FROM SKIN TO NEURONS: EXAMINING VARIATIONS IN REPROGRAMMING
EFFICIENCY
By
Sarah A Keaton

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Physiology- Master of Science
2013

ABSTRACT
FROM SKIN TO NEURONS: EXAMINING VARIATIONS IN REPROGRAMMING
EFFICIENCY
By
Sarah A Keaton

Cellular reprogramming is a newly emerging field with promising clinical applications.
The ability to generate non-dividing crucial cell types from rapidly proliferating cell types, the
potential to heal diseased people who do not have many treatment options, being able to bypass
immune rejection, and avoid invasive surgery has captured the media‘s attention. However, there
have been disparities in the efficiency of reprogramming and these needs to be addressed before
cellular reprogramming can be applicable in a clinical setting. To better understand the variations
of cellular reprogramming, human and mouse fibroblasts were converted into induced neuralcells
in an attempt to unveil the impact of disease state, tissue origin and genetics. The experimental
results indicatereprogramming efficiency was reproducible within a primary fibroblast line
however there was a dramatic difference between lines even from an isogenic source. Testing a
larger number of fibroblast lines, even lines with the identical genetic backgrounds and tissue
origins, is likely the most direct means of improving reprogramming efficiency and enabling this
procedure to be available for therapeutic use

Dedicated to my parents, Karen and Patrick Keaton, who have always supported me and
encouraged me to keep questioning.

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ACKNOWLEDGEMENTS

I would like to thank my committee members, Dr. Jose Cibelli, Dr. Susanne Mohr, Dr.
Steve Suhr, and Dr. Robert Wiseman. I would also like to acknowledge my fellow laboratory
members past and present in their support and for the production and maintenance of fibroblast
lines used in this thesis. Finally, I would like to thank the Physiology staff and fellow students
for always being there to help me advance myself educationally.

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TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………………………..vi
LIST OF FIGURES………………………………………………………………………...……vii
KEY TO ABBREVIATIONS……………………………………………………………………..x
INTRODUCTION……………………………………………………………………………...…1
CELLULAR REPROGRAMMING………………………………………………………………1
APPLICATION OF CELLULAR REPROGRAMMING………………………………………...1
GENERAL PROBLEMS IN CELLULAR REPROGRAMMING……………………………….2
GENERAL PURPOSE OF THIS STUDY……………………………………………………......3
DIRECT CELLULAR REPROGRAMMING……………………………………………….……3
iPSC CONVERSION……………………………………………………………………………..5
EFFICIENCY DIFFICULTY……………………………………………………………………..5
FIBROBLAST CELLS AS AN INPUT LINE……………………………………………………7
NEURONS AS AN OUTPUT CELL……………………………………………………………..7
CLINICAL APPLICATION AND NEUOLOGIC DISORDERS………………………………..8
PURPOSE OF THIS STUDY……………………………………………………………………11
METHODS………………………………………………………………………………………12
FIBROBLAST LINES……………………………………………………………………….…..12
FIBROBLAST CHARACTERIZATION…………………………………………………….….15
iNC INDUCTION……………………………………………...…………………….…………..19
IMMUNOHISTOCHEMISTRY AND IMAGING…………………...…………………………21
ELECTROPHYSIOLOGY………………………………………………………………………22
HUMAN NPC CULTURE AND NEURON DERIVATION…………………………………...23
QUANTIFICATION OF iNC CONVERSION…………..……………………………………...31
RESULTS………………………………………………………………………………………..31
CONCLUSIONS……………...………………………………………………………………....49
REFERENCES…………………………………………………………………………………..53

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LIST OF TABLES

Table 1: General Properties of Fibroblast Lines. Study ID, abbreviated line designation used this
report; Line ID, line common name; Donor, donor health status or strain; Sex, sex of donor
(male, female, or of mixed sex); Age, age of donor (years, unless otherwise noted); Tissue,
fibroblast tissue of origin. mRNA Expression: results of quantitative PCR analysis of multiple
markers. COL1A2, fibroblast marker collagen type I alpha 2; FNECTIN, fibroblast marker
fibronectin; FIBR1, fibroblast marker fibrillin I; FIBU5, fibroblast marker fibulin V; VIM,
fibroblast marker vimentin; KER14, keratinocyte marker keratin 14; PECAM, endothelial cell
marker Platelet endothelial cell adhesion molecule/CD31; FOXG1, neural progenitor marker
Forkhead box protein G1; SOX2, neural progenitor marker SRY (sex determining region Y)- box
2, MYOD, myogenic progenitor marker MyoD1; MYF5, myogenic progenitor marker myogenic
factor 5. Immunocytochemistry: summary of immunocytochemical analysis of fibroblastassociated (fibronectin, vimentin) and stem cell-associated markers (Sox-2, nestin) in fibroblast
lines. N/A, not applicable; ND, –not done; + , positive; – , weakly positive; - , negative.(From
Alicea et al. 2013 [54])…………………………………………………………………………..13
Table 2: Primers Used in Cloning and qRT-PCR Analysis. Cloning- primers used in the
amplification of cDNAs or plasmid DNA for construction of retroviral vectors; QPCR- primers
used in qRT-PCR quantification of gene expression. The species of the gene targeted by each
primer pair is shown on the right(From Alicea et al. 2013 [54])……………………………….15

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LIST OF FIGURES

Figure 1:Time Course of induced Neural Cell (iNC) Induction and Electrophysiological
Analysis. (A) Examples of untransduced fibroblasts or fibroblasts infected with a YFP-only
control vector (green) cultured under INC induction conditions for 4 weeks and stained for TUJ1
(red). (B) TUJ1-, SYN1-, and MAP2-positive mouse and human iNCs over time expressed as a
percentage of the maximum TUJ1 value. Infection was at time 0 (T0), T1 = 8–10 days, T2 = 18–
20 days, and T3 = 28–30 days. (C) Examples of human iNCs produced by different fibroblast
lines at the T1-T3 time points (labeled on left) and stained for TUJ1 (red). In the upper left panel,
arrows indicate early iNCs in a low-magnification field. In the upper right panel, the iNC boxed
at the left is shown magnified for each color channel and the merged image. In the center and
lower panels, iNCs at the T2 and T3 time points, respectively, are shown. Scale bars are 10 mm
unless otherwise noted. Insets depict the cell soma with the blue channel removed to more
clearly reveal iNC factor expression. (D) Representative electrophysiological recordings from
iNCs. Upper left panel - Isolated voltage-gated sodium current recorded from a representative
human iNC. Upper records show voltage steps from a holding potential of - 80mV to test
potentials of - 40, - 25, 0, and 25 mV. Lower records show leak-subtracted Na + current elicited
by these voltage steps. Note that the step to - 40mV is sub threshold and that the steps to - 25, 0
and 25mV are supra-threshold and elicit inward sodium current of amplitude and kinetics which
is voltage dependent. Inset shows the relationship of test potential and the maximum amplitude
of the Na + current elicited at each test potential in this cell. Upper right panel—Voltage-gated
inward Na+ current was blocked by TTX. Upper record: voltage step from - 80 to 0 mV, which
was repeated every 2.5 s during the recording. Lower records: current elicited by the membrane
depolarization at different times during the recording. Voltage-gated inward Na+ current (but not
outward K+ current) was reduced in a time-dependent manner after the application of TTX (5
mM) to the extracellular medium. Lower panels—Representative action potentials (APs)
recorded from NPC-neurons and INCs. Left - AP produced by an NPC-derived neuron.
Membrane potential change (upper record) elicited by application of membrane current step
(lower trace) while holding the membrane at - 72mV just before current step application.
Center—AP produced by an ADF iNC. Membrane potential changes (upper records, x and y)
elicited by application of membrane current step (lower trace) without (x) and with (y) holding
the membrane at - 90mV (x) or - 55mV (y) just before current step application. Right—Overlay
of the NPC and INC AP traces (NPC, green; ADF, red)Top Panel: Raw tracings for visual
reference only.(From Alicea et al. 2013 [54])For interpretation of the references to color in this
and all other figures, the reader is referred to the electronic version of this thesis..………….....24
Figure 2:Immunocytochemical Analysis of Human and Mouse Fibroblast Lines. (A)
Immunostaining of human cell lines with anti-vimentin (red) and anti-nestin (green) antibody.
Upper panels—both antibodies in all fibroblast cell lines as labeled. Middle panels—the green
channel only (nestin), displaying an absence of positive signal. Lower panels controls showing
the same antibodies on human NPCs as labeled. (B) As in A, with anti-fibronectin (red) and antiSox2 (green) antibodies. Insets in middle panels show magnified images, indicating that rare
spots of faint green fluorescence are not cell/ nucleus associated as with genuine Sox-2
immunopositivity observed in NPCs. (C, D) As in A and B for selected mouse fibroblast lines.
vii

The panels at the end show that as with human cells, rare patches of faint green fluorescence do
not co-localize with nuclei and do not indicate Sox-2 immunoreactivity. Blue color in all images
is nuclear DNA stain bis-benzimide.(From Alicea et al. 2013 [54])For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic version of
this thesis.………..…..…...…..…………………………………………………………………..27

