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G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts,which eventually blocks signal propagation and often induces receptor internalization. However,there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2,GRK3,GRK5,GRK6) simultaneously,but we have very limited knowledge about their interplay in primary immune cells. In particular,we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type,and (b) one specific GPCR between several immune cell subsets. To address this issue,we generated mouse models of single,combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils,T cells,B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes,many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands,whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types,and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly,we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis,which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together,our findings demonstrate the complexity of GRK functions in immune cells,which go beyond GPCR desensitization in specific leukocyte types. Furthermore,they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset. View Publication

Achievements in tissue engineering using mesenchymal stem cells (MSC) demand a clinically acceptable off-the-shelf" cell therapy product. Efficacy of cryopreservation of human bone marrow-derived MSC in clinically safe animal product-free medium containing 2% 5% and 10% dimethyl sulfoxide (DMSO) was evaluated by measuring cell recovery viability apoptosis proliferation rate expression of a broad panel of MSC markers and osteogenic differentiation. Rate-controlled freezing in CryoStor media was performed in a programmable cell freezer. About 95% of frozen cells were recovered as live cells after freezing in CryoStor solutions with 5% and 10% DMSO followed by storage in liquid nitrogen for 1 month. Cell recovery after 5 months storage was 72% and 80% for 5% and 10% DMSO respectively. Measurements of caspase 3 activity demonstrated that 15.5% and 12.8% of cells after 1 month and 18.3% and 12.9% of cells after 5 months storage in 5% and 10% DMSO respectively were apoptotic. Proliferation of MSC recovered after cryopreservation was measured during 2 weeks post-plating. Proliferation rate was not compromised and was even enhanced. Cryopreservation did not alter expression of MSC markers. Quantitative analysis of alkaline phosphatase (ALP) activity ALP surface expression and Ca deposition in previously cryopreserved MSC and then differentiated for 3 weeks in osteogenic medium demonstrated the same degree of osteogenic differentiation as in unfrozen parallel cultures. Cell viability and functional parameters were analyzed in MSC after short-term storage at 4°C in HypoThermosol-FRS solution also free of animal products. Hypothermic storage for 2 and 4 days resulted in about 100% and 85% cell recovery respectively less than 10% of apoptotic cells and normal proliferation marker expression and osteogenic potential. Overall our results demonstrate that human MSC could be successfully cryopreserved for banking and clinical applications and delivered to the bedside in clinically safe protective reagents. View Publication

Two monoclonal antibodies,BA16 and BA17,have been developed using a detergent-insoluble extract of human mammary epithelial organoids as immunogen. Indirect immunofluorescent staining of cultured cells showed that the component reacting with the antibodies was filamentous and the intensity of staining was stronger in mitotic cells. Immunoblotting of cell extracts showed that both antibodies react with only one band of 40 X 10(3) molecular weight,which was present in keratin-enriched extracts of cells or organoids. Furthermore,the tissue distribution of the component reacting with the antibodies was that predicted for human keratin 19. The antibodies showed differences in the intensity of staining of cells or tissue sections fixed and prepared in different ways indicating that they reacted with different epitopes. The pattern of expression of the 40 X 10(3) Mr keratin by normal mammary epithelial cells was investigated by immunoperoxidase staining of tissue sections,cultured milk cells,and organoids of different sizes cultured in collagen gels. It was found that basal or myoepithelial cells did not express this keratin. Some heterogeneity of expression of this component was seen in luminal epithelial cells,found almost exclusively in the smaller structures. These cells did,however,express other keratins characteristic of luminal cells. The distribution in the mammary tree of the luminal cells that did not express the 40 X 10(3) Mr keratin appears to be similar to that expected for cells with the proliferative potential to produce new terminal ductal lobular units or an increase in branching of existing terminal ductal lobular units. It is shown that these cells have considerable proliferative potential by the fact that they form large colonies in milk cell cultures. View Publication

Pluripotent stem cells display significant heterogeneity in gene expression,but whether this diversity is an inherent feature of the pluripotent state remains unknown. Single-cell gene expression analysis in cell subsets defined by surface antigen expression revealed that human embryonic stem cell cultures exist as a continuum of cell states,even under defined conditions that drive self-renewal. The majority of the population expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity,consistently high transcripts for all pluripotency-related genes studied,and no lineage priming. This subpopulation was characterized by its expression of a particular set of intercellular signaling molecules whose genes shared common regulatory features. Our data support a model of an inherently metastable self-renewing population that gives rise to a continuum of intermediate pluripotent states,which ultimately become primed for lineage specification. ?? 2014 The Authors. View Publication

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover,we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining,transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling. View Publication

The Mesenchymal-to-Epithelial Transition (MET) has been recognized as a crucial step for successful reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs). Thus,it has been demonstrated,that the efficiency of reprogramming can be enhanced by promoting an epithelial expression program in cells,with a concomitant repression of key mesenchymal genes. However,a detailed characterization of the epithelial transition associated with the acquisition of a pluripotent phenotype is still lacking to this date. Here,we integrate a panel of morphological approaches with gene expression analyses to visualize the dynamics of episomal reprogramming of human fibroblasts to iPSCs. We provide the first ultrastructural analysis of human fibroblasts at various stages of episomal iPSC reprogramming,as well as the first real-time live cell visualization of a MET occurring during reprogramming. The results indicate that the MET manifests itself approximately 6-12. days after electroporation,in synchrony with the upregulation of early pluripotency markers,and resembles a reversal of the Epithelial-to-Mesenchymal Transition (EMT) which takes place during mammalian gastrulation. View Publication

