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.

Cartilage regeneration is one of the most widely studied areas in tissue-engineering. Despite significant progress, most efforts to date have only focused on generating homogenous tissues whose bulk properties are similar to articular cartilage. However, anatomically and functionally, articular cartilage consists of four spatially distinct regions: the superficial, transitional, deep, and calcified zones. Each zone is characterized by unique extra-cellular matrix (ECM) compositions, mechanical properties, and cellular organization. The ECM is primarily composed of type II collagen and glycosaminoglycans (GAGs), whose relative concentrations vary between zones and therefore lead to distinctive mechanical properties. One of the major unsolved challenges in engineering cartilage has been the inability to regenerate tissue that mimics the zonal architecture of articular cartilage. Recent studies have attempted to imitate this spatial organization using zone-specific chondrocytes isolated from donor animal cartilage. Directed differentiation of a single stem population into zonally organized native-like articular cartilage has not yet been reported. This dissertation reports that hydrogels, incorporating both synthetic and natural polymers as well as cell-induced degradability, are suitable for generating zone-specific chondrogenic phenotypes from a single MSC population. Specifically, cues provided from the unique combinations of chondroitin sulfate (CS), hyaluronic acid (HA), and MMP-sensitive peptide (MMP-pep) within a PEG-based hydrogel, direct the chondrogenic differentiation of MSCs. The findings of this dissertation demonstrate the capability of creating native-like and mechanically relevant articular cartilage consisting of zone specific layers. This ability provides a new direction in cartilage tissue engineering and could be invaluable for cartilage repair if incorporated with current minimally invasive surgical techniques. / text

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mesenchymal stromal cells (MSC) are bone marrow-derived, non-hematopoietic progenitors that are amenable to genetic engineering, making them attractive delivery vehicles for therapeutic proteins. However, limited transplanted cell survival compromises the efficacy of MSC-based gene therapy. We hypothesized that co-implantation of insulin-like growth factor-1 (IGF-I)-overexpressing MSC (MSC-IGF) would improve MSC-based therapy of anemia by providing paracrine support to erythropoietin (EPO)-secreting MSC (MSC-EPO). Murine MSC were found to express the IGF-I receptor and be responsive to IGF-I stimulation. IGF-I also improved MSC survival in vitro. MSC were admixed in a bovine collagen matrix and implanted by subcutaneous injection in a murine model of chronic renal failure. Mice receiving MSC-EPO co-implanted with MSC-IGF experienced a greater and significantly sustained elevation in hematocrit compared to controls; heart function was also improved. Co-implantation of MSC-IGF therefore represents a promising new strategy for enhancing implanted cell survival, and improving cell-based gene therapy of renal anemia.

Orientador: Teresa Cristina Cardoso da Silva / Banca: Roberto Gameiro de Carvalho / Banca: Andréa Fontes Garcia / Resumo: As células mesenquimais derivadas da geleia de Wharton isoladas da matriz do cordão umbilical canino tem sido sugeridas como uma fonte promissora de MSCs para serem usadas nas aplicações clínicas em ciência veterinária, como uma ferramenta potencialmente efetiva na regeneração óssea. MicroRNA (miARN) é um regulador pós-transcricional da expressão gênica em várias condições fisiológicas, incluindo a osteogênese. Neste estudo, as MSCs caninos (cMSCs) isoladas da geléia de Wharton foram induzidos a osteogênese e a transcrição de miR-106b foi avaliada em 0, 7, 14 e 21 dias após a indução. Em outro experimento, as cMSC foram transfectadas com um imitador de miR106b e um inibidor e induzidos a osteogênese. Morfologicamente, cMSCs transfectadas com um inibidor de miR-106b produziram células semelhantes a osteócitos quando comparadas às mesmas células transfectadas com o mímico de miR-106b. cMSCs apresentaram transcrição de miR-106b após 7 dias de osteoindução em um nível baixo em comparação com o controle positivo, enquanto as células transfectadas com o mímico de miR-106b mostraram que o miR-106b deveria ser regulado positivamente. Após a inibição da expressão de miR-106b em cMSCs osteoinduzidas, a atividade da fosfatase alcalina (ALP) foi aumentada. A transcrição do mRNA de osteocalcina, osteopontina e RUNX2 foi regulada positivamente aos 21 dias após a osteoindução, após a inibição de miR106b. Esses achados, pela primeira vez, mostraram que a expressão de miR106b regula negativam... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Wharton's jelly derived-MSCs isolated from canine umbilical cord matrix have been suggested as a promising source of MSCs to be used for clinical applications in veterinary science, as a potentially effective tool in bone regeneration. MicroRNA (miRNA) is a post-transcriptional regulator of gene expression in several physiological conditions, including osteogenesis In this study, canine MSCs (cMSCs) isolated from Wharton's jelly were induced to osteogenesis and miR-106b transcription was measured at 0, 7, 14 and 21 days following induction. In another experiment, cMSCs were transfected with a miR106b mimic and an inhibitor and induced to osteogenesis. Morphologically, cMSCs transfected with an inhibitor of miR-106b appeared as osteocyte-like cells when compared to the same cells transfected with the mimic of miR-106b. cMSCs showed miR-106b transcription after 7 days of osteoinduction was at a low level compared to the positive control, whereas transfected cells with the miR-106b mimic showed miR-106b to be upregulated. After inhibition of miR-106b expression in osteoinduced cMSCs, alkaline phosphatase (ALP) activity was increased. Osteocalcin, osteopontin and RUNX2 mRNA transcription were upregulated at 21 days after osteoinduction following miR-106b inhibition. These findings have, for the first time, shown that the expression of miR-106b negatively regulates osteogenesis in canine MSCs derived from Wharton's jelly and seems to interfere with cell differentiation. / Mestre

