Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.

INTRODUÇÃO: As células-tronco hematopoiéticas (CTH) do sangue do cordão umbilical (SCU) têm sido utilizadas com sucesso para o tratamento de doenças malignas e não malignas. No entanto, algumas unidades de SCU podem apresentar baixa quantidade de células nucleadas totais (CNT). Algumas abordagens têm sido sugeridas para evitar problemas em relação à baixa concentração de CTH no transplante, como a administração de duas unidades de SCU para o paciente e a expansão ex vivo de CTH. OBJETIVO: Avaliar as taxas de proliferação celular na expansão ex vivo do SCU em sistema de cocultura com células-tronco mesenquimais (CTM) obtidos a partir de diferentes fontes com alta e baixa confluência e adicionando-se ou não coquetel de citocinas no meio de cultura. MÉTODOS: Este estudo foi aprovado pelo Comitê de Ética de Pesquisa (CAPPesq) do Hospital das Clínicas da Faculdade de Medicina da USP. A coleta do SCU (n =10) foi realizada após o nascimento do bebê e expulsão da placenta. O processamento foi realizado utilizando o método de redução de volume, o qual consiste em depleção de eritrócitos. As amostras de CTM provenientes do endotélio vascular do cordão umbilical foram obtidas de doadores diferentes (n=3) e o tecido adiposo (n=3) do inventário do LIM-31. A expansão das CNT e das células com expressão de marcadores CD133+/CD34+ foram observados depois de sete dias de cultura. Além disso, o ensaio para análise de unidades de formadoras de colônias (UFC) foi realizado em todas as amostras antes e depois da expansão do SCU. Para a expansão em sistema de cocultura foi separado dois grupos para ambas as fontes de CTM (Grupo I - cocultura com adição de coquetel de citocinas vs. Grupo II - cocultura sem citocinas). RESULTADOS: Após sete dias, no grupo I com cocultura confluente, a taxa de proliferação de CNT foi duas vezes maior ao comparar com cocultura subconfluente (35 vs. 16 vezes). No mesmo grupo também foi possível evidenciar elevada taxa de proliferação de células CD133+/CD34+. O índice de proliferação das UFC no grupo I aumentou até oito vezes. A cocultura subconfluente tanto do endotélio vascular do cordão umbilical como do tecido adiposo apresentou menor rendimento em comparação as CTM confluentes. A expansão das células na presença de citocinas apresentou maior proliferação celular ao comparar às coculturas sem adição de citocinas. CONCLUSÃO: Este estudo mostrou que para alto rendimento de células do SCU, o sistema de cocultura requer adição de coquetel de citocinas e CTM confluente independentemente da fonte utilizada / INTRODUCTION: Umbilical cord blood (UCB) hematopoietic stem cells have been successfully used for the treatment of both malignant and non-malignant diseases. Nevertheless, some UCB units could have low total nucleated cells (TNC) dose. Several approaches have been suggested to avoid inadequacy problems of hematopoietic stem cells (HSC) number for transplantation, such as administration of two UCB units to the patient and HSC ex vivo expansion. OBJECTIVE: Evaluate UCB ex vivo expansion proliferative rates in a high and low mesenchymal stem cells (MSC) confluence feeder layer obtained from different MSC sources and by adding or not cytokines cocktail into the medium. METHODS: This study was approved by the Research Ethic Committee (CAPPESQ) of Hospital das Clínicas da Faculdade de Medicina da USP. The collection of UCB (n=10) was made after delivery of the infant and the expulsion of placenta. Processing was performed using volume reduction method which consists in red blood depletion. MSC samples from umbilical cord endothelium were obtained from three different donors and adipose tissue (n=3) obtained from LIM31\'s pattern inventory. The total nucleated cell (TNC), expression of hematopoietic surface markers such as CD133+/CD34+ were observed after seven days of culture. Beyond that, colony forming unit assay (CFU) was performed before and after UCB expansion. The expansion by coculture method was observed in two groups (Group I - coculture with cytokines cocktail added vs. Group II- coculture without cytokines cocktail) for both MSCs sources. RESULTS: After seven days, analysis of confluent coculture showed that TNC proliferation rate ware almost 2 times higher than in subconfluent coculture (35 vs. 16-fold) in Group I and also revealed higher proliferative rate in CD133+/CD34+ cells considering. CFU showed similar increase after seven days of culture in comparison of day 0 (up to 8-fold). Subconfluent coculture for both umbilical cord endothelium and adipose tissue showed lower yield compared with those with high MSC confluence. The expansion in the presence of cytokines showed higher cell proliferation compared to the cocultures without addition of cytokines. CONCLUSION: This study showed that coculture system may require the addition of cytokines cocktail in the media and confluent MSC regardless of source for high yield of UCB cells

Surgical repair of torn rotator cuff tendons have a high rate of failure and does not address the underlying pathophysiology. Tissue engineering strategies, employing the use of multipotent progenitor cells or growth factors, represent potential therapies to improve the outcome of rotator cuff surgery. The use of glycosaminoglycan-based biomaterials in these therapies may enhance the effectiveness of cell and growth factor delivery techniques. Furthermore, understanding the cellular and molecular mediators in tendon overuse can help elucidate the causes of tendon degeneration. Thus the overall goals of this dissertation were to 1) develop heparin-based biomaterials to enhance cell pre-culture and maintain growth factor bioactivity and 2) characterize the histological and enzymatic changes in a supraspinatus tendon overuse model. To investigate the use of heparin in enhancing dynamic signaling, mesenchymal stem cells (MSCs) were encapsulated in heparin-containing hydrogels and evaluated for differentiation markers when cocultured with a small population of differentiated cells. To probe the effect of sulfation of heparin on the interactions with protein, selectively desulfated heparin species were synthesized and evaluated for their ability to bind and protect proteins. Finally, to develop a tendon overuse model that can become a test bed for testing future targeted therapeutics, an animal model was evaluated for tissue damage and protease activity. Together these studies represent a multi-pronged approach to understanding how tendon tissues become degenerative and for developing technologies to improve the biological fixation of tendon to bone in order to reduce the need for revision surgeries.

INTRODUÇÃO: As células-tronco hematopoiéticas (CTH) do sangue do cordão umbilical (SCU) têm sido utilizadas com sucesso para o tratamento de doenças malignas e não malignas. No entanto, algumas unidades de SCU podem apresentar baixa quantidade de células nucleadas totais (CNT). Algumas abordagens têm sido sugeridas para evitar problemas em relação à baixa concentração de CTH no transplante, como a administração de duas unidades de SCU para o paciente e a expansão ex vivo de CTH. OBJETIVO: Avaliar as taxas de proliferação celular na expansão ex vivo do SCU em sistema de cocultura com células-tronco mesenquimais (CTM) obtidos a partir de diferentes fontes com alta e baixa confluência e adicionando-se ou não coquetel de citocinas no meio de cultura. MÉTODOS: Este estudo foi aprovado pelo Comitê de Ética de Pesquisa (CAPPesq) do Hospital das Clínicas da Faculdade de Medicina da USP. A coleta do SCU (n =10) foi realizada após o nascimento do bebê e expulsão da placenta. O processamento foi realizado