Project managers:

• Lucie Guilbaud, MD, PhD

Associated Clinical team: Fetal Medicine Department, Trousseau Hospital, APHP.Sorbonne Université, Pr Jean-Marie Jouannic

The purpose of our research is to develop stem cell therapy as an adjuvant treatment in fetal myelomeningocele surgery to improve spinal cord repair and children’s prognosis.

Myelomeningocele (MMC) represents one of the most common congenital defects of the central nervous system prenatally diagnosed. This malformation leads to lifelong disabilities including lower extremity paralysis, sphincters deficiency, sexual dysfunction, and cognitive disabilities. In 2011, a randomized clinical trial, the MOM (Management of Myelomeningocele) Study demonstrated that in utero MMC surgical repair improves motor function compared to neonatal repair (Adzick, NEJM, 2011). Since then, several centers around the world have been offering this fetal surgery, with similar results than the MOM study (Zamlynski, J Matern Fetal Neonatal Med, 2014 – Moron, BJOG, 2018 – Möhrlen, Fetal Diagn Ther, 2020 – Guilbaud, article in press, 2021). The follow-up of the MOM study’s children at school-age demonstrates that motor improvement achieved after fetal surgery still exists over the years (Houtrow, Pediatrics, 2020). However, this benefit remains limited since 71% of children are not able to walk independently at an average age of 7.8 years. In addition, fetal surgery does not seem to provide an improvement in sphincter functions (Macedo, BJU Int, 2019 – Clayton, J Pediatr Urol, 2020). In this context, it thus appeared essential to develop new adjuvant therapies to fetal surgery in order to improve fetal spinal cord repair and children’s prognosis.

The purpose of our research is to develop cell therapy as an adjuvant treatment in MMC fetal surgery in the ovine model of MMC. Our first results demonstrated that fetal repair of MMC using allogenic ovine umbilical cord-derived mesenchymal stromal cells (UC-MSCs) patch provides a moderate improvement in motor functions and sphincter functions at birth, as well as gray matter and neuronal preservation. The use of UC-MSCs also seems to prevent from fibrosis development at the MMC scar level (Guilbaud, article in progress, 2021).

This research is the result of a collaboration between Team 7 of Unit 976 and the Fetal Medicine Department of Trousseau Hospital (APHP.Sorbonne Université, Paris), which also is a component center of the National Center for Rare Diseases MAVEM (Vertebral and spinal cord malformations). The Fetal Medicine Department of Trousseau Hospital is a pioneer in MMC fetal surgery in France. It started a fetal MMC surgery program in 2013 (PRIUM 1) in which fetuses are operated on by open surgery. After several years of experimental research in the ovine model of MMC (Guilbaud, Childs Nerv Syst, 2014 – Guilbaud, Childs Nerv Syst, 2017 – Guilbaud, Fetal Diagn Ther, 2019), they started a fetoscopic MMC surgery program in February 2021 (PRIUM2).

Project managers:

• Alexandra Fuchs,
• François Chatelain,
• Lousineh Arakelian,
• Elsa Mazari-Arrighi,
• Dmitry Ayollo,
• Wissam Farhat

Our team aims at developing engineering approaches – such as Cell Sheet Technologies, Micropatterning and Extrusion and Stereolithographic Bioprinting – to build tissues from cells and matrices. Two clinical applications are pursued: hepatic tissue, including biliary and vascular networks (iLiTE-RHU project), and an esophageal bioprinted substitute (Bio3DHE project)

1. Project iLiTE, RHU-PIA : Vascular, biliary and hepatic tissues https://ifbf-institute.org/projets/ilite-innovation-in-liver-tissue-engineering-2016-2021
   The RHU iLiTE project (innovations in Liver Tissue Engineering) brings together teams of the Paul-Brousse and Saint-Louis hospitals, University of Paris Saclay, CEA, INSERM, INRIA, ENS and three Life Science companies, and encompasses various approaches and applications of liver tissue engineering, both for clinical and in vitro purposes : external liver bioreactor, liver on chips and for transplantation.
   In this project, bioconstruction of the liver is considered as an assembly of building blocks: biliary network, vascular network and elementary units of hepatic tissue.Saint_Louis’ Stem Cell biotechnologies team is involved in the following approaches:
   • Bioprinting of vascular and biliary networks: We are exploring engineering approaches to build planar vascular and biliary networks by micropatterning and stereolithographic bioprinting. The latter uses light activated polymerization of hydrogels to build hydrogel structures encapsulating either cholangiocytes (for the biliary network) or endothelial cells (for the vascular network). Various network configurations are being tested as well as various types of hydrogel supports, either natural or artificial, with varying degrees of elasticity and topology, to induce these cells to self-organize into tubes.
   • Hepatic cell sheets: Our aim is to construct bioengineered hepatic tissue using cell sheet technologies. This method consists in culturing confluent hepatocyte layers on the thermoresponsive polymer poly-NIPAM. To detach cell sheets, temperature is reduced to below 32°C which makes the polymer more hydrophilic, inducing spontaneous cell detachment in sheets. Unlike enzymatic treatments, this technology allows preservation of intercellular junctions and polarity which are vital for hepatocytes’ viability and functionality.
   • Integration : Our ultimate goal is to combine these approaches by stacking the obtained planar constructs to control the association of the different types of cells and the final architecture, with the purpose to get as close as possible to physiological architecture of a liver, or more exactly of the hepatic lobule
2. Project Bio3DHE: Development of 3D bio-printing by extrusionIn this project funded by MSD-Avenir we are investigating the use of extrusion bio-printing to engineer 1) an esophageal substitute and 2) a vascularized patch:
   • 3D-extrusion printing allows the construction of complex shapes and structures using both natural and synthetic biocompatible bio-inks. In the case of esophagus, they can be exquisitely tailored to the patient’s pathology and morphology, while mimicking the tissue’s native characteristics and cellular microenvironments. This approach is conducted in parallel to the decellularization approach described below.
   • Similarly, 3D printing can control the position of cells in a fibrin patch according to the blueprint of a vascular network. During in vitro maturation, it is anticipated that endothelial cells will form cords and tubes in the pre-set pattern. The obtained vascularized fibrin patch can be used to promote long term survival of cells in bio-engineered constructs during maturation in vitro or after in vivo implantation.

