# CFP and YFP, but Not GFP, Provide Stable Fluorescent Marking of Rat Hepatic Adult Stem Cells

Authors: Rouzbeh R. Taghizadeh; James L. Sherley
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

The stable expression of reporter genes in adult stem cells (ASCs) has important applications in stem cell biology. The ability to integrate a noncytotoxic, fluorescent reporter gene into the genome of ASCs with the capability to track ASCs and their progeny is particularly desirable for transplantation studies. The use of fluorescent proteins has greatly aided the investigations of protein and cell function on short-time scales. In contrast, the obtainment of stably expressing cell strains with low variability in expression for studies on longer-time scales is often problematic. We show that this difficulty is partly due to the cytotoxicity of a commonly used reporter, green fluorescent protein (GFP). To avoid GFP-specific toxicity effects during attempts to stably mark a rat hepatic ASC strain and, therefore, obtain stable, long-term fluorescent ASCs, we evaluated cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), in addition to GFP. Although we were unable to derive stable GFP-expressing strains, stable fluorescent clones (up to 140 doublings) expressing either CFP or YFP were established. When fluorescently marked ASCs were induced to produce differentiated progeny cells, stable fluorescence expression was maintained. This property is essential for studies that track fluorescently marked ASCs and their differentiated progeny in transplantation studies.


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## Body

## 1. Introduction

Fluorescent
proteins have become widely used as markers for positively identifying and
tracking expressing cells in many in
vitro and in vivo studies. The most widely used, green fluorescent protein (GFP),
cloned fromAequorea victoria, does
not require substrates or cofactors and can be used in a variety of species [1, 2]. Among its various uses as a marker, GFP has been used for transient studies
of cell apoptosis [3], cytoskeletal dynamics [4], and inhibitory gene
expression [5]. Since no cofactors are needed for the native GFP protein to
develop fluorescence, it has been possible to use it as a marker in many
different organisms. For instance, transgenic Drosophila melanogaster
[6], zebrafish [7, 8], mice [9–12], and
rats [13, 14] have been successfully derived using wild-type GFP. Although
successful in obtaining stable GFP-expressing transgenic animals, attempts to
develop in vitrocell lines stably expressing GFP have
been largely unsuccessful [2, 3, 15–18].Currently,
in adult stem cell (ASC) research, there is a critical need for methods to
verify ASCs in vitro and track
their progeny in in vivo
repopulation studies. Since markers that uniquely identify ASCs are unknown,
the accepted method to confirm the “stemness” of a cell population is by
transplantation of cells into animals after injury to targeted tissues. If ASCs
are present at significant levels in the transplanted cell population, the
animal survives with repair of the damaged tissue. However, in these
experiments, there is uncertainty as to whether the tissue has been repaired by
the transplanted cells, by activated resident host ASCs, or by host cells
recruited from another tissue source altogether. To overcome these
uncertainties, methods to identify the transplanted cells and their descendents
have been applied. Ideally, these methods need to identify donor cell progeny
independent of subsequent differentiation status.Though
attractive as an in vivo tracking tool in ASC repopulation
assays, GFP has several drawbacks. One shortcoming of GFP is that it can induce
cell death. Intense excitation of the protein in vitro for extended periods can generate free radicals that
are quite toxic to cells [15]. The inability to obtain constitutively
expressing GFP cell strains may also be related to DNA methylation effects. In
the presence of an irreversible inhibitor of methyl transferase, C3A human
hepatoblastoma cells transfected with GFP showed a significantly greater
retention of GFP expression and exhibited higher levels of GFP production [19].
As a result, GFP has been more successfully used extensively as a viable marker
for mostly short-time scale experiments (hours), whereas attempts to establish
long-term (months) GFP-expressing cell strains have been mostly unsuccessful
[2, 3, 15–18]. The reported efficiency of establishing stable, constitutively
expressing cell lines is extremely low [18].GFP-expressing
transgenic mice are readily available [9–12] and are a possible source for
GFP-labeled cells. These mice are uniformly green with the exceptions of hair
and red blood cells. However, there are still barriers to their use as a source
of fluorescently marked ASCs. One major drawback is that, for many tissues,
methods do not exist for efficient isolation of ASCs. Additionally,
GFP-transgenic mice do not offer a solution for tracking ASCs derived from
other species for which transgenic technology has not been developed.Due to similar difficulties in
developing ASCs with constitutive GFP expression, we evaluated two
variants of GFP, cyan fluorescent protein (CFP) and yellow fluorescent protein
(YFP) that have different excitation-emission profiles and, therefore, may have
less toxicity associated with their free-radical byproducts. An early screen ofA. victoria mutants produced CFP
which has an emission spectrum below that of GFP due to a Tyr66 to
Trp66 substitution [20, 21]. Similarly, YFP has been rationally
designed on the basis of the GFP crystal structure to red-shift the absorbance
and emission spectra with respect to GFP. Based on these differences, we evaluated
whether CFP and YFP might allow for stable, long-term fluorescence in rat
hepatic ASCs derived in our laboratory. We were able to establish stable,
long-term expressing ASC strains. When these fluorescently marked ASCs were
induced to produce differentiated progeny cells, stable expression of
fluorescence was maintained. Our findings indicate that CFP and YFP are more
suitable markers for ASC studies in
vitro and predict that they will be better markers for in vivo studies, as well.