Figure 3:Morphology and Derivation of Fibroblast Lines Used in Analysis. (A) Morphology of
the seven human fibroblast lines used for analysis. (B) Outgrowth of fibroblasts and other cell
types from tissue fragments of eight mouse tissues at 1 week post plating. The dark mass in the
upper left corner is the adherent tissue fragment itself. (C) Morphology of the twelve mouse
fibroblast lines (at passage 6) used for analysis.………………………………………………....33
Figure 4:Fibroblast- and Nonfibroblast-associated mRNA Expression in Human and Mouse
Fibroblast Lines. Heat map color is proportional to expression level—bright green is high
expression and black is no detectable transcript.COL1A2, collagen type 1a2; FNECTIN,
fibronectin; FIBR1, fibrillin I; FIBU5, fibulin V; KER14, keratin 14. (From Alicea et al. 2013
[54])For interpretation of the references to color in this and all other figures, the reader is
referred to the electronic version of this thesis..…………………..………..………..………..…35
Figure 5:Schematic of the NITSC MMLV Retrovirus and YFP Fusion Factors. LTR, long
terminal repeat; Ψ+ , stem-cell permissive packaging sequence; NEO, G418-resistance gene; I,
IRES element; TTA, tet-transactivator; To, tetoperator. (From Alicea et al. 2013
[54])………………………………………………………………………………………………36
Figure 6:Induced Neural Cell (iNC) Conversion of Mouse and Human Fibroblast Lines. (A)
HEK cells transfected with YFP fusion protein for iNC conversion, as labeled. Phase-contrast
images are in the upper panels, and corresponding fluorescent images are in the lower panels. (B)
Mouse embryonic fibroblasts infected with the individual factors (as labeled) and stained for βIII-tubulin/TUJ1 in red (upper panels). Mouse (MEFs) or human fibroblasts (FET) infected with
combinations of Zic1/ Ascl1/Pou3f2 (ZAP) or Myt1L/Ascl1/Pou3f2 (MAP) + NeuroD1 (MAPN)
(lower panels). Green fluorescence indicates expression of reprogramming factor(s). Blue color
is bis-benzimide nuclear staining of DNA. Insets have the blue channel removed and the green
channel intensified to show YFP-factor expression in the nucleus of all iNCs. (C) Mouse iNCs
produced from MEFs by day 10–15 post-infection immune positive for multiple neural markers
(red), including MAP2, pan-neurofilament (NF), doublecortin (DCX), or synapsin I (SYN). iNCs
produced from adult mouse fibroblast lines (as labeled) with typical iNC morphology
immunostained for β-III-tubulin/TUJ1(red). (D) Human iNCs with typical morphology at day
24–30 post-infection and immunostained for multiple neuronal markers as in (C) in addition to
PSD95, GABA receptor b 3 (GABARB3), and GAD1 (as labeled). GABAR-B3 and GAD1 iNCs
were labeled using immunoperoxidase secondary antibody coupled with DAB staining. Scale
bars are 10 mm unless otherwise labeled. (E) Relative conversion of mouse fibroblast lines to
iNCs calculated as a function of cell number at the time of harvest. The highest efficiency of
conversion within each group was set to a value of 100. Bars indicate standard error of the mean
(SEM). (F) As in E, for the human fibroblast lines. (From Alicea et al. 2013 [54])For
interpretation of the references to color in this and all other figures, the reader is referred to the
viii

electronic version of this thesisTop Legend (Left to Right): VIMENTIN NESTIN DNA. DATA
(Left to Right): FET, NWB, RET, AUT, SAF, ADF, EAF, E2F. Middle Legend: NESTIN.
DATA (Left to Right): FET, NWB, RET, AUT, SAF, ADF, EAF, E2F. Bottom Legend:
HUMAN NPCS. DATA (Left to Right): VIMENTIN DNA,VIMENTIN, NESTIN DNA,
NESTIN. B) Top Legend (Left to Right): FIBRONECTIN SOX2 DNA. (Left to Right): FET,
NWB, RET, AUT, SAF, ADF, EAF, E2F. Middle Legend: Sox2. DATA (Left to Right): FET,
NWB, RET, AUT, SAF, ADF, EAF, E2F. Bottom Data (Left to Right): FIBRONECTIN DNA,
FIBRONECTIN, SOX2 DNA, SOX2. C) Top Legend (Left to Right): VIMENTIN NESTIN
DNA. Data (Left to Right): HE4, KI3, KI5, KI6, LI6, SM1, TA4, TE4, TE5. Middle Legend:
Nestin. Data (Left to Right): HE4, KI3, KI5, KI6, LI6, SM1, TA4, TE4, TE5. Bottom Legend:
Data (Left to Right): HE4, KI3, KI5, KI6, LI6, SM1, TA4, TE4, TE5.D) Top Legend: (Left to
Right): FIBRONECTIN SOX2 DNA. Data (Left to Right): HE4, KI3, KI5, KI6, LI6, SM1, TA4,
TE4, TE5. Bottom Legend: SOX2 Data (Left to Right): HE4, KI3, KI5, KI6, LI6, SM1, TA4,
TE4, TE5……………………..…………………………………………………………………..39
Figure 7: Representative images of β-III-tubulin/TUJ1 immuno-stained iNCs. (A) iNCs in
human lines as labeled. Magnified insets have the blue channel removed and the green channel
exaggerated to show factor expression in the iNC nucleus.All human lines produced iNCs,
including AUT and HSK, that were not incorporated in the analysis. The iNCs shown in the E2F
panel are a combination of multiple 1000X-magnification images. At the bottom right corner is a
characteristic neuron produced from a human neural progenitor cell (NPC) differentiated in
parallel with iNCs. (B) iNCs in mouse lines as labeled. Magnified insets have the blue channel
removed and the green channel increased to show factor expression in the iNC nucleus.(From
Alicea et al. 2013 [54])For interpretation of the references to color in this and all other figures,
the reader is referred to the electronic version of this thesis……………………………………..44
Figure 8: Relative Infection EfficiencyA) Showing relative conversion of mouse fibroblast lines
factoring in factor expression at day 4 post-infection. The highest efficiency of conversion within
each group was set to a value of 100. Bars indicate standard error of the mean (SEM). B) As in
A, for the human fibroblast lines.………………………………………………………………..47

ix

KEY TO ABBREVIATIONS

DNA

Deoxyribonucleic acid

DS

Donkey serum

HEK

Human Embryonic Kidney 293

iNC

Induced neuron cell

iPSC

Induced pluripotent stem cell

MECP2

Methyl-CpG-binding protein 2

MEFs

Mouse embryonic fibroblast

MMLV

Moloney murine leukemia virus

MOI

Multiplicity of infection

NPCs

Neural Progenitor Cells

ORFs

Open reading frames

PBST

PBS with 0.3% Triton X-100

PORN/lam

Polyornithine/laminin

PCR

polymerase chain reaction

qPCR

Quantitative polymerase chain reaction

RT-PCR

Quantitative real-time polymerase chain reaction

Vcon

Conditioning potential

Vh

Holding potential

Vtest

Testing potential

VSV

Vesicular stomatitis virus

YFP

Yellow fluorescent protein

ZAP

YZIC/YASCL/YPOU3F
x

INTRODUCTION
CELLULAR REPROGRAMMING
Cellular reprogramming is an important and relatively new field in medicine that has the
potential to heal diseased people whoface minimal treatment options. Cellular reprogramming
describes the process by which a fully differentiated, somatic (non-germ line) cell type is
induced to convert to a different cell type. If not for cellular reprogramming, this conversion
would not be observed under normal physiological conditions [1]. With the possibility of
converting an abundant, readily available cell type into a scarce, crucial cell type required to
maintain physiological homeostasis, the new and potentially auspicious field of cellular
reprogramming is deserving of research attention.
APPLICATION OF CELLULAR REPROGRAMMING
The concept of generating new, non-proliferating cell types from a patient‘s own cells
coupled with the ability to bypass the chance of immune rejection is a promising aspect of
cellular reprogramming research. Furthermore, this technique aids in the replenishment of crucial
and scarce host cells under a dissimilar, currently-existing genetic program. As a result,
application of potentially toxic and harmful pharmaceuticals may be bypassed.
Spinal cord injury serves as an example of the ways in which direct cellular
reprogramming could have therapeutic benefits for those with little hope due to the current
absence of curative therapy. Currently, care in the acute and sub-acute to chronic phases are
severely limited to high-dose corticosteroid treatment intended to reduce inflammation, surgical
stabilization and decompression to reduce further damage with focus placed on symptomatic
relief [2]. Spinal cord injury severs axons causing the distal segment of the axon to degenerate,
disrupting motor control and sensory input below the level of the lesion [3].
1

A taylored of the cellular reprogramming regimen could permit rehabilitation of a patient
who has suffered a severe injury resulting in paraplegia with the use of their own cells.
Hypothetically, a skin biopsy could be obtained from the patient for isolation of the fibroblasts
followed by reprogramming to induced neural cells (iNCs) via transcription factors in vitro. The
iNCs could be injected back into the patient at the site of injury. With proper establishment and
incorporation into the nervous system network, this process could aid in healing the patient.
Currently this practice is not clinically available. However with increased efficiency and research
in reprogramming techniques, this may be a viable option with clinical applications in the future.
The future clinical application of cellular reprogramming may revolutionize the ways in
which acute injures are currently treated. Immunosuppressive drugs will likely be mandatory
with rapid administration to evade immune flare-up. It may be possible or necessary to preprime these cells through exposure to stressful environments and cell repositories may be
available in case of injury.
The present study examines the process of direct cellular reprogramming and the
components that influence the efficiency of converting human and mouse fibroblasts into
functional iNCs. Research such as this may very well be a step toward future clinical
applications of cellular respecification with unprecedented medicinal benefit.
GENERAL PROBLEMS IN CELLULAR REPROGRAMMING
As stated previously, cellular respecification is not an available means of clinically
treating ailments at this time for various reasons. A primary issue with the process is conversion
efficiency. When exploring multiple reprogramming regimens -irrespective of the input or
desired output cell type- the efficiency of conversion is subpar. This issue must be addressed in
order to move toward clinical applications.
2

