Alla Gent.ma Prof. ssa Maria del Zompo
- Direttore del Dipartimento di Scienze Biomediche
Universita’ degli Studi di Cagliari
Cagliari, 3-6-2013
Resoconto delle ricerche svolte dal Dr. Roberto Loi presso il Weiss
Laboratory, Vermont Lung Center, Department of Medicine, University of
Vermont (USA) nel periodo di aspettativa per studio decorrente dal 1-42012 al 31-3-2013.
Durante la sua permanenza presso il laboratorio diretto dal Prof. Daniel J. Weiss
presso il Vermont Lung Center, il Dott. Loi si e’ occupato di studi concernenti
l’utilizzo di cellule staminali mesenchimali e di cellule staminali pluripotenti indotte
per il ripopolamento di “scaffolds” acellulari ottenuti mediante de-cellularizzazione
di polmoni umani e murini.
Gli studi condotti sono risultati nella pubblicazione di tre articoli su rivista per un
Impact Factor medio di 7.5 e in due manoscritti ancora in preparazione.
I risultati degli studi sono inoltre stati presentati a congressi internazionali.
Elenco delle pubblicazioni derivanti dall’attivita’ di ricerca svolta dal Dr. Roberto
presso il Vermont Lung Center – University of Vermont durante il periodo di
aspettativa.
Articoli su riviste
1) The effect of age and emphysematous and fibrotic injury on the recellularization of de-cellularized lungs. Sokocevic D, Bonenfant NR, Wagner DE,
Borg ZD, Lathrop MJ, Lam YW, Deng B, Desarno MJ, Ashikaga T, Loi R,
Hoffman AM, Weiss DJ. Biomaterials. 2013 Apr; 34(13):3256-69.
2) The effects of storage and sterilization on de-cellularized and re-cellularized
whole lung. Bonenfant NR, Sokocevic D, Wagner DE, Borg ZD, Lathrop MJ, Lam
YW, Deng B, Desarno MJ, Ashikaga T, Loi R, Weiss DJ. Biomaterials. 2013 Apr;
34(13):3231-45.
3) Endogenous Distal Airway Progenitor Cells, Lung Mechanics, and
Disproportionate Lobar Growth following Long-Term Post- Pneumonectomy in
Mice. Eisenhauer P, Earle B, Loi R, Sueblinvong V, Goodwin M, Allen GB,
Lundblad L, Mazan MR, Hoffman AM, Weiss DJ. Stem Cells. 2013 (In press).
Comunicazioni a congresso
1) Age and Injury Adversely Affect Re-Cellularization of De-Cellularized Lung.
Sokocevic D, Bonenfant N, Wagner DE, Borg ZD, Lathrop M, Lam YW, Deng B,
DeSarno M, Ashikaga T, Loi R, Hoffman AM, Weiss DJ. American Thoracic
Society International Conference. Philadelphia (USA), May 17-22, 2013.
2) Cyclic mechanical stretch activates YAP/TAZ to regulate SP-C expression in
distal lung epithelial cells. Wagner DE, Bonenfant NR, Borg Z, Sokocevic D, Loi
R, Weiss DJ. American Thoracic Society International Conference. Philadelphia
(USA), May 17-22, 2013.
3) Optimizing Lung De-Cellularization and Re-Cellularization: Effects of Storage
and Sterilization. Bonenfant NR, Sokocevic D, Wagner DE, Borg ZD, Lathrop M,
Lam YW, Deng B, DeSarno M, Ashikaga T, Loi R, Weiss DJ. American Thoracic
Society International Conference. Philadelphia (USA), May 17-22, 2013.
4) Derivation of normal and cystic fibrosis human induced pluripotent stem cells
(iPSCs) from airway epithelium. Loi R, Weiss DJ. 36th European Cystic Fibrosis
Conference. Lisbon (Portugal) June 12-15, 2013.
Sommario degli studi svolti dal Dott. Roberto Loi presso il Vermont Lung Center
– University of Vermont durante il periodo di aspettativa.
Abstract
Use of stem cells for repopulation of de-cellularized cadaveric lungs scaffolds for
ex vivo lung tissue generation offers a new potential therapeutic approach for
clinical lung transplantation. Mesenchymal stromal cells (MSCs) obtained from
bone marrow of adult mice can localize to lungs and acquire phenotypic and
functional characteristics of differentiated lung epithelial cells, therefore they
represent an option for re-cellularization of lung scaffolds.