## 2. Materials and Methods

### 2.1. Cells

Previously
derived [22] rat hepatic ASC strain, Lig-8, was maintained in Dulbecco’s
Modified Eagle Medium (DMEM; Life Technologies, Carlsbad, Calif, USA)
supplemented with 10% dialyzed fetal bovine serum (DFBS, JRH Biosciences,
Lenexa, Kan, USA), 1% Penicillin/Streptomycin (Life Technologies, Carlsbad, Calif,
USA), and 400μM xanthosine (Xs; Sigma-Aldrich, St. Louis, Miss, USA) in a 37°C
humidified incubator with a 5% CO2 atmosphere.


### 2.2. Plasmids

Amplified
plasmids for transfection were isolated by Qiagen (Valencia, Calif, USA) column
fractionation as specified by the supplier and further purified by cesium
chloride equilibrium density gradient centrifugation. Transfections were
performed using 5μg
of pEGFP-N3 vector plasmid (Clontech Laboratories, Palo Alto, Calif, USA) under
the control of a cytomegalovirus (CMV) promoter. In addition, the pEGFP-N3
vector contains a neomycin resistance gene insert under the control of the
simian virus-40 (SV40) promoter conferring resistance to the antibiotic
Genetecin™ (Life Technologies, Carlsbad, Calif, USA). The CFP and YFP
derivatives of pEGFP-N3 were prepared by digestion and complete removal of the
EGFP insert using BamH1 and Not1 endonucleases (New England Biolabs, Beverly, Mass,
USA). The respective CFP or YFP insert was ligated into the vector after gel
purification. Additional vectors used to attempt to derive stable GFP
expressing cells included pCX-EGFP (supplied by B. Engleward, Massachusetts
Institute of Technology) and pEGFP-N1 (Clontech Laboratories, Palo Alto, Calif,
USA). pCX-EGFP regulates EGFP (enhanced GFP) under a chicken beta-actin
promoter/CMV-IE enhancer and pEGFP-N1 is a sequence variant of pEGFP-N3.


### 2.3. Transfection

Lig-8
cells were seeded at 1/10 confluency in
a 75-cm2 flask (Corning, Corning, NY, USA) one day prior to
transfection. Lig-8 cells were then independently transfected with the CFP, YFP,
or GFP expression plasmids using Cytofectene (BioRad Laboratories, Hercules, Calif,
USA), per manufacturer’s suggested instructions. Approximately, 1.5×106 cells (1/5 confluency in 75-cm2 flask) were transfected for 16–20
hours and then given two phosphate buffer saline (PBS) washes, followed by
supplementation with regular growth medium. The transfected cells were cultured
for an additional 42–48 hours. After this time period, the transfected cells
were replated at 1/6 density in parallel in 10-cm diameter
plates (Corning, Corning, NY, USA). After overnight culture, the culture medium
was replaced with medium supplemented to 0.5 mg/ml Genetecin™ to select for
stably transfected cell clones.


### 2.4. Clonal Cell Viability

Propidium
iodide (PI; Sigma, St. Louis, Mo, USA) was added directly to media and cells at
5μg/ml to determine viability of clonal cells. A Nikon super high-pressure
mercury lamp was used to image PI cells using a Nikon epifluorescent
microscope.


