Biomimetic reconstruction of the hematopoietic stem cell niche for in vitro amplification of human hematopoietic stem cells.

Hematopoietic stem cell transplantation is successfully applied since the late 1950s; however, its efficacy still needs to be increased. A promising strategy is to transplant high numbers of pluripotent hematopoietic stem cells (HSCs). Therefore, an improved ex vivo culture system that supports proliferation and maintains HSC pluripotency would override possible limitations in cell numbers gained from donors. To model the natural HSC niche in vitro, we optimized the HSC medium composition with a panel of cytokines and valproic acid and used an artificial 3D bone marrow-like scaffold made of polydimethylsiloxane (PDMS). This 3D scaffold offered a suitable platform to amplify human HSCs in vitro and, simultaneously, to support their viability, multipotency and ability for self-renewal. Silicon oxide-covering of PDMS structures further improved amplification of CD34+ cells, although the conservation of naïve HSCs was better on non-covered 3D PDMS. Finally, we found that HSC cultivated on non-covered 3D PDMS generated most pluripotent colonies within colony forming unit assays. In conclusion, by combining biological and biotechnological approaches, we optimized in vitro HSCs culture conditions, resulting in improved amplification, multipotency maintenance and vitality of HSCs.

Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far-field and near-field optical microscopy.

A non-enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context 'low temperature' means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 degrees C. For LTFISH, the DNA probe had to be single-stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near-field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH-procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single-stranded DNA probes to double-stranded DNA targets are discussed.