Notably, the remodeling of chromatin structure and the alteration of epigenetic
marks that underlie lineage commitment, including histone methylation and
acetylation and DNA methylation, can be induced by environmental cues,
including by extracellular matrix (ECM)-dependent signaling. Optimal procedures
for handling of de-cellularized lungs prior to the re-cellularization process have
not been described to date. In fact, it is not yet clear how storage and sterilization
of de-cellularized lungs affect the composition and properties of the resulting
ECM, and the efficiency of the following re-cellularization process. Further, it is
unclear how time of post-mortem storage of the lungs affects tissue
characteristics and subsequent re-cellularization process, a relevant factor with
procurement of human lungs from autopsy. To investigate this, we assessed the
effects of advanced age, representative emphysematous and fibrotic injuries, and
the combination of advanced age and emphysematous injury and found
significant differences in both histologic appearance and in the retention of
extracellular
matrix
(ECM)
and
other
proteins,
as
assessed
by
immunohistochemistry and mass spectrometry, between the different conditions.
Furthermore, we assessed effects of delayed necropsy, prolonged storage (3
and 6 months), and of two commonly utilized sterilization approaches: irradiation
or final rinse with peracetic acid, on architecture and extracellular matrix (ECM)
protein characteristics of de-cellularized mouse lungs. These different conditions
resulted in significant differences in both histologic appearance and in retention
of ECM and intracellular proteins as assessed by immunohistochemistry and
mass spectrometry.
Finally, we assessed binding and proliferation of bone
marrow-derived mesenchymal stromal cells (MSCs) in de-cellularized scaffolds,
using mouse epithelial C10 cells as a reference. Despite the observed
differences among scaffold conditions, binding, retention and growth of bone
marrow-derived mesenchymal stromal cells (MSCs) over a one month period
following intratracheal inoculation was similar between experimental conditions.
In contrast, significant differences occurred with C10 mouse lung epithelial cells.
Therefore, age, lung injury, delayed necropsy, duration of scaffold storage,
sterilization approach, and cell type used for re-cellularization may significantly
impact the usefulness of this biological scaffold-based model of ex-vivo lung
tissue regeneration.
Introduction
Bone marrow-derived Mesenchymal Stem Cells (MSCs) from adult mice
can localize to recipient mouse lungs and acquire phenotypic characteristics of
airway or alveolar epithelial cells [1,2]. These findings raise the possibility that
abnormal lung epithelium can be repopulated with functional cells of bone
marrow origin. As an alternative to in vivo repopulation of diseased lung
epithelium, increasing interest in the use of de-cellularized complex whole organ
scaffolds for ex vivo tissue engineering has provided both opportunity and also
unique challenges.
Ex vivo tissue engineering has been successfully used for the
regeneration and clinical transplantation of tissues such as skin, cartilage, and
bone [3]. Engineering organs with more structural and cellular complexity such
as heart, lung, and liver is a more challenging endeavor, yet recent advances in
tissue engineering techniques and in regenerative medicine have established a
foundation upon which the functional replacement of these organs appears
possible [3-4]. One promising approach involves the use of naturally occurring 3dimensional extracellular matrix (ECM) obtained by the de-cellularization of
whole organs. The matrix serves as a biologic scaffold for ex vivo recellularization and generation of functional tissue with either differentiated adult
cells or potentially by stem/progenitor cells [5].
The unresolved issues which still require clarification include defining
optimal, organ specific approaches for de-cellularization and for sterilization and
storage of de-cellularized organs prior to re-cellularization [3-6]. A number of
recent publications have comparatively assessed different de-cellularization
protocols for trachea and lung. Notably, architecture and extracellular matrix
(ECM) protein composition of either trachea or lungs may differ substantially
between different de-cellularization regimens [7-9]. Whether this difference will
subsequently affect re-cellularization and generation of functional tissue remains
to be clarified [6,7].
Methods of optimal sterilization and storage have been
already developed for trachea [9,10] but not yet clearly established for decellularized lungs. One further consideration is that of post-mortem time prior to
lung harvest and de-cellularization, a practical issue for procurement of human
lungs. Several hours or even days may pass prior to post-mortem tissue harvest
and it is still unknown whether this delay will affect the suitability of the donor
lung for de-cellularization and subsequent re-cellularization.
To address these questions, we assessed architecture and ECM protein
content and distribution in mouse lungs obtained following a prolonged postmortem period prior to harvest compared to freshly procured lungs. We also
assessed lungs obtained immediately after euthanasia and then subsequently
stored after de-cellularization for prolonged periods (3 and 6 months). We further
evaluated effects of sterilization using either irradiation or final rinse with
peracetic acid, a commonly used protocol in storage of other biologic scaffolds
[11-15]. We then assessed growth of two different cell types, murine bone
marrow-derived mesenchymal stromal cells (MSCs) and C10 mouse lung type 2
alveolar epithelial cells, following intratracheal inoculation into the different decellularized lungs.