### 2.5. Derivation of Fluorescent Protein-Expressing Clones

After
14 days in culture, transfected cells from each 10-cm diameter plate were
trypsinized and each transferred into a 75-cm2 flask with selection
medium maintained thereafter. After two days, the transfected cells were
reseeded at 1000 cells each into five individual 10-cm diameter dishes.  Resistant colony formation was assessed after
10–14 days of culture in selection medium, with medium changes every 3
days.  After this time, colonies were
counted and scored as nonexpressing (B1; 0% of cells in the colony were
expressing fluorescent protein), semiexpressing (B2; estimated 25–75% of cells
in the colony were expressing fluorescent protein) or fullyexpressing (B3; ~100% of the cells
in the colony were expressing fluorescent protein). Several well-separated
colonies were isolated within cloning cylinders (Bellco Glass, Vineland, NJ,
USA), harvested by trypsinization, transferred to 25-cm2 flasks
(Corning, Corning, NY, USA), and allowed to expand for 10–14 days, until the
flask was >90% confluent. At that point, cells from the 25-cm2 flasks were trypsinized and transferred into a 75-cm2 flask. Within
3-4 days, the flasks were 90% confluent. After trypsinization, ~80% of the
cells in cultures of expanded clones were frozen in liquid nitrogen [22] for
early passage stocks. All expanded clones exhibited respective CFP or YFP
fluorescence expression for at least 15 population doublings before being
cryogenically stored. The remaining cells were maintained in culture and
passaged for at least 24 weeks (estimated 140 population doublings). Three of
the CFP-expressing Lig-8 clonal strains, B1, B2, and B3, were subsequently
evaluated for this study.  Population
doublings were determined based on the estimated total number of cells produced
over time with the exponential generation time of ~18 hours determined for
Lig-8 parent cells.


### 2.6. Flow Cytometry and Fluorescence Microscopy

A
FACStar Plus flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ,
USA) was used to quantify the fraction of fluorescent cells in populations. The
FACStar Plus was equipped with two coherent Innova 90 lasers for visible and
ultraviolet argon lines. Data acquisition and analyses were performed with CellQuest
software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and Summit
analyses software (Cytomation, Inc., Fort Collins, Colo), respectively. The
nontransfected parent Lig-8 ASC strain was used as a negative control for all
analyses to account for background cell autofluorescence. Cell populations were
analyzed using both flow cytometry and epifluorescence microscopy using a Nikon
TE300 microscope system with DAPI/GFP/CFP/YFP filters. A Hamamatsu digital camera
and OpenLab imaging system were used for digital imaging.  The Student’s t test was used to determine
the statistical confidence of observed differences in fluorescence.


### 2.7. Differentiation Induction

Cells
were treated for 9 days with 20 ng/ml epidermal growth factor (EGF) and 0.5 ng/ml transforming growth factor b (TGF-b) (Life Technologies, Carlsbad, Calif,
USA), in the same culture medium, except that the DFBS was reduced to 1%. The
details of the differentiation induction protocol will be reported elsewhere
[23].


## 2.1. Cells

Previously
derived [22] rat hepatic ASC strain, Lig-8, was maintained in Dulbecco’s
Modified Eagle Medium (DMEM; Life Technologies, Carlsbad, Calif, USA)
supplemented with 10% dialyzed fetal bovine serum (DFBS, JRH Biosciences,
Lenexa, Kan, USA), 1% Penicillin/Streptomycin (Life Technologies, Carlsbad, Calif,
USA), and 400μM xanthosine (Xs; Sigma-Aldrich, St. Louis, Miss, USA) in a 37°C
humidified incubator with a 5% CO2 atmosphere.