utilizando o método de redução de volume, o qual consiste em depleção de eritrócitos. As amostras de CTM provenientes do endotélio vascular do cordão umbilical foram obtidas de doadores diferentes (n=3) e o tecido adiposo (n=3) do inventário do LIM-31. A expansão das CNT e das células com expressão de marcadores CD133+/CD34+ foram observados depois de sete dias de cultura. Além disso, o ensaio para análise de unidades de formadoras de colônias (UFC) foi realizado em todas as amostras antes e depois da expansão do SCU. Para a expansão em sistema de cocultura foi separado dois grupos para ambas as fontes de CTM (Grupo I - cocultura com adição de coquetel de citocinas vs. Grupo II - cocultura sem citocinas). RESULTADOS: Após sete dias, no grupo I com cocultura confluente, a taxa de proliferação de CNT foi duas vezes maior ao comparar com cocultura subconfluente (35 vs. 16 vezes). No mesmo grupo também foi possível evidenciar elevada taxa de proliferação de células CD133+/CD34+. O índice de proliferação das UFC no grupo I aumentou até oito vezes. A cocultura subconfluente tanto do endotélio vascular do cordão umbilical como do tecido adiposo apresentou menor rendimento em comparação as CTM confluentes. A expansão das células na presença de citocinas apresentou maior proliferação celular ao comparar às coculturas sem adição de citocinas. CONCLUSÃO: Este estudo mostrou que para alto rendimento de células do SCU, o sistema de cocultura requer adição de coquetel de citocinas e CTM confluente independentemente da fonte utilizada / INTRODUCTION: Umbilical cord blood (UCB) hematopoietic stem cells have been successfully used for the treatment of both malignant and non-malignant diseases. Nevertheless, some UCB units could have low total nucleated cells (TNC) dose. Several approaches have been suggested to avoid inadequacy problems of hematopoietic stem cells (HSC) number for transplantation, such as administration of two UCB units to the patient and HSC ex vivo expansion. OBJECTIVE: Evaluate UCB ex vivo expansion proliferative rates in a high and low mesenchymal stem cells (MSC) confluence feeder layer obtained from different MSC sources and by adding or not cytokines cocktail into the medium. METHODS: This study was approved by the Research Ethic Committee (CAPPESQ) of Hospital das Clínicas da Faculdade de Medicina da USP. The collection of UCB (n=10) was made after delivery of the infant and the expulsion of placenta. Processing was performed using volume reduction method which consists in red blood depletion. MSC samples from umbilical cord endothelium were obtained from three different donors and adipose tissue (n=3) obtained from LIM31\'s pattern inventory. The total nucleated cell (TNC), expression of hematopoietic surface markers such as CD133+/CD34+ were observed after seven days of culture. Beyond that, colony forming unit assay (CFU) was performed before and after UCB expansion. The expansion by coculture method was observed in two groups (Group I - coculture with cytokines cocktail added vs. Group II- coculture without cytokines cocktail) for both MSCs sources. RESULTS: After seven days, analysis of confluent coculture showed that TNC proliferation rate ware almost 2 times higher than in subconfluent coculture (35 vs. 16-fold) in Group I and also revealed higher proliferative rate in CD133+/CD34+ cells considering. CFU showed similar increase after seven days of culture in comparison of day 0 (up to 8-fold). Subconfluent coculture for both umbilical cord endothelium and adipose tissue showed lower yield compared with those with high MSC confluence. The expansion in the presence of cytokines showed higher cell proliferation compared to the cocultures without addition of cytokines. CONCLUSION: This study showed that coculture system may require the addition of cytokines cocktail in the media and confluent MSC regardless of source for high yield of UCB cells

