Auswahl der wissenschaftlichen Literatur zum Thema „Mesenchymal stromal cells derivedfrom Wharton's Jelly“

Endometrial mesenchymal stromal cells (eMSCs), along with mesenchymal stromal cells (MSCs) isolated from other tissues, are promising for use in regenerative medicine. The benefits of eMSCs include their presence in adults, simplicity of isolation, high proliferative and differentiation capacity. In this study, we have employed the flow cytometry technique to assess expression of 28 molecular markers on the surface of two eMSCs cultures. The culture of MSCs isolated from Wharton's jelly of the umbilical cord (uMSCs) was used as a reference, because uMSCs were studied in details earlier and demonstrated their effectiveness in vivo. Both types of MSCs demonstrated similar expression profiles. They included stem cells surface molecules, cell adhesion molecules and their ligands, some receptor molecules responsible for cell metabolism and proliferation, as well as immunological response molecules.

La restauration de la continuité digestive suite à l'ablation d'une portion de l'œsophage consiste à la réalisation chirurgicale d’une anastomose œsogastrique intra-thoracique. Néanmoins des complications post-opératoires sont décrites telles que des atteintes pulmonaires, des fistules, des sténoses, des nécroses de plastie, et un reflux gastro-œsophagien. Le développement d'un substitut issu de l'ingénierie tissulaire dérivé de la matrice œsophagienne biologique décellularisée (MBD) est prometteur dans la perspective d’améliorer la prise en charge du traitement chirurgical pour le remplacement de l’œsophage. L'objectif principal de cette étude était d'optimiser la conception d’une MBD de porcs et de caractériser ses propriétés biologiques et mécaniques. Le second objectif était de cellulariser la MBD au moyen de cellules immuno-privilégiées, facilement disponibles : les cellules stromales mésenchymateuses humaines issues de la gelée de Wharton (CSM-GW).La décellularisation de l'œsophage était réalisée selon un protocole basé sur la perfusion dynamique de solutions chimiques et enzymatiques de la lumière de l’organe. L’analyse histologique et la quantification de l’ADN résiduel de la MBD permettaient de déterminer l’efficacité du protocole de décellularisation. L'ultrastructure de la MBD était analysée par des marquages immunohistochimiques (IHC), et la composition du contenu protéique de la matrice extracellulaire (MEC) était décrite par spectrométrie de masse. Les tests de cytotoxicité in-vitro de la MBD étaient réalisés conformément à la norme ISO 10993-5. L’évaluation de la force de rétention à la suture, la résistance à la traction et la pression à l’éclatement de la MBD consistait à décrire le comportement mécanique du substitut en regard de son utilisation clinique.Les CSM-GW utilisées pour la cellularisation de la MBD étaient extraites à partir de cordons ombilicaux humains et leur profilage par cytométrie en flux permettait de confirmer la pureté de la population cellulaire. La réponse immunitaire des CSM-GW était quantifiée après une co-culture avec des cellules mononucléées du sang périphérique (PBMC). Le phénotypage des PBMC permettait d’évaluer l’expression des marqueurs immunitaires au contact des MSC-GW, et l’étude du sécrétome, par une méthode immuno-enzymatique (ELISA), quantifiait le relargage des cytokines. La stratégie de cellularisation de la MBD proposée reposait sur le développement de feuillets cellulaires de MSC-GW. La validation du protocole de fabrication des feuillets consistait en la caractérisation du phénotype cellulaire par IHC et l’étude mécanique des feuillets permettait de mesurer leur résistance à la perforation.L’absence de contenu cellulaire et la quantification de l’ADN résiduel de la MBD confirmaient l’efficacité de la décellularisation selon les critères de validation en vigueur. L’ultrastructure et les composants biologiques de la MEC étaient préservés et l'analyse protéomique de la MEC mettait en évidence une complexité protéique. Le traitement de décellularisation n’induisait pas de toxicité de la MBD et le comportement mécanique de la MBD était adapté à son utilisation en tant que substitut œsophagien.La culture des CSM-GW sous forme de feuillets favorisait la cellularisation de la MBD. Une fois ensemencés, les feuillets avaient conservé leur phénotype cellulaire et leur caractéristiques immuno-privilégiées. Un remodelage tissulaire in-vitro était visible ainsi que la formation d’une nouvelle MEC produite par les CSM-GW.Les caractérisations de la MBD obtenue offraient une complexité biologique et un comportement mécanique favorable à son utilisation en tant que substitut œsophagien. La MBD était cellularisable avec des feuillets cellulaires de CSM-GW, pouvant favoriser ainsi l'intégration et le remodelage des tissus
Upon removal of a portion of the esophagus, the restoration of the digestive continuity involves the surgical creation of an intrathoracic esophagogastric anastomosis. However, postoperative complications such as lung impairments, fistulas, strictures, graft necrosis, and gastroesophageal reflux are reported. The enhancement of surgical procedures for esophageal replacement has made promising progress by the development of a substitute through tissue engineering that utilizes a decellularized biological esophageal matrix (DEM). The primary objective of this study was to optimize the design of porcine DEM and characterize its biological and mechanical properties. The secondary objective was to cellularize DEM using readily available immune-privileged human mesenchymal stromal cells derived from Wharton's jelly (hMSCs-WJ).Esophageal decellularization was performed according to a protocol based on the dynamic perfusion of chemical and enzymatic solutions through the organ lumen. Histological analysis and residual DNA quantification of the DEM were conducted to determine the efficiency of the decellularization protocol. The ultrastructure of the DEM was analyzed using immunohistochemical (IHC) labeling, and the composition of the extracellular matrix (ECM) protein content was described by mass spectrometry. In-vitro cytotoxicity tests of DEM were conducted following ISO 10993-5 standards. The evaluation of suture retention strength, tensile strength, and bursting pressure of DEM aimed to describe the mechanical behavior of the substitute for clinical use.hMSCs-WJ used for DEM cellularization were extracted from human umbilical cords, and their flow cytometry profiling confirmed the purity of the cell population. The immune response of hMSCs-WJ was quantified after co-culture with peripheral blood mononuclear cells (PBMCs). PBMCs phenotyping assessed the expression of immune markers in contact with hMSCs-WJ, while enzyme-linked immunosorbent assay (ELISA) quantified cytokine release. The proposed DEM cellularization strategy involved the development of cell sheets from hMSCs-WJ. The validation of the cell sheet production protocol involved the characterization of the cellular phenotype by IHC analysis, and the mechanical study of the sheets measured their resistance to perforation.The absence of cellular content and residual DNA quantification in DEM confirmed the efficacy of decellularization according to current validation criteria. The ultrastructure and biological components of the ECM were preserved, and proteomic analysis highlighted protein complexity. Decellularization treatment did not induce DEM toxicity, and the mechanical behavior of DEM was suitable for its use as an esophageal substitute.Culturing hMSCs-WJ as cell sheets promoted the cellularization of the DEM. Once seeded, the sheets retained their cellular phenotype and immune-privileged characteristics. In-vitro tissue remodeling was visible, along with the formation of a new ECM produced by hMSCs-WJ.Characterization of the obtained DEM offered biological complexity and favorable mechanical behavior for its use as an esophageal substitute. DEM was cellularizable with hMSCs-WJ cell sheets, potentially promoting tissue integration and remodeling

