Stem cell donor registry activities during the COVID-19 pandemic: a field report by DKMS.

The COVID-19 pandemic has serious implications also for patients with other diseases. Here, we describe the effects of the pandemic on unrelated hematopoietic stem cell donation and transplantation from the perspective of DKMS, a large international donor registry. Especially, we cover the development of PBSC and bone marrow collection figures, donor management including Health and Availability Check (HAC), transport and cryopreservation of stem cell products, donor recruitment and business continuity measures. The total number of stem cell products provided declined by around 15% during the crisis with a particularly strong decrease in bone marrow products. We modified donor management processes to ensure donor and product safety. HAC instead of confirmatory typing was helpful especially in countries with strict lockdowns. New transport modes were developed so that stem cell products could be safely delivered despite COVID-19-related travel restrictions. Cryopreservation of stem cell products became the new temporary standard during the pandemic to minimize risks related to transport logistics and donor availability. However, many products from unrelated donors will never be transfused. DKMS discontinued public offline donor recruitment, leading to a 40% decline in new donors during the crisis. Most DKMS employees worked from home to ensure business continuity during the crisis.

Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy.

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 x 10(-9) cm(2) s(-1) independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.

Stem cells and experimental leukemia can be distinguished by lipid raft protein composition.

The stable transfection of the canine CD34(-) multipotent cell line DO64 with retroviral constructs containing the cDNA for the canine major histocompatibility complex (MHC) class II DR genes led to the cell clone DO64#14, which is characterized by malignant transformation and tumor growth in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The additional expression of p27(kip-1) in the transformed cell clone partially reversed the malignant phenotype. Because several proteins associated with lipid rafts are involved in signal transduction and because changes of lipid raft composition are linked to the pathogenesis of leukemias, raft-associated proteins in DO64 cells and the deduced transformed cell clones were compared using a proteomic approach. Raft-associated proteins were separated by two-dimensional electrophoresis and identified by MALDI-TOF-MS. Here we show that the stem cell line DO64 and the deduced cell clones can clearly be distinguished by differences in the expression of a number of raft-associated proteins, namely caveolin-1, flotillin- 1, vimentin, galectin-3, and glyceraldehyde-3-phosphate dehydrogenase. All identified proteins play an important role in cellular functions and may therefore participate in raft-mediated leukemic transformation. Therefore, our study suggests that the analysis of lipid raft protein composition may be useful for the identification of molecular markers of the transformation process.

Increased association with detergent-resistant membranes/lipid rafts of apically targeted mutants of the interleukin-6 receptor gp80.

Interleukin (IL)-6 is an important cytokine in inflammatory processes, differentiation and growth. The IL-6 receptor complex comprises the specific IL-6 receptor (gp80) and two molecules of the signal tranducing component gp130 which transduces the signal into the nucleus via the Jak-STAT pathway. Both, gp80 and gp130 are sorted preferentially to the basolateral membrane of polarised Madin-Darby canine kidney (MDCK) cells. Previously, we have shown that gp130 partially localises to detergent-resistant membranes (DRMs)/lipid rafts and that lipid raft integrity is crucial for signalling to occur. Here we now demonstrate that wild-type gp80 is associated with DRMs only to a minor extent. However, gp80 mutants which lack parts of the cytoplasmic domain and therefore are more apically expressed than the wild type show an increased affinity for the liquid-ordered membrane domain. Studies with non-polarised MDCK cells suggest that the lipid raft association of the different mutants of gp80 precedes the establishment of cell polarity. Our findings suggest that lipid rafts play a role in the sorting of apically targeted gp80.

Polarity and lipid raft association of the components of the ciliary neurotrophic factor receptor complex in Madin-Darby canine kidney cells.

Ciliary neurotrophic factor (CNTF) signals via a tripartite receptor complex consisting of the glycosyl-phosphatidylinositol (GPI)-anchored CNTF receptor (CNTF-R), the leukaemia inhibitory factor receptor (LIF-R) and the interleukin-6 (IL-6) signal transducer gp130. We have recently reported that gp130 is endogenously expressed in the polarised epithelial model cell line Madin-Darby canine kidney (MDCK) and we have demonstrated a preferential basolateral localisation of this protein. In the present study we show that MDCK cells also express the LIF-R and respond to stimulation with human LIF by activation of tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT3), both however in an unpolarised fashion. This suggests that MDCK cells may be target cells for LIF. We have furthermore stably expressed the human CNTF-R in MDCK cells and by two different assays we found an apical localisation. Consistent with these findings, stimulation of CNTF-R-positive cells resulted only in an activation of STAT3 when CNTF was added apically. These data demonstrate that each subunit of the CNTF receptor complex has a distinct distribution in polarised cells which may reflect the different roles the respective cytokines play in vivo. Since it is currently believed that lipid rafts are involved in signal transduction as well as protein sorting we studied the association of the three receptor complex components with membrane rafts using different protocols. Whereas the CNTF-R cofractionated quantitatively with lipid rafts independently of the method used, gp130 and the LIF-R were found to associate with lipid rafts only partially when detergents were used for isolation. These findings could indicate that either the three receptor complex subunits are localised to the same kind of raft but with different affinities to the liquid-ordered environment, or that they are localised to different types of rafts. CNTF-, LIF-, and IL-6-dependent STAT3 activation was sensitive to the cholesterol-depleting drug methyl-beta-cyclodextrin (MCD) suggesting that the integrity of lipid rafts is important for IL-6-type cytokine-induced STAT activation.