Fluorescent labeling of human mesenchymal stem cells by thiophene fluorophores conjugated to a lipophilic carrier

Article abstract of DOI:10.1039/c0cc01918f

Fluorescent labeling of human mesenchymal stem cells (hMSCs) is essential for their study in vitro and in vivo. Here, we report a new chemical biodelivery method for labeling hMSCs using a series of very efficient and highly versatile thiophene-based fluorescent dyes. Such conjugates contribute to a stable and viable labeling of hMSCs and constitute a promising strategy for in vivo applications.

Full text of DOI:10.1039/c0cc01918f

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Fluorescent labeling of human mesenchymal stem cells by thiophene
fluorophores conjugated to a lipophilic carrierw
Maria Duca,*a Barbara Dozza,b Enrico Lucarelli,b Spartaco Santi,c Audrey Di Giorgioa and
Giovanna Barbarellad
Received 16th June 2010, Accepted 6th September 2010
DOI: 10.1039/c0cc01918f
Fluorescent labeling of human mesenchymal stem cells (hMSCs)
is essential for their study in vitro and in vivo. Here, we report a
new chemical biodelivery method for labeling hMSCs using a
series of very efficient and highly versatile thiophene-based
fluorescent dyes. Such conjugates contribute to a stable and
viable labeling of hMSCs and constitute a promising strategy for
in vivo applications.
Human mesenchymal stem cells (hMSCs) are currently widely
investigated for their potential use in cell-based therapies.1
These therapies have a great potential in the treatment of a
number of human diseases, such as cancer, Parkinson’s
disease, multiple sclerosis, heart diseases and many others.2
Despite a rapid transition of stem cell-based products from
animal studies to clinical trials, there are several unresolved
issues that limit their applications, such as the lack of methods
for labeling MSCs in order to monitor their fate and distribution
after implant.
Fig.
1
Structure and absorption/photoluminescence maximum
wavelengths of compounds 1–4 (TFs). lmax, lPL nm: 407, 554 (1);
403, 518 (2); 370, 476 (3); 438, 625 (4). 1–3: 10À6 M in phosphate buffer
solution pH 7.2; 4: 10À5 M in DMSO. From ref. 4.
With respect to traditional organic fluorophores such as
fluorescein or rhodamine and their derivatives, TFs have much
greater resistance to photobleaching and long-lasting emission.4
Furthermore, TFs have widely tunable emission, high quantum
efficiency and large Stokes shifts. Noteworthily, these dyes have
a robust aromatic backbone which can undergo a variety of
functionalizations, including bio-derivatization.4
Owing to its high sensitivity, fluorescent imaging is an ideal
tool to investigate the localization and the viability of labeled
cells.3 For example, living cells pre-labeled in vitro with a
fluorescent dye can be implanted subcutaneously in immuno-
compromised animals and, a few days after the implant, a
biopsy can be performed to verify whether and where labeled
cells can be detected by fluorescence microscopy in histological
sections.
In this work, we report an efficient fluorescent labeling of
living hMSCs using new modified TFs. The molecular structure
and the maximum absorption and photoluminescence wave-
lengths of the TFs used in this study are shown in Fig. 1
(compounds 1–4).
In order to be an effective tracer of live stem cells, a
fluorescent probe must have the capability to be internalized
into the cells with long-term retention as well as long-lasting
fluorescence. More importantly, the probe should be biologically
inert and non-toxic. Owing to these requirements and despite
the great number of dyes available today on the market, the
search for new fluorescent dyes easily uptaken by cells, bio-
compatible and bearing bright and long-lasting fluorescence is
still a priority.
Compounds 1–4 have been tested for their ability to
penetrate the cell membrane and label hMSCs. The results
indicated that none of them was able to pass the phospholipidic
bilayer and no fluorescence was observed inside cells
(representative results for compound 1 are shown in Fig. S1, ESIw).
