Ralf Huss

Targeting tumor stroma using engineered mesenchymal stem cells reduces the growth of pancreatic carcinoma

Objective:To analyze the efficacy of engineered mesenchymal stem cell based therapy directed towards pancreatic tumor stroma. Summary Background Data:Mesenchymal stem cells (MSC) are actively recruited to tumor stroma where they enhance tumor growth and metastases. Upregulation of chemotactic cytokine (CCL5) by MSCs within the tumor stroma has been shown to play a central role in this process. Murine MSCs were engineered to express reporter genes or therapeutic genes under control of the CCL5 promoter and adoptively transferred into mice with growing pancreatic tumors. The effect on tumor growth and metastases was then evaluated. Methods:MSCs isolated from bone marrow of C57/Bl6 p53−/− mice were stably transfected with red fluorescent protein (RFP), enhanced green fluorescent protein (eGFP), or herpes simplex virus (HSV) thymidine kinase (Tk) gene driven by the RANTES promoter. MSCs were intravenously applied once per week over 3 weeks to mice carrying an orthotopic, syngeneic pancreatic Panc02 tumor. Results:eGFP and RFP signals driven by the CCL5 promoter were detected by fluorescence in treated pancreatic tumor samples. The HSV-Tk therapy group treated intraperitoneal with the prodrug ganciclovir 5 to 7 days after stem cell application lead to a 50% reduction of primary pancreatic tumor growth (P < 0.0003, student t test) and reduced liver metastases (0% vs. 60%). Conclusion:The active homing of MSCs into primary pancreatic tumor stroma and activation of the CCL5 promoter was verified using eGFP- and RFP-reporter genes. In the presence of ganciclovir, HSV-Tk transfected MSCs led to a significant reduction of primary pancreatic tumor growth and incidence of metastases.

Multipotent Mesenchymal Stem Cells Acquire a Lymphendothelial Phenotype and Enhance Lymphatic Regeneration In Vivo

Background— The importance and therapeutic value of stem cells in lymphangiogenesis are poorly understood. We evaluated the potential of human and murine mesenchymal stem cells (MSCs) to acquire a lymphatic phenotype in vitro and to enhance lymphatic regeneration in vivo. Methods and Results— We assessed the lymphendothelial differentiation of human and murine MSCs after induction with supernatant derived from human dermal microvascular endothelial cells, isolated lymphatic endothelial cells, and purified vascular endothelial growth factor (VEGF)-C in vitro. We used human or murine progenitor MSC lines and then characterized the lymphatic phenotype by morphology, migratory capacity, and the expression of lymphatic markers such as Prox-1, podoplanin, Lyve-1, VEGF receptor-2, and VEGF receptor-3. Using a murine lymphatic edema model, we assessed the potential of these cells to form a functional lymphatic vasculature in vivo after injection of syngeneic MSCs. Incubation with supernatant from lymphatic endothelial cells induced an endothelium-like morphology and the expression of lymphendothelial markers in both human and murine MSCs in vitro. MSCs showed migratory activity along a VEGF-C gradient, which was enhanced by VEGF-C conditioning. In vivo, the local application of MSCs resulted in a significant decrease in edema formation (−20.1%; P<0.01 versus untreated tails) after 3 weekly cell injections and restored the drainage of intradermally injected methylene blue after 7 weekly injections. Conclusions— MSCs were capable of expressing a lymphatic phenotype when exposed to lymph-inductive media and purified VEGF-C. Migratory activity toward VEGF-C in vitro suggests homing capability in vivo. Restoration of lymphatic drainage after injection of MSCs in a lymphedema model indicates that MSCs play a role in lymphatic regeneration. The potential clinical application of MSC in wound healing and reduction of lymphatic edema warrants further research.

Use of human mesenchymal cells to improve vascularization in a mouse model for scaffold-based dermal regeneration.

