Mesenchymale Stromazellen (MSCs) werden beim Pferd bereits mit vielversprechenden Ergebnissen zur Behandlung von muskuloskelettalen Erkrankungen, insbesondere von Sehnenerkrankungen, eingesetzt. In bisherigen klinischen Studien lag das Hauptaugenmerk auf der Behandlung von Erkrankungen der Oberflächlichen Beugesehne bei Rennpferden, die jedoch in Deutschland nur einen verhältnismäßig kleinen Anteil des Patientenaufkommens darstellen. Die zu erwartenden Ergebnisse nach MSC-Behandlung von Fesselträgererkrankungen sind dagegen noch nicht bekannt. Darüber hinaus sind die grundlegenden Kenntnisse zur Biologie equiner MSCs noch unzureichend, was Verständnis und Optimierung des bestehenden Therapiekonzeptes erschwert. Häufig wird die Verwendung alternativer Gewebequellen für MSCs diskutiert, wobei jedoch nur wenige vergleichende Daten zu den jeweiligen zellulären Eigenschaften vorliegen. Ziel dieser Arbeit war es daher, zum einen mehr Kenntnisse über die zu erwartenden klinischen Ergebnisse nach MSC-Behandlung von Sehnenerkrankungen zu erlangen, einschließlich Erkrankungen des Fesselträgers, zum anderen den Wissensstand hinsichtlich der in-vitro-Charakterisierung equiner MSCs zu erweitern, wobei ein Vergleich klinisch relevanter Charakteristika zwischen MSCs aus verschiedenen Gewebequellen angestrebt wurde. In die klinische Studie wurden 98 Pferde, die aufgrund von Sehnen- und Banderkrankungen mit MSCs behandelt worden waren, einbezogen. Von 58 dieser Tiere konnten Langzeitergebnisse nach einem Beobachtungszeitraum von mindestens einem Jahr erhoben werden. Diese wurden hinsichtlich des Behandlungserfolges sowie möglicher Einflussfaktoren ausgewertet, wobei die Behandlung als erfolgreich bewertet wurde, wenn die Patienten nach dem Beobachtungszeitraum voll trainiert oder im Sport eingesetzt werden konnten und dabei kein Rezidiv aufgetreten war. Die Behandlung mit MSCs wurde bei 84,5 % der Pferde als erfolgreich eingestuft, wobei Erkrankungen der Oberflächlichen Beugesehne mit 84,2 % und Erkrankungen des Fesselträgers mit 83,3 % gleichermaßen gute Ergebnisse zeigten. Tendenziell beeinflussten Nutzungsdisziplin, Erkrankungsstadium und Patientenalter das klinische Ergebnis ebenso wie bei konventioneller Behandlung. Insgesamt war nach MSC-Behandlung das Auftreten von Rezidiven deutlich seltener zu beobachten als in der Literatur für die konventionelle Behandlung beschrieben wird. Für die in-vitro-Studie zur vergleichenden Charakterisierung equiner MSCs aus verschiedenen Quellen wurden Knochenmark, Fett- und Sehnengewebe sowie Nabelschnurblut und -gewebe gewonnen. Aus diesen Proben wurden jeweils die plastikadhärenten MSCs isoliert und hinsichtlich Zellausbeute, Proliferations- und Migrationseigenschaften, tripotentem Differenzierungspotential sowie der Expression der Sehnenmarker Kollagen 1A2 und Skleraxis vergleichend untersucht. Die Ausbeute an MSCs war bei allen soliden Geweben (Fett-, Sehnen-, und Nabelschnurgewebe) hochsignifikant höher (p < 0,001). Ebenso proliferierten MSCs aus Fett- und Sehnengewebe signifi-kant schneller als MSCs aus Knochenmark oder Nabelschnurblut (p < 0,01). Von letzteren wurden darüber hinaus etwa drei viertel aller Zellkulturen vor der achten Passage seneszent. Das höchste Migrationspotential zeigten wiederum MSCs aus Sehnen- und Fettgewebe, wobei hier MSCs aus Nabelschnurgewebe das ungünstigste Ergebnis erzielten (p < 0,01). Die adipogene Differenzierung gelang bei MSCs aus allen Quellen vergleichbar gut. Bei der osteogenen Differenzierung erreichten MSCs aus Knochenmark das beste Ergebnis, während MSCs aus Nabelschnurblut und –gewebe nur schwach osteogen differenzierten (Tag 21: p < 0,01; Tag 35: p < 0,05). Im Gegensatz dazu erreichten MSCs aus Nabelschnurblut bei der chondrogenen Differenzierung die meisten Scorepunkte, MSCs aus Knochenmark dagegen die wenigsten (p < 0,05). Kollagen 1A2 wurde von MSCs aus Fettgewebe am höchsten exprimiert, Skleraxis von MSCs aus Nabelschnurblut. MSCs aus Sehnengewebe exprimierten beide Sehnenmarker auf fast ebenso hohem Level. MSCs aus Knochenmark dagegen zeigten hier jeweils die niedrigste Expression (p < 0,05 für Kollagen 1A2). Basierend auf den Ergebnissen der klinischen Studie ist die MSC-Therapie nach wie vor als vielversprechende Behandlungsoption für Sehnenerkrankungen anzusehen und