Immune tolerance of tissue-engineered skin produced with allogeneic or xenogeneic fibroblasts and syngeneic keratinocytes grafted on mice

Accepted Manuscript Full length article Immune tolerance of tissue-engineered skin produced with allogeneic or xenogeneic fibroblasts and syngeneic keratinocytes grafted on mice Benjamin Goyer, Danielle Larouche, Dong Hyun Kim, Noémie Veillette, Virgile Pruneau, Vincent Bernier, François A. Auger, Lucie Germain PII: DOI: Reference:

S1742-7061(19)30244-2 https://doi.org/10.1016/j.actbio.2019.04.010 ACTBIO 6050

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

14 December 2018 28 March 2019 2 April 2019

Please cite this article as: Goyer, B., Larouche, D., Kim, D.H., Veillette, N., Pruneau, V., Bernier, V., Auger, F.A., Germain, L., Immune tolerance of tissue-engineered skin produced with allogeneic or xenogeneic fibroblasts and syngeneic keratinocytes grafted on mice, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio. 2019.04.010

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Immune tolerance of tissue-engineered skin produced with allogeneic or xenogeneic fibroblasts and syngeneic keratinocytes grafted on mice Benjamin Goyera, Danielle Larouchea, Dong Hyun Kima, Noémie Veillettea, Virgile Pruneaua, Vincent Bernierb, François A. Augera, Lucie Germaina,* a

Centre de recherche du CHU de Québec - Université Laval, Department of Surgery, Faculty of

Medicine, Université Laval and Centre de recherche en organogénèse expérimentale de l’Université Laval/LOEX b

Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine,

Université Laval.

* Corresponding author at: Centre de recherche du CHU de Québec – Université Laval, Hôpital Enfant-Jésus 1401, 18e rue LOEX, Aile-R Québec, Québec, Canada G1J 1Z4 E-mail address: [email protected] (L. Germain)

Short title: Allogeneic and xenogeneic fibroblasts are not rejected Keywords: Graft rejection, artificial skin, tissue engineering, transplantation tolerance, homologous transplantation, autologous transplantation

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Abstract Organs are needed for the long-term replacement of diseased or wounded tissues. Various technologies based on cells seeded in synthetic or biomaterial scaffolds, or scaffold-free methods have been developed in order to produce substitutes that mimic native organs and tissues. For cell-based approaches, the use of living allogeneic fibroblasts could potentially lead to the production of “off-the-shelf” bioengineered organs/tissues. However, questions remain regarding the outcome of allogeneic grafts in terms of persistence of allogeneic cells, tolerance and the host immune reaction against the tissue after implantation. To evaluate graft tolerance of engineeredtissues containing non-autologous fibroblasts, tissue-engineered skin substitutes (TESs) produced with syngeneic, allogeneic or xenogeneic fibroblasts associated with syngeneic, allogeneic or xenogeneic epithelial cells were grafted in mice as primary and secondary grafts. The immune response was evaluated by histological analysis and immunodetection of M2 macrophages, CD4and CD8-positive T cells, 15, 19, 35 and 56 days after grafting. Tissue-engineered skin composed of non-autologous epithelial cells were rejected. In contrast, TESs composed of non-autologous fibroblasts underlying syngeneic epithelial cells were still present 56 days after grafting. This work shows that TES composed of non-autologous fibroblasts and autologous epithelial cells are not rejected after grafting.

Statement of significance We found that tissue-engineered skin substitutes produced by a scaffold-free cell-based approach from allogeneic fibroblasts and autologous epithelial cells are not rejected after grafting and allow for the permanent coverage of a full-thickness skin wounds. In the field of tissue

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engineering, these findings open the possibility of selecting a human fibroblastic or stromal cell population based on its biological properties and adequate biosafety, banking it, in order to produce “ready-to-use” bioengineered organs/tissues that could be grafted to any patient without eliciting immune reaction after grafting. Our results can be generalized to any organs produced from fibroblasts. Thus, it is a great step with multiple applications in tissue engineering and transplantation.

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1. Introduction The loss of tissue can occur for many reasons, including trauma, chronic or acute wounds, genetic disorders and surgical interventions. The developing field of tissue engineering aims to repair, maintain and improve damaged tissues or whole organs. To accomplish this, different collagen-based biomaterials have been developed as replacement tissues for skin, cornea, bone, cartilage, blood vessels, hear valve, bladder or muscle, and some have proven effective in regenerative medicine in humans [1]. A good example is the skin. Several cultured living autologous skin substitutes containing both a dermis and an epidermis have been tested on humans to permanently replace damaged skin following acute full-thickness burn injuries [2-15], or to speed up the healing of large or lasting ulcers [16, 17]. The cell source is a key element in the successful implantation and long-term survival of a living tissue-engineered substitute. Cells must be accepted by the immune system of the host and provide the desired function. Depending on their tissue source, cells are classified as autologous (patient's own), syngeneic (genetically identical), allogeneic (same species but genetically different) or xenogeneic (different species). Specific challenges are associated with each of these sources. Autologous cells are the most appropriate because they are not rejected by the immune system. However, the availability of engineered tissues is delayed by the time necessary for the primary culture steps. For each patient, cells must be extracted and amplified from a biopsy of their own tissue. In contrast, the production of tissue-engineered substitutes from allogeneic or xenogeneic cells allows the establishment of biobanks, which can in turn be accessed to massively produce “off-the-shelf” engineered substitutes. However, allogeneic and xenogeneic cells could be immunogenic and may require post-graft immunotherapy for long-term tissue persistence.