Another relevant issue in the application of de-cellularized cadaveric lungs
scaffolds for ex vivo lung tissue generation is represented by the fact that some
of the donor lungs that might be utilized for de-cellularization and ex vivo
bioengineering may originate from aged donors, donors with pre-existing
structural lung diseases, or a combination of both age and lung disease. At
present it is unknown how these factors might affect either de-cellularization or
subsequent re-cellularization. Therefore, to assess these questions, we included
in our analysis the comparative assessment of architecture and ECM content in
de-cellularized mouse lungs from young (8-12 week) vs old (15-18 month) mice,
lungs from young mice after induction of either emphysematous lung injury
following intratracheal inoculation with elastase or of fibrotic injury following
intratracheal instillation of bleomycin, or in young mice injured with elastase and
allowed to age. Also in this case we assessed growth of murine bone marrowderived mesenchymal stromal cells (MSCs) in parallel with C10 mouse lung
epithelial cells following intratracheal inoculation into the different de-cellularized
lungs.
Materials and Methods
Mice
Adult C57BL/6J male mice aged 8-12 wks (young mice) or 15-18 months (old
mice), (Jackson Laboratories) were maintained at UVM in accordance with
institutional and American Association for Accreditation of Laboratory Animal
Care (AAALAC) standards and review.
Lung Injury
Emphysematous lung injury in mice was induced by oropharyngeal inoculation of
porcine pancreatic elastase (USB) at a dose of 135 IU/mg (1.5 IU PPE) per kg
body weight (Elastin Products). Mice were euthanized either one month later
(young mice) or approximately 43-68 weeks later (old mice) and the heart-lung
blocs subsequently de-cellularized. To induce fibrotic lung injury in mice,
0.075U/mouse
of
bleomycin
(APP
Pharmaceutical)
was
instilled
by
oropharyngeal inoculation. The heart-lung blocs were harvested 14 days postinstillation and subsequently de-cellularized.
Lung De-cellularization
Mice and rats were euthanized by lethal intraperitoneal injection of sodium
pentobarbital. After opening the chest, the trachea was cannulated with a blunted
Luer-lock syringe and the heart-lung bloc was harvested. The lungs underwent
de-cellularization and were subsequently stored for certain periods of time or
underwent specific sterilization techniques. The lungs were de-cellularized under
sterile conditions according to previously published protocols [7,8,16-18]. Lungs
were washed in de-ionized water (DI) containing 5X penicillin/streptomycin (
Cellgro) for one hour at 4°C. The lung was rinsed five times by injection of 3mL of
the de-ionized water solution through the cannulated trachea. The vasculature
was rinsed by injection of 15 cc total volume through the right ventricle. 3cc of
0.1% Triton X (Sigma) and 5X pen/strep in de-ionized water were then infused
through both the trachea and the right ventricle, and the lungs were submerged
in Triton X solution and incubated at 4° C for 24 hours. The following day, the
lungs were rinsed with pen-strep solution as described above. 3cc of 2% sodium
deoxycholate (Sigma) and 1X pen/strep in de-ionized water were then infused
through the trachea and right ventricle and the lungs incubated in this solution at
4° C for 24 hours. The lungs were then rinsed with the de-ionized water as
described above. 3cc of 1M NaCl and 5X pen/strep were then infused through
the trachea and right ventricle and the lungs incubated in the solution for 1 hour
at room temperature. The lungs were then removed from the NaCl solution and
rinsed with de-ionized water as described above. 3cc of 30ug/mL porcine
pancreatic DNase (Sigma), 1.3mM MgSO4 (Sigma), 2mM CaCl2 (Sigma), 5X
Pen/Strep were then infused through the trachea and right ventricle and the
lungs incubated in the solution for 1 hour at room temperature. Finally the lungs
were removed from the DNase solution and rinsed with 5x pen/strep in 1x PBS
as described above for the DI solution rinses. Lungs were stored in PBS/penstrep solution at 4° C until utilized.
To assess effects of prolonged storage of the de-cellularized lungs, lungs were
stored in sterile PBS with 5X penicillin/streptomycin at 4°C for either 3 or 6
months prior to analysis. To assess effects of two different sterilization
approaches, one set of de-cellularized whole lungs was rinsed three times,
through both the trachea and right ventricle, with 15mL of a 0.1% peracetic acid
in 4% ethanol solution and then incubated in this solution for two hours prior to
assessment [11,12]. Another set of de-cellularized whole lungs was irradiated for
12 minutes at a constant dose of 6 Gy/ minute using a RadSource 2000
Biological Irradiator prior to assessment [13-15]. To assess effects of delayed
harvest, mice were euthanized and then kept at 4°C for 72 hours prior to
necropsy and removal of the heart-lung bloc for subsequent de-cellularization
and re-cellularization.