ist auch für die Behandlung von Fesselträgererkrankungen geeignet. Zukünftige, kontrollierte klinische Studien müssen jedoch die Wirksamkeit der MSC-Therapie noch weitergehend bestätigen. Die in-vitro-Studie zeigte signifikante Unterschiede zwischen equinen MSCs aus verschiedenen Quellen auf, die bei der Auswahl einer Gewebequelle für die MSC-Isolierung für klinische Anwendungen berücksichtigt werden sollten. MSCs aus Fettgewebe erscheinen aufgrund ihrer sehr guten Proliferations- und zuverlässigen Differenzierungseigenschaften als eine gute Alternative zu MSCs aus Knochenmark für autologe Therapien. MSCs aus Sehnengewebe sind den hier vorliegenden Ergebnissen zufolge besonders gut für die Behandlung von Sehnenerkrankungen geeignet; vor einer routinemäßigen Anwendung dieser MSCs sollten jedoch ihre Eigenschaften weiterführend untersucht werden. / In horses, mesenchymal stromal cells (MSCs) are used for the treatment of musculoskeletal diseases, especially tendon injuries, with promising results. Previous clinical studies mainly focused on the treatment of superficial digital flexor tendon injuries in racehorses, which, however, represent only a relatively small percentage of the overall equine case load in Germany. Average outcome to be expected following MSC treatment of suspensory ligament injuries was not yet determined. Moreover, basic knowledge on equine MSC biology is still deficient, hampering the understanding and thus the optimisation of the existing treatment regime. The use of alternative MSC sources is frequently discussed, yet to date, only few data comparing the cellular properties of equine MSCs from different sources have been published. The aim of this study was, on the one hand, to gain more knowledge concerning the expected outcome after MSC treatment of tendon injuries, including injuries to the suspensory ligament. On the other hand, it was aimed at expanding the knowledge on equine MSC characterisation in vitro, thereby focusing on the comparison of clinically relevant properties of MSCs derived from different sources. In the clinical study, 98 horses were included, all of which had received MSC treatment for tendon or ligament injuries. In 58 of these horses, long term results after a follow-up period of at least one year could be collected. These data were analysed with respect to treatment outcome and potential influencing factors. Treatment was considered successful when horses were back to full training or competition after the follow-up period, without having suffered a re-injury. The overall success rate was 84.5 %. Success rates in horses suffering from superficial digital flexor tendon injuries and in horses suffering from suspensory ligament injuries were comparably good (84.2 % and 83.3 %, respectively). Similar to conventional therapies, the sports discipline in which the horses performed, age and disease stage tended to influence the outcome. Overall, re-injury rates after MSC treatment were considerably lower than those described in the literature following conventional treatment. For the comparative characterisation of MSCs from different sources in vitro, samples of bone marrow, adipose and tendon tissue, as well as umbilical cord blood and –tissue were collected. Plastic-adherent MSCs were isolated out of these samples and comparatively characterised focusing on cell yields, proliferation and migration properties, trilineage differentiation potential and the expression of the tendon markers collagen 1A2 and scleraxis. MSC yields were significantly higher in all solid tissues (adipose, tendon and umbilical cord tissue) (p < 0.001). Further, MSCs from adipose and tendon tissue proliferated significantly faster than MSCs from bone marrow or umbilical cord blood (p < 0.01). Moreover, approximately three quarters of the samples derived from the latter sources underwent senescence before reaching passage eight. The highest migration potential was found in MSCs derived from tendon and adipose tissue again, while MSCs from umbilical cord tissue showed the least (p < 0.01). The adipogenic differentiation potential was comparably good in MSCs from all different sources. The osteogenic differentiation was most distinct in MSCs from bone marrow, while MSCs from umbilical cord blood and tissue showed only weak evidence