In order to overcome the absence of cell membrane penetration of
the free fluorophore, to promote their cellular delivery and, more
importantly, to avoid their expulsion from the cytoplasm, we have
conjugated them to two different carriers based on the lipophilic
and cationic properties of the triphenylphosphonium (TPP) ion:5
(i) a stable carrier that should remain covalently linked to the
fluorophore after internalization (Scheme 1, compounds 7–10) and
(ii) a biolabile carrier that should be cleaved inside the cell releasing
the free fluorophore (Scheme 2, compound 13). TPP ion is a very
useful tool to deliver molecules inside cells since it allows the
cytoplasm uptake of non-permeable drugs, it is non-toxic for cells
and not expensive.5,6
Thiophene fluorophores (TFs) are the last entry in the
colourful field of organic dyes and they have been already used
for the efficient fluorescent labeling of proteins and DNA.4
a
´
LCMBA UMR6001 CNRS Institut de Chimie de Nice, Universite de
Nice Sophia Antipolis, Parc Valrose, 06100 Nice, France.
E-mail: maria.duca@unice.fr; Fax: +33 (0)492076151;
Tel: +33 (0)492076153
b Rizzoli Orthopedic Inst., via di Barbiano 1/10, 40136, Bologna, Italy
c Istituto di Genetica Molecolare, CNR, Bologna, Italy
d ISOF-CNR and Mediteknology srl, Consiglio Nazionale Ricerche,
Via P. Gobetti 101, 40129 Bologna, Italy
w Electronic supplementary information (ESI) available: Experimental
section, 1H- and 13C-NMR, HRMS and cellular experiments. See
DOI: 10.1039/c0cc01918f
First of all, we chose to synthesize conjugates 7–10
(Scheme 1). In order to do that, we performed the direct
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Scheme
1 Synthesis of conjugated compounds 7–10. Reagents:
(a) NH3 gas, MeOH, overnight; (b) Et3N, DMF, 4 h.
amination of commercially available (4-bromobutyl)triphenyl-
phosphonium bromide (5) in the presence of ammonia gas in
methanol leading to 4-triphenylphosphonium butyl amine (6)
in quantitative yield. Compound 6 was then coupled directly
to the succinimidyl ester of TFs 1–4. This reaction was
performed in DMF and in the presence of triethylamine,
leading to the desired compounds 7–10 in 70–75% yield, after
semi-preparative HPLC purification.
Fig. 2 Microscopic observation of the human mesenchymal stem
cells (hMSCs) labeled with compound 7. Living hMSCs were treated
with compound 7 (0.1 mg mLÀ1) for 60 minutes and then immediately
visualized (a). After the removal of extracellular conjugate, TF-labeled
hMSCs were continuously cultured and monitored at 24 (b), 48 (c) and
72 (d) hours. Cell nucleus was stained with Hoechst 33342 (blue color).
Compound 7 appeared in green color.
In order to assess the impact of the TPP carrier linked to the
TF on the labeling of hMSCs (penetration, expulsion,
concentration that effectively labeled hMSCs. Additionally,
an incubation time of 60 minutes was found to be effective in
labeling hMSCs. After removal of the unbound fluorophores,
we continued culturing the TF-labeled hMSCs for 72 hours.
Fig. 2 shows the fluorescent images of TF-labeled hMSCs
obtained with compound 7 immediately after dye incubation
(a) and 24, 48 and 72 hours after incubation (b, c, and d).
The comparison of conjugates 7 and 13 shows that a marked
difference is observed between the simplified carrier 6 and the
TBTP carrier (see Fig. S3 ESIw for hMSCs labeled with
compound 13). In fact, 7 is more efficient than 13 in labeling
hMSCs. Since the stability of all conjugates in the extracellular
medium has been studied and confirmed experimentally (data
not shown), we suggest that the internalization of TBTP-based
conjugate 13 is less favorable than the one of TPP-based
conjugate 7 in hMSCs.
localization), we also synthesized
a modified conjugate
bearing an intracellularly biodegradable carrier, based on the
4-thiobutyltriphenylphosphonium ion (TBTP),7 instead of the
TPP ion. Since the TPP ion is known to drive the accumulation
of linked molecules to mitochondria,8 a new TBTP-based
carrier (Scheme 2) has been developed recently.9 This
TBTP-based carrier is able, upon direct conjugation to a
NH2-containing drug, to release the unaltered drug via the
reduction of the disulfide bond and the formation of episulfide
and CO2.9 In order to synthesize the TF–TBTP carrier
conjugate 13 (Scheme 2) p-nitrophenyl carbamate activated TBTP
(11) has been coupled to the NH2-containing TF 1210 in the
presence of triethylamine. Desired compound 13 was obtained in
72% yield, after semi-preparative HPLC purification.
