 4 using AG20442 Parkinson's patientderived fibroblasts. Four attempts (two from each patient) resulted in the establishment of iPS
cell lines with DNA fingerprints matching the parent fibroblasts, one attempt resulted in a line
with a fingerprint matching the feeders, and in four of the nine attempts, no area of cell growth
was found, suggesting that the cell had either died or become senescent after the isolation and
plating process.
The successful generation of iPS cell lines from single adult fibroblasts with minimal
optimization of RNA dose and transfection frequency suggests that RNA reprogramming is a
reliable and efficient method for reprogramming single cells. In addition, these results allowed us
to directly calculate the efficiency of RNA reprogramming. While reprogramming efficiency is
typically defined as the number of colonies obtained as a fraction of the input population, we
found that attempting to calculate efficiency using this definition led to efficiencies greater than
100%. For example, in the adult fibroblast reprogramming experiment shown in Fig. 6.4, 371
colonies were obtained after a 1:50 passage, yielding an estimated total of 18,550 colonies from
10,000 starting cells (assuming all of the cells from the passage had been saved), and a
corresponding efficiency of 185.5%. Partially reprogrammed colonies are broken up during the
passage, which confounds calculations of reprogramming efficiency using the standard
definition. However, because the single cell-reprogramming experiments did not include a
.AGM
Figure 6.7. Temporal requirements of e-Myc expression during RNA reprogramming.
CCD- 1124Sk adult dermal fibroblasts were reprogrammed using RNA as described in Appendix
A. RNA encoding c-Myc-2 (T58A) was included for the indicated number of days, beginning on
day 0. Cells were passaged on day 5 using a split ratio of 1:50. A total of 10 transfections were
performed, and the cells were stained for Tra- 1-81 on day 14. Colony counts are shown in the
corner of each image.
Figure 6.8. Plating single fibroblasts for single-cell RNA reprogramming. A single CCD1124Sk adult dermal fibroblast was plated in the centre of each well of 3 6-well plates as
described in Appendix A. The cells were allowed to attach, and the cells were counted. A single
cell was found in 16 out of 18 wells. No cell was found in the remaining 2 wells.
54
Day 5
Day 8
Day 11
Figure 6.9. Reprogramming single cells using RNA. A single adult dermal fibroblast (CCD11 24Sk) was reprogrammed to pluripotency using RNA as described in Appendix A. The colony
shown was picked on day 14 to establish the CCD-1 124Sk-single-cell-iPS-1 line.
passage, the efficiency of RNA reprogramming can be defined as the fraction of single-cell
experiments that yielded a characterized iPS cell line. Using this definition, we obtained an
efficiency of 44%, which also includes those cells that did not survive the isolation and plating
procedure.
6.5 Summary
Developing therapies based on somatic cell reprogramming will require a method of generating
safe iPS cells. Here we have demonstrated efficient, rapid, and reliable reprogramming of
human cells using RNA. Fibroblasts transfected with in vitro-transcribed RNA encoding Oct4,
Sox2, Klf4, c-Myc, and Lin28 formed large colonies that, within two weeks, exhibited
morphology and gene expression consistent with pluripotent stem cells. Using this method, we
reprogrammed single adult fibroblasts (including from a 53 year-old Parkinson's patient) with
44% efficiency (4 stable lines generated out of 9 total attempts). Our results suggest that the low
efficiency and slow kinetics of existing reprogramming methods are consequences of the
limitations of the genetic expression systems used, and not fundamental limitations of
reprogramming using defined factors, as was previously thought. As a result of the high
efficiency, speed, and integration-free nature of RNA reprogramming, we anticipate that this
technique will quickly become the method of choice for generating disease- and patient-specific
pluripotent stem cells for both research and therapeutic applications.
Chapter 7
Characterization of RNAReprogrammed Cell Lines
When isolated from the primary culture, RNA-reprogrammed cells immediately
established lines under standard hES cell-culture conditions.
7.1 Gene Expression
RNA-reprogrammed induced pluripotent stem (iPS) cell lines derived from seven adult patients,
including five with idiopathic Parkinson's disease, ranging in age from 53 to 85 years, expressed
genes that are upregulated in ES cells, including the transcription factors Oct4, Sox2, and Nanog,
the de novo DNA methyltransferases Dnmt3a and Dnmt3b, and telomerase reverse transcriptase
(TERT) (Figs. 7.1, 7.2). Microarray gene expression analysis showed that RNA-reprogrammed
cells exhibited an hES cell-like pattern of gene expression (Fig. 7.3).