Submitted by João Paulo Ignacio Gonzaga null (jp-ignaciogonzaga@hotmail.com) on 2017-08-30T00:35:08Z No. of bitstreams: 2 Dissertação João Paulo 2017.pdf: 958263 bytes, checksum: 7ef6cdf74470df5e3235cc2a1783d5ea (MD5) Dissertação João Paulo 2017.pdf: 958263 bytes, checksum: 7ef6cdf74470df5e3235cc2a1783d5ea (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido está sem a ficha catalográfica e sem o certificado de aprovação. A versão submetida por você é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração em seu conteúdo após a aprovação. Corrija esta informação e realize uma nova submissão contendo o arquivo correto. Agradecemos a compreensão. on 2017-08-30T17:26:52Z (GMT) / Submitted by João Paulo Ignacio Gonzaga null (jp-ignaciogonzaga@hotmail.com) on 2017-08-31T18:19:05Z No. of bitstreams: 1 dissertação João Paulo 2017 Pronta.pdf: 1042134 bytes, checksum: 67115cf2cf469d731be2911085bc6b7a (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-01T13:24:56Z (GMT) No. of bitstreams: 1 gonzaga_jpi_me_araca.pdf: 1042134 bytes, checksum: 67115cf2cf469d731be2911085bc6b7a (MD5) / Made available in DSpace on 2017-09-01T13:24:56Z (GMT). No. of bitstreams: 1 gonzaga_jpi_me_araca.pdf: 1042134 bytes, checksum: 67115cf2cf469d731be2911085bc6b7a (MD5) Previous issue date: 2017-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As células mesenquimais derivadas da geleia de Wharton isoladas da matriz do cordão umbilical canino tem sido sugeridas como uma fonte promissora de MSCs para serem usadas nas aplicações clínicas em ciência veterinária, como uma ferramenta potencialmente efetiva na regeneração óssea. MicroRNA (miARN) é um regulador pós-transcricional da expressão gênica em várias condições fisiológicas, incluindo a osteogênese. Neste estudo, as MSCs caninos (cMSCs) isoladas da geléia de Wharton foram induzidos a osteogênese e a transcrição de miR-106b foi avaliada em 0, 7, 14 e 21 dias após a indução. Em outro experimento, as cMSC foram transfectadas com um imitador de miR106b e um inibidor e induzidos a osteogênese. Morfologicamente, cMSCs transfectadas com um inibidor de miR-106b produziram células semelhantes a osteócitos quando comparadas às mesmas células transfectadas com o mímico de miR-106b. cMSCs apresentaram transcrição de miR-106b após 7 dias de osteoindução em um nível baixo em comparação com o controle positivo, enquanto as células transfectadas com o mímico de miR-106b mostraram que o miR-106b deveria ser regulado positivamente. Após a inibição da expressão de miR-106b em cMSCs osteoinduzidas, a atividade da fosfatase alcalina (ALP) foi aumentada. A transcrição do mRNA de osteocalcina, osteopontina e RUNX2 foi regulada positivamente aos 21 dias após a osteoindução, após a inibição de miR106b. Esses achados, pela primeira vez, mostraram que a expressão de miR106b regula negativamente a osteogênese em MSCs caninos derivados da geléia de Wharton e parece interferir na diferenciação celular. / Wharton`s jelly derived-MSCs isolated from canine umbilical cord matrix have been suggested as a promising source of MSCs to be used for clinical applications in veterinary science, as a potentially effective tool in bone regeneration. MicroRNA (miRNA) is a post-transcriptional regulator of gene expression in several physiological conditions, including osteogenesis In this study, canine MSCs (cMSCs) isolated from Wharton´s jelly were induced to osteogenesis and miR-106b transcription was measured at 0, 7, 14 and 21 days following induction. In another experiment, cMSCs were transfected with a miR106b mimic and an inhibitor and induced to osteogenesis. Morphologically, cMSCs transfected with an inhibitor of miR-106b appeared as osteocyte-like cells when compared to the same cells transfected with the mimic of miR-106b. cMSCs showed miR-106b transcription after 7 days of osteoinduction was at a low level compared to the positive control, whereas transfected cells with the miR-106b mimic showed miR-106b to be upregulated. After inhibition of miR-106b expression in osteoinduced cMSCs, alkaline phosphatase (ALP) activity was increased. Osteocalcin, osteopontin and RUNX2 mRNA transcription were upregulated at 21 days after osteoinduction following miR-106b inhibition. These findings have, for the first time, shown that the expression of miR-106b negatively regulates osteogenesis in canine MSCs derived from Wharton´s jelly and seems to interfere with cell differentiation.

Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T08:29:05Z (GMT). No. of bitstreams: 1 Torello_CristianeOkuda.pdf: 8596128 bytes, checksum: e07457446c814c5f191e0c629491876d (MD5) Previous issue date: 2009 / Resumo: Neste trabalho, investigamos os efeitos do extrato padronizado de Rhodiola rosea L. (ERR) sobre a resposta imunohematopoética de camundongos infectados com Listeria monocytogenes. Os resultados demonstram que o ERR aumenta a resistência dos animais frente uma dose letal de Listeria quando administrado profilaticamente nas doses de 100 e 250 mg/kg por sete dias consecutivos anteriores à inoculação. Paralelamente, reversão da mielossupressão induzida pela infecção, concomitante ao aumento na atividade de células natural killers (NK) e na produção das citocinas TNF-a, IFN-? e fatores estimuladores de colônias foram observados. Os resultados obtidos demonstram que o ERR compartilha da habilidade de regular positivamente os desequilíbrios hematopoiéticos e imunológicos envolvidos nos estágios iniciais da infecção com L. monocytogenes. Estes efeitos podem ser atribuídos à recuperação no equilíbrio da resposta hematopoiética através da produção de IL-1a e IL-6 pelas células estromais no microambiente medular e também da produção de fatores estimuladores de colônias a partir das 24 horas de infecção, promovendo aumento no número de progenitores de macrófagos e granulócitos na medula óssea. Além disso, a eficácia do ERR também depende do aumento na produção das citocinas TNF-a e IFN-?, do aumento na atividade funcional das células NK e polarização da resposta celular para Th1. Juntos, estes efeitos contribuem para o aumento na resistência a L. monocytogenes. / Abstract: In this work, we have investigated the effects of Rhodiola rosea L. extract (ERR) in Listeria monocytogenes infected mice. Our results demonstrated that ERR protects mice from a lethal dose of L. monocytogenes, when administered prophylactically at 100 and 250 mg/kg, for seven consecutive days prior to the infection. In addition, prevention of myelosuppression induced by infection, concomitant to increasing natural killers (NK) cells activity and production of TNF-a, IFN-? and colony-stimulating factors were observed. The results showed that ERR share the ability of regulating positively the hematopoietic and immunological unbalance involved in the initial stages of infection with this pathogen. These effects could be attributed to recovering of the hematopoietic response through the production of IL-1a and IL-6 by stromal cells in the bone marrow microenvironment and the production of colony-stimulating factors at 24 hours of infection, promoting an increase in the number of granulocytes and macrophages progenitors in the bone marrow microenvironment. Moreover, the efficacy of ERR depends on high levels of TNF-a and IFN- ?, increased NK cells activity and polarization to Th1 response. Together, these effects contribute to increasing resistance to L. monocytogenes. / Universidade Estadual de Campi / Farmacologia / Doutor em Farmacologia

