Juliette Peltzer

The osteogenic differentiation improvement of human mesenchymal stem cells on titanium grafted with polyNaSS bioactive polymer.

Osseointegration of metallic implants used in orthopedic surgery requires that osteoprogenitor cells attach and adhere to the surface, then proliferate, differentiate into osteoblasts, and finally produce mineralized matrix. Because the ability of progenitor cells to attach to a scaffold surface during early stages is important in the development of new tissue structures, we developed in our laboratory, a strategy involving grafting of implants with a polymer of sodium styrene sulfonate (polyNaSS) used as a scaffold which enables human mesenchymal stem cells (hMSCs) interactions. In the present study, we investigated the cellular response of hMSCs to polyNaSS surfaces of titanium (Ti). In particular, cell proliferation, cell viability, cell differentiation, and cell spreading were evaluated. Results showed that cell proliferation and cell viability did not differ with any statistical significance between modified and unmodified Ti surfaces. Interestingly, culture of MSCs on polyNaSS surfaces resulted in a significant increase of cell spreading and cell differentiation compared with the other tested surfaces. These results suggest that titanium surface grafted with polyNaSS is a suitable scaffold for bone tissue engineering.

Heterogeneous functions of perinatal mesenchymal stromal cells require a preselection before their banking for clinical use.

Perinatal sources of mesenchymal stromal cells (MSCs) have raised growing interest because they are readily and widely available with minimal ethical/legal issues and can easily be stored for allogeneic settings. In addition, perinatal tissues are known to be important in mediating the fetomaternal tolerance of pregnancy, which confer upon perinatal-MSCs (P-MSCs) a particular interest in immunomodulation. It has been recently shown that it is possible to deeply modify the secreted factor profiles of MSCs with different cytokine stimuli such as interferon gamma or tumor necrosis factor alpha to license MSCs for a better immunosuppresive potential. Therefore, we aimed to compare adult bone marrow-MSCs with MSCs from perinatal tissues (cord blood, umbilical cord, amnion, and chorion) on their in vitro immunological and stromacytic efficiencies under different priming conditions. Our results showed that P-MSCs had a potential to modulate the in vitro immune response and be useful for hematopoietic progenitor cell ex vivo expansion. However, we showed contrasted effects of cytokine priming embedded in an important between-donor variability. In conclusion, our study highlights the importance to elaborate predicitive in vitro tests to screen between-donor variability of perinatal tissues for banking allogeneic standardized MSCs.

Preliminary In Vitro Assessment of Stem Cell Compatibility with Cross-Linked Poly(ε-caprolactone urethane) Scaffolds Designed through High Internal Phase Emulsions

By using a high internal phase emulsion process, elastomeric poly(ε-caprolactone urethane) (PCLU) scaffolds were designed with pores size ranging from below 150 μm to 1800 μm and a porosity of 86% making them suitable for bone tissue engineering applications. Moreover, the pores appeared to be excellently interconnected, promoting cellularization and future bone ingrowth. This study evaluated the in vitro cytotoxicity of the PCLU scaffolds towards human mesenchymal stem cells (hMSCs) through the evaluation of cell viability and metabolic activity during extract test and indirect contact test at the beginning of the scaffold lifetime. Both tests demonstrated that PCLU scaffolds did not induce any cytotoxic response. Finally, direct interaction of hMSCs and PCLU scaffolds showed that PCLU scaffolds were suitable for supporting the hMSCs adhesion and that the cells were well spread over the pore walls. We conclude that PCLU scaffolds may be a good candidate for bone tissue regeneration applications using hMSCs.

Intradermal Injection of Bone Marrow Mesenchymal Stromal Cells Corrects Recessive Dystrophic Epidermolysis Bullosa in a Xenograft Model