of differentiation (day 21: p < 0.01; day 35: p < 0.05). In contrast, following chondrogenic differentiation, MSCs from umbilical cord blood scored highest and MSCs from bone marrow scored lowest (p < 0.05). Collagen 1A2 was most highly expressed in MSCs from adipose tissue, highest scleraxis expression levels were found in MSCs from umbilical cord blood. MSCs from tendon tissue, however, expressed both markers at almost evenly high levels. Contrastingly, lowest expression levels of both markers were found in MSCs derived from bone marrow (p < 0.05 for collagen 1A2). Based on the results of the clinical study, MSC therapy can still be considered a very promising treatment option for tendon diseases and is also a suitable treatment for suspensory ligament injuries. In the future, controlled clinical studies will have to further confirm the efficacy of this treatment regime. The in-vitro-study showed significant differences between equine MSCs derived from different sources, which should be considered when choosing a MSC source for clinical applications. For autologous therapies, MSCs derived from adipose tissue appear to be a good alternative to MSCs derived from bone marrow, due to their remarkable proliferation and reliable differentiation capacities. Furthermore, according to this study, MSCs derived from tendon tissue are especially suitable for treating tendon injuries. Prior to routine clinical applicability of these MSCs, however, their properties should be further investigated.

Células reticulares fibroblásticas (FRCs, gp38+ e CD31-) e células duplo negativas (DNCs, gp38- e CD31-) são células estromais encontradas em órgãos linfoides secundários, como linfonodos. Enquanto as FRCs têm sido amplamente estudadas, pouco se sabe ainda sobre DNCs. Apesar da função estrutural das FRCs nos linfonodos já estar bem estabelecida, estudos recentes indicam que as FRCs também desempenham um papel fundamental em processos imunológicos, por exemplo, migração celular, ativação e qualidade da resposta imune, além da participação na tolerância periférica. Outra célula estromal em constante estudo são as células-tronco mesenquimais (CTMs), principalmente encontradas na medula óssea. Estas células compartilham similaridades, como por exemplo; são células estromais encontradas em órgãos linfoides, apresentam morfologia e características semelhantes quando cultivadas in vitro e estão envolvidas na resposta imune por mecanismos semelhantes. As CTMs são provenientes de um órgão linfoide primário, cuja função principal não está relacionada à resposta imunológica, entretanto, de acordo com inúmeros trabalhos, estas células possuem capacidade de interferir na ativação de várias células do sistema imunológico. Portanto, nossa hipótese é de que as FRCs e DNCs, que se encontram em um órgão linfoide secundário, cuja função principal remete a resposta imunológica, apresentem também um papel regulador, descrito na literatura como tolerância periférica e contração de uma resposta imunológica já estabelecida. Em nosso estudo mostramos que FRCs e DNCs foram isoladas a partir de linfonodos humanos e devidamente caraterizadas. Evidenciamos que FRCs e DNCs atendem todos os critérios mínimos propostos pela sociedade internacional de terapia celular para serem consideradas células-tronco estromais. Além disso, mostramos que FRCs e DNCs influênciam a proliferação e a expressão de moléculas de homing em linfócitos alogênicos in vitro. Portanto, contribuimos de forma inédita para o entendimento funcional das FRCs e DNCs, visto que estudos em humanos envolvendo estas células são escassos / Fibroblastic reticular cells (FRCs, gp38+ e CD31-) and double-negative cells (DNCs, gp38- e CD31-) are stromal cells found in secondary lymphoid organs, such as lymph nodes. While the FRCs has been widely studied, little is known about DNCs. Despite the structural function of FRCs on lymph nodes is well established, recent studies indicate that FRCs also play a key role in immunological processes, for example, cell migration, immune response activation and quality, beyond their involvement in peripheral tolerance. Another stromal cell type in constant study are mesenchymal stem cells (MSCs), mainly found in bone marrow. These cells share similarities with FRCs and DNCs, for example; they are