J-PS-1
CCD-1109Sk-IPS1
AG20442-IPS-1
CCD-11095k-IPS-Z
AG204424
CCD-1109Sk-iPS-3
AG204434PS-1
CD-1109Sk-iPS-4
AG20443-IPS-2e
CCD-1124Sk-iPS-1
AG20445-4PS-1
CCD-1124Sk-iPS-2
AG20445-iPS-2
CCD-1124k-IPS-3
AG20446-IPS-1*
CCD-11245k-iPS-4
AG20446-iPS-2
AG20442-single-cell-iPS-1*
AGO8395-iPS-1
AG20442-single-cell-iPS-2
AGO8395-APS-2
AG20442-single-cell-iPS-3
AG20442-Iso-iPS
CCD-1124Sk-singIe-cel-iPS-1
AG20446-Iso-iPS
CCD-1124Sk-single-ceII-IPS-2
Figure 7.1. Cells reprogrammed using RNA express pluripotency markers. Pluripotencymarker staining of twenty six RNA-reprogrammed iPS-cell lines. All lines uniformly expressed
the surface marker Tra- 1-81, and the transcription factors Oct4, Sox2, and Nanog.
102
* AG20442
OBJ-iPs-1
101
o A620442-iPS-1
S
*A62042iPS-2
10
D AG20442 iso-iWS
MA62042indI.-COPS2
0 AG20442-single-c~kPS- 3
1
104
10-
10-
10-4
OCT4 Sc8C2NANO(UN2S ALPE 03A
D3-
10-
TERT
* A620443
O A620443-iPS
1
MA620443-iPS.2*
OCT4 SMl MAWdOG
L1N28 ALP. PMA 039
101
TEAT
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1013
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1
SA620446 iPS 2
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101
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*AG089YA-P$ 2
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t
i
~
i
l
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0
LO
a. 10-' OC14 sOX2 NANOG ItiNR APS
CCP-1109%
1
OCCo 11095k PS 21
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DiA
03D
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CCD-1109SkiPS-3
CCO11095k W54
,
O10'
4,
10-2
.2 101
10
0c4
5032 NANOGUN28 ALPI D3A D38
s1-
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10 *CCO 1124Sk-iP5Z
20CCD1124SiPS3
*CD-1174SkiPSA4
OCCI) 1124Wk ig ce~lLP 1
*CCD)1124Sk snwIecOfS-2
n
AV C74 SMlONANOGUHiS ALR PMA D38
1811
Figure 7.2. Cells reprogrammed using RNA exhibit gene expression consistent with the
pluripotent stem-cell state. RT-PCR data showing expression of pluripotency-associated genes
in starting fibroblasts and RNA-reprogrammed iPS-cell lines, relative to expression in H9 hES
cells. (D3A, Dnmt3a; D3B, Dnmt3b.) Error bars: s.d.
1051
iTie
104 -
-10
04
3
+-Dnmt3b
U-
_cdh
103
+-Dnmt3a
102
Tert
Oct4-r
Lin28-+
Cdhi-+
101
"
101
102
-
1
Nodal
+-Nanog
Sox2 -+
103
H9
10 4
10s
106
101
102
13
H9
104
10
106
Figure 7.3. Cells reprogrammed using RNA exhibit ES cell-like gene expression patterns.
Microarray gene expression analysis of the CCD-l 124Sk-iPS-l line. CCD-l 124Sk fibroblasts,
H9 hES cells, and the CCD- 1124Sk-iPS- 1 line were analyzed using 44,000-probe geneexpression arrays. Each sample was analyzed using two identical arrays. Probes with a >2-fold
difference between arrays were discarded, and remaining probes were normalized to GAPDH.
Red lines indicate equivalent expression, and 5-fold up- and downregulation.
7.2 Teratoma and in vitro Directed Differentiation
When injected into immunocompromised mice, RNA-reprogrammed cells generated welldifferentiated teratoma-like structures containing elements of all three germ layers (Fig. 7.4). We
also generated neurons that stained positive for tyrosine hydroxylase, a marker of dopaminergic
neurons, from ten Parkinson's patient-derived iPS cell lines using a published protocol 6 3 (Fig.
7.5).