After the synthesis of these conjugates, compounds 7 and 13
were studied for their ability to pass the cellular membrane
and label hMSCs. It is worth mentioning that the optical
properties of TFs were not substantially modified upon
conjugation.
Finally, we evaluated the intracellular localization of
compound 7 by confocal microscopy. We examined whether
there was any co-localization between 7 and plasma membrane,
nucleus, mitochondria, or Golgi apparatus (Fig. 3a, c, e, and g,
respectively). We used a specific antibody against the cell
surface marker CD90 that was expressed by 99.3% of the cells
to visualize plasma membrane and another antibody against
the Golgi matrix protein of 130 kDa (GM130) as a marker of
cis-Golgi. Propidium Iodide (PI) and MitoTracker Orange
CMTMRos were used as nucleus and mitochondrial markers,
respectively. The fluorescence signal due to 7 was detectable
mainly in the cytoplasm with a diffused staining. This is
confirmed by planar and vertical single optical sections passing
through the nucleus; the values indicated in the figure represent
the percentage of co-localization of 7 with the structure under
study (Fig. 3b, d, f, h). The fact that 7 does not accumulate
exclusively in mitochondria, as it could have been expected
based on previous results using the TPP carrier,8 is very
interesting in our study. Whether this is due to TF playing a
We first optimized fluorophore concentrations and found
that 0.1 mg mLÀ1 for compound
7 was the lowest
Scheme 2 Coupling of the carrier 11 to the NH2-containing TF
fluorophore 12 and synthesis of compound 13. Reagents: (a) Et3N,
CH2Cl2, 4 h. lmax, lPL nm: 361, 438 (12).
c
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role in inhibiting the complete internalization in mitochondria
has still to be elucidated, but the overall results demonstrate the
efficiency of these conjugates and underline the applicability of our
strategy. The use of photostable TFs able to be internalized inside
cells once conjugated to a non-toxic lipophilic carrier and be
distributed in the cytoplasm demonstrated to be very efficient. TF-
labeled cells are viable suggesting that the presence of conjugated
TFs is cytocompatible and does not induce cell death.
In summary, these findings represent an original investigation
of the labeling of live hMSCs with conjugated thiophene-based
fluorophores. This is the first time that such small, very efficient
and highly versatile fluorophores are used in hMSCs labeling.
Furthermore, it is important to note that the carrier strategy
developed in this work is particularly straightforward and easily
applicable to a wide range of cell systems, thus rendering this
approach very promising for future applications in stem cell-
based studies.
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Fig. 3 Co-localization of compound 7 (green color) with plasma
membrane, nucleus, mitochondria, and Golgi apparatus (red color).
Living hMSCs were labeled with 7, fixed, and stained by using anti-
CD90 (a plasma membrane marker) (a, b), PI (a nucleus marker)
(c, d), MitoTracker orange CMTMRos (a mitochondrial marker)
(e, f), or anti-GM130 (a cis-Golgi marker) (g, h). Immunofluorescence
was conducted by using cyanin-3-labeled secondary antibody.
Confocal microscopy images document the fluorescence distribution
of the whole cell in 3D projections (a, c, e and g) or single optical
sections passing through the nucleus (b, d, f and h). The insets show
the vertical x–z single section obtained along the transverse x-axis
indicated with the yellow dashed line. The values (mean of 3 different
experiments) given show the amount of green signal (7) that
co-localized with red signal. Bars, 20 mm.
c
7950 Chem. Commun., 2010, 46, 7948–7950
This journal is The Royal Society of Chemistry 2010