Project managers:

• Briac Thierry,
• Lionel Faivre,
• Lousineh Arakelian,
• Pierre Cattan,
• Thomas Domet.

Tissue engineering is a credible alternative to conventional esophageal or tracheal reconstruction techniques. For that purpose, the use of specific extracellular matrix issued from the decellularization of native tissues is currently the preferred option because of their biocompatibity, their performance in promoting a tissue remodelling process and the existence of reliable decellularization process. Our team develops, qualifies and tests extracellular matrix in animal models for oesophageal and tracheal replacement, in view of a clinical trial.

This project consists of two branches dealing with two different organs, the esophagus and the trachea, which use the same principle of substitute manufacturing the decellularization of a native organ. Due to its physical, biochemical and biomechanical properties, the extracellular matrix plays a dynamic role in tissue regeneration (Badylack Anat Rec B New Anat 2005, Agmon Curr Opin Solid State Mater Sci 2016). There are specificities of organs that make an extracellular matrix from a given organ allow a specific arrangement of the stem cells that will colonize it. It therefore seems preferable to preserve this organ specificity in the choice of the tissue from which the matrix comes (Gattazzo Biochim Biophys Acta BBA 2014).

1. Œsophagus
   After esophagectomy for benign lesions (peptic stenosis, long atresia, caustic burns, etc.) or malignant (adenocarcinoma, squamous cell carcinoma), esophageal replacement usually requires gastric or colonic transplants. The same is true for the treatment of stenosis refractory to endoscopic dilations or esophageal atresia after failure of the lengthening techniques. In these latter situations, esophageal segmental pathology currently requires replacement of the entire esophagus. These reconstructions are associated with significant morbidity and mortality and their functional results are often altered by reflux, delay in emptying the oesophageal substitute or even dumping syndrome (Poghosyan J Visc Surg 2011). In addition, stenosis or transplant distention frequently appears in the long term, requiring re-intervention (Chirica Ann Surg 2010). Finally, these reconstructions, when they fail, lead to a therapeutic impasse with exclusive enteral nutrition by feeding jejunostomy.There is therefore a definite interest in the development of other esophageal substitutes which, while preserving the intra-abdominal organs, would allow esophageal replacements adapted to the pathology to be treated. The ideal would be to dispose of a tailor-made substitute. In animal models, the interposition of synthetic materials has given disappointing results in this indication, linked to anastomotic fistulas, chronic infection and extrusion of the material (Schuring Ann Otol Rhinol Laryngol 1966). Esophageal allograft requires immunosuppression and is difficult in practice due to the multiple origins of vascularization of this organ (Macchiarini J Thorac Cardiovasc Surg 1995). Tissue allograft, including an aortic graft, had mixed results (Gaujoux Surgery 2010). Currently, only esophageal replacement with biomaterials from tissue engineering seems to be a credible alternative (Poghosyan T et al. J Pediatr Gastroenterol Nutr 2011 and Poghosyan T. et al. J Visc Surg 2015). Multiple biomaterials have been tested in this context, including biological matrices, alone or in combination with synthetic materials (Takimoto, J Thorac Cardiovasc Surg 1998, Saito Surg Today 2000, Catry Cell Transplant 2017 and Kim J Visc Exp 2020). Decellularized tissues have particular interest in this indication.Our team developed a porcine esophageal decellularization protocol which allowed us to obtain a biocompatible, in vitro non-immunogenic, sterile, non-cytotoxic substitute, while structure and biomechanical property are preserved (Arakelian J Tissue Eng Regen Med 2019). In a porcine model, we then successfully used this matrix to perform the circumferential replacement of 5 cm of the thoracic esophagus, under the cover of an esophageal endoprosthesis. Ninety percent of the animals had nutritional autonomy after removal of the stent at the 3rd month. Analysis of the graft area revealed, from 3 months, the initiation of tissue remodeling towards an esophageal phenotype with the appearance of an epithelium, muscle cell islets and neurofilaments. Seeding the matrix with autologous mesenchymal stem cells or omental maturing of the substitute before esophageal replacement did not bring any benefit, either in clinical terms or in terms of tissue remodeling (Levenson article in progress 2020).Our project is ongoing by applying our decellularization protocol to human esophagus. The qualification of the human matrix obtained is currently in progress. According to our first results, we obtained a clinical grade (?) decellularized human esophagus which has retained its histological structure, which is non-cytotoxic, with no nuclei or residual cellular DNA. After finalization of matrix qualification, the transfer of technology for matrix production will pave the way to the first a clinical trial.