7.3 Short Tandem Repeat (STR) Analysis
We performed a short tandem repeat (STR) analysis to confirm the origin of the reprogrammed
cell lines (Fig. 7.6). Interestingly, while most lines originated from the parent fibroblasts, two
lines (one each from two of the five Parkinson's patients) exhibited a fingerprint matching the
feeders (feeder-derived lines are indicated with an asterisk "*"). We performed an experiment
without cells (only feeders), and obtained no colonies (Fig. 6.5), although two small areas of
cells undergoing morphological changes were found, suggesting that the feeder layer contained
only a very small number of non-inactivated cells. We were also able to derive iPS cell lines
from Parkinson's patient-derived fibroblasts using autologous feeders64 (AG20442-Iso-iPS and
AG20446-Iso-iPS in Figs. 7.1, 7.2, see Appendix A).
7.4 Karyotype
Fourteen of the 17 iPS cell lines that we tested (including one from BJ, four from two adult
patients, and seven from four Parkinson's patients) were karyotypically normal (Fig. 7.7). Of the
five Parkinson's patient-derived fibroblast cultures from which we derived iPS cell lines, one
(AG20442) is known to exhibit karyotypic mosaicism, with the abnormal cells containing
multiple translocations involving chromosomes 6,8,13,15, and 16 (Coriell Institute). These
translocations were found in two of the four iPS cell lines that we established from this
61
Figure 7.4. Parkinson's patient-derived RNA-reprogrammed cells form teratomas in vivo.
Teratomas formed by RNA-reprogrammed cells (BJ-iPS-1, AG20443-iPS-1, AG20445-iPS-1,
and AG08395-iPS-1) showing elements of all three embryonic germ layers: endoderm (columnar
epithelium, "C.E."), mesoderm (cartilage, "Ca"), and ectoderm (pigmented epithelium, "P.E.";
neuroepithelium, "N.E.").
62
Figure 7.5. Parkinson's patient-derived RNA-reprogrammed cells differentiate into
dopaminergic neurons in vitro. in vitro differentiation of Parkinson's patient-derived RNAreprogrammed cells to dopaminergic neurons: nuclei (Hoechst; blue), p-III tubulin (green), and
tyrosine hydroxylase (red). All 10 Parkinson's patient-derived lines yielded cultures containing
TH*/P-III tubulin* cells with a neuronal morphology.
63
1.4 N
Mf V
.4
f 4Y
rn
-
-
v4
N
Adult
Parkinson's Patients
1-4 1-"
V-4 V-4 94 V4
W-4 V-4
=~,
r
6
r4666
,i
a%
-
"
1-
q414
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e**t*-*e*
Figure 7.6. STR analysis of RNA-reprogrammed iPS cell lines. Genomic DNA was isolated
from samples of each cell line shown. Four STR loci and amelogenin were amplified, and the
amplification products were resolved by agarose gel electrophoresis. Asterisks (*) show lines
with a fingerprint matching that of the feeders. All other lines showed fingerprints matching the
corresponding parent fibroblasts.
BJ-iPS-1
1
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AG20442-IPS-2
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46,XY101
2.
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i
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1
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5
11
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16
15
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21
20
AG20443-IPS-2*
4
3
2
10
9
IS
46,Xt(6;8;16q13;q2224)t(13;1Kq12;q26,1)(18)
AG20443-PS-1
1
a
5
0
70
AG20442-single-ceI-iPS-3
1
51
10
2
13
14
n
19
20
21
16
4
46,01 201
22
17
13
X
Y
46,XY(201
Figure 7.7. G-banded karyotypes of seventeen RNA-reprogrammed cell lines, including two
lines derived from single, Parkinson's patient-derived fibroblasts. Two of the four lines
derived from AG20442 Parkinson's patient-derived fibroblasts showed karyotypic abnormalities
known to be present in a fraction of the parent fibroblasts (Coriell Institute). The AG20443-iPS- 1
line also exhibited karyotypic abnormalities. All other lines exhibited a normal karyotype.
65
patient. The karyotype of another patient from which we derived iPS cells (AG20443) has not
been published. The one line that we generated from this patient contained abnormalities
involving chromosomes 3 and X.
