Regenerative treatment of focal hyaline cartilage defects could prevent or delay the development of secondary osteoarthritis. Current surgical techniques result partly in i) the formation of mechanically inferior fibrous cartilage or ii) present the disadvantage of the donor site morbidity from harvesting cartilage biopsy as well as iii) the dedifferentiation of chondrocytes due to in vitro expansion and iv) the reduced re-differentiation potential of in vitro expanded chondrocytes. The self-healing capacities of injured and degenerated articular cartilage revealed a promising target cell population for a regenerative, autologous treatment of these defects using mesenchymal stromal cells (MSCs). Several case studies, randomized and controlled clinical studies showed the general ability of autologous, bone marrow-derived, expanded MSC transplantation to regenerate articular cartilage lesions [1]. However, these two-stage approaches are based on time- and cost-consuming expansion of MSCs under good manufacturing practice (GMP) conditions and hold a risk of contamination during this process. In 2010, CD271, the low-affinity nerve growth factor receptor, was described as a suitable surface marker to enrich MSCs from human bone marrow aspirate intraoperatively [2]. The aim of the present dissertation was to investigate the feasibility of generating cartilage grafts from either ovine (study no. 1) and human (study no. 2) non-expanded CD271+ bone marrow cells in a collagen type I hydrogel. Study no. 1 (“Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells - An in vitro proof-of-concept study”) investigated several surface marker candidates for the prospective MSC separation and examined their potential of resulting colony-forming units, respective their yield of potent MSCs [3]. This study was conducted with ovine bone marrow samples. CD271 was the most effective surface marker to isolate the target cell population. Subsequently, CD271+, CD271- and unseparated mononuclear cells (MNCs), containing the MSCs, were used to generate cartilage grafts without an expansion of these cells in monolayer culture. It could be proven, that ovine CD271+ cells were able to generate a potent hyaline cartilage graft. Study no. 2 (“Single-stage preparation of human cartilage grafts generated from bone marrow-derived CD271+ mononuclear cells”) was performed as the final translational step from animal-derived bone marrow to human donor material and is therefore strengthening the therapeutically focus of the entire work [4]. Briefly, eight bone marrow aspirates were used for MNC isolation and subsequent magnetic cell separation (MACS). The resulting CD271+ and CD271- MNCs were compared to unseparated MNCs. Subsequently, they were seeded in a clinically approved collagen type I hydrogel and cultivated for up to 5 weeks to investigate the progression of the chondrogenic differentiation processes. Graft analysis included cell viability visualization by live/dead staining, determination of the DNA and the secreted sulphated glycosaminoglycan (sGAG) content as well as the immunohistochemical staining for typical chondrogenic differentiation markers and the extracellular matrix molecules aggrecan and collagen type II. A proliferation of cells in the generated grafts was shown of CD271+ and unsep, but not CD271- MNCs. Hence, the cell number was 2.8-fold higher after 35 days compared to the first day for CD271+ MNCs grafts, while CD271- MNCs did not proliferate in the grafts and unsep MNCs showed only a slight increase in cell number. The chondrogenic potential was measured by quantification of freshly produced sGAGs and the expression of chondrogenic markers. In grafts with CD271+ MNCs, sGAG production increased over time and reached its maximum at day 35, whereas grafts with CD271- MNCs showed no measurable sGAG deposition. The amount of sGAG in unsep MNC grafts increased only slightly over the whole cultivation period. Aggrecan and collagen type II staining varied considerably between the MNCs donors. Collagen type II positive staining was observed in CD271+ MNC grafts (5/8 donors) and unsep MNC (2/8) grafts. In comparison to macroscopically healthy cartilage, three-dimensional grafts of the CD271+ group yielded a proceeding extracellular matrix production. In summary, CD271+ MNCs showed the highest proliferation rate, cell viability, sGAG deposition and cartilage marker expression compared to the CD271- or unseparated MNC fractions in in vitro generated three-dimensional cartilage grafts. Therefore, the presented work demonstrated the feasibility of generating a cartilage graft from CD271+ bone marrow-derived MNCs in a clinically approved collagen type I hydrogel without a previous monolayer expansion of these cells. This will enable the intraoperative purification of CD271+ MNCs, which contain the majority of colony-forming MSCs, by MACS technology. The clinical application will be possible with currently available and clinical approved cell separation devices. Providing a cartilage graft with non-expanded CD271+ MNCs by a fast and simple intraoperative therapeutic approach fulfils the need for a “single-step, tissue-engineered solution to focal cartilage defects, and elimination of the morbidity of the donor defect” as requested by the editors of the journal Arthroscopy [5]. References of the summary 1. Filardo G, et al. (2016). Stem cells in articular cartilage regeneration. J Orthop Surg Res 11:42. 