To investigate whether the maternal metabolic environment affects the DNA methylation of mesenchymal stromal/stem cells (MSCs) from umbilical cord (UC) Wharton’s Jelly (WJ), potentially rendering them unsuitable for clinical use in multiple recipients, a pilot study was conducted on fourteen UCs obtained post partum from healthy non-obese (BMI=19-25; n=7) and obese (BMI≥30; n=7) donors receiving elective Caesarean sections. The time of first WJ-MSCs outgrowth from UC explants was similar in samples from obese and non-obese donors. However, the cells from non-obese donors proliferated faster after 34 hours of culture than cells from obese donors. Differentiation into adipogenic, osteogenic and chondrogenic lineages was similar between obese and non-obese donor samples as demonstrated by tissue-specific staining and RT-PCR for lineage markers. However, WJ-MSCs from obese donors exhibited stronger immunosuppressive activity than those from non-obese donors. Genome-wide DNA methylation of triple-positive (CD73+CD90+CD105+) WJ-MSCs sorted from the first passage of a mixed population of cells was assessed. Samples from the obese and non-obese donors clustered separately, and 5,767 of the analysed CpG sites (1%) exhibited different methylation. Sixty-seven genes were observed with at least one CpG site with a methylation difference ≥0.2 in four or more obese donors. These 67 genes were further refined based on a list of polymorphic CpG sites and segmental duplications. In 18 of the 67 genes with a different CpG methylation pattern, the CpG sites were in non-polymorphic regions. However, two genes (DCAF6 and ZNF714) resided in segmentally duplicated regions. To determine whether methylation differences altered gene expression, the samples were analysed using a HumanHT-12 Expression BeadChip array and, of the 18 genes, only PNPLA7 was significantly affected at the mRNA level, which was confirmed independently by RT-PCR and Western blotting. Although the number of analysed donors was limited, the data suggest that an abnormal metabolic environment related to excessive body weight might alter the properties of WJ-MSCs used for cellular therapy.