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In the case of skin, cutaneous allografts are rejected by the immune system and the onset of necrosis usually appears within two weeks [18]. The phenomenon of skin allograft rejection was first described by Medawar in a rabbit model [19], and highlighted by Billingham who discovered the critical role of the major histocompatibility complex (MHC) in triggering the immune response [20]. The MHC is composed of cell surface proteins that allow the immune system to discriminate between its own cells and foreign cells. The immunogenicity of endothelial cells and keratinocytes (skin epithelial cells) is well known. These cell types have to be autologous for permanent integration of the graft into the host [21, 22]. In contrast, some indications suggest that allogeneic fibroblasts within cryopreserved skin do not lead to rejection after grafting [23, 24]. However, it is unclear whether allogeneic fibroblasts persist in grafted tissues, or if they are replaced by host cells after engraftment [16, 24-27]. Our group has developed a scaffold-free bilayered tissue-engineered skin (TES) produced from keratinocytes and fibroblasts that can be harvested from a small skin biopsy. Our cell-based approach, referred to as the self-assembly approach of tissue engineering [28], is based on the capacity of stromal cells to form their own extracellular matrix (ECM) in vitro in the presence of ascorbic acid [29]. After the addition of keratinocytes and culture at the air-liquid interface, TES presents a fully differentiated epidermis. This method, initially developed with human cells, can also be used to produce murine TESs [30]. Therefore, grafting this TES model on immunocompetent mice offers the possibility to study graft rejection of tissues produced from different cell sources (autologous, allogeneic or xenogeneic), while minimizing the inflammatory reaction that could be induced by an exogenous scaffold. The objective of this study was to determine whether TESs produced with allogeneic fibroblasts associated with autologous keratinocytes were tolerated or rejected after grafting.

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Different TESs produced with syngeneic, allogeneic or xenogeneic fibroblasts, associated with syngeneic, allogeneic or xenogeneic epithelial cells were grafted onto immunocompetent mice without immunosuppressive drug treatment. Tissue-engineered dermal substitutes (TEDs) were also grafted subcutaneously to evaluate the immune response against fibroblasts in the absence of epithelial cells. The results demonstrate that the integrity of TES composed of non-autologous fibroblasts and autologous epithelial cells, as well as TEDs composed of non-autologous fibroblasts is maintained 56 days after grafting without adverse effects on tissue regeneration. 2. Materials and methods 2.1. Ethics statement This study was conducted according to our institutions’ guidelines and the Declaration of Helsinki. All protocols were approved by the institution’s animal care and use committee (Comité de protection des animaux de l’Université Laval, Québec, Canada) and by the institution’s committee for the protection of human subjects (Comité d’éthique de la recherche du CHU de Québec - Université Laval). All patients provided their informed formal written consent, agreeing to supply biopsy tissue for this study. 2.2. Cell isolation and culture Human cells were obtained from healthy skin biopsies harvested from the scalp of two adult donors (37 and 55 years old). Briefly, skin fragments were digested with 500 µg/mL thermolysin (Sigma-Aldrich, USA) in the HEPES buffer [10 mM 4-(2-hydroxyethyl)-1piperazine ethane sulfonic acid (MP Biomedicals Inc., Canada), 6.7 mM potassium chloride, 142 mM sodium chloride, and 1 mM calcium chloride] at 4°C overnight. The dermis was then separated from the epidermis with forceps. Keratinocytes were dissociated in a trypsin/ethylenediaminetetraacetic acid (EDTA) solution [0.05% trypsin 1–500 (Intergen,