The lipophilic statin lovastatin decreases cholesterol synthesis and is a safe and effective treatment for the prevention of cardiovascular diseases. Growing evidence points at antitumor potential of lovastatin. Therefore,understanding the molecular mechanism of lovastatin function in different cell types is critical to effective therapy design. In this study,we investigated the effects of lovastatin on the differentiation potential of human embryonic stem (hES) cells (H9 cell line). Multiparameter flow cytometric assay was used to detect changes in the expression of transcription factors characteristic of hES cells. We found that lovastatin treatment delayed NANOG downregulation during ectodermal and endodermal differentiation. Likewise,expression of ectodermal (SOX1 and OTX2) and endodermal (GATA4 and FOXA2) markers was higher in treated cells. Exposure of hES cells to lovastatin led to a minor decrease in the expression of SSEA-3 and a significant reduction in CD133 expression. Treated cells also formed fewer embryoid bodies than control cells. By analyzing hES with and without CD133,we discovered that CD133 expression is required for proper formation of embryoid bodies. In conclusion,lovastatin reduced the heterogeneity of hES cells and impaired their differentiation potential. View Publication

Bone marrow-derived endothelial precursor cells incorporate into neovasculature and have been successfully used as vehicles for gene delivery to brain tumors. To determine whether systemically administered Sca1+ bone marrow cells labeled with superparamagnetic iron oxide nanoparticles can be detected by in vivo magnetic resonance imaging in a mouse brain tumor model,mouse Sca1+ cells were labeled in vitro with ferumoxides-poly-L-lysine complexes. Labeled or control cells were administered intravenously to glioma-bearing severe combined immunodeficient (SCID) mice. Magnetic resonance imaging (MRI) was performed during tumor growth. Mice that received labeled cells demonstrated hypointense regions within the tumor that evolved over time and developed a continuous dark hypointense ring at a consistent time point. This effect was not cleared by administration of a gadolinium contrast agent. Histology showed iron-labeled cells around the tumor rim in labeled mice,which expressed CD31 and von Willebrand factor,indicating the transplanted cells detected in the tumor have differentiated into endothelial-like cells. These results demonstrate that MRI can detect the incorporation of magnetically labeled bone marrow-derived precursor cells into tumor vasculature as part of ongoing angiogenesis and neovascularization. This technique can be used to directly identify neovasculature in vivo and to facilitate gene therapy by noninvasively monitoring these cells as gene delivery vectors. View Publication

OBJECTIVE: Various preparative protocols have been proposed for the acquisition and cultivation of mesenchymal stem cells (MSC). Whereas surface antigen markers have failed to precisely define this population,microarray analysis might provide a better tool for characterization of MSC. METHODS: In this study,we have analyzed global gene expression profiles of human MSC isolated from adipose tissue (AT),from umbilical cord blood (CB),and from bone marrow (BM) under two growth conditions and have compared them to terminally differentiated human fibroblasts (HS68). Profiles were compared using our Human Genome Microarray representing 51.144 different cDNA clones. RESULTS: Cultured with the appropriate conditions,osteogenic and adipogenic differentiation could be confirmed in all MSC preparations but not in fibroblasts. No phenotypic differences were observed by flow cytometry using a panel of 22 surface antigen markers. Whereas MSC derived from different donors using the same culture procedure yielded a consistent and reproducible gene expression profile,many genes were differentially expressed in MSC from different ontogenetic sources or from different culture conditions. Twenty-five genes were overlapping and upregulated in all MSC preparations from AT,CB,and BM as compared to HS68 fibroblasts. These genes included fibronectin,ECM2,glypican-4,ID1,NF1B,HOXA5,and HOXB6. Many genes upregulated in MSC are involved in extracellular matrix,morphogenesis,and development,whereas several inhibitors of the Wnt pathway (DKK1,DKK3,SFRP1) were highly expressed in fibroblasts. CONCLUSION: Our results have provided a foundation for a more reproducible and reliable quality control using genotypic analysis for defining MSC. View Publication

The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies,yet the molecular effectors of this process are relatively uncharacterized. For extravasation,studies based predominantly on static transwell assays support a general model,whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However,this model of chemotaxis cannot readily reconcile how chemokines influence intravasation,as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus,to analyze how T cells integrate perivascular chemokine signals under physiologic flow,we developed a novel transwell-based flow chamber allowing for real-time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment,transmigrated T cells did not undergo retrograde transendothelial migration (retro-TEM). However,when exposedto abluminal CXCL12,transmigrated T cells underwent striking retro-TEM and re-entered the flow stream [corrected]. This CXCL12 fugetactic (chemorepellant) effect was concentration-dependent,augmented by apical flow,blocked by antibodies to integrins,and reduced by AMD3100 in a dose-dependent manner. Moreover,CXCL12-induced retro-TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream. View Publication

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma,graft-versus-host disease,and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway,within a single day,without added cytokines,as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized,as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature,common to ECP-processed monocytes from normal subjects,and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway,this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances,ECP may potentially offer a general source of therapeutic DCs. View Publication