7.5 Copy-Number Variation (CNV) Analysis by HighResolution Array Comparative Genomic Hybridization
(aCGH)
iPS cells generated using DNA-based vectors are known to acquire copy number variations that
arise as a result of the reprogramming process 6 5-66. To determine whether RNA reprogramming
results in similar genetic aberrations, we analyzed two lines (CCD-1 109Sk-iPS-1 and CCD11 24Sk-iPS- 1) using high-resolution array comparative genomic hybridization (aCGH) with
244,000-probe oligonucleotide arrays having an 8.9kb median probe spacing (Fig. 7.8). No
aberrations with an interval of two or more consecutive probes were detected in CCD- 1109SkiPS- 1, and only a single aberration involving an amplification of a 1.4-megabase region of
chromosome 16 was detected in CCD- 1124Sk-iPS-1. Although the parent fibroblasts for each
line were used as the reference samples in this experiment, it cannot be determined from these
results whether this aberration occurred during reprogramming or whether it was present in a
fraction of the starting fibroblast population. However, the absence of aberrations in CCD1 109Sk-iPS-l suggests that the high efficiency of RNA reprogramming is not achieved at the
cost of increased copy number variations.
7.6 Summary
The characterization experiments described above are summarized in Table 7.1
1
0
CCD-1109Sk-iPS-1
0
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14 15 16 17 181920212
X
Y
5
6
7
8
9
10
11
12
13
14
X
Y
b
1
0
0
1
01
S.5
0
CCD-1124Sk-iPS-1
1
2
3
4
15 16 17 1819202122
chromosome 16
9x10 7
-1
chromosome 16q22
01
0
.5
Co-.5
-1
7.12x10 7
7.26x10 7
Figure 7.8. RNA reprogramming can generate pluripotent stem cells free of copy number
variations. aCGH analysis of CCD- 1109Sk-iPS- 1 (a) and CCD- 1124Sk-iPS- I (b), using
244,000-probe oligonucleotide arrays with an 8.9kb median probe spacing. The parent
fibroblasts were used as the reference sample in each case. No aberrations were detected in the
CCD- 1109Sk-iPS- 1 line. A single aberration consisting of an amplification of a 1.4Mb region of
chromosome 16 was detected in the CCD- 1124Sk-iPS- 1 line.
Table 7.1. Summary of iPS cell-line characterization experiments.
Teratoma
RT-
STR
in vitro
(tumours/
differentiation
injections)
Cell line
analysis
ICC
PCR
Karyotype
BJ-iPS-1
X
X
X
X
AG20442-iPS-1
X
X
X
X
X
(1/1)
AG20442-iPS-2
X
X
X
X
X
(1/1
AG20443-iPS-1
X
X
X
X
X
(1/1)
AG20443-iPS-2*
X
X
X
X
X
(0/1)
AG20445-iPS-1
X
X
X
X
X
(1/1)
AG20445-iPS-2
X
X
X
X
X
(0/1)
AG20446-iPS-1*
X
X
X
X
X
(0/1)
AG20446-iPS-2
X
X
X
X
X
(0/1)
AG08395-iPS-1
X
X
X
X
X
(1/1)
AG08395-iPS-2
X
X
X
X
X
(1/1)
CCD-1109Sk-iPS-1
X
X
X
X
CCD-1 109Sk-iPS-2
X
X
X
X
CCD- 1109Sk-iPS-3
X
X
X
CCD-1 109Sk-iPS-4
X
X
X
CCD-l124Sk-iPS-1
X
X
X
X
(1/I)
CCD-1124Sk-iPS-2
X
X
X
X
(1/1)
CCD-1 124Sk-iPS-3
X
X
X
CCD-J 124Sk-iPS-4
X
X
X
AG20442-Iso-iPS
X
X
AG20446-Iso-iPS
X
X
X
X
X
cell-iPS-2
X
X
X
X
AG20442-singlecell-iPS-3
X
X
X
X
single-cell-iPS-1
X
X
X
CCD- I124Sksingle-cell-iPS-2
X
X
X
AG20442-singlecell-iPS-1*
AG20442-single-
CCD-1 124Sk-
Microarray
I aCGH
(3/3)
X
(1/1)
X
X
Chapter 8
Conclusions
Somatic cell reprogramming can be used to generate a nearly unlimited source of patient-specific,
tissue-specific cells. However, all current methods for reprogramming somatic cells to
pluripotency are slow, inefficient, and most rely on the use of viruses or other DNA-based
vectors, which carry the risk of insertional mutagenesis. Because of these drawbacks, while
techniques for direct reprogramming by defined factors have been available for more than five
years, very little is known about the mechanisms underlying and controlling the reprogramming
process. In fact, the extremely low efficiencies of existing reprogramming methods has led the
field to question whether reprogramming efficiency may be fundamentally limited by some
unknown stochastic element. To explore the reprogramming process, we aimed to develop an
expression system that would enable the expression of reprogramming proteins, without the
drawbacks of DNA-based vectors.
in vitro-transcribed RNA-transfection can be used to express high levels of functional
protein in cells. However, because cells respond to transfection with in vitro-transcribed RNA as
they do to infection with an RNA virus (by halting growth, upregulating pattern-recognition
receptors, and secreting inflammatory cytokines), we first had to explore methods for
suppressing the pathways involved in this response to enable repeated RNA transfection. Here,
we describe a combinatorial siRNA screen showing that combined knockdown of the
69
inflammatory cytokine, interferon-p, the downstream signaling molecule, STAT2, and the
translation-inhibiting protein kinase, EIF2AK2, enables frequent transfection with in vitrotranscribed RNA.