Master of Science
Department of Anatomy and Physiology
Mark L. Weiss
Wharton's jelly is a non-controversial source of mesenchymal stromal cells. Isolation of the cells is non-invasive and painless. The cells have been shown to have a wide array of therapeutic applications. They have improved symptoms when transplanted in a variety of animal disease models, can be used in tissue engineering applications to grow living tissue ex vivo for transplantation, and can be used as drug delivery vehicles in cancer therapy. The cells have also been shown to be non-immunogenic and immune suppressive. This thesis focuses on optimizing isolation protocols, culture protocols, cryopreservation, and characterization of cells in different growth conditions. Results from the experiments indicate that isolation of cells by enzyme digestion yields cells consistently, a freezing mixture containing 90% FBS and 10% DMSO confers maximum viability, and the expression of mesenchymal stromal cell consensus markers does not change with passage and cryopreservation. The results of the experiments also show that cells grow at a higher rate in 5% oxygen culture conditions compared to 21% oxygen culture conditions, serum does not have an effect on growth of the cells, serum and oxygen do not have effects on the expression of mesenchymal stromal cell consensus markers and the cells are stable without nuclear abnormalities when grown in 5% oxygen and serum free conditions for six passages after first establishing in serum conditions.

Doctor of Philosophy
Department of Anatomy and Physiology
Mark L. Weiss
Graft Versus Host Disease (GVHD) is the major complication following hematopoietic stem cell transplantation. GVHD is activated by immunocompetent T cells presented in the donor grafted tissue. Due to the increased use of bone marrow transplantation to treat diverse malignancies, the incidence of GVHD has shown a notable increase. Depending of the degree of immunological mismatch between donor and host, 50-70% of patients develop GVHD after allogeneic Bone Marrow Transplantation (BMT). Once GVHD develops, mortality reaches up to 50% in humans. Several studies using Mesenchymal Stromal Cells (MSCs) to prevent and treat GVHD have produced controversial results. It is thought that distinct MSCs sources used in those studies might be an important factor that produces different outcomes. For cellular therapy, the most attractive characteristics of MSCs are their reduced immunogenic potential, and their abilities to modulate immune responses. This dissertation addressed the hypothesis that Wharton’s jelly cells (WJCs) would prevent the pathology and death associated with GVHD after BMT. To accomplish this, I created a clinically relevant model of GVHD by transplanting allogeneic bone marrow across minor histocompatibility antigen (HA) barriers in the rat. To enhance alloreactive T-cell stimulation, bone marrow (BM) was co-administered with a fraction of CD8[superscript]+ cells magnetically selected from spleen to induce GVHD. Bone marrow tissue was isolated from a donor rat Fischer 344 (F344, RT1lv) and transplanted into lethally irradiated (10 Gray) Lewis rat (LEW, RT1l). Once GVHD was induced, MSCs derived from umbilical cord WJCs were either co-transplanted at day 0 with bone marrow, or given on day 2 post-BMT intravenously. The prophylactic potential of WJCs in an in vivo GVHD model was assessed as survival time, clinical symptomatology occurrence, and histopathology injuries in target tissues. Results indicate that while co-administration of WJCs with hematopoietic cells on day 0 failed to alleviate GVHD associated symptomatology and mortality. WJCs administered on day 2 post-induction ameliorated GVHD-associated symptomatology, improved engraftment and survival.

Master of Science
Department of Clinical Sciences
Mary Lynn Higginbotham
Objectives – To isolate and maintain canine Wharton’s jelly mesenchymal stromal cells (WJMSCs) in culture, to determine the effects of micellar nanoparticles containing doxorubicin (DOX) on WJMSCs and canine osteosarcoma (OSA) D17 cell viability, and to determine the effects of conditioned media from WJMSCs loaded with micellar nanoparticles containing DOX on OSA D17 cell viability. Sample Population – Canine WJMSCs containing various concentrations of DOX micelles and canine OSA D17 cells. Procedures – WJMSCs were isolated from canine umbilical cords. Micellar nanoparticles containing DOX were prepared and added to culture plates containing canine OSA D17 cells to determine micelle effects on cell growth and viability. Conditioned media from culture plates containing canine WJMSCs incubated with various DOX micelle concentrations was added to OSA D17 cells for conditioned media experiments. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess OSA D17 cell viability. A trypan blue stain was also utilized to perform cell counts to determine the effect of the DOX micelles on stromal cell growth. Results – WJMSCs were successfully isolated and maintained in culture. Micellar nanoparticles containing DOX decreased OSA D17 cell viability. OSA D17 cell viability was also decreased following incubation with conditioned media from canine WJMSCs loaded with micellar nanoparticles containing DOX. Significant decreases with the conditioned media of canine WJMSCs loaded with 10μM micelles occurred at 48 hours (p < 0.005) and at 72 and 96 hours (p < 0.0001). Significant decreases were also observed with the 1 μM DOX micelles at 72 hours (p < 0.005) and 96 hours (p < 0.0001). WJMSC numbers decreased in a dose dependent manner following incubation with DOX micelles. Changes in WJMSC number was not caused by increased cell death as all variables produced similar percentages of dead cells. Conclusions – Canine WJMSCs were successfully isolated and maintained in culture. Stromal cells containing DOX micellar nanoparticles induced OSA D17 cell cytotoxicity while inducing an anti-proliferative, rather than cytotoxic effect, on the WJMSC. These data support future in vivo experiments utilizing canine WJMSCs and micellar nanoparticles.