## 2.2. Plasmids

Amplified
plasmids for transfection were isolated by Qiagen (Valencia, Calif, USA) column
fractionation as specified by the supplier and further purified by cesium
chloride equilibrium density gradient centrifugation. Transfections were
performed using 5μg
of pEGFP-N3 vector plasmid (Clontech Laboratories, Palo Alto, Calif, USA) under
the control of a cytomegalovirus (CMV) promoter. In addition, the pEGFP-N3
vector contains a neomycin resistance gene insert under the control of the
simian virus-40 (SV40) promoter conferring resistance to the antibiotic
Genetecin™ (Life Technologies, Carlsbad, Calif, USA). The CFP and YFP
derivatives of pEGFP-N3 were prepared by digestion and complete removal of the
EGFP insert using BamH1 and Not1 endonucleases (New England Biolabs, Beverly, Mass,
USA). The respective CFP or YFP insert was ligated into the vector after gel
purification. Additional vectors used to attempt to derive stable GFP
expressing cells included pCX-EGFP (supplied by B. Engleward, Massachusetts
Institute of Technology) and pEGFP-N1 (Clontech Laboratories, Palo Alto, Calif,
USA). pCX-EGFP regulates EGFP (enhanced GFP) under a chicken beta-actin
promoter/CMV-IE enhancer and pEGFP-N1 is a sequence variant of pEGFP-N3.


## 2.3. Transfection

Lig-8
cells were seeded at 1/10 confluency in
a 75-cm2 flask (Corning, Corning, NY, USA) one day prior to
transfection. Lig-8 cells were then independently transfected with the CFP, YFP,
or GFP expression plasmids using Cytofectene (BioRad Laboratories, Hercules, Calif,
USA), per manufacturer’s suggested instructions. Approximately, 1.5×106 cells (1/5 confluency in 75-cm2 flask) were transfected for 16–20
hours and then given two phosphate buffer saline (PBS) washes, followed by
supplementation with regular growth medium. The transfected cells were cultured
for an additional 42–48 hours. After this time period, the transfected cells
were replated at 1/6 density in parallel in 10-cm diameter
plates (Corning, Corning, NY, USA). After overnight culture, the culture medium
was replaced with medium supplemented to 0.5 mg/ml Genetecin™ to select for
stably transfected cell clones.


## 2.4. Clonal Cell Viability

Propidium
iodide (PI; Sigma, St. Louis, Mo, USA) was added directly to media and cells at
5μg/ml to determine viability of clonal cells. A Nikon super high-pressure
mercury lamp was used to image PI cells using a Nikon epifluorescent
microscope.


## 2.5. Derivation of Fluorescent Protein-Expressing Clones

After
14 days in culture, transfected cells from each 10-cm diameter plate were
trypsinized and each transferred into a 75-cm2 flask with selection
medium maintained thereafter. After two days, the transfected cells were
reseeded at 1000 cells each into five individual 10-cm diameter dishes.  Resistant colony formation was assessed after
10–14 days of culture in selection medium, with medium changes every 3
days.  After this time, colonies were
counted and scored as nonexpressing (B1; 0% of cells in the colony were
expressing fluorescent protein), semiexpressing (B2; estimated 25–75% of cells
in the colony were expressing fluorescent protein) or fullyexpressing (B3; ~100% of the cells
in the colony were expressing fluorescent protein). Several well-separated
colonies were isolated within cloning cylinders (Bellco Glass, Vineland, NJ,
USA), harvested by trypsinization, transferred to 25-cm2 flasks
(Corning, Corning, NY, USA), and allowed to expand for 10–14 days, until the
flask was >90% confluent. At that point, cells from the 25-cm2 flasks were trypsinized and transferred into a 75-cm2 flask. Within
3-4 days, the flasks were 90% confluent. After trypsinization, ~80% of the
cells in cultures of expanded clones were frozen in liquid nitrogen [22] for
early passage stocks. All expanded clones exhibited respective CFP or YFP
fluorescence expression for at least 15 population doublings before being
cryogenically stored. The remaining cells were maintained in culture and
passaged for at least 24 weeks (estimated 140 population doublings). Three of
the CFP-expressing Lig-8 clonal strains, B1, B2, and B3, were subsequently
evaluated for this study.  Population
doublings were determined based on the estimated total number of cells produced
over time with the exponential generation time of ~18 hours determined for
Lig-8 parent cells.


## 2.6. Flow Cytometry and Fluorescence Microscopy

A
FACStar Plus flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ,
USA) was used to quantify the fraction of fluorescent cells in populations. The
FACStar Plus was equipped with two coherent Innova 90 lasers for visible and
ultraviolet argon lines. Data acquisition and analyses were performed with CellQuest
software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and Summit
analyses software (Cytomation, Inc., Fort Collins, Colo), respectively. The
nontransfected parent Lig-8 ASC strain was used as a negative control for all
analyses to account for background cell autofluorescence. Cell populations were
analyzed using both flow cytometry and epifluorescence microscopy using a Nikon
TE300 microscope system with DAPI/GFP/CFP/YFP filters. A Hamamatsu digital camera
and OpenLab imaging system were used for digital imaging.  The Student’s t test was used to determine
the statistical confidence of observed differences in fluorescence.