Using these results on innate immune suppression, we next explored methods for
reprogramming human fibroblasts to pluripotency using RNA. We show that adult human
fibroblasts can be reprogrammed rapidly, efficiently, and reliably, using RNA. In fact, according
to the standard definition of reprogramming efficiency, this technique achieves efficiencies
greater than 100%, demonstrating that the low efficiencies of previous methods were caused by
limitations of the expression systems used, and were not the result of a fundamental stochastic
element of the reprogramming process, as was previously thought. By taking advantage of the
extremely high efficiency of this technique, we were further able to demonstrate reprogramming
of single cells, a method which can be applied to the study of the mechanisms underlying
reprogramming by enabling precise tracking and analysis of the cell at all stages of the
reprogramming process.
8.1 Proposed Directions for Future Research
RNA reprogramming was achieved through the combination of a low-immunogenicity RNA
design and immune suppression. Incorporation of the Cap I structure and complete substitution
of non-canonical pyrimidines were used to reduce the immunogenicity of in vitro-transcribed
RNA. However, the fundamental question of why in vitro-transcribed RNA elicits a potent
immune response, or rather, what mechanisms do cells use to distinguish endogenous from
exogenous RNA, remains unanswered. Exploring this question will likely lead to new
techniques for further reducing the immunogenicity of in vitro-transcribed RNA, and may one
day lead to non-immunogenic, in vitro-transcribed RNA. While the method presented here
tolerates the residual immunogenicity of RNA synthesized as described, RNA reprogramming
may be a special case due to the high rate of proliferation of the cells undergoing the process,
and thus, the dilution, through cell division, of activated immune-response proteins. Applying
RNA transfection to the problem of directed differentiation would likely lead to cell death caused
70
by accumulation of these proteins in slowly dividing or post-mitotic cell types, and may thus
require further advances in RNA design and cell-culture techniques.
Appendices
Appendix A
Methods
A.1 Chapters 2-4
A.1.1
Cell Culture
MEF cultures were prepared from E13 CF-I mice (Charles River Laboratories) according to an
approved protocol (MIT Committee on Animal Care #0307-023-10). Samples tested negative
for mycoplasmal contamination by both DNA fluorochrome staining and live-culture methods.
H9 human embryonic stem cells were obtained from the National Stem Cell Bank at passage 24,
and were cultured on irradiated MEFs as described 67- 68. Cells from frozen stocks (in hES-Cell
Media + 10% DMSO + 30% Defined FBS) were seeded on plates coated with basement-
membrane extract (Pathclear, Trevigen), and cultured in media conditioned for 24 hours on
irradiated MEFs. Primary human fibroblasts from normal fetal lung tissue (MRC-5) or from
normal adult skin (CCD- I109Sk) were obtained from the ATCC and were cultured according to
their recommendations.
A.1.2
in vitro transcription
dsDNA templates were prepared as described' 2 . Briefly, total RNA was extracted from H9 hES
cells and enriched for poly(A)+ mRNA (Oligotex, Qiagen). Oct4, Sox2, Klf4, c-Myc, Utfl,
Nanog, Lin28, MyoD 1, and Aicda coding sequences, and p-globin UTRs were reverse
transcribed using an RNase H- reverse transcriptase (MonsterScript, Epicentre). Template
73
components were amplified with a high-fidelity polymerase (Phusion Hot Start, NEB or KAPA
HiFi, Kapa Biosystems) and ligated with E.coli DNA ligase (NEB). Capped, poly(A)+ RNA
was synthesized using the mScript mRNA Production System (CELLSCRIPT). The temperature
and duration of the in vitro-transcription reaction were optimized for specificity and yield as
described12. Transcripts were analyzed both before and after poly(A) tailing by denaturing
formaldehyde-agarose gel electrophoresis. Primers used for assembly of in vitro-transcription
templates are given in reference 12.