Lung Histology
De-cellularized
lungs
were
fixed
by
gravity
(20
cm
H2O)
with
4%
paraformaldehyde for 10 minutes at room temperature, embedded in paraffin,
and 5-µm sections mounted on glass slides.
Following deparaffinization,
sections were stained with hematoxylin & eosin, Verhoeff’s Van Gieson (EVG),
Masson’s Trichrome, or Alcian Blue, and were assessed by standard light
microscopy [7,16].
Immunohistochemical (IHC) Staining
Deparaffinization was performed with three separate 10 min incubations of
xylenes, followed by sequential descending ethanols and rehydration in water.
Antigen retrieval was performed by heating tissue sections in 1x sodium citrate
buffer (Dako) at 98⁰C for 20 minutes followed by a 15 min cooling step at room
temperature. Tissue sections were permeabilized in 0.1% Triton-X solution
(Sigma Aldrich) for 15 min. Triton-X was removed with two 10 min washes in 1%
BSA solution. Blocking was performed with 10% goat serum for 60 min. After
blocking, primary antibody was added and tissue sections were incubated
overnight at 4°C in a humidified chamber. Tissue was washed three times with
BSA solution for 5 min each. Secondary antibody was added and incubated for
60 min at room temp in a humidified chamber in the dark. Tissue was washed
three times in BSA solution for 5 min each in the dark. DAPI nuclear stain was
added for 5 min at room temperature in the dark followed by 2 washes in BSA
solution for 5 min each. Tissue was submerged in Aqua Polymount (Lerner
Laboratories), and a cover slip was added.. Slides were stored at 4°C in the dark
to preserve fluorescence. Primary antibodies used were: Laminin antibody
polyclonal (ab11575 – 1:100 – Abcam), Smooth muscle myosin heavy chain 2
polyclonal (ab53219 – 1:100 – abcam), Purified Mouse Anti-Fibronectin
monoclonal (10/Fibronectin – 1:100 – BD Transduction Laboratories), Collagen I
polyclonal (ab292 – 1:100 – abcam), Ki67 Proliferation marker polyclonal
(ab16667 - 1:50 - abcam), Mouse clone anti-human Actin polyclonal (1A4 1:10,000 - Dako), Rabbit polyclonal to alpha elastin (ab21607 – 1:100 – abcam),
Cleaved Caspase-3 polyclonal (Asp175 – 1:100 – Cell Signaling Technology).
Secondary antibodies used: Alexa Fluor 568 goat anti-rabbit IgG (H+L) (1:500,
Invitrogen), Alexa Fluor 568 F(ab’)₂ fragment of goat anti-mouse IgG (H+L)
(1:500, Invitrogen) [7,16].
Mass Spectrometry
Six samples (three duplicate pieces, of the same approximate volume and
weight, were obtained from similar parenchymal regions of lungs de-cellularized
with the Triton/SDC, SDS, and CHAPS protocols, respectively, and processed
according to standard protocol [7, 16]. Each sample was loaded in triplicate onto
a fused silica microcapillary LC column packed with C18 reversed-phase resin.
Peptides were separated at a flow rate of 250 nL/min for 45 min. Nanospray ESI
was used to introduce peptides into a linear ion trap quadrupole (LTQ) Orbitrap
mass spectrometer (Thermo Electron). Mass spectrometry data were acquired in
a data-dependent acquisition mode, in which a full orbitrap-MS scan (from m/z
400-2000, resolution r=30,000 at m/z 400) was followed by 10 LTQ-MS/MS
scans of the most abundant ions.
After an LC-MS run was completed and spectra obtained, the spectra were
searched against the IPI Mouse protein sequence databases (V 3.75) using
SEQUEST (Bioworks software, version 3.3.1; Thermo Electron, San Jose, CA),
with search parameters detailed in Supplemental Methods. Proteins that were
identified by two or more peptides in each of the six samples were regarded as
identified. Proteins that were found at least in 2 out of 3 LC-MS/MS replicates
were included in the analysis.
Cells and Cell Inoculation
Mesenchymal stromal cells (MSCs) derived from bone marrow of adult male
C57BL/6 mice were obtained from the NCRR/NIH Center for Preparation and
Distribution of Adult Stem Cells at Texas A and M University [19]. MSCs were
cultured on cell-culture treated plastic at 37°C and 5% CO2 in MSC basal
medium consisting of Iscove’s Modification of Dulbecco’s Medium supplemented
with 2 mM L-glutamine, 100 U/ml penicillin and 100μg/ml streptomycin (Fisher),
10% fetal bovine serum (Atlanta Biologicals) and 10% horse serum (HS,
Invitrogen). Cells were used at passage 9 or lower and maintained in culture at
confluency no greater than 70%. Purity was determined by expression of Sca-1,
CD106, CD29, absence of CD11b, CD11c, CD34, and CD45 expression, and the
ability to differentiate into osteoblasts, chondrocytes and adipocytes in vitro [19].