Without increased efficiency, the quantity of input cell type required for harvest would be
far too large to allow the diseased patient to return to physiological homeostasis. Without the
knowledge and proper understanding of this phenomenon, application of this respecification
regimen on human subjects cannot be justified due to the risk of detrimental effects. What is
priming certain cells to be capable of conversion to a different cell type, while others are unable
to reprogram at all? In addition, if the cells are capable, why is the conversion at such low
efficiency?
GENERAL PURPOSE OF THIS STUDY
The current study attempts to address the issue of conversion efficiency. If the causes of
variable results and generally low observed efficiency rates can be identified, clinical availability
of reprogramming regimens might be attainable. Variation exists each time a protocol is
performed, whether it is an extra second in the incubator, genetics or specimen origin. In the
current study, components of the cell lines will be examined closely including: disease states,
genetics and tissue origin to better address what is influencing the variable conversion results.
DIRECT CELLULAR REPROGRAMMING
Direct cellular reprogramming is the process by which a fully differentiated, somatic cell
type is induced to convert to a different cell type without reverting to a stem cell-like state. This
induced cellular conversion would not occur under normal physiological conditions directly.
This specialized area involves the application of transcription factors and/or small molecules to
the input cell to induce phenotypic changes.
The majority of cells in an organism differ both, morphologically and functionally from
one another in varying degrees. However all cells originate from a single cell, the zygote, and
3

through multiple cell divisions, the zygote gives rise to all cell types present in a given organism.
Most cells in an organism contain exactly the same deoxyribonucleic acid (DNA) sequence, and
epigenetics are impacting the gene expression among different cell types [4]. Epigenetics
describes ―the interactions between genes and the cellular environment that produces a change in
the cell phenotype‖ [5].
In a eukaryotic cell, the DNA is condensed into chromatin within the nucleus.
Transcription factors are proteins that are involved in gene transcription regulation. A
transcription factor can interact with DNA nonspecifically to find its binding site where the
transcription factor alters conformation and induces transcription by interacting with other
proteins. The process of transcription requires transcription factors to bind to regulatory
elements of the DNA located near the target genes. Once the transcription factor binds,
transcription of the target gene starts [6]. This process can lead to changes in cellular
morphology and function. By utilizing the knowledge of transcription factors, it is possible to
apply certain transcription factors to reprogram a somatic cell into a different pathway that it
would not normally express in physiological homeostasis (i.e. remaining a fibroblast cell).
One of the earliest studies on direct cellular reprogramming achieved success by
transfecting the transcription factor MyoD to convert mouse fibroblasts into skeletal muscle
myotubes [7]. This study became the first documented demonstration of a single transcription
factor acting as a master switch for changing the cell‘s identity by activating the whole genetic
program of muscle differentiation in a non-muscle cell type. Since this first discovery,
laboratories have been able to generate functional cardiomyocytes [8], iNCs[9] and induced
pluripotent stem cells (iPSCs)[10] from fibroblast cells.

4

iPSC CONVERSION
Yamanaka was awarded the Nobel Prize for Physiology or Medicine in 2012 for
reprogramming human fibroblasts into induced pluripotent stem cells (iPSCs). An increased
understanding of the mechanisms by which cellular conversion functions, and the possibilities
for the future via his research were gained [10]. One issue with reprogramming into a stem cell
is related with the observed genetic instability. A transcription factor used (cMyc) has been
reported to promote carcinogenesis [11], and once the cell is converted into a pluripotent state it
needs to be converted back to the desired cell type using a variety of reagents that are not present
in normal physiology [12]. Therefore, although the generation of iPSCsfrom a fully
differentiated cell type is promising and can generate a self-renewing cell type, it has its
downfalls.
EFFICIENCY DIFICULTY
With direct cellular reprogramming, the cell is converted from one input cell directly into
the desired output cell, consequently saving time and possible error during conversion to a stem
cell-like state. An overarching issue for both reprogramming into an iPSC and direct cellular
reprogramming is a low conversion rate. There are two subcategories within this issue: the low
conversion rate in general, making clinical application less attainable and controversy between
different laboratories reporting different results.
Multiple laboratories have reported success with reprogramming cells into different cell
types, all demonstrating low efficiency of conversion. Although successful conversion has been
demonstrated through both phenotypic and functional data, the amount of the input cells

5

successfully converted is very low, with only a few input cells converting into output cells while
the rest remain in a fibroblastic phenotype [13-16].
Current publications report various conversion rates with the same input cells, and from
the same type of input animals to the same type of output cell, causing discrepancy and
controversy between differing publications and data. Cell type, species of origin, and age of the
donor subject are known variables that could influence reprogramming efficiency. However,
even when these properties and technical aspects of the reprogramming regimen are held
constant, variation has been observed using direct methods such as infection with recombinant
retroviruses or plasmid transduction expressing transcription factors [13-16]. Even one of the
first direct cellular reprogramming successes noted reprogramming was not uniform among all
cell lines [7]. A comparison of five mouse fibroblast cell lines, C3H10T1/2, NIH3T3, Swiss 3T3,
Swiss 3T3 clone 2, andL Cells, transfected with a MyoD expression plasmid and selected to
produce colonies of stably transduced cells, yielded colonies of both the input and the conversion
phenotype. The conversion efficiency varied dramatically from a maximum of 53% myoblastic
colonies in C3H10T1/2 cells to a minimum of 3% myoblastic colonies in L cells all harvested
from the same species.Likewise, a published report from Lattanzi et al. [18] that compared the
conversion of fibroblasts to myogenic cells from different tissue sources infected with a high titer
multiplicity of infection (MOI 2,000) MyoD adenovirus vector found that murine dermis-,
muscle-, and bone marrow-derived fibroblasts converted at efficiencies of 59%, 43%, and 7%,
respectively, and human fibroblasts derived from the same tissues at efficiencies of 54%, 36%,
and 6% respectively.
Together, these reports indicate that conversion variation may be observed irrespective of
whether vector delivery is relatively highly efficient (adenoviral infection) or inefficient (plasmid
6

transduction). More recently, dissimilarity in the input cell population was proposed to account
for the conversion disparity of fibroblasts to functional cardiomyocytes as reported by several
groups [16, 19-21]. The reason why there is great variation between the results obtained from
different groups using the same cell line to convert into a different phenotype is poorly
understood.
FIBROBLAST CELLS AS AN INPUT LINE
Fibroblast cells are an ideal cell type for cellular reprogramming due to their high
abundance throughout the human body. Fibroblasts are the basic structural unit present in all
organs including the skin, and they are involved in the wound healing process. The skin is a
superb location clinically to harvest cells because it is easily accessible, therefore less invasive
than many surgical procedures. There is also less clinical risk involved in both the healing and
harvesting aspects. Fibroblasts are also ideal for use as an input cell because they are easily
cultured and are adherent to the plate during experimentation.
Fibroblasts have markers that can be used to confirm their fibroblastic identity which
includes vimentin and fibronectin. Vimentin is an intermediate filament protein and is expressed
by normal fibroblasts. Vimentin is known to preserve cellular integrity and provide resistance
against stress [22]. Fibronectin is another protein, known to play roles in cell adhesion, growth,
migration, differentiation and wound healing in adults when expressed [23]. Through
examination of these markers, a cell type, which is fibroblastic in nature, may be identified.
NEURONS AS AN OUTPUT CELL
Neurons are specialized cells that are primed for sending and receiving signals, a function
that is directly related to structure. The uniquely shaped neuron contains four domains: the cell
7

body (soma), dendrites, axon, and presynaptic terminals. The cell body surrounds the nucleus,
and has been reported to perform many of the neuronal housekeeping functions including
synthesis and processing of proteins. The cell body also receives information from outside
stimuli. Dendrites are also responsible for receiving information from outside stimuli. The
dendrite membranes are covered with receptors that bind and respond to the neurotransmitters
released by neighboring cells. The chemical messages of these neighboring cells are translated
by membrane receptors into an electrical event that impacts the excitability of the receiving
neuron. The cytoplasm of the dendrites contains a dense system of microtubules, making the
dendrites plastic and easily remodeled. The axon is similar to the dendrites, but is responsible for
the input and output of action potentials to and from the cell body [6].
The axon is longer than dendrites, and the cytoplasm is dense in parallel arrays of
microtubules and microfilaments, providing structural stability and rapid movement of materials
back and forth between the cell body and the axon terminus. Axons are the message-carrying
portion of the neuron, and they are known to carry action potentials to specific target zones.
These electrical events (action potentials) can be recorded in a laboratory via patch clamp
analysis. The presynaptic terminals are located at the target zone where the axon terminates in
multiple endings. These terminals are designed for rapid conversion of the neurons‘ electrical
signal into a chemical signal for the target zones cell to process and act upon [6]. There are
multiple types of neurons; however the present study examines iNCs, which can be thought of as
an unspecialized neuron-like cell. Neurons are an ideal output cell because they have a
reportable and drastic phenotypic change from fibroblasts like the formation of somas, and their
change can be reported via functional data of patch clamp analysis.
CLINICAL APPLICATION AND NEUROLOGIC DISORDERS
8

In the present study, direct reprogramming of fibroblasts from multiple origins into iNCs
is explored. This process is clinically relevant due to the prevalence of diseases involving
neurons including: Alzheimers, Parkinson‘s and Hunington‘s disease. These neurological
disorders are characterized by the loss of neurons or neuron functionality, and injections of iNCs
at the site of depletion may benefit patients with such neurological disorders. iNCs injections
may also be clinically useful at the site of injury for paraplegic patients to reestablish a normal
neural network and heal as addressed above.
This study uses two fibroblast lines from people with neurologic disorders. This should
provide some insight, but not conclusive evidence, to the capacity of fibroblasts from neurologic
disorder patients to iNCs. The two cell lines used came from one patient will Rett Syndrome and
another patient with Schizophrenia.
Rett Syndrome is a severe neurodevelopmental disorder affecting primarily females with
a ratio of 1:10000 female births. Rett Syndrome is one relatively common genetic cause for
severe mental retardation in females. Normal development is observed until 6-18 months of age
at which time fine and gross motor skills and social interaction is lost [24]. Most patients also
exhibit breathing problems [25] and depending on the case, patients may exhibit seizures and
speech problems. Patients undergo devastating motor deterioration, generalized rigidity,
dystonia, and worsening of scoliosis. Most patients with Rett Syndrome lose mobility, and are
often wheelchair-bound during their teenage years. As patients grow older, they often develop
characteristics of Parkinson‘s disease [26,27].
With this neurodevelopmental disorder, there is an overall decrease in brain and
individual neuron size. Autopsy studies have demonstrated a 12%–34% reduction in brain

