Nicole A. Hofmann

Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo

Endothelial progenitor cells are critically involved in essential biologic processes, such as vascular homeostasis, regeneration, and tumor angiogenesis. Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic, and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum-free large-scale clinical-grade propagation of mesenchymal stromal cells, we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of more than 30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation, and genomic stability were maintained. For the first time, proliferative, functional, and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.

Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation

In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype, and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription, and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord, and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a BM cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13, and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs), as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age, with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment

The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.

Chemokine receptors in gastric MALT lymphoma: loss of CXCR4 and upregulation of CXCR7 is associated with progression to diffuse large B-cell lymphoma

Chemokine receptors have a crucial role in the development and progression of lymphoid neoplasms. To determine the chemokine receptor expression profile in gastric mucosa-associated lymphoid tissue (MALT) lymphoma, we performed an expression analysis of 19 chemokine receptors at mRNA levels by using real-time RT-PCR, as well as of five chemokine receptors—CCR8, CCR9, CXCR4, CXCR6 and CXCR7—by immunohistochemistry on human tissue samples of Helicobacter pylori-associated gastritis, gastric MALT lymphoma and gastric extranodal diffuse large B-cell lymphoma originating from MALT lymphoma (transformed MALT lymphoma). Following malignant transformation from H. pylori-associated gastritis to MALT lymphoma, an upregulation of CCR7, CXCR3 and CXCR7, and a loss of CXCR4 were detected. The transformation of gastric MALT lymphomas to gastric extranodal diffuse large B-cell lymphoma was accompanied by upregulation of CCR1, CCR5, CCR7, CCR8, CCR9, CXCR3, CXCR6, CXCR7 and XCR1. Remarkably, CXCR4 expression was exclusively found in nodal marginal B-cell lymphomas and nodal diffuse large B-cell lymphomas but not at extranodal manifestation sites, ie, in gastric MALT lymphomas or gastric extranodal diffuse large B-cell lymphomas. Furthermore, the incidence of bone marrow infiltration (16/51 with bone marrow involvement vs 35/51 with bone marrow involvement; Spearman ρ=0467 P<0.001) positively correlated with CXCR4 expression. CXCL12, the ligand of CXCR4 and CXCR7, was expressed by epithelial, endothelial and inflammatory cells, MALT lymphoma cells and was most strongly expressed by extranodal diffuse large B-cell lymphoma cells, suggesting at least in part an autocrine signaling pathway. Our data indicate that CXCR4 expression is associated with nodal manifestation and a more advanced stage of lymphomas and hence, might serve as useful clinical prognostic marker.

Oxygen Sensing Mesenchymal Progenitors Promote Neo-Vasculogenesis in a Humanized Mouse Model In Vivo

Despite insights into the molecular pathways regulating hypoxia-induced gene expression, it is not known which cell types accomplish oxygen sensing during neo-vasculogenesis. We have developed a humanized mouse model of endothelial and mesenchymal progenitor co-transplantation to delineate the cellular compartments responsible for hypoxia response during vasculogenesis. Mesenchymal stem/progenitor cells (MSPCs) accumulated nuclear hypoxia-inducible transcription factor (HIF)-1α earlier and more sensitively than endothelial colony forming progenitor cells (ECFCs) in vitro and in vivo. Hypoxic ECFCs showed reduced function in vitro and underwent apoptosis within 24h in vivo when used without MSPCs. Surprisingly, only in MSPCs did pharmacologic or genetic inhibition of HIF-1α abrogate neo-vasculogenesis. HIF deletion in ECFCs caused no effect. ECFCs could be rescued from hypoxia-induced apoptosis by HIF-competent MSPCs resulting in the formation of patent perfused human vessels. Several angiogenic factors need to act in concert to partially substitute mesenchymal HIF-deficiency. Results demonstrate that ECFCs require HIF-competent vessel wall progenitors to initiate vasculogenesis in vivo and to bypass hypoxia-induced apoptosis. We describe a novel mechanistic role of MSPCs as oxygen sensors promoting vasculogenesis thus underscoring their importance for the development of advanced cellular therapies.

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion >1x10(8) ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. (3,4) This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

TRPV1 mediates cellular uptake of anandamide and thus promotes endothelial cell proliferation and network-formation

ABSTRACT Anandamide (N-arachidonyl ethanolamide, AEA) is an endogenous cannabinoid that is involved in various pathological conditions, including cardiovascular diseases and tumor-angiogenesis. Herein, we tested the involvement of classical cannabinoid receptors (CBRs) and the Ca2+-channel transient receptor potential vanilloid 1 (TRPV1) on cellular AEA uptake and its effect on endothelial cell proliferation and network-formation. Uptake of the fluorescence-labeled anandamide (SKM4-45-1) was monitored in human endothelial colony-forming cells (ECFCs) and a human endothelial-vein cell line (EA.hy926). Involvement of the receptors during AEA translocation was determined by selective pharmacological inhibition (AM251, SR144528, CID16020046, SB366791) and molecular interference by TRPV1-selective siRNA-mediated knock-down and TRPV1 overexpression. We show that exclusively TRPV1 contributes essentially to AEA transport into endothelial cells in a Ca2+-independent manner. This TRPV1 function is a prerequisite for AEA-induced endothelial cell proliferation and network-formation. Our findings point to a so far unknown moonlighting function of TRPV1 as Ca2+-independent contributor/regulator of AEA uptake. We propose TRPV1 as representing a promising target for development of pharmacological therapies against AEA-triggered endothelial cell functions, including their stimulatory effect on tumor-angiogenesis.

PRMT1-mediated methylation of MICU1 determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake in immortalized cells.

Recent studies revealed that mitochondrial Ca2+ channels, which control energy flow, cell signalling and death, are macromolecular complexes that basically consist of the pore-forming mitochondrial Ca2+ uniporter (MCU) protein, the essential MCU regulator (EMRE), and the mitochondrial Ca2+ uptake 1 (MICU1). MICU1 is a regulatory subunit that shields mitochondria from Ca2+ overload. Before the identification of these core elements, the novel uncoupling proteins 2 and 3 (UCP2/3) have been shown to be fundamental for mitochondrial Ca2+ uptake. Here we clarify the molecular mechanism that determines the UCP2/3 dependency of mitochondrial Ca2+ uptake. Our data demonstrate that mitochondrial Ca2+ uptake is controlled by protein arginine methyl transferase 1 (PRMT1) that asymmetrically methylates MICU1, resulting in decreased Ca2+ sensitivity. UCP2/3 normalize Ca2+ sensitivity of methylated MICU1 and, thus, re-establish mitochondrial Ca2+ uptake activity. These data provide novel insights in the complex regulation of the mitochondrial Ca2+ uniporter by PRMT1 and UCP2/3.

Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum.

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

Endothelial Colony-Forming Progenitor Cell Isolation and Expansion

Vessel wall-derived somatic endothelial colony-forming progenitor cells (ECFCs) are key players in vascular homeostasis and regeneration. Due to their robust proliferative potential and profound vessel-forming capacity, ECFCs are considered to represent an attractive tool for vascular regenerative medicine and a promising target for antiangiogenic tumor therapy. Here, we describe an easily applicable method for isolating ECFCs directly from unmanipulated adult human blood and an animal protein-free large-scale expansion system to generate more than 100 million functional ECFCs.