C10 mouse lung type 2 alveolar epithelial cells were obtained courtesy of
Matthew Poynter Ph.D., University of Vermont and cultured under standard
conditions [20]. The right lobes were tied off using sterile suture under sterile
conditions, and then then removed. 2x106 MSCs or C10 cells suspended in 1 mL
MSC or C10 basal media, respectively, were mixed with 1ml of low-melting
temperature agarose (Cambrex) and the 2mL cell suspension injected through
the cannulated trachea into the left lung. The inoculated lung was then incubated
for 30 minutes at 4°C until the agarose hardened and the lobe sliced with a
sterile razor blade to yield transverse sections of approximately 1mm in
thickness. Each slice was placed in a well of a sterile 24-well dish, covered with
sterile cell media and placed in a standard tissue culture incubator at 37°C until
agarose melted out of the tissue. The lungs were then submerged overnight in
basal MSC or C10 media at 37°C and 5% CO2. The next day, medium was
changed to either fresh basal medium [7]. Individual slices were harvested at 1,
3, 7, 14, 21, and 28 days post-inoculation, fixed for 10 minutes at room
temperature in 4% paraformaldehyde, and mounted 5 μm paraffin sections were
assessed by H and E staining for presence and distribution of the inoculated
cells.
Statistical Analyses
Heat maps for the natural log of unique peptide hits for each positively identified
protein in the mass spectrometric analyses of lungs de-cellularized under each
experimental condition were generated using the 'pheatmap' package for 'R'
statistical software version 2.15.1. Two group comparisons were done using the
non-parametric exact permutation test with p<0.05 considered statistically
significant [21].
Non-parametric Spearman correlations were also done with
concordance considered significant at p <0.05 [21]. The exact permutation tests
and correlations were done using SAS statistical software, version 9.2.
Results
Architecture and ECM composition of the decellularized mouse lungs
Histologic evaluation with H&E, Verhoeff’s Van Gieson (EVG), and Masson’s
trichrome stains demonstrates, as it’s been shown by us and others [7,8,1618,22-26], that freshly de-cellularized lungs, compared to native lung, maintain
the architecture of the extracellular matrix. Glycosaminoglycans (GAGs) were
less evident by Alcian Blue staining in freshly de-cellularized lungs, likely
representing in large part loss of cell-associated GAGs during the decellularization process [7,16,18].
Overall, delayed necropsy appeared to minimally affect the histologic
appearance and presence of collagens (Trichrome), elastin (EVG), and GAGs
(Alcian Blue). Following 3 months of storage, scattered areas of atelectasis were
observed particularly in central regions of the de-cellularized lungs. Following 6months storage, the de-cellularized lungs were markedly atelectatic, showing
very different morphology compared to native or freshly de-cellularized lungs.
Peracetic treated lungs had a similar appearance to native or freshly decellularized lungs although some central regions appeared more atelectatic. In
contrast, irradiated lungs demonstrated an abnormal appearance with scattered
heterogenous pattern of thickened alveolar septa, and large emphysematousappearing alveolar spaces.
The lung architecture of de-cellularized lungs obtained from the 3 month
storage, peracetic acid, and to some degree the irradiated lungs better
resembled native or freshly de-cellularized lungs. In contrast, no significant
improvement was observed in the 6 month storage lungs.
As we and others have previously demonstrated, type 1 collagen and laminin
were largely retained in freshly de-cellularized lungs whereas elastin was
significantly decreased.
Fibronectin was mostly retained but became
fragmented-appearing [7,16].
As previously demonstrated, some cellular
proteins, including smooth muscle actin & smooth muscle myosin were also
retained in freshly de-cellularized lungs [7,16]. Neither delayed necropsy, 3 or 6
month storage, or peracetic acid treatment had any apparent effect on the
presence of these proteins although the 6 month storage lungs remained
abnormal appearing.
In irradiated lungs, staining for laminin and collagen-1
appeared to be more intense, likely due to the clumping and thickened tissue.
Fibronectin, smooth muscle actin, and smooth muscle myosin appeared to be
present in the same patterns and intensities observed in native or freshly decellularized lungs.
Residual protein composition of de-cellularized lung scaffolds
Mass spectrometry was utilized to detect differences in residual protein content
under the different storage and sterilization procedures. Freshly de-cellularized
right lower lobes were used as controls. Proteins were assigned to one of five
groups; ECM, cytoskeletal, intracellular cytosolic, intracellular nuclear, and
membrane associated. Heat maps were generated with each positively identified
protein and its corresponding number of unique peptide hits. The delayed
necropsy lungs contained statistically significant increases in a large number of
cellular associated proteins (non ECM) as compared to freshly de-cellularized
lungs.