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.:Inhaltsverzeichnis I Abbildungsverzeichnis III Tabellenverzeichnis IV Bibliographische Beschreibung V Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 CIK-Zellen (CIK) 3 1.1.1 Merkmale von CIK-Zellen 3 1.1.2 Wirkungsmechanismen von CIK-Zellen 3 1.1.3 Studienlage 4 1.1.4 Bisherige Ansätze zur Verbesserung der Kultivierungsbedingungen 6 1.2 Mesenchymale Stammzellen (MSC) 7 1.2.1 Allgemein 7 1.2.2 Differenzierung von MSC 7 1.2.3 Heterogenität und Einflussfaktoren der MSC - Identitätsproblematik 8 1.2.4 Charakterisierung von MSC 9 1.2.5 Therapeutische Einsatzmöglichkeiten von MSC 11 2 Zielformulierung 15 3 Material und Methoden 16 3.1 Tiere 16 3.2 Materialien 17 3.2.1 Materialien für Zellkultur 17 3.2.2 Materialien für FACS-Analyse 18 3.2.3 Materialien für Zytotoxizitätsassay 19 3.2.4 Materialien für CFU-F-Assay 20 3.3 Methoden 21 3.3.1 Statistische Auswertung 21 3.3.2 Zellkultur 22 3.3.3 FACS (Fluorescence Activated Cell Sorting) 26 3.3.4 Markierung der MSC mit DiD 28 3.3.5 Zytotoxizitätsassay (LDH-Freisetzungsassay) 29 3.3.6 CFU-F-Assay 32 4 Ergebnisse 34 4.1 Beeinflussung der Wachstumskurve 34 4.1.1 Der Wachstumskurvenverlauf von CIK-Zellen (Kontrollen) 34 4.1.2 Der Wachstumskurvenverlauf von CIK-Zellen in der Kokultur mit MSC 35 4.1.3 Der Wachstumskurvenverlauf in MSC-konditioniertem Medium 37 4.1.4 Der Wachstumskurvenverlauf bei Restimulierung an Tag 14 38 4.2 Beeinflussung des Oberflächenphänotyps 40 4.2.1 Der Oberflächenphänotyp von CIK-Zellen 40 4.2.2 Vergleich Oberflächenphänotyp Kontrollen mit kokultivierten CIK 43 4.3 Beeinflussung der Vitalität 46 4.4 Beeinflussung der Zytotoxizität 48 4.5 Identifizierung der MSC 49 4.5.1 Adhärenz an Plastikoberflächen 50 4.5.2 Fibroblastenähnliche Wachstumsmorphologie 50 4.5.3 Wachstum in Colony-Forming-Units 51 4.5.4 Der Oberflächenphänotyp von MSC 53 4.6 Schicksal der MSC in der Kokultur 54 4.6.1 Der Oberflächenphänotyp der adhärenten Zellen nach Kokultur 54 4.6.2 Kokultur mit DiD gelabelten MSC 57 5 Diskussion 59 5.1 Beeinflussung der Wachstumskurve 60 5.1.1 Mechanismen der Beeinflussung des Wachstumskurvenverlaufs 60 5.1.2 Fehlerbetrachtung 68 5.2 Identifizierung der CIK sowie Beeinflussung von Phänotyp und Vitalität 69 5.3 Beeinflussung der Zytotoxizität 70 5.3.1 Vergleich Zytotoxizität Kontrollen mit Kokulturen 70 5.3.2 Fehlerbetrachtung 71 5.4 Identifizierung der MSC 72 6 Schlussfolgerung 75 7 Ausblick 77 8 Zusammenfassung 79 Literaturverzeichnis 83 Danksagung I

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As interações epitélio-mesênquima podem influenciar o desenvolvimento do tumor no sítio primário e no linfonodo comprometido de pacientes com câncer de mama. Nosso objetivo foi avaliar a taxa de proliferação e perfil gênico de fibroblastos de linfonodos comprometidos e não comprometidos obtidos de pacientes com câncer de mama e determinar a influência de células epiteliais mamárias normais (MCF10A) ou malignas (MDA-MB-231) na expressão gênica destes fibroblastos. Foram estabelecidas culturas primárias de fibroblastos de linfonodos (3 comprometidos e 3 não comprometidos) de 6 diferentes pacientes com câncer de mama e não houve diferença na taxa de proliferação destes fibroblastos. Co-culturas de células MCF10A ou MDA-MB-231 com fibroblastos, separadas fisicamente por membranas porosas, foram realizadas por 72 horas. O RNA total dos fibroblastos foi extraído, amplificado e o perfil gênico foi analisado usando-se uma lâmina de cDNA microarray. Fibroblastos de linfonodos comprometidos e não comprometidos apresentaram perfil gênico similar, pois apenas 13 genes foram modulados, sendo que maior expressão de PGBD3 e PTBP2 em fibroblastos de linfonodos comprometidos foi confirmada em ensaios de RT-PCR em tempo real. Em