estromal cells found in lymphoid organs, they present similar morphology and characteristics when cultured in vitro and they are involved in the immune response by similar mechanisms. MSCs are derived from a primary lymphoid organ which the major function is not related to immune response, but according to numerous studies these cells have the capacity of the interfere on activation of various immune cells. Consequently, our hypothesis is that FRCs and DNCs, usually found in secondary lymphoid organ, display immune regulatory roles, which were described in the literature as peripheral tolerance and immune response contraction. In our study we showed that FRCs and DNCs were isolated from human lymph nodes and adequately characterized. We evidenced that FRCs and DNCs meet all minimum criteria proposed by the International Society of Cell Therapy to be considerate a stromal stem cell. Therefore, we contributed in an unpublished manner to the functional understanding of FRCs and DNCs, since human studies involving these cells are scarce

INTRODUÇÃO: As células-tronco mesenquimais são capazes de regenerar diferentes tipos de tecidos, no entanto, a maioria dos métodos para sua obtenção são invasivos. Recentemente, foi descoberta a existência destas células no sangue menstrual. OBJETIVO: Padronizar as técnicas de coleta e transporte do fluido menstrual, bem como a caracterização, isolamento, expansão e criopreservação de células-tronco do fluido menstrual e avaliar a disponibilidade de células tronco mesenquimais no fluido menstrual. MÉTODOS: No período de agosto de 2011 a março de 2012 foram selecionadas 20 voluntárias com ciclo menstrual regular, sem doença ginecológica. O fluido menstrual foi coletado no dia de maior fluxo e submetido a imunofenotipagem e cultivo celular. Foram realizadas duas passagens em meio de cultura até atingir semi-confluência das células-tronco, as quais foram, em seguida, criopreservadas. RESULTADOS: Os parâmetros analisados apresentaram os seguintes valores médios: volume de fluido menstrual 6,90±5,60mL; tempo de transporte 17,20±5,50h; número de células totais 3,95 x106±3,88 x106 com 76,05%±24,57 de células viáveis. Após a cultura, as células mesenquimais aumentaram de 0,14%±0,26 para 96,19%±2,14. Na primeira passagem de cultura, após 15 a 21 dias, as colônias formaram grupos que atingiram a confluência, que a partir da segunda passagem ocorreu em cerca de 3 dias. As células-tronco mesenquimais criopreservadas eram viáveis. CONCLUSÃO: As células-tronco do fluido menstrual podem ser obtidas sem métodos invasivos. O fluido menstrual pode ser transportado em condições ideais de temperatura até 24 horas após a coleta. As células tronco mesenquimais podem ser caracterizadas por imunofenotipagem, isoladas, cultivadas e expandidas e, em seguida, criopreservadas. O fluido menstrual contém células tronco mesenquimais viáveis e apropriadas para cultivo / INTRODUCTION: Mesenchymal stem cells may renovate different tissues, but techniques to obtain these cells are invasive. Recently, those cells were detected in menstrual blood. OBJECTIVE: Patterning techniques of collection, transportation, characterization, isolation, expansion and cryopreservation of stem cells in menstrual fluid. METHODS: From August 2011 to March 2012 twenty volunteers were selected with regular menstrual cycle without gynecological diseases. They collected menstrual fluid on the most intense flux day to analysis by immunophenotyping and cellular culture. Culture was made in 2 stages until reached semi-confluence of stem cells and these cells were cryopreserved. RESULTS: Average of menstrual fluid volume was 6,90±5,60mL, transportation time was 17,20±5,50h, and total number of cells was 3,95 x106±3,88 x106 witch 76,05%±24,57 were viables. After culture, mesenchymal stem cells increased from 0,14%±0,26 to 96,19%±2,14. After 15 to 21 days of culture in first passage, colonies formed clusters that reached confluence. In second passage, it happens after 3 days of culture and stem cells were cryopreserved. CONCLUSION: Stem cells of menstrual fluid may be easily obtained without invasive methods. Menstrual fluid can be transported in good conditions of temperature up to 24 hours of collection. Mesenchymal stem cells of menstrual fluid may be characterized by immunophenotyping, as well as it is possible to isolated, cultivate and cryopreserved them. Menstrual fluid has viable and proper for culture mesenchymal stem cells