Notably, several proteins associated with erythrocytes including
hemoglobins A and B and the erythrocyte membrane protein Slc4a were
markedly increased in the delayed necropsy as compared to fresh decellularized
lungs. Peracetic acid-treated lungs contained a significantly higher number of
ECM components compared to the controls. Lung scaffolds which had been
stored in PBS for 3 months at 4ºC contained several ECM components, such as
laminins, aggrecan, fibrillin, and myosins, which were significantly increased
compared to freshly de-cellularized scaffolds. There were no ECM proteins which
achieved statistically significant differences in scaffolds which had been stored
for 6 months or between the 3mos vs 6mos or irradiated vs. acid-treated group
comparisons.
Mass spectrometry was also utilized for assessment of residual protein content
and composition in decellularized lungs obtained from young, old, elastase, and
bleomycin-injured mouse lungs
We hypothesized that residual protein content would differ between young and
old mice, and normal (young or old) vs. injured lungs. Lungs (right lower lobes)
which had been freshly de-cellularized from young naïve mice were utilized as
controls. Comparisons included old vs. young naïve mice, old elastase vs. young
elastase-injured mice, old vs. old elastase-injured mice, and young naïve mice
vs. young bleomycin-injured mice. Heat maps were generated with proteins
broadly categorized as cytoskeletal, extracellular matrix (ECM), intracellular
cytoplasmic, intranuclear and membrane proteins. The majority of statistically
significant differences appeared between groups in the ECM proteins. As
compared to controls, de-cellularized lungs from old mice, or either young or old
elastase-treated mice, contained statistically fewer overall ECM proteins. Decellularized lungs from bleomycin-injured mice contained significantly more
residual ECM proteins, with increases in residual collagen 6a, fibrillin, fibrinogen,
and fibronectin. De-cellularized lungs from young elastase-treated mice
contained more residual overall ECM proteins compared to those from old
elastase treated mice.
Similarly there were higher levels of residual ECM
proteins in de-cellularized lungs from old vs old elastase-treated mice.
While
there were statistically significant differences in levels of residual proteins in the
other categories (cytoskeletal, cytosolic, membrane, nuclear) between the
experimental groups, there was no clear or obvious association between
experimental group and residual protein content.
Growth of MSCs and C10 cells in de-cellularized lungs
To assess the impact of the different storage and sterilization procedures on the
re-cellularization of lung scaffolds, two cell types were inoculated into separate
de-cellularized lungs via an intratracheal route (1x106 of each cell type per lung)
and engraftment and survival were assessed in lung slices at 1, 3, 7, 14, 21, and
28 days. Similar initial localization and distribution (day 1) of both C10s and
MSCs throughout the lungs were observed with the different storage and
sterilization conditions as that seen in freshly de-cellularized scaffolds.
As
previously observed [7,16,17], many of the MSCs that initially lodged in
parenchymal lung regions developed a bipolar elongated appearance over time.
Also as previously observed, C10 cells develop an elongated phenotype over
time as they grow along alveolar septa [7]. The length of time in which the cells
remained viable in the scaffolds was variable, dependent upon both the cell type
and the treatment of the de-cellularized scaffold.
In the 6-month storage
condition, no viable cells were observed after 7 to 14 days in culture. In all other
conditions, MSCs survived robustly through 28 days of culture. The cells were
localized throughout the tissue and retained their characteristic bipolar elongated
phenotype. We had previously found strong Ki67 staining and minimal caspase3 staining of MSCs at both 1 and 28 days when cells were inoculated into freshly
de-cellularized lungs [7,16]. In the current studies, Ki67 staining demonstrated
the presence of actively proliferating cells throughout the lung slices for the
different storage and sterilization conditions both at day 1 and day 28 after
inoculation. Minimal apoptosis was observed by caspase-3 staining at days 1 or
28 for any condition except the 6 month storage in which increased caspase-3
staining was observed at 7 and 14 days. Similarly, we had previously observed
sustained growth and spreading of intratracheally inoculated C10 cells along
alveolar walls following either 1 or 14-28 days in culture [7]. In parallel, robust
Ki67 and minimal caspase-3 staining was observed [7]. In contrast, C10 cells
inoculated into different sterilization and storage conditions were largely nonviable or absent at different times ranging between 7 and 14 days in culture. At
the last viable time point for each condition, the C10 cells were largely localized
on the periphery of the tissue or lining the major airways. Ki-67 and caspase-3
staining demonstrated active proliferation and minimal early apoptosis 1 day after
seeding but significant increase in apoptosis at or before the last viable time point
at which cells were observed for each storage and sterilization condition.