TO THE EDITOR Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-offunction mutations in COL7A1 encoding type VII collagen (C7) that forms anchoring fibrils (AFs), structures essential for dermal-epidermal adherence (Uitto et al., 2017). Patients with RDEB suffer from skin and mucosal blistering and develop severe complications including invasive squamous cell carcinoma, resulting in a poor prognosis (Guerra et al., 2017). Different therapeutic strategies have been explored, including gene-, protein-, cell-based, and pharmacological therapies that have shown promising preclinical or transitory clinical benefits (Rashidghamat and McGrath, 2017). To date, there is no specific treatment for RDEB. Bone marrowemesenchymal stromal cells (BM-MSCs) have shown therapeutic potential for RDEB patients through BM transplantation and intradermal (ID) and intravenous injections (Conget et al., 2010; El-Darouti et al., 2016; Petrof et al., 2015; Tamai et al., 2011; Tolar et al., 2009; Wagner et al., 2010). Human bone marrowe mesenchymal stromal cells (hBMMSCs) form a heterogeneous cell population that can self-renew or differentiate into mesenchymal lineages (Caplan, 1991). hBM-MSCs display properties that could potentially improve wound healing in RDEB: immunomodulation; anti-inflammatory, angiogenic, and antifibrotic properties; secretion of trophic factors; improvement of tissue repair; and the capacity to induce protein expression in the host tissues through a paracrine effect (Nuschke, 2014; Qi et al., 2014). Herein, we assessed the long-term capacity of hBM-MSCs to survive, produce, and deposit C7 at the dermalepidermal junction (DEJ) after ID injection into human RDEB skin equivalents (SEs) transplanted onto immune-deficient nude mice that reproduce the skin defect observed in RDEB (Titeux et al., 2010). Phenotypic analyses of hBM-MSCs from healthy donors showed that they were positive for well-established surface markers (CD105, CD90, CD73, CD29, and CD44) and negative for potential hematopoietic contaminants (HLA-DR and CD45) (see Supplementary Figure S1 online). The capacity of these hBM-MSCs to differentiate in vitro into osteoblasts, adipocytes, and chondrocytes was previously shown (Peltzer et al., 2015). Then, we compared COL7A1 expression in hBM-MSCs from several donors cultured with 5% human platelet lysate or 10% fetal calf serum (see Supplementary Figure S2a and b online). C7 expression by hBM-MSCs from donor 1 was similar to human healthy fibroblasts when grown in human platelet lysate, whereas hBM-MSCs from other donors showed lower amounts of C7 (see Supplementary Figure S2c, d, and e). We next tested the capacity of hBMMSCs to synthesize C7 able to form AF structures in vivo in a xenograft model. We ID injected hBM-MSCs beneath RDEB SE completely devoid of C7 expression (see Supplementary Figure S3 online), thus excluding any possible paracrine effect of hBM-MSCs on hRDEB fibroblasts and/or keratinocytes, leading to increased production of endogenous mutant C7. The dose of 2 10 of hBM-MSC was chosen based on a previous preclinical study (Kuhl et al., 2015). We collected SE samples from the injected area at 1, 2, 4, and 6 months after treatment. Immunofluorescence staining of SE sections showed linear staining of human C7 (see Supplementary Figure S4 online) along the DEJ up to 6 months in healthy SEs and in hBM-MSCeinjected RDEB SEs, whereas vehicle-injected RDEB SEs

Mesenchymal Stromal Cells Based Therapy in Systemic Sclerosis: Rational and Challenges

Systemic Sclerosis (SSc) is a rare chronic disease, related to autoimmune connective tissue diseases such as Systemic Lupus Erythematosus and Sjögrens Syndrome. Although its clinical heterogeneity, main features of the disease are: extensive tissue fibrosis with increase matrix deposition in skin and internal organ, microvascular alterations and activation of the immune system with autoantibodies against various cellular antigens. In the diffuse cutaneous scleroderma subtype, the disease is rapidly progressive with a poor prognosis, leading to failure of almost any internal organ, especially lung which is the leading cause of death. Primary trigger is unknown but may involve an immune process against mesenchymal cells in a genetically receptive host. Pathophysiology reveals a pivotal role of fibrosis and inflammation alterations implicating different cell subtypes, cytokines and growth factors, autoantibodies and reactive oxygen species. Despite improvement, the overall survival of SSc patients is still lower than that of other inflammatory diseases. Recommended drugs are agents capable of modulating fibrotic and inflammatory pathways. Cellular therapy has recently emerged as a credible option. Besides autologous hematopoietic stem cell transplantation which demonstrated remarkable improvement, mesenchymal stromal cells (MSCs) represent promising therapeutic candidates. Indeed, these cells possess anti-inflammatory, antiproliferative, antifibrotic, and immunomodulary properties especially by secreting a large panel of bioactive molecules, addressing the most important key points of the SSc. In addition, these cells are very sensitive to their environment and are able to modulate their activity according to the pathophysiological context in which they are located. Autologous or allogeneic MSCs from various sources have been tested in many trials in different auto-immune diseases such as multiple sclerosis, Crohns disease or systemic lupus erythematosus. They are characterized by a broad availability and no or low acute toxicity. However, few randomized prospective clinical trials were published and their production under ATMP regulatory procedures is complex and time-consuming. Many aspects have still to be addressed to ascertain their potential as well as the potential of their derived products in the management of SSc, probably in association with other therapies.