All engineered bioartificial structures developed for tissue regeneration require oxygen and nutrients to establish proper physiological functions. Aiming to improve vascularization during dermal regeneration, we combined the use of a bioartificial collagen scaffold and a defined human mesenchymal cell (MC) line. This cell line, termed V54/2, exhibits typical morphologic and immunohistochemical characteristics of MC. V54/2 cells seeded in the scaffold were able to survive, proliferate, and secrete significant amounts of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) during 2 weeks in vitro. To induce dermal regeneration, scaffolds with or without cells were transplanted in a nude mice full skin defect model. After 2 weeks of transplantation, scaffolds seeded with V54/2 cells showed more vascularization during the dermal regeneration process than controls, and the presence of human cells in the regenerating tissue was detected by immunohistochemistry. To confirm if local presence of angiogenic growth factors is sufficient to induce neovascularization, scaffolds were loaded with VEGF and bFGF and used to induce dermal regeneration in vivo. Results showed that scaffolds supplemented with growth factors were significantly more vascularized than control scaffolds (scaffolds without growth factors). The present work suggests that combined use of MC and bioartificial scaffolds induces therapeutic angiogenesis during the scaffold-based dermal regeneration process.

Genetically Engineered Stem Cells for Therapeutic Gene Delivery

Stem cell and gene therapy approaches have held out much hope for the development of new tools to treat disease. Therapeutic approaches based on these methods have only rarely found their way into the clinic. The linking of stem cell therapy with selective gene therapy enhances therapeutic options for the regeneration or replacement of diseased or missing cells. This review focuses on the rationale and preliminary results of combining stem cell and gene therapy. Special emphasis is placed on various molecular techniques currently used to genetically engineer stem cells. Viral and nonviral genes delivering technologies are detailed as are techniques for the modulation of gene expression in the context of stem cell recruitment and differentiation. Finally potential clinical applications for this new therapeutic strategy are discussed.

Modulation of mesenchymal stromal cell characteristics by microcarrier culture in bioreactors

Mesenchymal stromal cells (MSCs) are promising candidates for cell therapy. Their therapeutic use requires extensive expansion to obtain a sufficiently high number of cells for clinical applications. State‐of‐the‐art expansion systems, that is, primarily culture flask‐based systems, are limited regarding scale‐up, automation, and reproducibility. To overcome this bottleneck, microcarrier (MC)‐based expansion processes have been developed. For the first time, MSCs from the perinatal sources umbilical cord (UC) and amniotic membrane (AM) were expanded on MCs. This study focuses on the comparison of flask‐ and Cytodex 1 MC‐expanded MSCs by evaluating the influence of the expansion process on biological MSC characteristics. Furthermore, we tested the hypothesis to obtain more homogeneous MSC preparations by expanding cells on MCs in controlled large‐scale bioreactors. MSCs were extensively characterized determining morphology, cell growth, surface marker expression, and functional properties such as differentiation capacity, secretion of paracrine factors, and gene expression. Based on their gene expression profile MSCs from different donors and sources clearly clustered in distinct groups solely depending on the expansion process—MC or flask culture. MC‐ and flask‐expanded MSCs significantly differed from each other regarding surface markers and both paracrine factors and gene expression profiles. Furthermore, based on gene expression analysis, MC cultivation of MSCs in controlled bioreactor systems resulted in less heterogeneity between cells from different donors. In conclusion, MC‐based MSC expansion in controlled bioreactors has the potential to reliably produce MSCs with altered characteristics and functions as compared to flask‐expanded MSCs. These findings may be useful for the generation of MSCs with tailored properties for clinical applications. Biotechnol. Bioeng. 2014;111: 2290–2302.

Bone marrow-derived mesenchymal stem cells migrate to healthy and damaged salivary glands following stem cell infusion