9

weight and volume in patients suffering from Rett Syndrome [28]. The Rett Syndrome brain
shows no obvious degeneration, atrophy, or inflammation, and there are no signs of neuronal
migration defects [29,30]. These observations strongly suggest that Rett Syndrome is a disorder
of postnatal neurodevelopment, rather than a neurodegenerative process.
Mutations of the X-linked gene methyl-CpG-binding protein 2 (MECP2) are involved in
most Rett Syndrome patients. However precise functional consequences resulting from the
mutation of MECP2 and the protein interactions surrounding this mutation remains unknown.
The array of mutations includes missense, nonsense, frame shift, and deletions encompassing
whole exons. Eight missense and nonsense mutations account for 70% of all mutations [25]. The
actual mutation of the patient used in this study is unknown. However, the inclusion of a female
patients‘ fibroblast cell line may provide insight on the potential for a patient with a genetic
disorder to produce a cell type that is not functioning properly within their own body.
Fibroblasts harvested from a patient with Schizophrenia are also included in this study.
Schizophrenia is a disabling and chronic brain disorder that is still poorly understood, despite its
prevalence in society [31]. People with Schizophrenia may exhibit hallucinations, delusions, an
increased tendency to perform violent acts, and disordered thinking and behavior. The onset of
Schizophrenia is usually post birth. In men, the onset occurs during the teen years into the
twenties, while the onset in women occurs somewhat later, typically between twenty and thirty
years of age. The cause of onset time is unknown, however it‘s hypothesized that a multitude of
causes are involved, including environment and genetics [32]. Neuroimaging can demonstrate
changes within a Schizophrenia patient‘s brain in comparison with a non-disease type. The loss
of cortical gray matter in the brain with evidence of progression over time is one such example
of a demonstrable change [33].
10

PURPOSE OF THIS STUDY
The primary purpose of the present study is to investigate the possible differences in
cellular reprogramming capacity between disease subtypes, genetic backgrounds, and tissue
origin. One set of cell lines analyzed in this study are primary mouse fibroblasts from various
organs. These fibroblasts were all derived from the same mouse, resulting in an identical genetic
background among the harvested cells. Through examination of these cell lines, it is possible to
discern whether or not genetics plays a role in the ability to reprogram. By removing genetic
variation from the equation, it is possible to investigate differences in reprogrammability based
solely on the origin of input tissue. In the present study, human fibroblast lines from multiple
patients are examined to allow for comparison of the same genetic background to a diverse array.
Through examination of two cell lines, derived from patients with neurologic disorders, it
is possible to compare the reprogrammability of a person with a neurodegenerative disorder to
that of a healthy individual. The overall goal is to not generate concise conclusions from the data
gathered, due to a lack of diseased cell lines applied to the study. However, through such
examination, some light may be shed on capacity for reprogrammability. This could be further
researched in a future study.
With this knowledge at hand, a better understanding regarding the importance of harvest
locations of input cells may be obtained. It is possible that certain harvest locations are primed
to exhibit superior efficiency of conversion while other locations are not. Furthermore, multiple
questions can begin to be addressed including those regarding the capacity of fibroblasts
harvested from a patient with a neurologic disorder to convert to iNCs as well as those regarding
variation in genetic background as it relates to conversion efficiency.

11

METHODS
FIBROBLAST LINES
Human primary fibroblasts were acquired by skin punch biopsies or gingival explants
under MSU-approved IRB protocols (ADF, E2F, EAF, and HSK). Commercial sources,
including the ATCC (FET and HDNF) and the Coriell Institute (RET, SAF, and AUT), were also
applied to the study. Additional information regarding these cell lines is located in Table 1
below. Mouse fibroblast lines were harvested from an individual, 5-month-old nu/nu mouse that
was sacrificed by CO2 overdose. The mouse carcass was disinfected with ethanol, and multiple
3

tissues were removed for dissection. 125mm fragments of each target organ were removed and
segmented. Individual tissue segments were placed in the wells of a six-well plate for outgrowth.
Primary outgrowths were plated in fibroblast medium [Dulbecco‘s modified Eagle medium
(DMEM), 10% fetal bovine serum (FBS), and 1 · antibiotic/antimycotic (Invitrogen)], and were
then left undisturbed (excluding medium changes) for 2 weeks, and then passaged 1:1 to a new
well. After an additional week of growth, the lines were passaged 1:2. This process was repeated
to passage 5 at which time the wells were compared to positively identify those with typical
fibroblast morphology, an absence of nonfibroblastic cells, and similar growth features. Thirteen
mouse fibroblast lines were chosen for two additional rounds of passage and expansion. These
cell lines were frozen as multiple aliquots for use in these experiments.

12

Table 1: General Properties of Fibroblast Lines. Study ID, abbreviated line designation used this report; Line ID, line common name;
Donor, donor health status or strain; Sex, sex of donor (male, female, or of mixed sex); Age, age of donor (years, unless otherwise
noted); Tissue, fibroblast tissue of origin. mRNA Expression: results of quantitative PCR analysis of multiple markers. COL1A2,
fibroblast marker collagen type I alpha 2; FNECTIN, fibroblast marker fibronectin; FIBR1, fibroblast marker fibrillin I; FIBU5,
fibroblast marker fibulin V; VIM, fibroblast marker vimentin; KER14, keratinocyte marker keratin 14; PECAM, endothelial cell
marker Platelet endothelial cell adhesion molecule/CD31; FOXG1, neural progenitor marker Forkhead box protein G1; SOX2, neural
progenitor marker SRY (sex determining region Y)- box 2, MYOD, myogenic progenitor marker MyoD1; MYF5, myogenic
progenitor marker myogenic factor 5. Immunocytochemistry: summary of immunocytochemical analysis of fibroblast-associated
13

Table 1 (cont‘d)

(fibronectin, vimentin) and stem cell-associated markers (Sox-2, nestin) in fibroblast lines. N/A, not applicable; ND, –not done; + ,
positive; – , weakly positive; - , negative. (From Alicea et al. 2013 [54])

14

FIBROBLAST CHARACTERIZATION
Fibroblast RNAs from the mouse lines at passage 6–7 and the human lines at ‗‗passages‘‘
8–10 were purified using Trizol (Invitrogen) or the RNeasy kit (Qiagen). Following the
manufacturer‘s guidelines, 2 mg of purified RNA was converted to cDNA using Superscript II
(Invitrogen). Quantitative polymerase chain reaction (qPCR) was executed on an ABI Prism
7000 analyzer using 1 mL of cDNAand normalizing against nuclear lamin A or ARHGAP
mRNAs as internal controls. Other genes used as internal controls (RPL27A, EED, and GR)
generated comparable results. Primers for qPCR analysis are shown below in Table 2.
PRIMER
CLONING
ASCL1 F
ASCL1 R
MYF5 F
MYF5 R
MYF6 F
MYF6 R

5'-3' SEQUENCE
GAGAGAACGCGTGGCATGGAAAGCTCTGCC
ACACACATCGATTCAGAACCAGTTGGTGAAGTCG
GAGAGAACGCGTATGGACGTGATGGATGGCTGCC
GTGTGTAATCGATTCATAGCACATGATAGATAAGCC
GAGAGAACGCGTATGATGATGGACCTTTTTGAAACTGG
GTGTGAATCGATTTACTTCTCCACCACTTCCTCCACGC

SPECIES
Human
Human
Mouse
Mouse
Mouse
Mouse

Table 2:Primers Used in Cloning and qRT-PCR AnalysisPrimers Used in Cloning and qRT-PCR
Analysis. Cloning- primers used in the amplification of cDNAs or plasmid DNA for construction
of retroviral vectors; QPCR- primers used in qRT-PCR quantification of gene expression. The
species of the gene targeted by each primer pair is shown on the right. (From Alicea et al. 2013
[54])

15

Table 2 (cont‘d):
MYOD F
MYOD R
MYOG F
MYOG R
MYT1L F
MYT1L R
NEUROD1 F
NEUROD1 R
POU3F2 F
POU3F2 R
YFP F
YFP R
ZIC1 F
ZIC1 R
QPCR
PANL27 F
PANL27 R
ARHGAP1 F
ARHGAP1 R
COL1A2 F
COL1A2 R
EED F
EED R
FIBR1 F
FIBR1 R
FIBU5 F
FIBU5 R
FNECTIN F
FNECTIN R
FOXG1 F
FOXG1 R
KER14 F
KER14 R
LAMIN F
LAMINR
MYF5 F
MYF5 R
MYOD F
MYOD R

GAGAGAACGCGTGGTATGGAGCTTCTATCGCCGCCAC
ACACACATCGATTCAAAGCACCTGATAAATCGC
GAGAGAACGCGTATGGAGCTGTATGAGAC
GTGTGTAATCGATTCAGTTGGGCATGGTTTCATC
GAGAGAGGCGCGCCCGATGGAGGTGGACACCGAGG
CACACAATCGATTCAGACCTGAATTCCTCTCACAGCC
GAGAGAACGCGTGGTATGACCAAATCGTACAGCG
GTGTGTGTTTAAACCTAATCATGAAATATGGCATTGAG
CTG
GAGAGAGGCGCGCCCAATGGCGACCGCAGCGTCTAAC
C
ACACACTATCGATTCAACGCGTCTGGACGGGCGTCTGC
AC
CACAGGCCGCCTGGGCCATGGTGAGCAAGGGCG
AAACTTAACGCGTCTTGTACAGCTCGTCCATG
GAGAGAGGCGCGCCCGGGAATGCTCCTGGACGCCGG
ACACACATCGATTAAACGTACCATTCGTTAAAATTGGA
AGAGAGCGCAC