Le tissu osseux a la capacité de se régénérer suite à une fracture. Cependant, des consolidations incomplètes concernent aujourd’hui environ un million de personnes par an. L’approche d’ingénierie tissulaire associant des cellules stromales mésenchymateuses (CSM) à des biomatériaux émerge comme une stratégie prometteuse pour réparer ces défauts. Par ailleurs, l’efficacité de la régénération osseuse semble varier en fonction du donneur mais aussi du support associé. L’objectif de mon travail de thèse a été de comprendre les mécanismes à l’origine de ces variabilités. Pour cela, nous nous sommes intéressés à deux mécanismes : (i) l’impact des biomatériaux sur le devenir et les fonctions des CSM et (ii) l’impact de l’hétérogénéité inter-donneur des CSM sur les mécanismes moléculaires in vitro et in vivo, à l’origine des différences du potentiel ostéoformateur de ces cellules.Dans un premier temps, deux types de biomatériaux largement utilisés en clinique ont été comparés dans un modèle murin de greffe ectopique : une céramique biphasique d’hydroxyapatite/béta-tricalcium-phosphate (HA/bTCP) et une matrice osseuse humaine gamma-irradiée (Tutoplast® process Bone [TPB]). Nos résultats ont montré une meilleure formation osseuse lorsque les CSM sont combinées au TPB par rapport au HA/bTCP. Ceci est associé à une meilleure adhésion des CSM in vitro et in vivo ainsi qu’à une différentiation ostéoblastique supérieure sur le TPB. La contribution directe des CSM à former l’os est associée à un effet paracrine sur la chémoattraction et/ou la différentiation ostéogénique des cellules de l’hôte. Cet effet est indépendant des chimiokines PDGF et SDF-1 mais semble être régulé par la voie d’IGF-1. Un autre effet paracrine des CSM est observé sur l’activité ostéoclastique, qui est plus importante sur la céramique HA/bTCP et qui semble être régulée par le facteur RANKL. L’augmentation de l’activité ostéoclastique pourrait être à l’origine d’un déséquilibre de la balance résorption/formation osseuse sur le HA/bTCP.Ainsi, nos résultats montrent que le support impacte la persistance des CSM au niveau du site de la greffe ainsi que leur rôles directs et paracrines, l’ensemble étant à l’origine d’une variabilité de la formation osseuse.Cette formation osseuse a été observée avec seulement 70% des donneurs de CSM. Nous avons donc décidé dans la deuxième partie de ce projet d’évaluer les différences entre ces deux groupes de donneurs. In vitro aucune différence de prolifération, de différentiation ou de phénotypie des CSM n’a été observée. De plus, les analyses transcriptomique et sécrétomique réalisées in vitro n’ont montré aucune variabilité entre les deux groupes. In vivo, la persistance des CSM sur le site de la greffe est donneur dépendante et n’est pas liée à un défaut de vascularisation ou à une mort cellulaire par apoptose augmentée. Par ailleurs, nos résultats soutiennent l’existence d’une corrélation entre la formation osseuse et la capacité des CSM greffées à adhérer au support, survivre ainsi qu’à participer à la formation osseuse via une action directe et un effet paracrine.En conclusion, ce travail montre que l’efficacité de la formation osseuse dépend du devenir et des fonctions des CSM greffées. L’origine (donneur) ainsi que le microenvironnement (support associé) impactent l’adhésion, la survie et les mécanismes d’action de ces cellules au cours de la régénération osseuse. De plus, nous avons constaté que le potentiel ostéogénique des CSM est dû à leur participation directe à former l’os qui est synergique à un effet paracrine de chémoattraction et/ou de différentiation ostéogénique des cellules de l’hôte. / Despite bone capacity to regenerate after injury, incomplete consolidation concerns 1 million persons per year. Bone tissue engineering involving human bone marrow mesenchymal stromal cells (hBMSCs) loaded on biomaterials emerges as a new strategy to repair large bone defects. Nevertheless, efficacy of bone regeneration seems variable depending on the donor as well as on the associated scaffold. The aim of my PhD work was to understand mechanisms responsible for these variabilities. To this end, we