fibroblastos, a cocultura com células MCF10A alterou a expressão de maior número de genes que a co-cultura com células MDA-MB-231. Em fibroblastos originários de linfonodos não comprometidos mantidos em co-cultura com células MDA-MB-231 foram modulados 151 genes, em relação aos mesmos fibroblastos cultivados na ausência de células epiteliais, sendo que oito deles apresentaram variação de expressão superior a três vezes (BET1, ENTPD1, USP7, DAPK1, ERBB2 e NCF2). As células MDA-MB-231 modularam algumas vias de sinalização em fibroblastos não comprometidos, como vias do cálcio, insulina, hormônio liberador de gonadotrofina (GnRH) e da regulação do citoesqueleto de actina, mas o mesmo não ocorreu em fibroblastos de linfonodos comprometidos. Duzentos e quarenta e nove genes foram diferencialmente expressos em fibroblastos originários de linfonodos comprometidos mantidos em co-cultura com células MDA-MB-231 e quatro genes apresentaram variação superior a três vezes (ACLY, AXUD1, CLCN5 e PDE6D). Nestes fibroblastos, algumas funções biológicas foram reguladas apenas por influência da célula MDA-MB-231, entre as quais metabolismo de aminoácidos, excreção, transporte intra Golgi e regulação da forma celular. As vias de sinalização e funções biológicas reguladas em fibroblastos pela interação com células epiteliais malignas podem estar implicadas no desenvolvimento de metástases regionais no câncer de mama. / Tumor development may be influenced by epithelial-mesenchimal interactions in the primary site as well as in the involved lymph node from breast cancer patients. Our aim was to evaluate the proliferation rate and gene profile of fibroblasts obtained from involved and uninvolved lymph nodes from breast câncer patients and to determine the influence of normal (MCF10A) or malignant (MDA-MB-231) mammary epithelial cells on the gene expression of these fibroblasts. Primary culture from fibroblasts obtained from 3 involved and 3 uninvolved lymph nodes (ILF and NILF) from 6 different breast cancer patients was established and no difference in their proliferation rate was detected. These fibroblasts were co-cultured with MCF10A or MDA-MB-231 cells, physically separated by a porous membrane for 72 hours. Total RNA was extracted from the cells, amplified and the gene profile from fibroblasts cultured alone or with epithelial cells was analyzed by cDNA microarray slides. Fibroblasts from involved and uninvolved lymph nodes present a similar gene profile as only 13 genes were differentially expressed between them, including PGBD3 and PTBP2, which higher expression in fibroblasts from involved lymph nodes was confirmed by real time RT-PCR. In fibroblasts, more genes seem to be regulated upon co-cultured with MCF10A than with MDA-MB-231 cells. In fibroblasts from uninvolved lymph nodes co-cultured with MDA-MB-231 cells, 151 genes were modulated as compared with fibroblasts cultured alone, and eight of them, presented an expression variation superior to three times (BET1, ENTPD1, USP7, DAPK1, ERBB2 e NCF2). In these fibroblasts, MDA-MB-231 could modulate some signaling pathways as calcium, insulin, gonadotrophin releasing hormone (GnRH) and actin cytoskeleton regulation signaling pathways. Two hundred forty nine genes were differentially expressed by fibroblasts from involved lymph nodes co-cultured with MDA-MB-231 cells, four of which with an expression variation superior to three times (ACLY, AXUD1, CLCN5 e PDE6D). In the last condition, fibroblasts had some biological functions regulated upon MDA-MB-231 cells influence, among which amino acid metabolism, excretion, intra-Golgi transport and regulation of cell shape. Signaling pathways and biological functions regulated in fibroblasts after interaction with malignant epithelial cells might be implicated in the development of regional metastasis in breast cancer.