## 2.7. Differentiation Induction

Cells
were treated for 9 days with 20 ng/ml epidermal growth factor (EGF) and 0.5 ng/ml transforming growth factor b (TGF-b) (Life Technologies, Carlsbad, Calif,
USA), in the same culture medium, except that the DFBS was reduced to 1%. The
details of the differentiation induction protocol will be reported elsewhere
[23].


## 3. Results

Using CFP, YFP, or GFP as
independent fluorescent protein markers, we transfected respective expression
plasmids into a previously described rat hepatic ASC strain, Lig-8 [22].
Asymmetric selfrenewal associated with production of differentiated progeny
cells is the defining feature of ASCs [22, 24]. Lig-8 cells were derived based
on their asymmetric cell kinetics [22, 24]. Lig-8 cells asymmetrically
self-renew and produce differentiated progeny with mature hepatocyte properties
[22, 23, 25]. The differentiated progeny cells express transcription factors
and protein antigens that are specific for hepatocyte development and
maturation, respectively. Hepatocyte-specific phenotypes include binucleation,
albumin secretion, and expression of inducible cytochrome P450s [22, 23, 25].
Based on well-defined ASC properties, Lig-8 cells were ideal for evaluation of
effects of GFP, CFP, and YFP in ASCs.We found that transfections
with GFP gene constructs yielded transfected colonies ~50-fold less efficiently
than transfections with the analogous CFP- or YFP-gene constructs (Table1). In
addition, colonies of GFP-transfected cells could not be propagated as stable
cell strains, whereas both CFP- and YFP-transfected cell colonies had 100%
cloning efficiency (Table 1). Furthermore, we determined that cells that were
transiently-expressing GFP appeared to undergo cell death, as GFP-expressing
cells were also positive for propidium iodide (PI) (Figure 1). PI is
impermeable to intact membranes but readily penetrates the membranes of
nonviable cells and binds to DNA and RNA, causing red fluorescence. The cells
eventually rounded up and detached from the culture dish, while still showing
GFP and PI fluorescence (Figure 1, colonies 6, 9, and 13). Similar observations
were made with Lig-8 cells transfected with pCX-EGFP and pEGFP-N1 plasmids. All
observed GFP-expressing colonies yielded this same fate (data not shown).Table 1
Relative transfection efficiency
of fluorescent gene markers and cloning efficiency of selected transfected cell
colonies. Transfection efficiency is defined as the
average number of colonies/estimated number of cells transfected/μg DNA.
Transfections included ∼1.5×106 cells. Cloning efficiency is defined as the number of stable cell strains
derived/number of colonies cultured.
Fluorescent markerAverage colony number/10-cm dishRelative transfection efficiencyNumber of clones pickedNumber of Stable cell strains derivedCloning efficiencyGFP1.3 (n=10)0.02800%CFP64 (n=3)1.0066100%YFP61 (n=3)0.9566100%Figure 1
Transient, short-term GFP fluorescence expression by the rat hepatic ASC
strain, Lig-8, is associated with cell death. Cell strain, Lig-8, was
transfected with the pEGFP-N3 expression vector. Shown are images of 5
independent colonies transiently expressing GFP fluorescence at 72–96 hours
posttransfection. Shown are the phases (a)–(e), GFP-fluorescence (f)–(j), propidium
iodide (PI) fluorescence (k)–(o), and merged GFP and PI fluorescence (p)–(t)
images. Arrows highlight specific examples of cells double-positive for GFP and
PI fluorescence in Colony no. 12. Scale bar is equivalent to 100 μm.Expanded CFP and YFP clones
expressed the respective fluorescent proteins stably for at least 24 weeks in
culture (~140 doublings; e.g., clone B3 in Figure2). Clones that were
successfully derived exhibited a range of CFP- or YFP-expressing cell
fractions. As a result, these clones were characterized as nonexpressing (B1;
0% of cells in the colony express fluorescent protein, datanot shown),
semiexpressing (Figure 3, B2; at least 25–75% of cells in the colony expressed
fluorescent protein, but not all); or fully expressing (Figure 3, B3;
approximately 100% of the cells in the colony expressed fluorescent protein).
Although cell strains were derived from both CFP and YFP expressing colonies,
only CFP cell strains were further evaluated.Figure 2
Stable, long-term expression of CFP fluorescence in stably transfected
rat hepatic ASC clones. Three cell clones expanded from CFP-transfected
colonies, B1, B2, and B3 (as described in text), were propagated and serially
analyzed for CFP expression. At the indicated number of population doublings,
the percentage of fluorescence-expressing cells was determined by flow
cytometry for each specified clone.  The
cells have been passaged for a maximum of 140 population doublings.Figure 3
Fluorescent protein (CFP or YFP)-expressing colonies from rat hepatic ASCs. Rat hepatic ASC strain, Lig-8, was stably
transfected with either a CFP or YFP expression vector. Shown are colonies with
approximately 20–75% of the cells expressing (B2) and colonies with essentially
100% of the cells expressing (B3).  Shown
are the phases (a)–(d), fluorescent (e)–(h), and merged (i)–(l) images of CFP- and
