The trabecular extracellular matrix (ECM) forms a three-dimensional scaffold to stabilize the bone marrow, provide substrates for cell-matrix interactions and retain, present or release signals to modulate hematopoietic stem and progenitor cell development. However, the impact of trabecular ECM components on hematopoiesis has been poorly studied. Using collagen IX alpha1 - deficient (Col9a1 ) mice, we revealed that a lack of collagen IX alpha1 results in a disorganized trabecular network enriched in fibronectin, and in a reduction in myeloid cells, which was accompanied by a decrease in colony-stimulating factor 1 receptor expression on monocytes from the bone marrow. In contrast, B-cell numbers in the bone marrow and T-cell numbers in the thymus remained unchanged. Alterations in the bone marrow microenvironment may not only reduce myeloid cell numbers, but also have long-term implications for myeloid cell function. Mice were infected with Listeria moncytogenes to analyze the function of myeloid cells. In this case, an inadequate macrophage-dependent clearance of bacterial infections was observed in Col9a1 mice in vivo. This was mainly caused by an impaired interferon-gamma/tumor necrosis factor-alpha-mediated activation of macrophages. The loss of collagen IX alpha1 therefore destabilizes the trabecular bone network, impairs myeloid cell differentiation, and affects the innate immune response against Listeria. Stem Cells 2018;36:1752-1763.

The recent successes in gene therapy for the treatment of rare blood disorders such as primary immunodeficiencies and hemoglobinopathies have highlighted the need to generate robust and scalable manufacturing processes. Here we compare large scale lentiviral transductions of hemopoietic stem cells performed on the CliniMACS Prodigy® to manual processes in terms of efficacy and viability of the end product. In our first series of experiments, we used a low, non-saturating MOI of 30 and the results showed that transduction efficiency on the CliniMACS Prodigy® was significantly increased from an average of 23.3% (manual) to 50.5% (CliniMACS Prodigy®). These results were further confirmed when we employed an MOI of 100 and showed that the average transduction efficiency was 74% from the CliniMACS Prodigy® compared to 54.1% from the manual steps. Moreover, the total viability of the cells cultured on the CliniMACS Prodigy® remained unaffected after two days of cultivation with an average recovery of 105% but with significantly less variability (SD: 15.4) compared to the manual steps (SD: 35.7). Finally there was no difference to the average VCN which was 2.2 from the manual steps and 2.4 from the CliniMACS Prodigy®. Overall, our results indicate that the CliniMACS Prodigy® generates higher transduction rates, combined with high viability compared to the manual process, but most importantly, with significantly lower variability, suggesting that it represents a closed system able to automatically perform complex processes as successfully as when manual handling steps are performed, but with higher predictability, efficiency and with minimal user interaction. Disclosures Papanikolaou: Miltenyi Biotec: Employment. Bissels:Miltenyi Biotec: Employment. Johnston:Miltenyi Biotec: Employment. Reinartz:Miltenyi Biotec: Employment. Brams:Miltenyi Biotec: Employment. Aivazidou:Miltenyi Biotec: Employment. Krenz:Miltenyi Biotec: Employment. Bomhard:Miltenyi Biotec: Employment. Knöbel:Miltenyi Biotec: Employment. Bosio:Miltenyi Biotec: Employment.