A.1.3 RNA Transfection
Lipid-mediated transfections (TransIT-mRNA, Mirus) were performed according to the
2
manufacturer's instructions. Electroporation was performed as described . Briefly, cells were
trypsinized, washed once in Opti-MEM (Invitrogen), and resuspended in a total volume of 50pL
of Opti-MEM in a standard electroporation cuvette with a 2mm gap. A 150uF capacitor charged
to 150V was discharged into the cuvette to electroporate the cells. Warm media was added, and
the cells were plated and cultured using standard protocols.
A.1.4 Quantitative RT-PCR
TaqMan Gene Expression Assays (Applied Biosystems) were used in one-step RT-PCR
reactions (iScript One-Step RT-PCR Kit, Bio-Rad) consisting of a 50C, I0min reverse
transcription step, followed by an initial denaturation step of 95C for 5min, and 45 cycles of 95C
for 15sec and 55C for 30sec.
A.1.5
siRNA-Mediated Knockdown
Cells were electroporated in Opti-MEM containing the indicated siRNAs (Silencer Select or
Anti-miR, Applied Biosystems), each at a final concentration of 100-800nM (Table 3.1).
A.1.6 Immunocytochemistry
Cells were rinsed in TBST and fixed for 10 minutes in 4% paraformaldehyde. Cells were then
permeabilized for 10 minutes in 0.1% Triton X-100, blocked for 30 minutes in 1% casein, and
incubated with appropriate antibodies (reference 56).
A.1.7
Western Blot
Whole-cell lysates (Qproteome Mammalian Protein Prep Kit, Qiagen) were separated on a 12%
polyacrylamide gel (ProSieve 50, Lonza) under reducing, denaturing conditions. Proteins were
transferred onto a PVDF membrane (Immobilon-P, Millipore) in CAPS buffer, pH 11.
Membranes were blocked in 5% skim milk, and probed with appropriate antibodies (reference
56).
p-actin was used
as a loading control.
A.2 Chapters 5-7
A.2.1 Cell Culture
Parkinson's patient-derived fibroblasts were obtained from the Coriell Institute. BJ neonatal
fibroblasts, CCD-1 109Sk adult dermal fibroblasts, and CCD- 1124Sk adult dermal fibroblasts
were obtained from the ATCC. H9 hES cells were obtained from Wicell. Human fibroblasts,
hES cells, and iPS cells were cultured using standard techniques. hES cells and iPS cells were
maintained on basement membrane extract-coated plates (Pathclear, Trevigen) in hES cell
medium (DMEM/F 12 + 20% Knockout Serum Replacer (Invitrogen) + 1mM L-glutamine +
50pM
P-mercaptoethanol
+ 1X non-essential amino acids) conditioned for 24 hours on irradiated
MEFs. 20ng/mL bFGF and 10ptM Y-276326 8 were added to the medium after conditioning.
A.2.2
RNA Synthesis
Templates for in vitro transcription were prepared as we previously described. 2
56.
Briefly,
templates encoding the reprogramming factors were cloned into the pCR-Blunt II-TOPO vector,
and propagated in TOP 10 chemically competent cells using the Zero Blunt TOPO PCR Cloning
Kit (Invitrogen). Plasmids were extracted using EndoFree Plasmid Maxi Kits (Qiagen), digested
with either EcoR I-HF or Notl-HF (NEB), and the templates were amplified using 12 cycles of
high-fidelity PCR (Table A. 1). The templates were separated by agarose gel electrophoresis, and
were extracted using a QiAquick Gel Extraction Kit (Qiagen). Cap 1-capped, human beta globin
UTR-stabilized, poly(A)+ RNA was synthesized using the mScript mRNA Production System
(CELLSCRIPT, Inc., Table A.2). The RNA was purified using RNeasy Midi Kits (Qiagen), and
the concentration was adjusted to 200ng/ptL. Superase-In RNase Inhibitor (Ambion) was added
at a concentration of 1 tL/20ptg of RNA, and the RNA was stored at 4C for up to several months.
The quality of the RNA was assessed by denaturing agarose gel electrophoresis. To prepare a
reprogramming mixture, RNA encoding Oct4, Sox2, Klf4, c-Myc-2 (T58A), and Lin28 was
mixed at a ratio of 40:12:16:15:8. For experiments using c-Myc-2 (wt), RNA encoding this
protein was substituted for RNA encoding c-Myc-2 (T58A) on a 1-to-I mass basis. Phosphatasetreated, ARCA-capped RNA was a generous gift of Stemgent, Inc.