YFP-expressing colonies. Scale bar is equivalent to 100μm.Although, as colonies, the
CFP-expressing cell strains exhibited the fluorescent properties described,
with further propagation in culture, the fluorescence for one of the
transfected clones decreased (B2 in Figure2). Moreover, cells derived from an
initially non-expressing B1 colony began to express CFP (Figure 2; B1) at
levels comparable to continuously, fully-expressing B3 clones (Figure 2; B3)
during clonal propagation. Qualitatively, the B2 and B3 CFP-expressing clones
maintained their initially observed fluorescent properties. Although there was
some variation in expression seen in the early stages of clonal analysis,
expression stabilized with propagation, and low variability was observed for at
least 24 weeks (estimated 140 population doublings). The B2 cell clone
exhibited the greatest fluctuation in fluorescence expression (Figure 2), but
did not change in its basic character of expressing CFP.Stable
fluorescent protein expression did not alter the essential properties of the
parent hepatic ASC strain, Lig-8 (data
not shown). We have found that, because of their asymmetric self-renewal
property, the parent Lig-8 hepatic ASCs are resistant to complete
differentiation by TGF-β,
EGF, and serum deprivation [23]. Under conditions of TGF-b supplementation,
Lig-8 cells produce differentiated progeny cells by asymmetric cell divisions
that allowed them to retain their undifferentiated ASC state [23]. To evaluate
CFP expression in differentiated progeny cells, the three CFP-expressing
fluorescent ASC strains (B1, B2, and B3) were examined after culture under
EGF/TGF-β-induced
differentiation conditions. All strains exhibited similar morphological and
cell kinetic properties observed for the nontransfected parental Lig-8 strain (data not shown). As shown in Figure 4,
under normal conditions, the B3 cell clone exhibited uniform cell morphology
(a)–(c), whereas under differentiation conditions, a heterogeneous collection of
varying morphological cell types appeared. Some differentiated cells had a
noticeable larger size and altered morphology (Figure 4, (d)–(f); arrows), compared
to cells under normal culture conditions.Figure 4
Qualitative comparison of
CFP-fluorescent B3 cells under control and differentiating culture conditions. Clone B3 cells were cultured under routine (control) (a)–(c) or differentiating
(d)–(f); 20 ng/ml epidermal growth factor, 0.5 ng/ml transforming growth factor b,
1% serum) culture conditions for 9 days. Shown are phases (a), (d), fluorescent
(b), (e), and merged (c), (f) images. Arrows indicate morphologically
differentiated cells. Scale bar is equivalent to 100μm.After
induction of differentiated progeny, the CFP-fluorescent cell fraction of B1
cells did not vary significantly relative to the routine (undifferentiated) culture conditions (Table2). However,
although exhibiting stable fluorescence expression under differentiating
conditions, B1 and B3 cell clones displayed a statistically significant
increase (60%, P<.01; and 91%, P<.002, resp.) in
median fluorescence per cell under differentiating conditions (see also Figure 5
for FACS histogram of B3 clone). The B2 and B3 cell clones showed only modest
(15% and 9%, resp.), albeit statistically significant (P<.03
and P<.02, resp.), reductions in fluorescent cell fractions
(Figure 5; Table 2).  Thus, although the
three cell strains were derived from three independent clones and displayed
differing fluorescent cell fractions (Figure 3), their fluorescence fraction
did not vary by more than 15% when differentiated progeny cells were produced.
The estimated fraction of differentiated progeny under these conditions is ≥80% [23], indicating that a majority of differentiated cells retain a high
level of fluorescence.Table 2
Quantitative comparison of the
CFP-fluorescent cell fractions of cultures under undifferentiated and
differentiated conditions. Flow cytometry quantification of the R2
region of flow histograms (see, e.g., in Figure5) for three fluorescent cell
clones (same as Figure 2). Data are the average % fluorescent cells at 24 weeks
in cultures ± standard deviation (SD). Cells were analyzed under normal culture
conditions (undifferentiated) and under conditions that increase differentiated
progeny (differentiation), as described in the text.
% CFP fluorescence (mean ± SD;n=2) conditionStrainUndifferentiatedDifferentiatedB194
± 0.0294
± 0.88B233
± 1.628
± 0.87B387
± 0.3279
± 2.3CFP-fluorescence expression of the rat hepatic ASC clone B3 is stable after
induction of differentiation. Flow
cytometry analysis was performed with cultures of the nontransfected parental
rat hepatic ASC strain, Lig-8 (a), and CFP-fluorescent clone B3 hepatic ASC
cultures. The B3 cells were evaluated under routine (b) and differentiating (c)
culture conditions. Histograms plot the relative numbers of cells as a function
of the log-relative CFP fluorescence per cell. The background fluorescence, as
defined by nontransfected Lig-8 cells is depicted as the R1 region, and
positive fluorescence is denoted by the R2 region. Numbers indicate the percent
of cells positive for CFP fluorescence expression.
(a)(b)(c)