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Canada) and 0.01% EDTA/ disodium salt (Sigma-Aldrich) prepared in phosphate-buffered saline (PBS)] at 37°C for 15 min, while fibroblasts were dissociated with 0.125 U/mL collagenase in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Canada) at 37°C for three hours. Cells were amplified in culture flasks until passage four and cryopreserved with freezing medium consisting of 90% fetal calf serum (FCS; Hyclone) and 10% dimethyl sulfoxide (DMSO; SigmaAldrich). Mouse cells were obtained from healthy C3H/HeN and BALB\c adult and newborn mice (Charles River Laboratories, Canada). Fibroblasts were isolated from dorsal skin of three onemonth-old females. Epithelial cells were isolated from the entire skin of one- to two-day-old newborn mice. For fibroblast isolation, dorsal skin was shaved and an area of 10 cm2 was harvested, washed in 70% ethanol (Les Alcools de commerce, Canada) and successive sterile PBS baths. The skin sample was placed in a Petri dish and incubated at 4°C overnight in a 0.25% trypsin solution with 40 µg/mL of DNAse (Sigma-Aldrich). The day after, the epidermis was peeled off and the dermis was gently scratched with a scalpel to eliminate remaining epithelial cells. The dermis was then incubated in the collagenase/DMEM solution at 37°C for three hours. Dissociated cells were cultured in DMEM with 10% FCS until passage three and cryopreserved. Mouse epithelial cells were extracted from the epidermis using the method of Yuspa and Harris [31] with slight modifications. Briefly, after being euthanized, newborn mice were washed in 70% ethanol and in 10% proviodine (Rougier Pharma, Canada). After amputation of the limbs and the tail, a longitudinal incision was performed on the ventral side; the skin was first peeled laterally, and then flayed away. The skin pieces were then washed in sterile PBS and placed in a 0.25% trypsin solution with 40 µg/mL DNAse at 4°C overnight. The day after, the epidermis was

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peeled off from the dermis using forceps and gently scraped in keratinocyte medium [DMEM: Ham’s F12 (Invitrogen), ratio 3:1 (Invitrogen, Canada), supplemented with 5% Fetal Clone II serum (HyClone, Logan, UT), 5 μg/ml insulin, 0.4µg/ml hydrocortisone, 10-10 M cholera toxin, 10 ng/ml murine epidermal growth factor (mEGF)], and antibiotics to detach epithelial cells. Epithelial cells were then layered onto a Lympholyte-M cushion (Cedarlane Laboratories, Canada) and centrifuged (1800 g, 30 min). Cells in the middle of the Lympholyte-M were collected, suspended in 0.1% trypsin, transferred into a trypsinization unit (Celstir suspension culture flask; Wheaton Sciences Products, USA) and incubated under agitation at 37°C for 10 min. The cell suspension was centrifuged, and recuperated after trypsin inactivation with fresh keratinocyte medium to obtain the dissociated epithelial cells. 2.3. Tissue-engineered skin production Murine (C3H/HeN or BALB\c mice) or human fibroblasts between passage two and four were used for the production of tissue-engineered dermal substitutes (TED). Briefly, fibroblasts were cultivated 28 days in fibroblast medium (DMEM with 10% FCS) supplemented with 50 µg/mL of ascorbic acid (Sigma Aldrich) according to the self-assembly approach of tissue engineering [28, 32, 33]. To produce TED, four fibroblast-derived ECM sheets were superimposed; using surgical forceps, a first fibroblast-derived ECM sheet was carefully detached from the culture flask and placed in a Petri dish. Small 1g-weights (15mm x 4mm stainless steel pieces (16G; Acier Inoxydable Den-Mar, Québec, QC, Canada) were used to placed around the tissue and to keep the sheet in place after extension. In the same manner, a second tissue sheet was detached and stacked on the first one. The procedure was repeated two additional times. A Merocel sponge (Medtronic, Instruments Ophtalmiques INNOVA), which was cut to fit the construct, was placed on top and maintained in place with 1g-weights. After two

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days, the Merocel sponge was removed and the TED was cultured for five more days. TEDs were then produced from C3H/HeN, BALB\c and human fibroblasts in order to obtain syngeneic, allogeneic and xenogeneic TEDs (referred to as syngeneic-Fk, allogeneic-Fd and xenogeneic-FH, respectively) (see Table S1). To produce TESs, 200,000 human keratinocytes per cm2 or 600,000 freshly extracted mouse epithelial cells (C3H/Hen or BALB\c) per cm2 were seeded onto TEDs within a seeding ring. The resulting constructs were cultured submerged in keratinocyte medium containing 50 µg/mL of ascorbic acid for seven days. For tissues with human keratinocytes, mEGF was replaced by 10 ng/ml human epidermal growth factor. Then, using forceps, the tissue was detached carefully from the bottom of the culture dish and placed on an anchoring paper and transferred onto an air-liquid stand. The tissue was cultured at the air–liquid interface in epidermal growth factor-free keratinocyte medium containing 50 µg/mL of ascorbic acid for 14 days. Syngeneic (C3H/HeN fibroblasts and epithelial cells, referred to as syngeneic-FkKk), allogeneic (BALB\c fibroblasts and epithelial cells, referred to as allogeneic-FdKd), xenogeneic (human fibroblasts and keratinocytes, referred to as xenogeneic-FHKH) and chimeric (C3H/HeN fibroblasts and BALB\c epithelial cells [referred to as mouse chimeric-FkKd], BALB\c fibroblasts and C3H/HeN epithelial cells [referred to as mouse chimeric-FdKk] and C3H/HeN epithelial cells and human fibroblasts [referred to as human chimeric-FHKk]) TESs were then obtained (see Table S2). 2.4. Grafting in mice To evaluate primary graft rejection, substitutes were grafted onto the dorsal muscular fascia of female C3H/HeN mice. TEDs were grafted subcutaneously, while TESs, were grafted within a Fusenig’s silicone chamber made in our laboratory with an inner diameter of 2.5 cm (Fig 1A). Such device is used to block the epithelium from the surrounding mouse skin to epithelialize