2. Jones E, et al. (2010). Large-scale extraction and characterization of CD271+ multipotential stromal cells from trabecular bone in health and osteoarthritis: implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells. Arthritis Rheum. 62:1944–1954. 3. Petters O, et al. (2018). Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells-An in vitro proof-of-concept study. J Tissue Eng Regen Med. 4. Petters O, et al. (2018). Single-Stage Preparation of Human Cartilage Grafts Generated from Bone Marrow-Derived CD271+ Mononuclear Cells. Stem Cells Dev 27:545–555. 5. Lubowitz JH and GG Poehling. (2009). Saving our cells: Advances in tissue engineering for focal cartilage defects. Arthroscopy: the journal of arthroscopic & related surgery: official publication of the Arthroscopy Association of North America and the International Arthroscopy Association 25:115–116.:1 Introduction 1 1.1 Articular cartilage 1 1.2 Cartilage lesions 2 1.3 Self-healing capability of articular cartilage 3 1.4 Treatment option for cartilage lesions 4 1.5 Mesenchymal stromal cells in cartilage regeneration 6 2 Rationale 8 3 Publication manuscripts 9 Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells - An in vitro proof-of-concept study 9 Single-stage preparation of human cartilage grafts generated from bone marrow-derived CD271+ mononuclear cells 21 4 Summary 33 5 References 35 6 Appendix 42 7 Declaration of Authorship 47 8 Curriculum vitae 48 9 Publications 49 10 Acknowledgements 49

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Multipotent mesenchymal stromal cells (MSC) were first discovered in bone marrow and can be isolated from “virtually all organs”. They could participate in tissue maintenance and self-renewing process. They are able to adhere to plastic surfaces and acquire a fibroblastic shape when isolated. They are characterized by a particular phenotype and are able to differentiate into several cell types if cultivated in a specific induction medium. These characteristics were defined on MSC in culture and do not represent how they may be in situ.MSC present particular properties. They can secrete growth factors and several cytokines that give them a trophic activity on one hand and the ability to modulate the immune system on the other hand. They are also able to differentiate. These different properties make them an attractive candidate for cell therapy.MSC are already the focus of several pre-clinical and clinical studies. Nevertheless, the results of these studies are difficult to interpret due to limited understanding of their basic biology. MSC are poorly defined in situ and are heterogeneous. Their heterogeneity is dictated by their tissue of origin and cell preparation. To date, there is no standard protocol for MSC isolation and culture. This leads to numerous questions regarding patient safety, and these questions require answers.The first part of the work deals with the methods used to optimize the extraction of MSC and purification from adipose tissue, one of the main sources of autologous MSC with bone marrow. Classical methods require an enzymatic digestion step. The enzyme used and the duration of adipose tissue digestion time can induce cellular alterations and modify cell functions. Moreover, the addition of a xenobiotic increases the risk of contamination and complicates the monitoring of good manufacturing practices (GMP). We propose a method that does not require this enzymatic digestion step while being easier, safer, faster, gentler and less expensive. Compared to the classical enzymatic method, our method yields an equivalent number of MSC from adipose tissue while preserving their properties.The second part of this work focuses on the characterization of the MSC subpopulations from adipose tissue and compares them to those from bone marrow, which are the historical gold standard. The study made it possible to deepen the knowledge of MSC surface markers in situ from these 2 sources. It also evaluated the various properties of the isolated subpopulations thanks to the cell surface markers CD271, SUSD2, MSCA-1, CD44 and CD34. We showed that MSC from bone marrow express MSCA-1, CD271 and SUSD2 markers in situ. We also found that a population clearly positive for the CD34 does exist in situ with different properties compared to those of the unselected populations or the negative counterpart. 