Improved proliferation and osteogenic differentiation of human mesenchymal stem cells on a titanium biomaterial grafted with poly(sodium styrene sulphonate) and coated with a platelet-rich plasma proteins biofilm

In order to replace damaged or lost bone in the human body, it is necessary to produce ‘spare body parts’ which are dependent on the use of biomaterial and stem cells and are referred to as ‘tissue engineering’. Surface modification and stem cell interaction of orthopaedic implants offer a promising approach and are investigated here specifically to improve osseointegration of the biomaterial. Osseointegration of titanium implants used in orthopaedic surgery requires that osseo-progenitor cells attach and adhere to the surface, proliferate, then differentiate into osteoblasts and, finally, produce a mineralised matrix. The surface modification of titanium with anionic polymer combined with coating of platelet-rich plasma is provided to create a favourable environment to promote early and strong fixation of implants. The ability of progenitor cells to attach to the surface during early stages is important in the development of new tissue structures; therefore, we developed in our laboratory a strategy involving the grafting of titanium implants with a polymer of sodium styrene sulphonate (poly(sodium styrene sulphonate)) and a biofilm coating of platelet-rich plasma which enables human mesenchymal stem cell interactions. The resulting biomaterial, titanium-poly(sodium styrene sulphonate) and coating of platelet-rich plasma, Ti-poly(sodium styrene sulphonate)–platelet-rich plasma was developed in order to further improve the biomaterial. In this work, we studied and characterised the ‘in vitro’ response of human mesenchymal stem cells to titanium biomaterial grafted with poly(sodium styrene sulphonate) bioactive polymer and coated with platelet-rich plasma proteins (Ti-poly(sodium styrene sulphonate)–platelet-rich plasma). This study shows an increased cell proliferation with Ti-poly(sodium styrene sulphonate)–platelet-rich plasma compared to foetal calf serum and an enhancement of the Ti-poly(sodium styrene sulphonate)–platelet-rich plasma effects on osteoblast differentiation. The results suggest that Ti-poly(sodium styrene sulphonate)–platelet-rich plasma would be a suitable scaffold for bone tissue engineering.

Les cellules souches mésenchymateuses : des cellules pour la médecine régénérative du futur ?

Resume Les cellules stromales mesenchymateuses (CMS) sont des cellules capables d’autorenouvellement et de differenciation en cellules des lignees osteoblastiques, chondrocytaires et adipocytaires a minima. Leur capacite d’adressage vers un tissu lese (homing), leur faible immunogenicite permettant d’envisager une utilisation allogenique, et l’absence de considerations ethiques liees a leur origine expliquent l’interet que suscitent ces cellules en medecine regenerative depuis maintenant deux decennies. Recemment les notions portant sur les communications qu’elles etablissent au sein des niches hematopoietiques ont ete etendues a un nombre important de types cellulaires. Une partie du benefice therapeutique lie a l’utilisation de ces cellules a pu etre expliquee par leurs proprietes trophiques, qui ont de plus ete a l’origine d’un changement du paradigme concernant leurs utilisations cliniques potentielles. Parallelement aux premiers essais cliniques il y a une dizaine d’annees, la question des risques lies a l’embolisation vasculaire apres injection de ces cellules, ainsi que celle, plus inquietante, de la possible transformation cancereuse des cellules amplifiees en culture ont suscite de vifs debats, sans que le rapport benefice-risque ne penche pour l’instant en defaveur de l’utilisation therapeutique des CSM. Au cours de cette revue nous rappellerons la definition admise pour qu’une population cellulaire puisse etre denommee CSM, les origines tissulaires de ces cellules en privilegiant celles accessibles en clinique, les essais cliniques en cours ainsi que les perspectives therapeutiques.