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the grafted tissue [34-36]. To prevent infections, all mice received intraperitoneal ceftazidime antibiotic (3 mg/mouse, Glaxo, Toronto, ON, Canada) 48 and 24 hours before the surgery. Mice were injected with Buprenorphine (0.05-0.1 mg/kg, Champion Alstoe, Whitby, ON, Canada) before surgery and Carprofen (5-10 mg/kg, Pfizer, Kirkland, QC, Canada) daily for 48 hours post-operatively to provide analgesia. A 2.5 cm2-circular piece of skin was removed on the mid back of the animal and the Fusenig’s chamber was inserted into the opening. The base (3.5-cmdiameter) of the Fusenig’s chamber was attached to the mouse skin using four stitches. The paniculus carnosus was removed, and the TES (about 2.5 cm2) was deposited onto the muscular bed (Fig 1A-C). The Fusenig’s chamber was closed with a HEPA-filtered lid allowing gas exchange. The lid was attached to the skin with four additional stitches. TEDs were grafted subcutaneously. A surgical incision was performed, the paniculus carnosus was removed and the TED was placed onto the muscular bed and fixed with four stiches (Fig 1 J). A simple continuous suture was performed to close the skin. For each tissue substitute type, at least 13 mice were grafted; except for mouse chimericFkKd TESs, which were grafted on six mice (see table S1 and table S2). Postoperative care consisted of watching the general state of health and monitored the weight after the surgery, the following two to three days, and once weekly when bedding was changed. For the first 19 days, animal that received grafts with allogeneic or xenogeneic cells were observed regularly to detect early signs of graft rejection (necrosis, colour change). However, the absence of desquamation within silicone chamber impaired the macroscopic evaluation of rejection due to corneocyte accumulation. To promote mouse confort, the Fusenig’s chamber was removed 21 days after grafting. On days 15, 19, 35 and 56, animals were euthanized and biopsies were collected to monitor the survival of the grafts.

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To evaluate secondary graft rejection caused by pre-existing exposure to allogeneic antigens, one million newborn BALB\c epithelial cells were injected subcutaneously onto female C3H/HeN mice three weeks before grafting syngeneic-FkKk, allogeneic-FdKd and chimeric-FdKk TESs (Table S3). Biopsies were taken after 19 days. For each tissue type, three mice were grafted. 2.4. Histological analysis Tissue samples were fixed in 3.7% formaldehyde (ACP Chemicals, Canada) and were processed for paraffin embedding. Sections (five µm) were stained with hematoxylin and eosin. Digital images were acquired using Axio Imager.Z2 microscope (Carl Zeiss Canada Ltd, Canada). A dermatologist and a pathologist performed the histological evaluations. 2.5. Immunofluorescence analysis Samples were embedded in Tissue-Tek OCT Compound (Miles, Inc., IN), cooled in liquid nitrogen, and conserved at -80°C. Direct immunofluorescence assays were performed on 12 µmthick cryosections. For the detection of immune cells, cryosections from mouse spleen and xenogeneic-FHKH TESs eight days after grafting were used as positive control and native C3H/HeN skin as negative control. For the detection of CD206, CD4 and CD8a, cryosections were fixed 10 minutes in 4% paraformaldehyde (Sigma-Aldrich). A mouse IgG blocking reagent (Cedarlane Laboratories, Canada) diluted in a normal mouse serum (Cedarlane Laboratories, Canada) was added for 30 minutes. Fc receptors were blocked using anti-mouse CD16/32 (TruStain fcX, Biolegend, USA) for 30 minutes. Antibodies were incubated two hours. The following antibodies were used: anti-mouse CD206 conjugated to Alexa Fluor 647 (Clone C068C2), anti-mouse CD4 conjugated to Alexa Fluor 594 (Clone GK1.5) and anti-mouse CD8a conjugated to Alexa Fluor 594 (Clone 53-6.7). For the detection of H-2Kk, H-2Dd and HLA-ABC