2 populations that are negative and positive for CD44 also exist with similar properties.In contrast to bone marrow MSC, only one selection was able to effectively isolate MSC from adipose tissue by a positive selection based on the expression of CD34. We also isolated a CD271+ population but only from lipoaspirate samples and not from abdominoplasty samples. Collectively, our results suggest that MSCA-1 seems to be the best marker through which to isolate MSC from bone marrow and that CD34 is the only marker able to positively isolate cells from adipose tissue. Thus, we show that the MSC from the different sources share similar properties although they have specific characteristics. The choice of the source and of the marker with which to isolate a particular subpopulation is important depending on their intended clinical use. / Les cellules stromales mésenchymateuses multipotentes (CSM) ont été mises en évidence dans la moelle osseuse et peuvent être isolées de « virtuellement tous les organes ». Elles participeraient à la maintenance et au renouvellement des tissus. Une fois isolées, elles sont capables d’adhérer à des surfaces en plastique en prenant une forme fibroblastique. Elles sont caractérisées par un phénotype particulier et peuvent se différencier en divers types cellulaires lorsque cultivées dans un milieu d’induction spécifique. Ces caractéristiques ont été définies sur les CSM en culture et ne reflètent pas forcément ce qui se passe in situ.Les CSM présentent des propriétés particulières. Elles peuvent sécréter des facteurs de croissance ainsi que de nombreuses cytokines qui leur permettent d’une part d’avoir une activité trophique et d’autre part de moduler le système immunitaire. Elles sont aussi capables de se différencier. Ces différentes propriétés les rendent particulièrement attractives pour la thérapie cellulaire.Les CSM font déjà l’objet de nombreuses études pré-cliniques et cliniques dont les résultats sont difficilement interprétables car nous n’avons à l’heure actuelle qu’une compréhension limitée de leur biologie de base. Les CSM sont encore mal définies in situ et sont hétérogènes. Cette hétérogénéité provient de leur différence d’origine et de leur préparation cellulaire :il n’existe aucune standardisation des protocoles d’isolation et de culture. Cette hétérogénéité entraine de nombreuses questions relatives à la sécurité du patient qui doivent être élucidées.La première partie de ce travail cherche à optimiser les méthodes d’extraction et de purification des CSM du tissu adipeux humain, la principale source de CSM autologues avec la moelle osseuse. Les méthodes classiques requièrent une étape de digestion enzymatique dont l’enzyme utilisée et le temps de digestion du tissu adipeux peuvent induire des altérations cellulaires et modifier leurs fonctions. De plus, l’adjonction de xénobiotiques augmente le risque de contamination et complique le suivi des bonnes pratiques de fabrication (BPF). Nous proposons une méthode qui s’affranchit de cette étape de digestion enzymatique tout en étant plus facile, plus sûre, plus rapide, moins chère et moins traumatisante pour les cellules. Elle permet d’obtenir un nombre tout aussi important de CSM du tissu adipeux que la méthode enzymatique classique en préservant leurs propriétés.La deuxième partie de ce travail vise à caractériser les sous populations de CSM du tissu adipeux humain en les comparant à celles de la moelle osseuse, source de référence historique. Cette étude a permis d’approfondir la connaissance des marqueurs de surface des CSM de ces 2 sources in situ, tout en évaluant les différentes propriétés des sous-populations isolées grâce aux marqueurs de surface CD271, SUSD2, MSCA-1, CD44 et CD34. Nous avons montré que les CSM de la moelle osseuse expriment les marqueurs MSCA-1, CD271 et SUSD2 in situ et qu’il existait une sous-population clairement positive pour le CD34 avec des propriétés différentes de celles de la population non sélectionnée ou négative pour ce marqueur. Il existe aussi 2 sous-populations positive et négative pour le CD44 avec des propriétés similaires.Contrairement aux CSM de la moelle osseuse, une seule sélection a permis d’isoler efficacement les CSM du tissu adipeux par une sélection positive sur base de l’expression du CD34. Nous avons pu aussi isoler une population CD271+ mais seulement des prélèvements de lipoaspirations et non des abdominoplasties.Au vu de nos résultats, MSCA-1 semble le meilleur marqueur pour isoler les CSM de la moelle osseuse tandis que le CD34 est le seul marqueur capable d’isoler positivement celles du tissu adipeux. Ainsi, nous montrons que les CSM issues de différentes sources partagent des propriétés similaires avec cependant des caractéristiques propres. Le choix de la source et du marqueur pour isoler une sous-population sont donc importants en fonction de leur utilité clinique envisagée. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

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