A.2.3 Autologous Feeder Preparation
Autologous feeders were prepared as previously described 64 . Briefly, sub-confluent cultures of
primary human fibroblasts were incubated for 3-4 hours with 12pig/mL mitomycin-c in DPBS
(with Ca, Mg). Cells were rinsed twice with DPBS before re-plating. Feeders were cultured for at
least 24 hours before use to eliminate all traces of mitomycin-c.
A.2.4 RNA Reprogramming
Human fibroblast feeders (GlobalStem or autologous feeders prepared as described above) were
plated in 6-well plates at a density of 6 x 105 cells/well in DMEM + 10% FBS. The following
day, the target cells were plated in wells containing feeders at a density of between 1 x 104 and 5
x
104 cells/well. The cells were allowed to attach (typically 2-6 hours), and the culture medium
was replaced with RNA reprogramming medium (Table 5.1) containing the immunosuppressant
B 18R (eBioscience). From this point onward, cells were incubated at 5% CO 2 and 5% 02. The
following day (day 0), cells were transfected with 2pg/well of reprogramming RNA using
6pt/well of Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's
instructions. The total transfection volume was 120p L/well. The culture medium was replaced
after 4 hours. Transfections were repeated every 24 hours for 12 days. A single passage was
performed on day 5-6, approximately 8 hours after transfection. To passage the cells, the
medium in each well was replaced with lmL of 0.25% trypsin + 1.25mM EDTA. After the cells
detached (approximately 2-3 minutes), 4mL of soybean trypsin inhibitor was added. The cells
were pelleted for 5min at 200 x g, and resuspended in 1mL of soybean trypsin inhibitor. A
fraction of the cells ( 1 /2 0th to 1 / 5 0 th based on the cell density at the time of the passage) was
added to a well of a 6-well plate containing fresh feeders. On day 12, the culture medium was
replaced with MEF-conditioned medium containing 20ng/mL bFGF and 1OpM Y-27632. iPScell colonies were picked on day 14 and plated on basement membrane extract-coated plates
(Pathclear, Trevigen) in MEF-conditioned medium + 20ng/mL bFGF + 1OpM Y-27632. iPS
cells were passaged using trypsin for the initial expansion, and by traditional methods (colony
picking) after passage 3.
A.2.5
Single-Cell RNA Reprogramming
Fibroblasts were suspended by trypsinization and plated at low density in a well of a 6-well
plate. A single cell was isolated using a 10 L pipet tip. The presence of a single cell was
confirmed by directly imaging the contents of the tip. The cell was then plated in the centre of a
well of a 6-well plate containing 600,000 feeders. After 6-8 hours, the medium was replaced with
RNA reprogramming medium, and reprogramming was carried out exactly as described above,
but without passaging.
A.2.6
qRT-PCR
Oct4, Sox2, Nanog, and Lin28 mRNA was amplified using primers and molecular beacons
designed to detect only the endogenous transcripts (Table A.3). All other transcripts were
detected using TaqMan Gene Expression Assays (Applied Biosystems). Amplification reactions
were performed using the iScript One-Step RT-PCR Kit for Probes (Bio-Rad), and consisted of a
50C, 10min reverse transcription step, followed by an initial denaturation step of 95C for 5min,
and 30-45 cycles of 95C for 15sec and 55C for 30sec. GAPDH was used as a loading control.
A.2.7
Immunocytochemistry
Immunocytochemistry was performed as we previously described5 6 using antibodies listed in
Table A.4. Cells were imaged on a Nikon TE2000 fluorescence research microscope using a
Photometrics CooISNAP HQ 2 cooled CCD camera.
A.2.8 Short Tandem Repeat (STR) Analysis
The D1 8S51, D8S 1179, THO1, FGA, and amelogenin loci were amplified using the PowerPlex
S5 System (Promega). Amplification products were resolved by agarose gel electrophoresis.
A.2.9 Karyotyping
G-banded karyotyping was performed by Cell Line Genetics, LLC.
A.2.10 Microarray
Total RNA was analyzed in duplicate using Agilent 44k gene expression arrays (Empire
Genomics). Probes yielding >2-fold difference between replicates were discarded, and
expression was normalized to GAPDH.
A.2.11 aCGH
Genomic DNA was analyzed using Agilent 244k CGH arrays (Empire Genomics). Aberrations
were detected using the ADM- I algorithm (threshold = 25).