focused on two objectives: (i) the impact of biomaterials on hBMSCs behavior and (ii) the impact of hBMSCs heterogeneities on molecular mechanisms in vitro and in vivo, resulting in variable bone-forming potential of these cells.First, two scaffolds widely used clinically were compared in an ectopic mouse model: the synthetic hydroxyapatite/beta-tricalcium-phosphate bioceramic (HA/βTCP) and the gamma-irradiated-processed human bone allograft (Tutoplast® Process Bone [TPB]). Our results showed that bone formation is higher when hBMSCs are loaded on TPB compared to HA/bTCP. This was correlated to a better hBMSCs adhesion in vitro as well as in vivo and to their higher osteoblastic differentiation on TPB. The direct participation of hBMSCs to form bone was associated to a paracrine effect of hBMSCs by inducing host cell chemoattraction and/or osteogenic differentiation, mediated probably by the IGF-1 pathway but independently from PDGF or SDF-1. Another paracrine effect was also observed on osteoclastic activity which was more important on HA/bTCP and could be RANKL dependent. This may impact the bone resorption/formation balance. Taken together, our results show that the associated scaffold impact MSCs persistence on the graft site, as well as their direct and paracrine effects leading to a variability in new bone amount.As bone formation was observed with only 70% of our donors, we then evaluated differences between the two groups. In vitro, no differences in cell proliferation, differentiation or phenotype were detected. Furthermore, transcriptomic and secretomic analysis in vitro did not identify any variability. The assessment of cell behavior in vivo showed that persistence of grafted cells was donor dependent and was not linked to a vascularization failure or a higher apoptosis. However, our results highlighted a correlation between bone formation and the ability of hBMSCs to attach and survive on the biomaterial as well as to contribute to bone formation by direct and paracrine effects.In conclusion, this work show that the efficacy of bone formation depend on the behavior of grafted hBMSCs. The origin (donor) and the microenvironment (associated scaffold) will impact their adhesion, survival and mechanisms of action during bone regeneration. Moreover, we identified that the osteogenic potential of hBMSCs act through a direct contribution that is synergic to a paracrine effect for host cell chemoattraction and differentiation.

Grâce à leurs propriétés de différenciation, les cellules stromales mésenchymateuses (CSM) constituent aujourd’hui un outil en médecine régénérative. La moelle osseuse reste une des plus utilisées. Une diminution de la capacité de prolifération et de différenciation des CSM-MO, au cours des passages, a été montrée. En parallèle, certaines études montrent que l'impact de l'âge du donneur sur les propriétés de CSM-MO reste encore controversé. Le but de notre étude était de mieux comprendre l'effet de l'âge du donneur mais aussi des passages en culture sur la capacité de prolifération et de différenciation des CSM de moelle osseuse. Les échantillons ont été séparés en 4 groupes en fonction de l’âge des donneurs (<20 ans; 20-40 ans; 40-60 ans; >60 ans) et les analyses ont été réalisés lors de la culture de cellules pendant 5 passages. Les résultats obtenus montrent que la capacité de prolifération de CSM-MO obtenues à partir de donneurs jeunes est supérieure à celle de cellules des donneurs âgés. De plus, cette capacité de prolifération diminue en fonction des passages en culture. En parallèle, la capacité des cellules à former des colonies, mesurée par le test CFU-F, diminue légèrement en fonction de l’âge des donneurs mais de façon importante en fonction du passage. Enfin, la capacité de différenciation des CSM-MO vers les trois types cellulaires étudiés, diminue en fonction des passages de cellules mais également en fonction de l’âge des donneurs. Notre étude montre que les propriétés des CSM issues de moelle osseuse sont modifiées lors de l’amplification in vitro mais aussi en fonction de l’âge des donneurs / Today with their properties of differentiation into specific cells types, mesenchymal stromal cells (MSC) can be used in regenerative medicine. Bone marrow (BM) is the better characterized one. The researchers