## 4. Discussion

This
report is a first study to look at GFP-, CFP-, and YFP-transgenes in
side-by-side experiments in the same ASC strain. We evaluated the use of these
transgenes for the derivation of stable, long-term fluorescence-expressing rat
hepatic ASC clones. We were able to attain transient GFP-expressing cells, but
due to either the toxicity and/or methylation associated with GFP, were unable
to propagate these clones as stable, long-term GFP-expressing hepatic ASC
strains. Given the failure to even establish clones from colonies with
extinguished GFP fluorescence, cytotoxicity seems to be the primary problem.GFP gene transfections gave rise to transient expressing
cells for up to 72–96 hours posttransfection. As culture continued, intact,
adherent cells positive for GFP-expression began to round up, detach, and lose
viability, as indicated by propidium iodide (PI) staining. Eventually, all adherent,
GFP-expressing cells rounded up, detached, and became positive for PI staining.
These experiments were performed with three different GFP plasmid constructs
(pEGFP-N3, pCX-EGFP, and pEGFP-N1). However, in no case were stable
GFP-expressing cell strains obtainable.
These observations, cumulatively, indicate that the GFP protein is toxic
to the cells.Other studies [15, 16] have obtained similar results using
various GFP expression plasmids. One group in particular [15] examined several
variants of GFP plasmids resulting in many of the GFP-expressing cells
contracting, rounding up, and dying, which was confirmed by decreasing
luciferase activity and increasing CPP32-activity, a cysteine protease that
plays a direct role in the proteolytic digestion of cellular proteins
responsible for progression to apoptosis.Our work with GFP confirms that the GFP protein product has
toxic side-effects in at least one type of ASC, whereas the CFP and YFP
protein, translated from the same plasmid vector construct is well tolerated by
these cells.  This conclusion can also
explain the transfection efficiency and cloning efficiency data from our
analyses. Since stable transfection efficiency is an indicator of the success
for the transfer and integration of genes into cells, it is likely that due to
GFP-related toxicity, both CFP- and YFP-genes transfected the hepatic ASCs
~50-fold better than the analogous GFP gene. Cloning efficiency data further
confirms the difficulties observed with stable, long-term transduction with
GFP; since 100% of CFP- and YFP-clones gave rise to cell strains, whereas none
of the GFP-derived colonies gave rise to stable clones. Examined CFP-expressing
clones B1, B2, and B3 retained a high level of fluorescent expression at 24
weeks of culture (approximately 140 population doublings), even though one of
the clones (B1) initially showed decreasing expression. Altogether, these
observations suggest further evidence that due to GFP-dependent toxicity, GFP
cannot be utilized as a stable fluorescent reporter in these hepatic ASCs.When
the CFP-expressing cell strains (B1, B2, and B3) were placed under
differentiation conditions after 120 doublings, either no or only modest
reductions in fluorescence cell fractions were observed relative to normal
culture conditions. However, although exhibiting stable fluorescence expression
under differentiating conditions, B1 and B3 cells displayed a statistically
significant increase in the median fluorescence of positive cells. This
increase in median fluorescence may be directly related to an increase in the
median cell size of the population, since it has been observed that as Lig-8
cells differentiate, they produce large hepatocyte-like cells [23].
Nonetheless, this stability in fluorescence is important, since it suggests
that CFP is expressed independent of the differentiation state of progeny
cells. Stable fluorescence expression in our in vitro differentiation