A.2.12 Teratoma
Approximately one million cells were injected either subcutaneously between the shoulder
blades or intramuscularly into a hind limb of a SCID-Beige or NOD-SCID mouse according to
an approved protocol (MIT Committee on Animal Care protocol #0710-057-13). Tumours were
removed after 4-10 weeks. Tumours were sectioned and stained with H&E by the Histology
Core Facility, Swanson Biotechnology Center, David H. Koch Center for Integrative Cancer
Research, MIT.
A.2.13 Directed Differentiation
Parkinson's patient-derived, RNA-reprogrammed cells were differentiated to dopaminergic
neurons using a previously described protocol6 3 . Cells were fixed and stained on day 48.
Table A.1. in vitro-transcription template synthesis.
Component
Concentration
5X GC Buffer
1x
10mM dNTP Mix
300pM
5uM HBB 5-UTR Forward Primer
.3 pM
5uM HBB 3-UTR Reverse Primer
.3 pM
ivT Template Digestion Reaction
500ng
KAPA HiFi Hot-Start DNA Polymerase
1OU
The cycling parameters were, 95C for 5min, 12 cycles of 98C for 20sec and 68C for 1min, 68C
for 5min. Sequences of primers are given in reference 12.
Table A.2. RNA synthesis.
Volume per 20UL Reaction (uL)
in vitro Transcription
1.2
RNase-Free Water
2
mScript iOX Transcription Buffer
100mM adenosine-triphosphate
1.8
100mM guanosine-triphosphate
1.8
100mM pseudouridine-triphosphate57'69
0.9
100mM 5-methylcytidine-triphosphate 57
1.8
100mM DTT
ScriptGuard RNase Inhibitor
2
0.5
mScript T7 Enzyme Mix
2
T7 Template (1 ig)
6
5'-Capping
lOX ScriptCap Capping Buffer
Volume per 1OOL Reaction (pL)
10
20mM GTP
5
20mM SAM
I
ScriptGuard RNase Inhibitor
2.5
mScript Capping Enzyme
4
mScript 2'-O-Methyltransferase
4
Heat-denatured RNA (60pg)
3'-Poly(A)-Tailing
73.5
Volume per 123.5pjL Reaction (1L)
mScript lOX Tailing Buffer
12
ScriptGuard RNase Inhibitor
0.5
20mM ATP
6
mScript Poly(A) Polymerase
5
5'-Capped ivT RNA (60ig)
100
Table A.3. Primers for RT-PCR of endogenous transcripts.
Oct4 Endo RT Forward Primer: AACCTGGAGTTTGTGCCAGGGTTT
Oct4 Endo RT Reverse Primer: AACTTCACCTTCCCTCCAACCAGT
Oct4 Endo Beacon: FAM-ACCCACCAGGAATTGGGAACACAAAGGGTGGGGG-BHQ- 1
Sox2 Endo RT Forward Primer: ATCCCATCACCCACAGCAAATGAC
Sox2 Endo RT Reverse Primer: TTTCTTGTCGGCATCGCGGTTT
Sox2 Endo Beacon: FAM-ACGTGAGTGAACACCAATCCCATCCACACTCACG-BHQ- 1
Nanog Endo RT Forward Primer: AGTCTGGACACTGGCTGAATCCTT
Nanog Endo RT Reverse Primer: TCCATGAGATTGACTGGATGGGCA
Nanog Endo Beacon: FAM-ATTTGGAAACCACGTGTTCTGGTTTCCATGA-BHQ- 1
Lin28 Endo RT Forward Primer: ATTGGGTGGTGTGTGTCTGATCCT
Lin28 Endo RT Reverse Primer: AGGCCACCTAGCAATCAACGTAGT
Lin28 Endo Beacon: FAM-AGAGCCCCCCAAGTTACTGTATTTGTCTGGGCTCT-BHQ- I
Table A.4. Antibodies.
Immunogen
Host
Vendor
Part Number
Dilution
Tra-1-81
mouse
Stemgent
09-0069
1:500 (1pg/mL)
Oct4
rat
R&D
MAB 1759
1:200 (2.5pg/mL)
Sox2
rabbit
Millipore
AB5603
1:1000 (1pgg/mL)
Nanog
goat
R&D
AF1997
1:200 (0.5pg/mL)
p-III tubulin
mouse
R&D
MAB 1195
1:200 (2.5pg/mL)
Tyrosine Hydroxylase
sheep
Pel-Freez
P60101
1:1000
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