have proven that with increasing passage number in culture the proliferation and differentiation potential of MSC decrease. In parallel many researchers have showed the impact of donor age on MSC properties remains controversial. The aim of our study was to better understand the effect of donor age but also culture passages on the proliferation and differentiation ability of bone marrow mesenchymal stromal cells. The samples were separated into 4 groups depending on the donor age (<20 years; 20-40 years; 40-60 years; > 60 years) and The samples were cultured for 5 passages. The results obtained show that the MSC proliferative capacity obtained from young donors is greater than that of cells from older donors. In addition, the proliferative capacity decreases with increasing passage number in culture. In parallel, the ability of colony-forming unit-fibroblast, measured by the CFU-F assay, decreases slightly depending on the age of the donors but significantly depending on the passage. Finally, the MSC differentiation ability decreases according to the passage of the cells but also depending on the donor age. Our study shows that the properties of bone marrow derived MSC are modified not only during amplification in vitro but also in terms of donor age

Diese Arbeit verdeutlicht die Wirkung von Chondroitinsulfat auf die Synthese von Prostaglandin E2 in humanen mesenchymalen Stromazellen in Abhängigkeit ihres Sulfatierungsgrades. MSC zeichnen sich durch ihre antiinflammatorischen Eigenschaften aus und haben damit einen modulierenden Effekt auf Wundheilungsprozesse. Als Vorläuferzellen von Osteoblasten sind sie direkt an der Knochenneubildung beteiligt. Eine persistierende Entzündung hat eine kontinuierliche Freisetzung von Zytokinen, wie IL-1 zur Folge. Es konnte gezeigt werden, dass IL-1 in hMSC zu einer Freisetzung von PGE2 führt. Unter kurzzeitiger Wirkung stimuliert PGE2 die Knochenneubildung. Eine langanhaltende Präsenz leitet dagegen die Bildung des Faktors RANKL, einen die Osteoklastogenese stimulierenden Faktor, ein. Seit langem ist der positive Effekt von Chondroitinsulfat in chronischen Entzündungsprozessen, wie Rheumatoider Arthritis, bekannt. Zudem werden sie in aktuellen Studien als Beschichtungsbestandteile von Knochenimplantaten verwendet. Sie führten hier zu einer besseren Bioinduktivität und Biokonduktivität. Bisher ist dennoch der molekulare Wirkmechanismus nicht genau beschrieben. Die Schwierigkeit besteht darin, dass die molekularen Signalkaskaden für die einzelnen Kulturmoldelle Unterschiede aufweisen und kein ubiquitärer Mechanismus dargestellt wird. In hMSC führte die Stimulation mit IL-1 unter vorheriger Zugabe von Chondroitinsulfat zu einer Reduktion der PGE2 Freisetzung. Der Effekt des hochsulfatierten sCS3 war gegenüber dem nativen C4S verstärkt. Die reduzierende Wirkung von C4S setzte verzögert ein. Es ist bereits bekannt, dass die negative Ladung der CS zu einer Bindung von Zytokinen führt. Dadurch wird eventuell die Konzentration der Zytokine, wie IL-1 im Bereich der Zellrezeptoren erniedrigt und führt zu einer verringerten Stimulation der Zelle. Denkbar ist auch die Beeinflussung der intrazellulären Signaltransduktionskaskade durch die Bindung der CS an einen speziellen, bisher unbekannten, Membranrezeptor. Die entscheidenden Enzyme der PGE2 Synthese sind die Cyclooxygenase-2 (Cox-2) und die mikrosomale Prostaglandin E Synthase 1 (mPGES1). Die mRNA beider Enzyme war unabhängig vom Sulfatierungsgrad der CS reduziert. Dieser Effekt konnte auf Protein-ebene nicht belegt werden. Die produzierte Proteinmenge an mPGES1 wird durch IL-1 induziert, bleibt aber auch durch Zugabe von CS unverändert. Somit kann von einer erhöhten Translationseffizient und mRNA Stabilität der mPGES1 RNA ausgegangen werden. MAPK Kinasen sind entscheidende Schnittstellen bei der Regulation der mRNA Stabilität als auch der Aktivität von Transkriptionsfaktoren. In dieser Studie konnte die MAPK p38 als entscheidendes Enzym bei der Wirkung von CS auf die PGE2 Synthese ermittelt werden. Dabei führten sowohl das natürliche C4S als auch das hochsulfatierte sCS3 zu einer verringerten Aktivierung. Der Transkriptionsfaktor NfkB ist einer von mehreren, die an den Promotorbereichen der beiden induzierbaren PGE2 Enzyme, Cox-2 und mPGES1, binden. Es ist