To assess re-cellularization in the injured or aged de-cellularized lungs, 1x106
MSCs or C10 epithelial cells were separately inoculated and engraftment and
survival were evaluated in lung slices cultured for 1, 3, 7, 14, 21, and 28 days.
On day 1, both MSCs and C10s were observed to primarily engraft in alveolar
spaces.
After one day of culture, MSCs acquired a characteristic spindle-shaped
phenotype, and could be found scattered throughout the different de-cellularized
lungs. In contrast, while C10 cells could be found growing throughout the 28 day
period in de-cellularized lungs obtained from young, old, and bleomycin-injured
mice, elastase-injured lungs retained no viable cells past 14 days in lungs
obtained from either young or old mice.
To determine the proliferation and apoptosis rate for C10s and MSCs
during the culture period, Ki67 and caspase-3 expression was assessed after
one day of culture and at the last time point at which viable cells were observed
for each condition. In the current study, robust Ki67 expression and minimal
caspase-3 expression was observed in MSCs after one day of culture under
each condition. Following 28 days in culture, less evident Ki67 expression but
increased caspase-3 expression was observed, especially in de-cellularized
lungs obtained from elastase or bleomycin-injured lungs.
Discussion
Use of de-cellularized whole lung scaffolds for ex vivo generation of functional
lung tissue may provide in the future a viable option for clinical lung
transplantation [6-8,16-18,22-27].
As already shown for other tissue types
including skin, muscle, bladder, successful use of biologic scaffolds has already
entered clinical practice [3-6]. However, the complex 3-dimensional structurefunctional biology of the lung makes this a more difficult task. A number of recent
reports have evaluated lung de-cellularization, re-cellularization, and implantation
in rodent and primate models [7,8,16-18,22-26]. While these reports show the
viability of this approach, a number of unanswered questions remain.
For
example, there is no consensus on the optimal ways of producing clinically useful
de-cellularized lungs, including the different detergent and physical approaches
to be applied. Recent studies demonstrate significant differences between the
structure and protein content and also the mechanical properties of decellularized lungs produced using different approaches [7,8,26]. However,
whether these differences will significantly affect subsequent re-cellularization
and also the potential immunogenicity of the de-cellularized scaffolds remains
unclear.
Recent data suggests that initial binding and short term growth of
stromal and epithelial cells inoculated into mouse lungs de-cellularized using
different detergent-based approaches is similar [7].
However, more data on
longer term growth and also on growth of other cell types to be inoculated into
the de-cellularized lungs, including vascular endothelial cells is necessary.
Other practical issues need to be considered for use of de-cellularized lung
scaffolds. Biologic scaffolds such as bone, cartilage, and skin can be stored for
prolonged periods of time prior to use, particularly if treated with irradiation or
final rinse in peracetic acid for sterilization [11-15].
However, it is yet to be
clarified whether these approaches or long term storage will be applicable for decellularized whole lungs. In this respect, recent data demonstrates that significant
tissue breakdown can occur in de-cellularized tracheas stored for up to one year
[10].
To address this issue, we initially evaluated freshly de-cellularized lungs that
were stored under refrigerated sterile conditions for up to 6 months. Most of the
lungs remained sterile with only infrequent episodes of bacterial or fungal
contamination. Histologic assessment of the stored lungs demonstrated
development over time of lung atelectasis and loss of native architecture. These
changes were partly reversible with inflation in lungs stored for 3 months, but
became irreversible with inflation following 6 months storage.
Therefore our
results suggest that de-cellularized lungs should not be stored beyond 3 months.
Irradiation, even at a dose under the one commonly generally recommended for
biologic materials according to International Standard of Organizations (1525kGy) [13-15], produced significant distortion that was only partly responsive to
subsequent lung re-inflation. Peracetic acid, a denaturing agent used both for
sterilization and to rinse out residual detergents and other reagents utilized
during tissue de-cellularization [11,12], had less effect on the resulting
architecture.
Between the different storage and sterilization procedures examined in our study
there were significant differences in residual protein content, as assessed by
mass spectrometry. Compared to freshly de-cellularized lungs, the most relevant
differences were observed in the scaffolds following delayed necropsy and with
use of peracetic acid sterilization of freshly de-cellularized lungs. The presence of
proteins characteristic of erythrocytes together with other intracellular proteins in
the delayed necropsy group suggests that autolysis of red blood cells and other
cells present in the lungs occurred over time, despite cold storage, and that the
proteins released from autolysed cells are not completely removed by the decellularization approach utilized. Additionally, peracetic acid can act as a protein
denaturing agent and in this respect it is commonly utilized to solubilize ECM
components for protein detection.