Mouse
Mouse
Mouse
Mouse
Human
Human
Human
Human

CCATCCAGACTGAGGAAGACCCGGAAAC
GGGCAGAAGCTCTGGTTCCTC
TGCTGTGGGCCAAGGATGCG
GGTCCGGGCTTGGGAACAGC
CAGGGGCTCTGCGACACAAGG
TCCGGCTGGGCCCTTTCTTAC
GGAAGGAGCCAGGAAGCCGC
ACTGTCGCAAATCGCGCCCA
AGGAAACCAGAGCCAGTCGGG
GGAATGCCGGCAAATGGGGACA
GTGTGTGAACCAGCCCGGCA
ACGTCTGCTGCAGGTTGCACG
CGCCCTGGTGTCACAGAGGCTA
TGGGGTGTGGAAGGGTTACCAG
ACGGGGAGATCCCGTACGCC
CCGCGAGCAGGTTGACGGAG
GCAGCGGCCTGCTGAGATCAA
CATTGGCATTGTCCACTGTGGCT
GTGCGCTCAGTGACTGTGGTTGA
CGAGCGCAGGTTGTACTCAGCG
TCTCCCCATCCCTCTCGCTGC
CCACTCGCGGCACAAACTCGT
CTCCAACTGCTCCGACGGCA
TCGACACCGCCGCACTCTTC

Human/Mouse
Hu/Mo
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human

16

Human
Human
Other
Other
Human
Human

Table 2 (cont‘d):
PECAM F
PECAM R
SOX2 F
SOX2 R
VIMENTIN F
VIMENTIN R
ARHGAP1 F
ARHGAP1 R
COL1A2 F
COL1A2 R
EED F
EED R
FIBR1 F
FIBR1 R
FIBU5 F
FIBU5 R
FNECTIN F
FNECTIN R
FOXG1 F
FOXG1 R
KER14 F
KER14 R
LAMIN F
LAMINR
MYF5 F
MYF5 R
MYOD F
MYOD R
PECAM F
PECAM R
SOX2 F
SOX2 R
VIMENTIN F
VIMENTIN R
ARHGAP1 F
ARHGAP1 R
COL1A2 F
COL1A2 R
EED F
EED R
FIBR1 F
FIBR1 R
FIBU5 F

TCCACATCAGCCCCACCGGA
TGGGCCACAATCGCCTTGTCC
GGGGGAAAGTAGTTTGCTGCCTC
CTGCCGCCGCCGATGATTGT
GAGCAGGATTTCTCTGCCTCTTCC
TCGTGATGCTGAGAAGTTTCGTTGA
TTTGCCGAGCTTTGACAGGCG
AATGGAGGCCAGCTTCAACTGG
CAGGGGCTCTGCGACACAAGG
TCCGGCTGGGCCCTTTCTTAC
GGAAGGAGCCAGGAAGCCGC
ACTGTCGCAAATCGCGCCCA
AGGAAACCAGAGCCAGTCGGG
GGAATGCCGGCAAATGGGGACA
GTGTGTGAACCAGCCCGGCA
ACGTCTGCTGCAGGTTGCACG
CGCCCTGGTGTCACAGAGGCTA
TGGGGTGTGGAAGGGTTACCAG
ACGGGGAGATCCCGTACGCC
CCGCGAGCAGGTTGACGGAG
GCAGCGGCCTGCTGAGATCAA
CATTGGCATTGTCCACTGTGGCT
GTGCGCTCAGTGACTGTGGTTGA
CGAGCGCAGGTTGTACTCAGCG
TCTCCCCATCCCTCTCGCTGC
CCACTCGCGGCACAAACTCGT
CTCCAACTGCTCCGACGGCA
TCGACACCGCCGCACTCTTC
TCCACATCAGCCCCACCGGA
TGGGCCACAATCGCCTTGTCC
GGGGGAAAGTAGTTTGCTGCCTC
CTGCCGCCGCCGATGATTGT
GAGCAGGATTTCTCTGCCTCTTCC
TCGTGATGCTGAGAAGTTTCGTTGA
TTTGCCGAGCTTTGACAGGCG
AATGGAGGCCAGCTTCAACTGG
CTCATACAGCCGCGCCCAGG
CGGTTGGCTAGCAGGCGCAT
CGCCGGCGGGAACAGACATG
TATTTGTGGGCGTGTCCGGGC
AGGCCCCCTGCAGTTACGGT
CCTCGGCCCATGCCCATTCC
ACAACCCGATACCCTGGTGCCT
17

Human
Human
Human
Human
Human
Human
Mouse
Mouse
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse

Table 2 (cont‘d):
FIBU5 R
FNECTIN F
FNECTIN R
FOXG1 F
FOXG1 R
KER14 F
KER14 R
LAMIN F
LAMINR
MYF5 F
MYF5 R
MYOD F
MYOD R
PECAM F
PECAM R
SOX2 F
SOX2 R
VIMENTIN F
VIMENTIN R

CGAGGCCCTTTGATGGGGCG
GAGCGACATGCTCTACAAAGTGCT
CTGGGGGTGAGTCTGCGGTTG
CGATCGCGGCTACCGGCTTC
CACTCCCAGAGTCGCGCTCAC
ACAGCCCCTACTTCAAGACCATCG
CGCAGGCTCTGCTCCGTCTC
GCCTTCGCACCGCTCTCATC
GCCGCTGCAGTGGGAACC
CCCCAACCTCAGCCACTGACC
GCCAGCAAATCCAGGCGGAGC
GGAGATCCTGCGCAACGCCA
GCAGCGGTCCAGGTGCGTAG
ACGAGAGCCACAGAGACGGTG
AGGGACGTGCACTGCCTTGAC
GCTGCCTCTTTAAGACTAGGGCTG
GCCGCCGCGATTGTTGTGAT
GTCGAGGTGGAGCGGGACAAC
CCGTTCAAGGTCAAGACGTGCCA

Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse

Human and mouse fibroblasts were processed for immunocytochemical analysis to
inspect marker expression at the individual cell-level, following the methods described below for
iNC analysis. Primary antibodies were applied to the experiment as follows: anti-vimentin
1:1250 (Millipore AB5733), anti-fibronectin 1:750 (BD, 610077), anti-nestin 1:250 (Santa Cruz
Biotechnologies;H-85), and anti-Sox2 1:250 (Santa Cruz Biotechnologies; Y-17). Multiple
images of each immunostained line were gathered. Approximately 1x10³ cells were inspected at
high magnification for the presence or absence of those markers consistent with stem cell (Sox2/nestin) or fibroblast identity (vimentin/fibronectin). Neural progenitor cells (NPCs) used for
comparison were fixed in parallel with fibroblasts, and were generated as described below for
iNC analysis.
5

The relative infectivity for factor expression was calculated by infecting 1x10 actively
growing cells with concentrated NITSC-NLS-YFP retrovirus at an MOI of approximately 0.5. 4
18

days post-infection, the yellow fluorescent protein (YFP) positive cells were counted as a
fraction of all cells over three replicates. The precise cellular age of each cell line was partially
unknown. Therefore, verification was required to confirm that none of the analyzed lines were
approaching cellular senescence, which could influence the reprogramming process.
During the verification procedure, each line was continuously passaged and counted for
four rounds (post-experiments) to confirm that all lines were still actively proliferative. One of
the 13 mouse lines, LU3, stopped proliferating by the third trial passage and was removed from
the final analysis. Two human lines, AUT and HSK, produced iNCs that were partially
characterized, but were not included in the final analysis. Prior to completion of various
experiments, the laboratory experienced freezer failure, resulting in the loss of ―matching‖ frozen
stock. Similarly, mouse lines TA6, KI2, and LU6 were not included in the experiments shown in
Figure 2 due to freezer failure.
iNC INDUCTION
cDNAs encoding human ASCL1, POU3F2, and ZIC1 were obtained from Open
Biosystems. Myt1L and NeuroD1 open reading frames (ORFs) were obtained from cDNA
produced from human brain reference RNA (Applied Biosystems). NITSC was produced by
introducing the BstEII-ClaI fragment encompassing Neo-IRES-TTA-TetO from NIT
(GenbankAcc# AF311318) into BstEII-ClaI cut pMSCVneo (Clontech) and a polylinker for
transgene expression, SfiI-MluI-PmeI-ClaI. Primers used for cloning factors into NITSC are
shown in Table 2. The amplified YFP ORF with a MluI site inserted immediately prior to the
stop codon was digested with SfiI-PmeI and introduced into NITSC to produce the control vector
NITSC-YFP. Remaining factor ORFs were polymerase chain reaction (PCR) amplified with
compatible MluI or Asc1 sites at the 5‘ end and ClaI at the 3‘ end for cloning into NITSC-YFP

19

to create the fusion protein constructs. NITSC recombinant moloney murine leukemia virus
(MMLV) particles were made by three-way calcium phosphate transfection of Human
Embryonic Kidney 293 (HEK) cells with gag-pol and Vesicular stomatitis virus (VSV) encoding
plasmids to produce replication defective virus particles. Two days post-transfection, viral
supernatants were harvested, filtered and introduced into fibroblast cultures via the carrier
polybrene (8 mg/mL) to increase infection efficiency as described for lentiviral vectors in Suhr et
al. [34]. Viral supernatants were frozen as aliquots and used on mouse embryonic fibroblasts
(MEFs) to provide a rough titer, and to allow viral preparations to produce iNCs before use with
target cells. The YZIC/YASCL/YPOU3F (ZAP) combination appeared the most potent on both
mouse and human fibroblasts in preliminary experiments, and was used for conversion unless
otherwise noted. Equal volumes of each viral supernatant were applied (i.e., for ZAP, typically
5mL of each viral supernatant for a total of 15mL infectious medium/ 10-cm plate).