Endothelzell-basierte Therapien vermitteln regenerative Effekte hinsichtlich der Revaskularisierung von ischämischen Geweben. Doch ist die Verfügbarkeit von autologen Endothelzellen aufgrund einer krankheitsbedingt reduzierten Frequenz im peripheren Blut oder einer verminderten Integrität der endogenen Endothelzell-Populationen eingeschränkt. Hingegen ist es möglich, allogene endotheliale Vorläuferzellen aus der Nabelschnur in zelltherapeutisch relevanten Mengen zu isolieren. In der vorliegenden Arbeit wurden zunächst die Eigenschaften allogener humaner Nabelschnur (NS)-abgeleiteter sog. Endothelial Colony-Forming Cells (ECFCs) mit denen von venösen NS-abgeleiteten Endothelzellen verglichen. Aufgrund der nachgewiesenen Immunogenität von allogenen ECFCs wurde eine weiterführende Strategie zur Reduktion dieser immunogenen Eigenschaften durch die Koapplikation mit Mesenchymalen Vorläuferzellen (MSCs) verfolgt. Humane ECFCs wurden mit MSCs desselben Spenders kombiniert und in funktionellen in vitro- und in vivo-Assays untersucht. Dadurch konnte nachgewiesen werden, dass IFNγ-stimulierte ECFC/MSC-Kokulturen eine reduzierte Expression von HLA-Molekülen zeigen. Entsprechend induzierten spezifische CD8+ T-Zellen eine reduzierte Lyse der kokultivierten ECFCs und MSCs. Die Kokultur von ECFCs und MSCs mit allogenen Immunzellen führte zu einer nahezu vollständigen Inhibition der T-Zell-Proliferation. Um die reduzierte Immunogenität von ECFC und MSC in vivo zu verifizieren, wurden die Zellen in immundefiziente Mäuse injiziert, welche nachfolgend mit humanen PBMCs rekonstituiert wurden. So konnte nachgewiesen werden, dass die Koapplikation von ECFCs und MSCs nicht nur die Entstehung von stabilen Gefäßnetzwerken begünstigt, sondern zudem in den Transplantaten zu einer verringerten Immunzell-Infiltration führte. Die Koapplikation von ECFCs mit MSCs könnte daher eine klinische Nutzung dieser allogenen Quelle für die therapeutische Unterstützung der Vaskularisierung ermöglichen. / Endothelial cell-based therapies promote tissue regeneration and vascularization after ischemic damage. The availability of autologous endothelial progenitor cells is restricted in diseased patients, however therapeutically relevant numbers of allogeneic Endothelial Progenitor Cells can be isolated from an umbilical cord (UC). In the present study, the immunogenic properties of these Endothelial Colony Forming Cells (ECFCs) were first compared to human umbilical vein endothelial cells (HUVECs). Both cytokine-treated endothelial cells induced CD4+ and CD8+ T cell proliferation after coculture with allogeneic immune cells. So far, the potential interactions between ECFCs and Mesenchymal Stem/Progenitor Cells (MSCs) concerning their immunological features is poorly understood, but we hypothesize that MSCs might improve the immune compatibility and vessel building characteristics of ECFCs. Therefore, human UC-derived ECFC and MSC cocultures from the same donor were analyzed using various functional in vitro and in vivo assays. Stimulation of these cocultures with IFNγ caused strongly reduced expression levels of HLA-molecules compared to ECFC monocultures. The decreased molecular density on the cocultured ECFCs resulted in reduced cytotoxic CD8+ T cell-mediated lysis. Further, during IFNγ stimulation, the combination of ECFCs with MSCs prevented initiation of allogeneic T cell proliferation. To verify this concept in vivo, ECFCs and MSCs were co-transplanted in a humanized allograft mouse model in immunodeficient mice in order to effectively induce stable microvessels. These experiments demonstrate that when MSCs are co-applied with ECFCs, they not only support the formation of stable blood vessels, but also lead to fewer HLA-DR+ human vascular structures and fewer infiltrating human leukocytes. The data presented indicate that crosstalk between UC-derived ECFCs and MSCs might lower the risk of allogeneic ECFC rejection.