studies is a predictor of stable expression in in vivo differentiation, since TGF-b is a ubiquitous
differentiation growth factor, especially in the liver [26]. The high stability
in fluorescence expression under both normal and differentiating conditions can
ideally be used in transplantation studies to evaluate the in vivo repopulation properties of
these cells.The
importance of this report is underscored by reports concluding statistically
significant amplification of hematopoietic stem cells (HSCs) by forced
expression of specific genes. For instance, the gene transfer of theHOXB4 gene into human hematopoietic stem
cells was reported to result in an overall approximately 2-fold increase in
total and CD34+ cells, normalized to a transfer of a control EGFP
gene construct [27]. This 2-fold increase and eventual significant overall
increase in in vivo
repopulation efficiency caused by HOXB4 regulation could be interpreted as the result of increased cell death in
EGFP-controls due to fluorescent toxicity and not due to the expansion of HSCs
using HOXB4 regulation, as suggested.
Similarly, in another report using recombinant HIV transactivating (TAT)-HOXB4
protein [28], TAT-GFP was used as a control for the in vivo expansion and pluripotency of HSCs. It was concluded
that TAT-GFP was ineffective in supporting HSC expansion, whereas TAT-HOXB4
resulted in a net expansion of 20-fold over control values. Again, this data
could result from the toxicity-dependent effects of the GFP gene, used as the
control for gene transfer.Availability
of stable, long-term marked ASCs has important applications in advancing ASC
research. Currently, there are no exclusive ASC markers that allow for easy
characterization and validation. One example is the expansion of HSCs in
culture, currently a major challenge in the field of HSC research. Although
markers have been found that promote enrichment of ASCs from specific tissues
[29, 30], these are not sufficient for determining their “stemness” in any
general sense. Currently, the main method used to establish the “stemness” of
an ASC population is transplantation of cells into animals and subsequent
determination of whether the cells can regenerate damaged tissues.In
some tissue models, determining repopulation efficiency is simpler than in
others. For bone marrow repopulation studies, the output metric is
reconstitution of viable recipient animals after donor cell transplant. Few ASC
studies have this ideal feature of functional reconstitution. Most of these
studies depend on in situ cell
histology to indicate effective tissue repopulation. Studies of this sort have
led to debates regarding issues of ASC plasticity [31]. If a faithful cell
marker is not tracked in transplanted ASCs, then uncertainty arises; since it
is not clear whether the transplanted cells or host cells are responsible for
the observed results. In some tissue models, such as the liver, where the
tissue has the capacity for active proliferation, tracking of transplanted
cells is even more crucial. Our findings suggest that CFP and YFP are better
reporters for the development of stable, long-term fluorescence-expressing ASCs
in culture. Their choice for future ASC research may help to resolve current
controversial issues, including ASC plasticity in animal repopulation assays.Notwithstanding
the current controversy regarding ASC plasticity and cell fusion [31–33], our
findings with GFP call for reevaluation of conclusions based on the
transduction of GFP-transgenes into manipulated ASC populations. Additionally,
our findings establish important quality control concepts for developing and
implementing methods and tools for future ASC therapeutics that employ gene
transfer. Our experience highlights the importance of careful in vitrocharacterization of genetically marked cell populations before in vivo transplantation.



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