anzunehmen, dass die hier aufgezeigte verringerte Aktivität von NfkB als auch die verhinderte Translokation in den Zellkern eine reduzierte Transkription der jeweiligen mRNA bedingten. Abhängig vom untersuchten Modell und den verwendeten Kulturbedingungen können diese Prozesse moduliert sein. Die Erkenntnisse dieser experimentellen Arbeit liefern einen weiteren wichtigen Baustein zum Verständnis der molekularbiologischen Abläufe während entzündlicher Prozesse. Die Verwendung von Chondroitinsulfat, insbesondere hochsulfatiertes CS, in Kombination mit hMSC kann gezielt zu einer Verringerung der Entzündungsreaktion während der Implantateinheilung führen. Die durch PGE2 hervorgerufenen Symptome, wie erhöhte Gefäßpermeabilität, Schwellung und verstärktes Schmerzempfinden begründen diese positiven Effekte.

No description available.

Mesenchymal stromal cells (MSC) represent a promising treatment option for tendon disorders and joint diseases, primarily osteoarthritis. Since MSC are highly context-sensitive to their microenvironment, their therapeutic efficacy is influenced by their tissue-specific pathologically altered targets. These include not only cellular components, such as resident cells and invading immunocompetent cells, but also components of the tissue-characteristic extracellular matrix. Although numerous in vitro models have already shown potential MSC-related mechanisms of action in tendon and joint diseases, only a limited number reflect the disease-specific microenvironment and allow conclusions about well-directed MSC-based therapies for injured tendon and joint-associated tissues. In both injured tissue types, inflammatory processes play a pivotal pathophysiological role. In this context, MSC-mediated macrophage modulation seems to be an important mode of action across these tissues. Additional target cells of MSC applied in tendon and joint disorders include tenocytes, synoviocytes as well as other invading and resident immune cells. It remains of critical importance whether the context-sensitive interplay between MSC and tissue- and disease-specific targets results in an overall promotion or inhibition of the desired therapeutic effects. This review presents the authors’ viewpoint on disease-related targets of MSC therapeutically applied in tendon and joint diseases, focusing on the equine patient as valid animal model.

Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine adipose-derived (AD-) MSC culture so far, as it supported AD-MSC proliferation and basic characteristics. The aim of the current study was to further analyze the functional properties of equine AD-MSC cultured with the same ePL, focusing on cell fitness, genetic stability and pro-angiogenic potency. All experiments were performed with AD-MSC from n = 5 horses, which were cultured either in medium supplemented with 10% FBS, 10% ePL or 2.5% ePL. AD-MSC cultured with 2.5% ePL, which previously showed decreased proliferation potential, displayed higher apoptosis but lower senescence levels as compared to 10% ePL medium (p < 0.05). Non-clonal chromosomal aberrations occurred in 8% of equine AD-MSC cultivated with FBS and only in 4.8% of equine AD-MSC cultivated with 10% ePL. Clonal aberrations in the AD-MSC were neither observed in FBS nor in 10% ePL medium. Analysis of AD-MSC and endothelial cells in an indirect co-culture revealed that the ePL supported the pro-angiogenic effects of AD-MSC. In the 10% ePL group, more vascular endothelial growth factor (VEGF-A) was released and highest VEGF-A concentrations were reached in the presence of ePL and co-cultured cells (p < 0.05). Correspondingly, AD-MSC expressed the VEGF receptor-2 at higher levels in the presence of ePL (p < 0.05). Finally, AD-MSC and 10% ePL together promoted the growth of endothelial cells and induced the formation of vessel-like structures in two of the samples. These data further substantiate that buffy-coat-based ePL is a valuable supplement for equine AD-MSC culture media. The ePL does not only support stable equine AD-MSC characteristics as demonstrated before, but it also enhances their functional properties.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellularmatrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of Nacetyl- galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.