Abstract

Abstract 1442

Poster Board I-465

We previously characterized the interaction between human hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) in a 2D-coculture-system and demonstrated that MSC-feeder-layer is capable of maintaining “stemness” in HSC. In order to develop a more physiological in vitro surrogate model for the stem cell niche, we established a 3D-coculture-system using MSC-spheroids. In this model system we analyzed the role of major adhesion molecules such as N-cadherin and the CXCR4/SDF-1 axis upon migration, adhesion and proliferation of HSC.

MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were isolated from umbilical cord blood by magnetic cell sorting of CD34+ mononuclear cells. MSC were cultured as “hanging drops” with initially around 500 cells. Alternatively, MSC were seeded on a layer of 1% agarose in 96 well plates (500-1000 cells per well). In both cases after 24-48h forming of spheroid-like cell aggregations could be observed. Formation of spheroids was obviously independent upon different MSC culture conditions. MSC-spheroids were cocultured with HSC and continuously monitored with time-lapse microscopy. After 24-48 hours of cocultivation, we stained the cellular contacts with a panel of antibodies specific for various components of tight, gap and adherens junctions. Using advanced confocal laser scanning microscopy in combination with deconvolution and volume rendering software, we were able to produce 3D-images of intercellular junctions between HSC and MSC. Additionally, thin sections of the spheroids were analyzed by electron microscopy.

In 2D-cocultures, intercellular connections between HSC and MSC are mainly characterized by podia formation of the HSC linking to the adjacent MSC. At the intimate contact zone to the MSC, the cytoplasmic plaque proteins alpha- and beta-catenin, as well as the transmembrane glycoprotein N-cadherin could be identified. Upon 3D-coculture of HSCs with MSC-spheroids, HSCs showed directed locomotion towards MSC-spheroids. After 12h several HSC showed directed locomotion towards MSC-spheroids and connected to the outer cell layer by podia formation. At the contact zone cadherin-catenin-based junctions could be identified. After additional 12-24h a further integration of HSC into the inner mass of the spheroid was observed. Cell division kinetic of HSC was increased when cocultured with MSC and the rate of CD34+ cells remained higher compared to monoculturing of HSC in the same culture-medium. Culturing HSC on MSC feeder-layer resulted in a reduced CXCR4 expression, possibly due to an internalization triggered by direct cell contact to MSC.

This novel 3D-setup of an in vitro surrogate model for the human stem cell niche provides deeper inside into the cellular interaction between HSC and stromal cells and demonstrates that direct cellular contact influences cell division kinetics and CXCR4 expression of HSC.

Disclosures

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.