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staining, cryosections were immunostained without prior fixation. Finally, cell nuclei were counterstained with Hoechst reagent 33258 (Sigma-Aldrich). Fluorescence was observed using a LSM700 confocal microscope equipped with an Axiocam digital camera (Carl Zeiss Canada Ltd). Images were captured and processed with Adobe Photoshop CC 2015 software (Adobe System, USA). To count CD206-, CD4- and CD8a-positive cells, 20 μm-thick cryosections were immunostained. Under a confocal microscope, whole sections were photographed and their surface area was measured with Image JW software (NIH, Bethesda, MD, USA). The number of CD206-, CD4- and CD8a-positive cells on each section was counted manually and depicted as the number of positive cell per mm2 of tissue. 2.6. Statistical analysis Statistical significance was determined using the bilateral Wilcoxon rank-sum test (GraphPad Prism version 5, Inc., San Diego, CA). A p-value under 0.05 was considered significant. 3. Results 3.1. Histological appearance of tissue-engineered substitutes cultured in vitro Before grafting, all TESs appeared histologically as homogenous tissues with an epidermis covering the dermal component (Fig 2A-F). The four typical layers of the epidermis (stratum basale, spinosum, granulosum and corneum) were observed in all TESs. Of note, human epithelial cells produced a thicker epidermis (Fig 2C) compared with mouse epithelial cells (Fig 2A, B, D-F), reflecting the differences in native tissues between these two species. The dermal component of all TESs (Fig 2A-F) and TEDs (Fig 2G-I) appeared as a homogeneous ECM-rich connective tissue containing fibroblasts.

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3.2. Engraftment of engineered tissues in C3H/HeN mice In order to test the hypothesis that allogeneic stromal cells allowed for long-term engraftment of tissue-engineered substitutes, C3H/HeN mice were chosen as graft recipient and different TESs and TEDs were produced with fibroblasts and epithelial cells from C3H/HeN and BALB\c, which are inbred strains of mice. Human fibroblasts and keratinocytes were also used to produce TESs and TEDs to evaluate rejection following exposition to xeno-MHC peptides. The primary immune response leading to skin graft rejection occurs between 9 to 18 days [37, 38]. In the first design course of the experiment, different post-graft time-points were explored: 15 and 19 days to evaluate the acute rejection, and 35 to 56 days to evaluate the long-term graft survival (see table S1 and table S2). Each TES was transplanted onto the dorsal muscular fascia of a C3H/HeN adult mouse. Grafts were surrounded by a Fusenig’s silicon chamber to prevent wound healing from the recipient skin (Fig 1A). As, histologically, there was no difference between 15 and 19 days, and 35 and 56 days, the experiment was repeated at 19 and 56 days to increase the number of replicates. Fifteen (data not shown) and 19 days after grafting, histologic analysis revealed a complete absence of epidermis in all allogeneic-FdKd (Fig 3B), xenogeneic-FHKH (Fig 3C) and mouse chimeric-FkKd (Fig 3D) TESs. This observation was associated with immune cell infiltrate within the connective tissue, which indicated rejection. Interestingly, all chimeric TESs produced with syngeneic epithelial cells and allogeneic (Fig 3E, mouse chimeric-FdKk) or xenogeneic fibroblasts (Fig 3F, human chimeric-FHKk) diplayed a histologically conform epidermis and were similar to TESs produced with syngeneic epithelial cells and fibroblasts (Fig 3A, syngeneicFkKk). Microvessels and few immune cells were observed in syngeneic-Fk, allogeneic-Fd and xenogeneic-FH TEDs grafted subcutaneously (Fig 3G-I). No sign of TED rejection was noted 15

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(data not shown) and 19 days (Fig 3G-I) after grafting. Collectively, these results suggest that, unlike epithelial cells, non-autologous fibroblasts included in tissue-engineered substitutes – either dermis or skin – do not lead to acute tissue rejection. The Fusenig’s chamber was removed 21 days post-grafting. Despite the loss of the epithelium in TESs which originally contained non-autologous epithelial cells, the engineereddermis of the TESs was still identifiable at the graft site (data not shown). After 35 and 56 days, host epithelial cells re-epithelialized the TESs that initially contained allogeneic (allogeneic-FdKd and mouse chimeric-FkKd TESs) or xenogeneic (xenogeneic FHKH and human chimeric FkKH TESs) epithelial cells and a decrease of immune cell infiltrate was observed in these tissues (data not shown and Fig 4B, C). TESs that were not rejected had a normal skin appearance, with the presence of blood vessels distributed throughout the engineered dermis, without gross evidence of immune cell infiltrate (Fig 4A, D, E). The persistence of allogeneic and xenogeneic fibroblasts within the dermal portion of allogeneic-FdKd, mouse chimeric-FdKk or xenogeneic FHKH TESs was evaluated by labelling the haplotype respective to each species. Allogeneic and xenogeneic fibroblasts were present in engineered tissues 19 and 56 days after grafting (Fig 5 and Fig 6) indicating that they are not rejected. Syngeneic-Fk and allogeneic-Fd TEDs grafted subcutaneously seemed stable after 35 (data not shown) and 56 days (Fig 4F, G) without signs of immune cell infiltrate or ECM tissue degradation. 3.3. Evaluation of immune cell markers in engineered tissues The innate immune response was evaluated by direct immunofluorescence labelling of CD206, also known as MRC1 (c-type mannose receptor 1), a receptor present on the surface of M2 macrophages, while the adaptive immune response was assessed by immunolabelling of CD4-, and CD8- positive T cells.