In order to determine the superlative conditions for iNC conversion during preliminary
6

studies, approximately 1x10 mouse or human fibroblasts (growing in fibroblast medium at
equal confluency) were infected with viral medium followed by an overnight wait-period to
allow for infection. Virus-infected cultures were then passaged by trypsin treatment to six-well,
12-well, or 35mm tissue culture plates followed by a period of 12-24 hours in fibroblast growth
medium to support plate attachment and cellular growth. The fibroblast medium was then
aspirated and replaced with iNC medium (DMEMF12 with N2 supplement and
penicillin/streptomycin at 50 m/mL) (Invitrogen). The iNC medium was changed at 4–5 day
intervals for the duration of the experiment, and the cells were kept in a 5% CO2 environment at
37° C. Cell culture plates coated with polyornithine/laminin (PORN/lam), or without coating,

20

were used for preliminary experiments interchangeably with little noticeable impact on the
formation of iNCs.
To determine the optimal time for counting iNCs, factor-infected MEFs and adult mouse
and human fibroblast cultures were fixed and immunostained with the neural TUJ1 antibody at
5–6 day intervals post-infection. iNC conversion (for the purpose of reprogramming efficiency
across mouse and human cells lines) was executed fundamentally as described previously.
6

However, 1x 10 target cells in one well of a six-well plate were infected with 3mL of the iNC
(ZAP) viral mixture and 24 hours later, passaged to three wells of a 12-well plate. The passaged
cells were allowed to rest an additional day, followed by a transition to iNC medium.
Subsequent medium changes occurred every 3–4 days until fixation and immunoprocessing on
day 12 for mouse iNCs and day 24 for human iNCs. Experiments for quantification of
reprogramming efficiency were performed in three distinct replicates.
IMMUNOHISTOCHEMISTRY AND IMAGING
Cells were fixed with 4% paraformaldehyde for 10 minutes followed by 3 phosphatebuffered saline (PBS) washes for 10 minutes each. PBST (PBS with 0.3% Triton X-100) with
3% donkey serum (DS) was used for 30–60 minutes at room temperature to block, and was then
replaced with PBST + 1% DS containing added primary antibody overnight at 4° C. The primary
antibodies were used at the following dilutions: TUJ1-1:3000 (Santa Cruz; Cat# sc-58888),
MAP2ab - 1:300 (Sigma;Cat#M1406), Synapsin 1–1:400 (Millipore; Cat#AB1543P), panneurofilament–1:1000 (Covance; SMI311), Doublecortin– 1:400 (Santa Cruz, Cat#sc-8066),
GAD–1:250 (Santa Cruz; Cat#sc-7513), PSD95–1:250 (NeuromAb), and GABAR3–1:250
(NeuromAb). After incubating overnight with the primary antibody, wells were washed with
PBST + 1%DS 3 times for 10 minutesand incubated with PBST +1%DS with the
21

applicablesecondary antibody ( JacksonImmunoResearch) for 30–60 minutes. Wells were
subsequently washed with PBS 3 times for 10 minutes to remove excess secondary, stained
briefly with PBS + 1 mg/mL bis-benzamideto label nuclear DNA, and then rinsed again. All
plates and wells were held at 4° C in the dark until imaging procedures were completed on a
Nikon Eclipse TE2000 inverted stage fluorescence microscope.
ELECTROPHYSIOLOGY
Infected NPC neurons or iNCs were cultured on PORN/lam-coated 35mm plates at a low
5

density (2.5x10 cells/35mm plate) as described previously for electrophysiological recordings.
All recordings obtained were generated using the entire cell configuration of the patch-clamp
technique [35]. The patch glass pipette electrodes were double pulled and heat polished. The
electrode was brought into contact with visually identified iNC targets to produce a highresistance seal between electrode tip and the cell membrane. The entire cell configuration was
attained by applying suction to the back of the electrode. For voltage clamp experiments,
electrode capacitance was compensated before attaining the whole cell configuration, and
membrane capacitance and series resistance were compensated after accomplishing this
configuration. Membrane current and potential signals were amplified (List Electronic EPC-7),
digitized (Digidata 14140A; Molecular Devices), and recorded on a computer. Voltage steps and
current injection pulses were generated and potential and current signals were evaluated using
software written by Dr. John Dempster (Dept. Physiology, University of Strathclyde).
In every voltage clamp recording, the holding potential (Vh) was - 80 mV. The existence and
properties of voltage gated current was analyzed during positive voltage steps (30 to 250 ms
depending on the experiment) to test potentials (Vtest) between - 75mV and + 50 mV. To inspect
voltage dependent, steady-state inactivation of voltage gated Na+ channels, a double voltage step
22

was used: A step to a conditioning potential (Vcon, - 130mV to + 40 mV, 50 ms) was applied
directly before the step to the test potential (0 mV). To evaluate whether or not cells had the
ability to produce action potentials, membrane potential was measured under current clamp.
The extracellular solution used was comprised of NaCl 135mM, KCl 5mM, glucose
10mM, MgCl2.6H2O 1mM, CacCl2.2H2O 2mM, and HEPES 20mM (pH 7.3). The recordings
of isolated voltage-gated Na+ current, the electrode solution comprised of CsCl 20mM, cesium
methanesulfonate 130mM, MgCl2.6H2O 2mM, glucose 10mM, EGTA 10mM, and HEPES
10mM (pH 7.3). The recordings of mixed voltagegated Na+ and K+ current, and for the
recordings of membrane potential and action potentials, the electrode solution comprised ofKCl
20mM, potassium methansulfonate 130mM, MgCl2.6H2O 2mM, glucose 10mM, EGTA
0.01mM, and HEPES 10mM (pH 7.3).
HUMAN NPC CULTURE AND NEURON DERIVATION
For the control human neurons shown in Figure 1 below, H9 human ES cells were
differentiated to NPCs as described in[36]. H9-NPCs were propagated to passage 5 in iNC
medium with 20 ng/mL FGF-2 added.NPCs were plated on PORN/lam plates, and FGF-2 was
gradually withdrawn to a final concentration of 2 ng/Mlby day 20–24 for differentiation. Cells
were then processed for immunostainingto confirm neuronal identity and electrophysiological
analysis was performed. As controls in the experiments undifferentiated NPCs at 30%–50%
confluencywere fixed and used as shown below in Figure 2.

23

Figure 1: Time Course of induced Neural Cell (iNC) Induction and Electrophysiological
24

Figure 1 (cont‘d):

25

Figure 1 (cont‘d):Analysis. (A) Examples of untransduced fibroblasts or fibroblasts infected with
a YFP-only control vector (green) cultured under INC induction conditions for 4 weeks and
stained for TUJ1 (red). (B) TUJ1-, SYN1-, and MAP2-positive mouse and human iNCs over
time expressed as a percentage of the maximum TUJ1 value. Infection was at time 0 (T0), T1 =
8–10 days, T2 = 18–20 days, and T3 = 28–30 days. BLUE: TUJ1, RED: MAP2, GREEN: SYN1.
(C) Examples of human iNCs produced by different fibroblast lines at the T1-T3 time points
(labeled on left) and stained for TUJ1 (red). In the upper left panel, arrows indicate early iNCs in
a low-magnification field. In the upper right panel, the iNC boxed At the left is shown magnified
for each color channel and the merged image. In the center and lower panels, iNCsatthe T2 and
T3 time points, respectively, are shown. Scale bars are 10 mm unless otherwise noted. Insets
depict the cell soma with the blue channel removed to more clearly reveal iNC factor expression.
(D) Representative electrophysiological recordings from iNCs. Upper left panel - Isolated
voltage-gated sodium current recorded from a representative human iNC. Upper records show
voltage steps from a holding potential of - 80mV to test potentials of - 40, - 25, 0, and 25 mV.
Lower records show leak-subtracted Na + current elicited by these voltage steps. Note that the
step to - 40mV is sub threshold and that the steps to - 25, 0 and 25mV are supra-threshold and
elicit inward sodium current of amplitude and kinetics which is voltage dependent. Inset shows
the relationship of test potential and the maximum amplitude of the Na + current elicited at each
test potential in this cell. Upper right panel—Voltage-gated inward Na+ current was blocked by
TTX. Upper record: voltage step from - 80 to 0 mV, which was repeated every 2.5 s during the
recording. Lower records: current elicited by the membrane depolarization at different times
during the recording. Voltage-gated inward Na+ current (but not outward K+ current) was
reduced in a time-dependent manner after the application of TTX (5 mM) to the extracellular
medium. Lower panels—Representative action potentials (APs) recorded from NPC-neurons and
INCs. Left - AP produced by an NPC-derived neuron. Membrane potential change (upper
record) elicited by application of membrane current step (lower trace) while holding the
membrane at - 72mV just before current step application. Center—AP produced by an ADF iNC.
Membrane potential changes (upper records, x and y) elicited by application of membrane
current step (lower trace) without (x) and with (y) holding the membrane at - 90mV (x) or 55mV (y) just before current step application. Right—Overlay of the NPC and INC AP traces
(NPC, green; ADF, red).Top Panel: Raw tracings for visual reference only.(From Alicea et al.
2013 [54])For interpretation of the references to color in this and all other figures, the reader is
referred to the electronic version of this thesis.