Xerostomia is a severe side effect of radiation therapy in head and neck cancer patients. To date, no satisfactory treatment option has been established. Because mesenchymal stem cells (MSCs) have been identified as a potential treatment modality, we aimed to evaluate stem cell distribution following intravenous and intraglandular injections using a surgical model of salivary gland damage and to analyse the effects of MSC injections on the recruitment of immune cells. The submandibular gland ducts of rats were surgically ligated. Syngeneic adult MSCs were isolated, immortalised by simian virus 40 (SV40) large T antigen and characterized by flow cytometry. MSCs were injected intravenously and intraglandularly. After 1, 3 and 7 days, the organs of interest were analysed for stem cell recruitment. Inflammation was analysed by immunohistochemical staining. We were able to demonstrate that, after intravenous injection, MSCs were recruited to normal and damaged submandibular glands on days 1, 3 and 7. Unexpectedly, stem cells were recruited to ligated and non-ligated glands in a comparable manner. After intraglandular injection of MSCs into ligated glands, the presence of MSCs, leucocytes and macrophages was enhanced, compared to intravenous injection of stem cells. Our data suggest that injected MSCs were retained within the inflamed glands, could become activated and subsequently recruited leucocytes to the sites of tissue damage.

Alkaline phosphatase, glutathione-S-transferase-P, and cofilin-1 distinguish multipotent mesenchymal stromal cell lines derived from the bone marrow versus peripheral blood.

Multipotent mesenchymal stromal cells (MSCs) can be isolated from bone marrow or peripheral blood. To identify phenotypical and functional differences between MSCs derived from these sources, the human bone marrow-derived, fibroblast-like cell line L87/4 was compared with the peripheral blood-derived, fibroblast-like cell line V54/2. Both cell lines expressed similar levels of SH3+, CD45(-), CD68(-), CD133(-), and HLA-DR(-). The bone marrow-derived cells expressed higher surface levels of CD105, CD10, and CD117 and preferentially expressed alkaline phosphatase, glutathione S-transferase P, and cofilin-1. The peripheral blood-derived line showed a higher number of CD34+/CD105+ double-positive and side population (SP) cells. The results demonstrate the more multipotent, yet quiescent, stromal phenotype of bone marrow MSCs, whereas MSCs isolated from the circulation display more hematopoietic-lineage characteristics. Importantly, potential marker genes that distinguish the two stages of MSCs are defined.

Chemokine directed homing of transplanted adult stem cells in wound healing and tissue regeneration

A major challenge in stem cell biology is to study the underlying mechanisms of tissue specific homing and differentiation. Recent results suggest that bone marrow derived stem cells can give rise to multiple cell types. Because chemokines and chemokine receptors are associated with development, differentiation and homing of immune cells, we undertook efforts to study the chemokine receptor expression profile of human adult stem cells to identify their potential role in tissue specific homing prior to transdifferentiation. Using human bone marrow-derived stem cell lines, we could demonstrate functional chemokine receptor expression of various chemokine receptors. The expression of CXCR5 and CCR7, associated with secondary lymphoid organ homing as well as CXCR4 and CCR10, involved in organ specific homing and CXCR3, CCR5 and CCR1, which are involved in inflammation events, suggested a role of chemokine receptors in tissue specific homing of stem cells. To proof the specific homing of stem cells in vivo, we used murine stem cell lines, stably introduced green fluorescent protein under control of CMV promotor into the cells and injected them intravenously into mice. We demonstrate the homing of these stem cells to lymphnode and thymus as well as mucosal tissue, while stem cells home exclusively to a site of lesion during wound healing and tissue regeneration. Our data suggest that chemokine biology may play a pivotal role in the homing of stem cells to specific tissues and niches prior to (trans)differentiation, while the homing changes during tissue damage and other adequate lesions.

Spindle Cell Metaplastic Breast Carcinoma with Leiomyoid Differentiation: A Case Report

Background: Metaplastic carcinoma of the breast is a rare but distinct entity within the group of undifferentiated invasive carcinomas. This entity accounts for less than 0.5% of all breast cancers and contains elements of epithelial (ductal), mesenchymal, and intermediate forms of differentiation. Of these metaplastic carcinomas, there have been reports of chondroid, squamous, osseous, and spindle differentiation. Case Report: We describe the clinical course of a 52-year-old female patient with an unusual histopathology of a spindle cell carcinoma of the breast, discuss the literature, and recommend an approach to diagnosis and treatment. The patient initially presented to an outside hospital with a rapidly growing breast mass that was originally diagnosed as a malignant phylloides tumor. She presented 11 months after the primary treatment with a local subcutaneous relapse. She later developed a local relapse of her metaplastic carcinoma in the chest wall. Extensive histopathological analysis lead to the diagnosis of a spindle cell metaplastic breast carcinoma with leiomyoid differentiation, which has not been described previously. Conclusions: Overlap in morphology can lead to a misinterpretation or underdiagnosis of metaplastic carcinomas. However, the prognosis is similar to more common types of breast adenocarcinoma.