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

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T22:22:55Z (GMT). No. of bitstreams: 1 Baratti_MarianaOzello_D.pdf: 9890862 bytes, checksum: f7eb8503519cc264f616a79dc48baf4f (MD5) Previous issue date: 2011 / Resumo: As síndromes mielodisplásicas (SMDs) constituem um grupo heterogêneo de desordens hematopoéticas, caracterizadas por exibirem hematopoese ineficaz com evidências de displasia da medula óssea resultando em citopenias no sangue periférico. Tecnologia de microarranjo tem permitido o refinado mapeamento da atividade transcricional do genoma humano. RNAs não codificadores (ncRNAs) transcritos de regiões intrônicas de genes conhecidos estão envolvidos em vários processos relacionados com controle transcricional e pós-transcricional da expressão gênica, interações com cromatinas, modificação de histonas e também estão se tornando evidentes em vários tipos de cânceres. Caracterização de ncRNAs em células progenitoras e células estromais de pacientes com SMD representa uma estratégia aparentemente importante para o entendimento da regulação gênica nesta doença. Neste estudo, o perfil de expressão gênica de células CD34+ e estromais de pacientes com SMD do subgrupo anemia refratária com sideroblastos em anel (ARSA) foi comparado com o de indivíduos saudáveis, usando oligoarranjos de 44 kilobases contendo íntrons e éxons, o qual incluiu sequências para genes codificadores, RNAs sense e antisense totalmente e parcialmente intrônicos. Em células CD34+ de pacientes com SMD-ARSA, 216 genes foram diferencialmente expressos (q-value ? 0,01) em comparação com indivíduos saudáveis, dos quais 65 (30%) eram transcritos não codificadores. O gene DMT1, um transportador de ferro, foi encontrado hiperexpresso em células CD34+ de SMD-ARSA. Em medula óssea total de 34 pacientes, a expressão foi mais evidente no subgrupo de pacientes com SMD de baixo risco/INT-1. Ensaios de imuno-histoquímica corroboram os dados encontrados na análise de expressão gênica e demonstram que DMT1 se encontra mais expresso nas células eritroblásticas. A hiperexpressão de DMT1 pode estar relacionada com o homeostase do ferro anormal nas SMDs. Em células estromais de SMD-ARSA, 12 genes foram diferencialmente expressos (q-value ? 0,05) em comparação com indivíduos saudáveis, dos quais 3 (25%) eram transcritos não codificadores. O gene SEMA3A, um membro secretado da família das semaforinas, foi encontrado hiperexpresso em células estromais de SMD-ARSA e na medula óssea total de 34 pacientes; sua hiperexpressão foi mais evidente em pacientes com SMD de alto risco/INT-2 e em pacientes com leucemia mieloide aguda (n=19). Ensaios funcionais demonstraram que SEMA3A está envolvido com aumento da adesão, diminuição da diferenciação e apoptose de células leucêmicas cocultivadas com células estromais HS27 hiperexpressando SEMA3A e age de maneira parácrina sobre as células precursoras. Pela primeira vez, o perfil diferencial de ncRNA em células CD34+ e células estromais entre SMD-ARSA e indivíduos saudáveis foi demonstrado, sugerindo que ncRNA pode ter um importante papel durante o desenvolvimento das síndromes mielodisplásicas / Abstract: Myelodysplastic syndromes (MDS) are a group heterogeneous of hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Noncoding-RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and chromatins interaction, and histone modification and they are becoming evident in several cancers. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. In this study, gene expression profiles of CD34+ and stromal cells of MDS patients with refractory anemia with ringed sideroblasts (RARS) subgroup were compared those of healthy individuals, using 44 kilobases combined introns and exons oligoarrays, which included probes for protein-coding genes, for sense and antisense strands of totally and partially intronic noncoding RNAs. In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ? 0.01) in comparison to healthy individuals, of which 65 (30%) were noncoding transcripts. The DMT1 gene, an iron-transporter, was found up-regulated in CD34+ cells of MDS-RARS. In the total bone marrow of 34 patients, the expression of DMT1 was more evident in the subgroup of low risk/INT-1 MDS patients. The immunohistochemistry assay confirms the data obtained in the gene expression assay and show that DMT1 is more expressed in erythroid cells. The higher expression of DMT1 can be related with abnormal iron homeostasis in MDS. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ? 0.05) in comparison to healthy individuals, of which 3 (25%) were noncoding transcripts. The SEMA3A gene, a secreted member of the semaphorins family, was found up-regulated in stromal cells of MDS-RARS and in the total bone marrow of 34 patients; further, the higher expression was more evident in high risk/ INT-2 subgroup of MDS patients and acute myeloid leukemia patients (n = 19). Functional assays demonstrated that SEMA3A is related to adhesion increase, differentiation decrease, and apoptosis of leukemia cells cocultivated with HS27 stromal cells higher expressing SEMA3A, also acting in a paracrine fashion in the precursors cells. These results demonstrated, for the first time, in CD34+ cells and stromal cells the differential ncRNA expression profile between MDSRARS and healthy individuals, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

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.