26

Figure 2:Immunocytochemical Examination of Mouse and Fibroblast Lines.(A,B) Mouse
fibroblast lines analyzed for immunocytochemical components. The panels at the end show that
as with human cells, rare patches of dim green fluorescence do not co-localize with nuclei and do
not indicate Sox-2 immunoreactivity. Blue color in all images is nuclear DNA stain bisbenzimide. (C) Anti-fibronectin (red) and anti-Sox2 (green) antibodies. Insets in middle panels
display magnified images, demonstrating that rare spots of dim green fluorescence are not cell/
Figure 2 (cont‘d): nucleus associated as with genuine Sox-2 immunopositivity observed in
NPCs. (D) Immunostaining of human cell lines with anti-vimentin (red) and anti-nestin (green)
antibody. Upper panels—both antibodies in all fibroblast cell lines. Middle panels—the green
channel only (nestin), exhibiting an absence of positive signal. Lower panels --- controls
demonstrating the same antibodies on human NPCs as labeled.(A) Top Legend (Left to Right):
VIMENTIN NESTIN DNA. DATA (Left to Right): FET, NWB, RET, AUT, SAF, ADF, EAF,
E2F. Middle Legend: NESTIN. DATA (Left to Right): FET, NWB, RET, AUT, SAF, ADF,
EAF, E2F. Bottom Legend: HUMAN NPCS. DATA (Left to Right): VIMENTIN DNA,
27

Figure 2 (cont‘d):

28

Figure 2 (cont‘d):

29

Figure 2 (cont‘d):

VIMENTIN, NESTIN DNA, NESTIN. B) Top Legend (Left to Right): FIBRONECTIN SOX2
DNA. (Left to Right): FET, NWB, RET, AUT, SAF, ADF, EAF, E2F. Middle Legend: Sox2.
DATA (Left to Right): FET, NWB, RET, AUT, SAF, ADF, EAF, E2F. Bottom Data (Left to
Right): FIBRONECTIN DNA, FIBRONECTIN, SOX2 DNA, SOX2. C) Top Legend (Left to
Right): VIMENTIN NESTIN DNA. Data (Left to Right): HE4, KI3, KI5, KI6, LI6, SM1, TA4,
TE4, TE5. Middle Legend: Nestin. Data (Left to Right): HE4, KI3, KI5, KI6, LI6, SM1, TA4,
TE4, TE5. Bottom Legend: Data (Left to Right): HE4, KI3, KI5, KI6, LI6, SM1, TA4, TE4,
TE5.D) Top Legend: (Left to Right): FIBRONECTIN SOX2 DNA. Data (Left to Right): HE4,
KI3, KI5, KI6, LI6, SM1, TA4, TE4, TE5. Bottom Legend: SOX2 Data (Left to Right): HE4,
KI3, KI5, KI6, LI6, SM1, TA4, TE4, TE5.
30

QUANTIFICATION OFiNC CONVERSION
Quantification of iNC conversion was accomplished by using Hoechst33342 staining for
nuclei/DNA (Blue), YFP fluorescence of the tagged proteins to indicate relative factor
expression (Green), and β-IIITubulin/TUJ1 (iNCs) immunostaining to indicate phenotypic
conversion (Red). Relative reprogramming efficiency was calculated by dividing the red
fluorescence value by the blue fluorescence or by dividing the red/blue value by the green
fluorescence value to contain a factor-expression component in the calculation. iNC conversion
was measured as the number of red fluorescent cells that had fibers of at least three soma lengths.
For each well, five fields were imaged at 100X magnification for each separate channel and
stored as a merged RGB image. The fields included one in the center of each well, and one at
each compass point (approximately 1 cm from the well edge). To measure fluorescence, the
RGB image was separated into individual black and white channels followed by quantification
with NIH ImageJ. The highest relative conversion value for iNCs for each group was set at 100
in the graphs, and the remaining values were calculated as a fraction of that maximum.
RESULTS
In an attempt to limit technical variance, all procedures were performed in parallel on all
cell lines within a group with a minimum of three distinct experiments. Fibroblasts were selected
as the input cell type for a variety of reasons. Fibroblasts are naturally adherent and capable of
undergoing cryogenic preservation. Furthermore, the ability of fibroblasts to be passed and
established with little difficulty has been previously recognized, and these cells have been used
in many studies published by various authors reporting a large variance of reprogramming
efficiency to a desired output cell type. Human fibroblasts were selected as an input cell type
due to their ability to be directly translated into clinical application for human medicine. An
31

assortment of ages was selected for human fibroblasts with numerous older subjects due to a
higher probability for older subjects to serve as the primary target for cellular reprogrammingbased therapy. Mouse fibroblasts were selected as an input cell type because all lines were
derived from a single donor, therefore being isogenic, and multiple lines could be harvested from
the same isogenic organ for comparison. As illustrated below in Figure 3, human and mouse
fibroblasts lines were grown in cell culture, underwent over eight passages while still exhibiting
homologous morphology, and were a minimum of four passages from mitotic senescence.

32

Figure 3: Morphology and Derivation of Fibroblast Lines Used in Analysis. (A)Morphology of
33

Figure 3 (cont‘d): the seven human fibroblast lines used for analysis. (B) Outgrowth of
fibroblasts and other cell types from tissue fragments of eight mouse tissues at 1 week post
plating. The dark mass in the upper left corner is the adherent tissue fragment itself. (C)
Morphology of the twelve mouse fibroblast lines (at passage 6) used for analysis.(From Alicea et
al. 2013 [54])

Quantitative real-time polymerase chain reaction (RT-PCR) analysis was performed to
confirm an abundance of fibroblast-associated mRNAs, and the absence of significant signals for
indicators of other differentiated or progenitor cell types as illustrated below in Figure 4. All
fibroblast lines displayed an abundance of the fibroblast-associated markers collagen type 1α2,
vimentin, fibronectin, fibrillin I, and fibulin V with mouse lines HE4 and KI3 testing negative for
mouse collagen type 1α2. HE4 also displayed weak vimentinimmunopositivity, but
immunopositivity none the less. Because both lines were positive for most fibroblast markers,
and negative for stem cell type mRNA indicators, both lines remained in the study. mRNAs for
other common contaminating cell types such as keratinocytes and endothelial cells were not
detected via RT-PCR displayed on both Table 1 and Figure 4.

34

Figure 4:Fibroblast- and Nonfibroblast-associated mRNA Expression in Human and Mouse Fibroblast Lines.Heat map color is

35

Figure 4 (cont‘d): proportional to expression level—bright green is high expression and black is
no detectable transcript.COL1A2, collagen type 1a2; FNECTIN, fibronectin; FIBR1, fibrillin I;
FIBU5, fibulin V; KER14, keratin 14.(From Alicea et al. 2013 [54])
Immunohistochemical analysis of fluorescence for Sox-2 and nestin was performed to
further characterize the fibroblasts at the individual cell level of each line. These markers are not
expressed at significant levels in fibroblast [37-43], however they are expressed robustly in
multiple stem cell classes including neural stem cells. As demonstrated in Figure 2 and
summarized in Table 1, no cells displayed immunopositivity for either stem cell marker nestin or
Sox-2.
iNCs were chosen as the output cell type due to reports of successful direct
reprogramming from multiple laboratories using similar protocols [15, 44-47]. Also, conversion
can be measured by morphological changes and functional changes divergent from input
fibroblast characteristics, therefore making conversion easily identifiable. For iNC conversion,
all fibroblasts were infected in parallel with combinations of an MMLV-based retroviral vector
encoding the neurogenic transcription factors ASCL1, POU3F2, ZIC1, MYT1L, or NeuroD1
[15,45] of human origin fused to YFP as shown schematically below in Figure 5.

Figure 5: Schematic of the NITSC MMLV Retrovirus and YFP Fusion Factors. LTR, long
36

Figure 5 (cont‘d):terminal repeat; Ψ+ , stem-cell permissive packaging sequence; NEO, G418resistance gene; I, IRES element; TTA, tet-transactivator; To, tetoperator.(From Alicea et al.
2013 [54])
YFP-fusion proteins localized primarily to the nucleus (Figure 6A). Cells infected with a
YFP vector only and cultured under identical conditions (used as a control) produced no strongly
TUJ1-positive cells or displayed neuron-like morphology (Figure 6A). Cells infected with
ASCL1, POU3F2, and ZIC1 transcription factors alone generated very small amount of iNCs
(Figure 6B, upper panel) whereas a combination of ASCL1, POU3F2, and ZIC1 generated many
TUJ1-positive iNCs for both human and mouse fibroblasts lines (Figure 6B, lower panel and
Figure 7).
The primary difference between the generation of mouse and human iNCs was the
maturation rate [15, 44-47]. Mouse iNCs reached mature morphology by day12 post-infection,
whereas human iNCsreached mature morphology at day 24 post-infection. Mature iNCs that
displayed processes greater than three cell body lengths were selected to be indicative of mature
neurons. The criterion of three soma-lengths as an indicator of bona-fide iNCs was originally
described by Vierbuchen et al. [15], and is based on the observation that iNCs with fibers of this
length or greater frequently also expressed multiple markers associated with mature neurons. In
our human and mouse iNCs displaying long processes, mostwere positive for multiple markers
of mature neurons in addition to TUJ1, including MAP2a/b, synapsin 1, doublecortin,
neurofilament 300 kDa as shown in Figure 6C and Figure 6D.iNCs also exhibited
electrophysiological properties such as TTX-sensitive Na+ currents and action potentials that
were essentially indistinguishable from human neurons generated under identical conditions
from human ES cell-derived human neural progenitor cells (NPCs) analyzed via patch clamp
analysis (Figure 1D).