[Cell-based strategies for salivary gland regeneration].

Xerostomia as a side effect of radiotherapy or due to Sjögrens disease leads to considerable impairment of the quality of life of the affected patients. Preventive treatment approaches such as intensity-modulated radiotherapy, surgical transfer of a submandibular gland to a site outside the radiation field or administration of amifostin during radiation treatment are not yet completely established in clinical practice and are not applicable for all patients. Symptomatic treatment with pilocarpin or synthetic saliva leads to an improvement of the symptoms only in some patients, and in the case of pilocarpin significant systemic anticholinergic side-effects might occur. Because large numbers of patients are affected and current treatment options are not satisfactory, it is essential to develop new treatment options. In parallel with the in vitro production of functional salivary gland constructs by means of tissue engineering techniques, attempts are currently under way to experimentally restore salivary gland function by genetic treatment approaches such as transfection of the affected salivary glands with aquaporins or pro-angiogenic factors. In addition, the in vivo application of stem cells is under investigation. In the present paper, we discuss the clinical and radiobiological background of xerostomia and highlight possible innovative future treatment options.ZusammenfassungDie Xerostomie nach Bestrahlung oder bei M.xa0Sjögren führt zu schwerwiegenden Einschränkungen der Lebensqualität der betroffenen Patienten. Präventive Therapieansätze wie die intensitätsmodulierte Strahlentherapie, die chirurgische Verlagerung einer Gl.xa0submandibularis aus dem Strahlenfeld oder die Gabe von Amifostin während der Bestrahlung sind noch nicht klinisch etabliert und auch nicht für alle Patienten einsetzbar. Die symptomatische Therapie mit Pilocarpin oder künstlichem Speichel führt nur bei einem Teil der Patienten zur Besserung der Symptome und weist im Falle von Pilocarpin bedeutsame systemische anticholinerge Nebenwirkungen auf. Aufgrund der Vielzahl der betroffenen Patienten und der ungenügenden Therapieoptionen ist die Entwicklung neuer Therapieformen von großer Bedeutung. Neben der In-vitro-Herstellung von funktionellen Speicheldrüsenkonstrukten mit Tissue-Engineering-Verfahren wird gegenwärtig versucht, durch gentherapeutische Ansätze, die die Transfektion der betroffenen Speicheldrüsen mit Aquaporinen oder gefäßwachstuminduzierenden Faktoren umfasst, die eingeschränkte Speicheldrüsenfunktion experimentell zu therapieren. Darüber hinaus wird die Applikation von Stammzellen in vivo als weitere Therapieoption diskutiert und analysiert. Im vorliegenden Artikel wird der klinische und strahlenbiologische Hintergrund der Xerostomie erläutert und die angesprochenen neuen Therapieansätze dargestellt und diskutiert.AbstractXerostomia as a side effect of radiotherapy or due to Sjögren’s disease leads to considerable impairment of the quality of life of the affected patients. Preventive treatment approaches such as intensity-modulated radiotherapy, surgical transfer of a submandibular gland to a site outside the radiation field or administration of amifostin during radiation treatment are not yet completely established in clinical practice and are not applicable for all patients. Symptomatic treatment with pilocarpin or synthetic saliva leads to an improvement of the symptoms only in some patients, and in the case of pilocarpin significant systemic anticholinergic side-effects might occur. Because large numbers of patients are affected and current treatment options are not satisfactory, it is essential to develop new treatment options. In parallel with the in vitro production of functional salivary gland constructs by means of tissue engineering techniques, attempts are currently under way to experimentally restore salivary gland function by genetic treatment approaches such as transfection of the affected salivary glands with aquaporins or pro-angiogenic factors. In addition, the in vivo application of stem cells is under investigation. In the present paper, we discuss the clinical and radiobiological background of xerostomia and highlight possible innovative future treatment options.