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In normal C3H/HeN skin, the basal number of M2 macrophages ranged from 67 to 106 cells/mm2 (median: 77 cells/mm2; n=3) (Fig 7). No CD4-expressing cell was observed (n=3) while the number of CD8-expressing cells ranged from 0 to 23 cells/mm2 (median: 19; n=3) (Fig 7). During the rejection process of xenogeneic-FHKH TESs (eight days post-grafting), the number of CD4-expressing cells was going up to 686 cells/mm2 (minimum: 441 cells/mm2; median: 441 cells/mm2, n=3), while CD8-expressing cells were rarely observed (minimum: 0, maximum: 21 cells/mm2; median: 0 cells/mm2, n=3). Nineteen days after grafting, the number of M2 macrophages ranged from 104 to 813 cells/mm2 (median: 287 cells/mm2; n=7) within syngeneic-FkKk, from 156 to 568 cells/mm2 (median: 257 cells/mm2; n=7) within allogeneic-FdKd and from 64 to 588 cells/mm2 (median: 177 cells/mm2; n=7) within mouse chimeric-FdKk TESs (Fig 8). A few CD4-expressing cells were found within tissues. CD4 ranged from 15 to 122 cells/mm2 (median: 51 cells/mm2; n=7) within syngeneic-FkKk, from 0 to 173 cells/mm2 (median: 44 cells/mm2; n=7) within allogeneic-FdKd, and from 15 cells/mm2 to 159 cells/mm2 (median: 21 cells/mm2; n=7) within mouse chimericFdKk, but no statistical difference was detected (Fig 8). The number of CD8-expressing cells was relatively low but tended to be higher in allogeneic-FdKd compared with syngeneic-FkKk TESs (Fig 6, p ≤0.05). They ranged from 0 to 69 cells/mm2 in syngeneic-FkKk (median: 6 cells/mm2; n=7), from 14 to 257 cells/mm2 in allogeneic-FdKd (median: 27 cells/mm2; n=7) and from 0 to 108 cells/mm2 in mouse chimeric-FdKk (median: 14 cells/mm2; n=7) TESs (Fig 8). Within TEDs grafted subcutaneously for 19 days, mostly M2 macrophages were observed within syngeneic-Fk (range: 0 to 329 cells/mm2; median: 159 cells/mm2; n=7), allogeneic-Fd (range: 24 to 282 cells/mm2; median: 181 cells/mm2; n=7) and xenogeneic-FH (range: 30 to 759 cells/mm2; median: 75 cells/mm2; n=7) TEDs. CD4- and CD8-positive cells (Fig 8) were

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seldomly observed. Indeed, the number of CD4-T cells ranged from 0 to 32 cells/mm2 in syngeneic-Fk TEDs (median: 0 cells/mm2; n=7), from 0 to 33 cells/mm2 in allogeneic-Fd TEDs (median: 0 cells/mm2; n=7) and from 0 to 69 cells/mm2 in xenogeneic-FH TEDs (median: 2 cells/mm2; n=7) (Fig 8). The number of CD8-expressing cells ranged from 0 to 20 cells/mm2 in syngeneic-Fk TEDs (median: 0 cells/mm2; n=7) and from 0 to 60 cells/mm2 in allogeneic-Fd TEDs (median: 0 cells/mm2; n=7). No CD8-expressing cells were found in xenogeneic-FH TEDs (n=7). After 56 days, the number of M2 macrophages was higher in allogeneic-FdKd TESs (range: 457 to 546 cells/mm2, median: 497; n=3) when compared with syngeneic-FkKk TESs (range: 245 to 377 cells/mm2, median: 350; n=3) and mouse chimeric-FdKk TESs (range: 263 to 379 cells/mm2, median: 269; n=3) TESs (Fig 9). A few CD4-positive cells were found in syngeneic-FkKk TESs (range: 7 to 20 cells/mm2, median: 14; n=3), allogeneic-FdKd TESs (range: 0 to 5 cells/mm2, median: 5; n=3) and mouse chimeric-FdKk TESs (range: 6 to 8 cells/mm2, median: 7; n=3). No CD8-expressing cells were observed in syngeneic-FkKk, allogeneic-FdKd TESs and chimeric-FdKk TESs (Fig 9). In tissue grafted without epidermis, by day 56, the number of M2 macrophages ranged from 9 to 1009 cells/mm2 in syngeneic-Fk TEDs (median: 313 cells/mm2; n=3), from 119 to 629 cells/mm2 in allogeneic-Fd TEDs (median: 460 cells/mm2, n=3) and from 116 to 244 cells/mm2 in xenogeneic-FH TEDs (median 184 cells/mm2, n=3) but no statistical difference was detected. CD4-positive cells were observed only in xenogeneic-FH TEDs (range: 4 to 226 cells/mm2, median: 71; n=6), while no CD8-expressing cells were observed in syngeneic-Fk, allogeneic-Fd and xenogeneic-FH TEDs (Fig 9).