Mesenchymal stromal cells (MSC) are adherent, clonogenic, fibroblast-like cells endowing with unique multipotent differentiation potential and immunosuppressive properties. They are considered as promising candidates for regenerative medicine and immunotherapy.

MSC can be isolated from different tissue sources including bone marrow (BM), adipose tissue (AT) and Wharton’s Jelly (WJ). Although fulfilling the ISCT criteria required to be recognized as MSC, MSC from these different sources could disclose some differences taking into account their different anatomical origin and ontogeny as well.

In the present work, we investigated the influence of MSC source on their immunosuppressive as well as differentiation properties. We further extended our study to the role of the microenvironment (infection and inflammation) on these features.

We show that BM-MSC express Toll-like receptors (TLR) from TLR1 to TLR6. In an inflammatory environment, TLR2, 3 and 4 are significantly upregulated. By upregulating TLR3 and TLR4 transcription, inflammation increases BM-MSC responsiveness to LPS (TLR4 ligand) and poly(I:C) (TLR3 ligand) leading to a pro-inflammatory shift of their cytokine profile. The effect of TLR ligation on BM-MSC osteogenic potential is donor dependent. Inflammation as well as stimulation with LPS and poly(I:C) result in a decrease of BM-MSC immunosuppressive capabilities.

We further observed that BM-, AT- and WJ-MSC do not have the same pattern of TLR expression and consequently do not respond the same way to bacterial or viral infection. WJ-MSC do not express TLR4 and although TLR3 is present at the protein level it is not functional as its ligation do not trigger cytokine expression. Inflammation modulates this TLR pattern expression by upregulating TLR3 in all three MSC types and TLR4 only in BM-MSC. TLR ligation increases the production of inflammatory cytokines in BM- and AT- but not in WJ-MSC and augments anti-inflammatory cytokines in AT-MSC. Although inflammation increases in all MSC types the secretion of inflammatory cytokines, additional TLR triggering does not further affect WJ-MSC. The immunosuppressive potential of WJ-MSC on mixed leucocytes reaction (MLR) is not affected either by inflammation or by TLR triggering.

On the differentiation side, WJ-MSC has the lower potential to differentiate into osteoblast as compared to BM- and AT-MSC, as revealed by alkaline-phosphatase (ALP) activity and by measuring extracellular Ca2+ deposits. However, inflammation is able to strongly increase the osteogenic differentiation of WJ-MSC as calcification and ALP activity appears as early as at day 7. However this latter enzymatic activity remains much lower than that disclosed by BM-MSC. TLR3 or TLR4 triggering does not affect the osteogenesis of WJ-MSC while it increases it in AT- and also, although to lesser extent, in BM-MSC.

Our work establishes that the source from which MSC is derived is of major importance for the design of MSC based immunointervention. WJ-MSC appear to be the most attractive cell type when an immunosuppressive action is required in an inflammatory or infectious context. Although WJ-MSC are poorly osteogenic, a complete osteogenic differentiation can be obtained under inflammatory conditions. Taking into account their easy accessibility as well as their huge proliferative potential, these data open an avenue for using these cells in regenerative medicine particularly in clinical settings where chronic inflammation or infection have to be considered.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Despite tremendous progress in medicine, injuries of the adult central neural system remain without satisfactory solution. Regenerative medicine employs tissue engineering, cellular therapies, medical devices, gene therapy, or growth factors with the aim to bridge the lesion, re-establish lost connections and enhance endogenous repair in order to restore neural function. The aim of my thesis was to evaluate therapeutic potential of two approaches, transplantation of human mesenchymal stromal cells (hMSCs) and biological scaffolds derived from extracellular matrix (ECM) for neural regeneration, particularly in models of spinal cord injury (SCI). First, hMSCs from various sources - bone marrow (BM), adipose tissue (AT) and Wharton's jelly (WJ) - were isolated and characterized in vitro. All cell types met the minimal criteria for MSC phenotype and displayed similar properties in terms of their surface marker expression, differentiation potential, migratory capacity, and secretion of cytokines and growth factors. On the other hand, the cell yield from WJ and AT was significantly higher, and MSCs isolated from these tissues proliferated better than from BM. Therapeutic effect of intrathecal application of hWJ-MSCs was then evaluated in SCI compression model in rats. The effect of low (0.5 million) and...