2. Trachea
   Tracheal replacement is one of the greatest challenges in airway surgery. In complex tracheal pathologies, after failure of classical surgical strategy for benign or malignant pathologies, or for recurrent stenosis, it could be an efficient alternative strategy.Few overriding tracheal replacement goals have been described over time (Belsey, 1950): lateral rigidity, longitudinal elasticity and flexibility, adequate air-tight lumen, uninterrupted lining of ciliated columnar epithelium, absence of collapse during breathing stages. Replacing the trachea while respecting these fundamental principles is a challenge, especially since the cellular tissues of the trachea are numerous and have very different biomechanical characteristics.In clinical practice, five replacement techniques have been proposed: synthetic prostheses (Pepper et al., 2019), tracheal allotransplantation (Delaere & Van Raemdonck, 2016), bioprotheses using aortic allografts (Martinod et al., 2017), reconstruction using autologous composite tissues (Kolb et al., 2018) and tissue bioengineering. High morbi-mortality rates have been reported with synthetic prostheses and allotransplantation. Aortic allograft is being used with success but requires long term stenting, which is not compatible with tracheal replacement in children, due to the narrow trachea’s diameter. Surgery reconstruction with autologous tissue requires an excellent surgical level and iterative postoperative endoscopies. Moreover, this is not possible before the age of 12 years, i.e. a tracheal diameter of around 10 mm.Therefore, tissue bioengineering remains the best technique, especially in pediatric tracheal pathology, which our domain of interest. Obtaining a biocompatible decellularized tracheal matrix would be our first step in this research.The main objective and the expected result of this work is the validation of a protocol for fast tracheal decellularization which maintains the biomechanical properties. The pig is the animal model of the pediatric trachea and will be retained for our study.Different tracheal decellularization protocols will be developed and analyzed by varying the decellularizing agents as well as their associations.The following data will be collected and analyzed for each protocol: duration, efficacy (validation criteria), histological study, structural analysis by electron microscopy, biomechanical resistance tests, vitality tests (i.e. possibility of colonization of the decellularized tissue by mesenchymal stem cells) and absence of toxicity.

Project managers:

• Eric Gabison,
• Benoit Souquet,
• Benoit Chapellier,
• Damien Guindolet

his translationnal research program targets epithelial and stromal compartments of the corneal stem cell niche in order to modulate corneal wound healing or restore homeostasis in acquired and congenital stem cells deficiencies.

PAX6, a transcription factor involved in eye development is a key player of corneal epithelial and stromal stem cells. The limbus, located at the corneal periphery, is the proposed niche of both corneal epithelial and stromal stem cells.
Modulation of PAX6 (or other transcription factors) in the stem cell niche in corneal epithelial stem cell or through mesenchymal stem cell may represent an alternative treatment for Limbal stem cell deficiencies.

Project managers:

• Audrey Cras,
• Miryam Mebarki

Development of umbilical cord-derived mesenchymal stromal cells-based therapy for immune-mediated inflammatory diseases such as graft versus host disease, autoimmune disease, traumatic brain injury or severe acute respiratory syndrome. The aim is to translate this technology to a clinical use, as it has already been done for the treatment of Covid-19 related severe acute respiratory syndrome.

Nowadays, there is no treatment allowing controlling severe and refractory immune-mediated inflammatory diseases. Mesenchymal stromal cells (MSC) represent a therapeutic perspective due to their capacity to home to inflammatory sites and to their anti-inflammatory, anti-fibrotic and immunomodulatory properties. MSC derived from the Wharton’s jelly of the Umbilical Cord (UC-MSC) feature several attractive characteristics: ease of procurement, high proliferation and low immunogenicity. Our project consists of three axes. The first aim is to develop a manufacturing process of UC-MSC that can be scaled-up to generate MSC-based therapies for clinical applications. The second axis is to characterize produced UC-MSC at basal state and in pro-inflammatory conditions, depending of the studied pathology. The characterization will include studies of:

• Security: assessment of long-term proliferation capacity, senescence, genetic instability, tumorigenicity…
• Identity and purity: expression of UC-MSC markers, trophic and immunomodulatory markers, absence of hematopoietic and endothelial markers.
• Functionality: evaluation of immunomodulatory, anti-inflammatory, angiogenic and neurotrophic properties of UC-MSC.

The last axis of our project aims to study the UC-MSC mechanism of action in the immune-mediated inflammatory diseases and to define biological parameters associated with the therapeutic efficacy of MSCs and clinical response.