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Collectively, these observations suggest that from the 19th day after grafting, the adaptative immune response mediated by CD4- and CD8-expressing cells was turned off in engineered tissues composed of allogeneic fibroblasts alone or in combination with autologous epithelial cells. 3.4. Engraftment of engineered tissues in pre-immunized C3H/HeN mice To evaluate the secondary immune response raised from a second contact with antigens corresponding to the genetic background of the allogeneic fibroblasts present in the TES, BALB\c epithelial cells were injected subcutaneously in C3H/HeN mice three weeks before grafting the syngeneic-FkKk, allogeneic-FdKd and mouse chimeric-FdKk TESs. By day five, no macroscopic sign of tissue graft rejection was observed (data not shown). On day 19, histological analysis revealed that the epithelium of allogeneic-FdKd TESs was completely rejected (Fig 10B), while both mouse chimeric-FdKk (Fig 10C) and syngeneic-FkKk TESs (Fig 10A) presented a fully differentiated epithelium. 3.5. Evaluation of immune cell markers in engineered tissues grafted in pre-immunized C3H/HeN mice At day 19, M2 macrophages were observed in syngeneic-FkKk (range: 300-684 cells/mm2; median 314 cells/mm2; n=3), allogeneic-FdKd (range: 204-594 cells/mm2; median 273 cells/mm2; n=3) and mouse chimeric-FdKk (range: 287-973 cells/mm2; median: 621 cells/mm2; n=3) TESs, but no statistical difference was detected. A few CD4-positive cells were counted in TESs. Their number ranged from 4 to 16 cells/mm2 in syngeneic FkKk TESs (median: 5 cells/mm2), from 27 to 41 cells/mm2 in allogeneic-FdKd TESs (median: 36 cells/mm2; n=3) and from 8 to 31 cells/mm2 in chimeric-FdKk (median: 12 cells/mm2; n=3). CD8-expressing cells were only

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detected in allogeneic-FdKd TES and ranged from 18 to 166 cells/mm2 (median: 157 cells/mm2; n=3) (Fig 10D-F). 4. Discussion The present study demonstrates that TESs composed of allogeneic fibroblasts and syngeneic epithelial cells allow the permanent coverage of full-thickness skin wounds. No rejection occurred in immunocompetent mice in the absence of immunosuppressive drugs. We also observed that a stromal tissue produced from allogeneic cells did not induce graft rejection. These findings open the possibility of using allogeneic fibroblasts within their ECM for the production of living substitutes with multiple applications in regenerative medicine. Notably, ready to use tissue-engineered stromal substitutes, which could be implanted or serve as stromal support for patient’s epithelial cells. In our study, we observed that the mouse chimeric-FdKk TESs composed of allogeneic fibroblasts and syngeneic epidermis induced a low inflammatory response of the same magnitude as the fully syngeneic-FkKk TESs after grafting. As expected, and also reported in a porcine animal model [39], the epithelium of the fully allogeneic-FdKd TESs was rejected. Besides, similar to the syngeneic-FkKk TES, the histologic features of mouse chimeric-FdKk TES was characterized by the persistence of a dense ECM and an epidermis that self-renewed and differentiated normally over the 8-week follow-up period. Major histocompatibility complex (MHC) antigens are known as initiators of allograft rejection through the allorecognition process [40]. The direct pathway of allorecognition is the ability of T cells to “directly” recognize nonself MHC molecules present on the surface of donor dendritic cells [40]. In our experiments, only cultured keratinocytes and fibroblasts colonized TESs, dendritic cells were absent [37]. Therefore, the allorecognition is expected to be limited to the indirect pathway which is the

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ability of T cells to “indirectly” recognize donor MHC molecules that are processed and presented as peptides by self-MHC [40]. In this situation, after being exposed by antigenpresenting cells, donor MHC class II and class I peptides activate CD4+ and CD8+ T cells, respectively [40-42]. Without prior stimulation, cultured keratinocytes express MHC class I and a low level of MHC class II at their cell surface [43-47] while cultured fibroblasts express MHC class I at low level but do not express MHC class II antigens [48]. In both cell types, MHC class II expression is up-regulated in response to the pro-inflammatory gamma-interferon [48]. We observed that the epidermis of allogeneic-FdKd TESs was completely rejected 19 days after grafting as expected. However, allogeneic fibroblasts survived within the dermal component of allogeneic-FdKd and mouse chimeric-FdKk TESs after 56 days. The absence of immune cell infiltrate and the negligible number of CD8- and CD4-expressing cells in mouse chimeric-FdKk TESs or allogeneic-Fd TEDs 19 days after grafting suggested that the adaptative immune response had occured and that fibroblasts were spared. ECM has been described as an important negative modulator of the fibroblast gamma-interferon response in vitro using scaffold-based threedimensional cultures [48]. Therefore, the self-assembled ECM surrounding allogeneic fibroblasts in our tissue-engineered substitutes could contribute to their low immunogenicity by limiting MHC class II induction in vivo. To explore this hypothesis, it would be very interesting to compare the inflammatory/immune response against allogeneic fibroblasts when self-assembled ECM are utilized compared with a traditional pre-made collagen scaffold. Allogeneic fibroblasts have been shown to induce T-cell proliferation if T-cells are previously primed with alloantigens [49]. In our study, mouse chimeric-FdKk TESs grafted on mice that have been previously exposed to MHC antigens specific to the genetic background of