Thus, as espected, freshly de-cellularized
peracetic acid-treated lungs contained a significantly increased number of ECM
components compared to non-treated lungs. The increase in ECM components
is therefore most likely not indicative of an absolute increase in ECM
components, but rather of an increased solubilization of ECM components which
were then more readily detected using mass spectrometry. Similarly, scaffolds
stored for 3 months had higher ECM protein levels than controls, but these
increases were absent in the scaffolds stored in the same conditions for 6
months.
Survival and proliferation of mesenchymal stem cells (MSCs) inoculated into the
airways was comparable between the delayed necropsy, 3 month storage,
peracetic acid, and irradiated de-cellularized lungs, suggesting that appropriate
preservation of ECM necessary for binding and subsequent cell growth and
proliferation were preserved.
Markers of apoptosis were observed following
MSC culture in de-cellularized lungs stored for either 3 or 6 months, suggesting
that prolonged storage of de-cellularized lungs may not support sustained cell
growth.
Viability of a type 2 alveolar epithelial cell line (C10) was diminished time in all
the experimental conditions tested compared to survival and proliferation of C10
cells inoculated into freshly de-cellularized lungs [7]. Markers of apoptosis were
observed at early time points in culture, particularly in C10 cells seeded into
freshly de-cellularized lungs stored for 3 or 6 months.
These results suggest that commonly utilized approaches for storage and
sterilization of other de-cellularized tissues and other types of biologic scaffolds
may not be suitable for de-cellularized lungs.
Another relevant issue for the potential applicability of de-cellularized lung
scaffolds is represented by the fact that some cadaveric lungs may come from
aged donors or from donors with previously existing structural lung diseases
such as emphysema or pulmonary fibrosis. While advanced age or severe cases
of either type of disease would not be suitable for consideration, moderately
affected lungs could conceivably be utilized for de-cellularization and subsequent
re-cellularization and clinical use. To evaluate this possibility, we assessed decellularization and initial re-cellularization of lungs obtained from aged mice and
from mice with experimentally-induced emphysematous or fibrotic lung injury.
The resulting ECM scaffolds for each condition were consistent with the
underlying injury and also showed preservation of the characteristic injury
patterns, as reflected by both histologic architecture and by proteomic
assessment using mass spectrometry. This demonstrates that successful decellularization can be achieved in aged and injured lungs and that the resulting
lung scaffold will reflect that original disease state. These findings are consistent
with recent description of de-cellularized cadaveric lungs obtained from patients
with idiopathic pulmonary fibrosis.
Initial binding and subsequent survival and proliferation of a stromal cell
line (MSCs) inoculated into the airways was robust across the different conditions
and comparable to that observed following inoculation into de-cellularized lungs
obtained from young healthy mice. The only exception was lack of initial cell
engraftment and subsequent growth in the more densely fibrotic regions of the
bleomycin-injured lungs, suggesting that appropriate preservation of the ECM
structures necessary for initial binding and subsequent growth and proliferation
were preserved. Similarly, engraftment and viability of an immortalized type 2
alveolar epithelial cell line (C10) was similar in aged and bleomycin injured lungs
compared to that observed in freshly de-cellularized normal lungs. In contrast,
despite good initial engraftment, survival of the C10 cells was diminished in
emphysematous lungs produced by elastase treatment in both young and old
mice. The reasons for this are not yet clear but one explanation could be that,
despite preservation of ECM proteins in the elastase-injured lungs, more subtle
and yet unidentified changes in the ECM scaffold do not support longer term
proliferation and survival of cells. Notably, there was minimal residual elastin in
young naïve de-cellularized lungs and no significant differences were detected
between those compared to either young or old elastase-injured de-cellularized
lungs.
These results suggest that de-cellularized lungs obtained from aged lungs
may be appropriate for ex vivo lung bioengineering approaches utilizing decellularization and re-cellularization strategies. Our data suggest that fibrotic
lungs support prolonged growth of inoculated cells but whether these lungs will
be useful for long-term regeneration yet needs to be determined. Recent data
suggest that fibroblasts cultured in vitro on scaffolds consisting of pieces of decellularized lungs obtained from patients with idiopathic pulmonary fibrosis are
induced to acquire a myofibroblast phenotype. This suggests that the specific
scaffold obtained from de-cellularization of lungs from different disease states
can significantly affect cell growth and differentiation. Accordingly, we found that
de-cellularized emphysematous lungs may not support long term viability of
epithelial cells.
Our studies do not address the impact of the different condition tested on a wider
range of cells including both mature pulmonary vascular endothelial cells as well
as a range of stem and progenitor cells that might be utilized for recellularization. This will need to be done in a rigorous manner in future studies.
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