37

Maximum conversion efficiency (calculated by percent of output cells versus
unconverted cells) for mouse fibroblasts to iNCs was 0.72% – 0.08% (TA4), and 1.07% –
0.18% for human cells (RET). This is lower than some other published studies that reported 2%–
18% conversion using the same or different combinations of factors [15,44–46]; however, the
lower efficiency maybe caused by using YFP-linked transcription factors. Reprogramming
efficiency was calculated by dividing the red fluorescence value (iNC conversion indicated by
TUJ1 immunostaining) by the Hoechst 33342 fluorescence value (indicating stained nuclei), as
shown in Figure 6 E and F. As demonstrated in this figure, there was a wide array of
reprogramming efficiency among different cell lines,even in mouse cell lines that were isogenic
and in some cases from the same organ (i.e. the KI cell lines).

38

Figure 6: Figure 6: Induced Neural Cell (iNC) Conversion of Mouse and Human Fibroblast Lines. (A) HEK cells transfected with
YFP fusion protein for iNC conversion, as labeled. Phase-contrast images are in the upper panels, and corresponding fluorescent
images are in the lower panels. (B) Mouse embryonic fibroblasts infected with the individual factors (as labeled) and stained for

39

Figure 6 (cont‘d):

40

Figure 6 (cont‘d):

41

Figure 6 (cont‘d):

42

Figure 6 (cont‘d): β-III-tubulin/TUJ1 in red (upper panels). Mouse (MEFs) or human fibroblasts
(FET) infected with combinations of Zic1/ Ascl1/Pou3f2 (ZAP) or Myt1L/Ascl1/Pou3f2 (MAP)
+ NeuroD1 (MAPN) (lower panels). Green fluorescence indicates expression of reprogramming
factor(s). Blue color is bis-benzimide nuclear staining of DNA. Insets have the blue channel
removed and the green channel intensified to show YFP-factor expression in the nucleus of all
iNCs. (C) Mouse iNCs produced from MEFs by day 10–15 post-infection immune positive for
multiple neural markers Figure 6 (cont‘d): (red), including MAP2, pan-neurofilament (NF),
doublecortin (DCX), or synapsin I (SYN). iNCs produced from adult mouse fibroblast lines (as
labeled) with typical iNC morphology immunostained for β-III-tubulin/TUJ1(red). (D) Human
iNCs with typical morphology at day 24–30 post-infection and immunostained for multiple
neuronal markers as in (C) in addition to PSD95, GABA receptor b 3 (GABARB3), and GAD1
(as labeled). GABAR-B3 and GAD1 iNCs were labeled using immunoperoxidase secondary
antibody coupled with DAB staining. Scale bars are 10 mm unless otherwise labeled. (From
Alicea et al. 2013 [54])

43

Figure 7: Representative images of β-III-tubulin/TUJ1 immuno-stained iNCs. (A) iNCs in
human lines as labeled. Magnified insets have the blue channel removed and the green channel
exaggerated to show factor expression in the iNC nucleus.All human lines produced iNCs,
including AUT and HSK, that were not incorporated in the analysis. The iNCs shown in the E2F
panel are a combination of multiple 1000X-magnification images. At the bottom right corner is a
characteristic neuron produced from a human neural progenitor cell (NPC) differentiated in
parallel with iNCs. (B) iNCs in mouse lines as labeled. Magnified insets have the blue channel
removed and the green channel increased to show factor expression in the iNC nucleus.(From
Alicea et al. 2013 [54])

44

Figure 7 (cont‘d):

45

Not all cells had the capacity to take up the virus. Due to this limitation, we utilized
another control for viral uptake. Each line was infected with a nuclearlocalized YFP virus at an
MOI of approximately 0.5, and then counted to determine the number of cells with yellow
fluorescent nuclei as a fraction of all cells at the time point of day 4 post-infection. By taking the
previous calculation for reprogramming efficiency (red fluorescence value divided by the
Hoechst 33342 fluorescence value), and dividing this by the YFP fluorescence value (indicative
of cells that were able to uptake the virus at 4 days post-infection), the number of cells that
would uptake the virus and change their phenotype into an iNC morphology could be compared
with the number of cells with the ability to uptake the virus at all. As demonstrated in Figure 8
below, even when the variable of ability for a cell to uptake a virus is accounted for, a wide
range of reprogramming efficiencies exists between different cell lines. It is important to note
that each cell lines reprogramming regimen was repeated three times and those individual results
were reproducible.

46

A

Figure 8: Relative Infection Efficiency A) Showing relative conversion of mouse fibroblast lines factoring in factor expression at day
4 post-infection. The highest efficiency of conversion within each group was set to a value of 100. Bars indicate standard error of the
mean (SEM). B) As in A, for the human fibroblast lines.

47

Figure 8 (cont‘d):

B

48

CONCLUSIONS
Here we strived to address several fundamental questions regarding the existence of
variation in the cellular conversion efficiency between disease subtypes, genetic backgrounds,
and tissue origin via conversion of fibroblasts to iNCs. We were able to demonstrate that the
direct reprogramming capacity of independent primary fibroblast cell lines was reproducible
within a single line from experiment to experiment, however the capacity varied dramatically
from line to line. Through analysis of multiple cell lines derived from the same mouse, drastic
differences of conversion efficiency displayed between different tissue types and even within the
same tissue of origin were discovered. A certain genetic background does not necessarily play a
large role in conversion efficiency into iNCs. The two human cell lines isolated from patients
with neurological disease (RET and SAF) displayed reprogramming efficiencies that were at
opposite ends of the spectrum with regard to iNC conversion. However, until a much higher
number of lines from multiple patients are compared directly, these differences are likely to arise
by chance.
Varying conversion rates are prompting the scientific community question what truly
defines a ―stem cell‖ and what is a truly ―differentiated cell‖. Further questions continue to be
raised regarding how some of these lines do not show any positivity for stem cell characteristics,
however the fibroblasts included may have the intrinsic ability to go down a different path and
therefore may not be ―forced‖ down a program but altering the displayed program to another
intrinsic program. Comi et al. reports that ASCL1 expression pushes human astrocytes down a
neural phenotypic path in vitro and in vivo with methylation analysis indicative of epigenetic
modification being the cause [50]. Via DNA Methylation Find-Peaks analysis they found regions
of enriched methylation. It is possible that future definitions of cell types will rely on epigenetic
49

marks instead of phenotypic characteristics of the cells. The terminology is in question and
further advances must be made to determine how fibroblasts are similar and variable to iNCs. Is
there really homogeneity of fibroblast lines at an individual fibroblast cell level? With different
microenvironments, this is unlikely.
Here we analyzed iNCs as having processes that are 3 cell bodies in length based on the
findings of Vierbuchen described above [15]. Through application of this specification, we are
excluding cells with shorter processes that may have required more time to mature. We are also
excluding the possibility that those shorter processes may have equal or greater functionality in
comparison with those processes of conventional length. Further longitudinal studies must be
conducted to truly analyze the ability of fibroblasts to convert and function as iNCs.
Applied here was a viral delivery method. As a result, clinical application becomes less
of a reality in the future because these vectors were once active viruses that were used to infect
an animal or bacteria with a harmful disease. This is not a large risk to the patients; however
there are alternative ways to introduce transcription factors into target cells that may be worth
exploration. Adler et al. reports the use of plasmids encoding neuronal transcription factors to
primary mouse embryonic fibroblasts with bioreducible linear poly(amino amine), stating there
is low toxicity and high transfection efficiency. Hence repeated doses of these transcription
factors can be delivered. However, their conversion rate measured was only 7.6% of cells were
Tuj1 positive and only a subset of these was MAP2 positive [50]. An alternate delivery method
of transcription factors may be required for translational use of conversion. Nevertheless, other
options currently like the one reported by Adler et al. are average at best, if not subpar in
comparison with the current viral delivery method.

50

There have been published reports on the ability to generate multipotent neural stem cells
from mouse and human fibroblasts [51, 52]. Thier et al. reported conversion to multipotent
neural stem cells with the ability to undergo over 50 passages while maintaining their marker
expression profile and differentiation capacity. They discovered that the neural stem cells
appeared to be activated by an intrinsic neural stem cell transcriptional program without
dependence on sustained transgene expression [51]. Ring et al. reported using only the single
transcription factor Sox2 to generate functional neurons that can integrate, survive, and be
multipotentin vivo without tumor formation [52]. The capability to generate a neural stem cell
has advantages related with the ability to produce multiple cell types including manifold types of
mature neurons, astrocytes, and oligodendrocytes. Furthermore, the capacity to self-renew
becomes a possibility. However the generation of the neural stem cells is at a very low
efficiency and has not been thoroughly tested on multiple cell lines from non-embryonic
fibroblast sources. Thus, exhibiting the same difficulties direct conversion of fibroblasts into
neurons has shown.
The media portrays cellular reprogramming as a process that will be clinically available
in the near future. In reality however there are basic mechanisms that are not understood, and
must be addressed regarding conversion efficiency. Great strides have been made in an attempt
to make this therapy viable and readily available to the masses. Torper et al. reported the
successful direct neural conversion of transplanted human and endogenous mouse cells in vivo in
a rodent brain, thereby substantiating for the first time that neural conversion is possible in vivo
[53]. Despite the findings by Torper et al., many hurdles still exist and must be addressed. In
reality, we are at a standstill because we have not yet identified the precise differences between
different fibroblasts that are responsible for variation in the efficiency of cellular reprogramming
51

into a different phenotype. Until this is addressed and understood, cellular reprogramming is not
yet ready for clinical deployment to the masses as a treatment option.
Until populations of input cells such as fibroblasts are better characterized as both
individual cells and cell populations, there will likely continue to be issues with regard to the
reliability of reprogramming. Clinical application is pending answers to these questions and
labor intensive work has to be completed in order to achieve medical application.

52

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