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allogeneic fibroblasts composing the dermal portion of the TES were not rejected. This result indicates that several consecutive batches of TESs composed of allogeneic fibroblasts and autologous epithelial cells can be transplanted without inducing a secondary immune response leading to the loss of the graft. Engrafted stromal tissues, produced from allogeneic cells and implanted subcutaneously, remained after 56 days with negligible immune cell infiltrate. However, the ECM of xenogeneicFH TEDs appeared reduced in volume and a few CD4-positive cells were counted within these tissues. The ECM composing TEDs produced by the self-assembly approach is a completely natural collagen-rich material that can be fabricated from fibroblasts or other stromal cell types [28, 29, 50-54]. Even if collagen is considered to be a weak antigen, inherent immunity is possible between species and could explain the observed ECM resorption [55]. Nevertheless, in a clinical set-up for patient treatment, there is no advantage in using non-human cells to produce stromal reconstructed tissues since stromal or mesenchymal stem cells are easily accessible from human skin, have excellent capability for expansion in vitro and are associated to few ethical issues. The main limitation of our model of autologous TES is the production time. With the method presented here that we adapted for mouse cells, 56 days were required to produce fullymature mouse TESs. We recently published a protocol which allows to produce a human TES suitable for the permanent coverage of full-thickness wound in 31 days after the expansion of cells [56]. When cultivating autologous human TESs with our method to cover large wounds, the initial isolation and expansion of cells from a small biopsy ranging from 2 to 10 cm2 delays the availability of the first grafts [15]. While the expansion of keratinocytes takes 15 days [57], whereas it takes 17 days of culture on average to obtain enough fibroblasts to start the production

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of TED [15]. Thus, the first autologous human TESs are available after 48-62 days. A preestablished biobank of allogeneic fibroblasts allows seeding at higher density resulting in the production of TEDs in less time [58]. Using allogeneic fibroblasts, allogeneic TEDs could be produced on a regular basis and since they are stable for weeks in an incubator when culture medium is regularly refreshed, they would be ready to use when the patient's keratinocytes are amplified. Alternatively, decellularized human TEDs could also allow for the production of a skin substitutes in less time [59, 60]. When decellularized, the ECM of fibroblast-derived tissue sheets is minimally disrupted [52] and if dried, it conserves its biological architecture after rehydratation [54]. Thus, dried TEDs or TEDs preserved in a physiological saline could also serve as “off-the-shelf” scaffolds to receive allogeneic fibroblasts upon request. Therefore, TESs would be available after a period of four to five weeks; two to three weeks for keratinocytes expansion plus two weeks for keratinocyte proliferation and differentiation on TEDs. 5. Conclusion The results presented here demonstrate that allogeneic fibroblasts included in tissueengineered substitutes do not elicit an immune rejection after grafting. In the field of tissue engineering, this opens the possibility of selecting a human fibroblastic or stromal cell population based on its biological properties (doubling time, size, ECM assembly) and adequate biosafety (pathogen-free, non-tumorigeneic, genetically stable), in order to standardize the manufacturing process of living tissue-engineered substitutes. Moreover, ready-to-use stromal tissue produced from biobanks will shorten the production time of skin substitutes and provide faster treatment for the patients. Conflicts of interest The authors declare no conflict of interest.

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Author contributions BG, DL, LG contributed to conception, design and development. BG carried out experiments. BG, NV, VP, DL, DHK, VB have collected data or have contributed to data analysis. BG wrote the original draft. DL, DHK, FAA, LG finalized the writing of the paper. LG supervised the experiments. All authors have approved the final version of the article. Acknowledgments The authors gratefully thank Drs. Alphonse Roy, Félix-André Têtu, and Maurice Bouchard for providing skin biopsies, as well as Israël Martel, Amélie Lavoie, Francis Bisson, Carolyne Simard-Bisson, Rina Guignard and Anne-Marie Moisan for their technical assistance. This research was funded by the Fondation des Pompiers du Québec pour les Grands Brûlés (FPQGB), the Réseau de thérapie cellulaire, tissulaire et génique du Québec (ThéCell) funded by the Fonds de la recherche du Québec - Santé (FRQS), Canada Research Chair (Tier 1) from Canadian Institutes of Health Research (CIHR) on Stem Cells and Tissue Engineering (LG), Research Chair on Tissue-Engineered Organs and Translational Medicine of the Fondation de l'Université Laval (LG), CIHR grants MOP-12087 (LG), FDN-143213 (LG) and MOP-115093 (FAA). BG has received fellowships from the Réseau de Recherche en santé de la vision. NV has received fellowships from the Institute of Musculosqueletal Health and Arthritis (IMHA) of the CIHR. References [1] [2]

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