TLR4-Agonist Stimulated Human Multipotent Mesenchymal Stromal Cells Enhance Both Hematopoietic Progenitors and Myeloid Differentiation

Human mesenchymal stromal cells (MSCs) can replicate as undifferentiated cells in vitro and are capable of differentiating into multiple mesenchymal tissues. In the bone marrow, MSCs are thought to contribute to nurturing stroma involved in maintaining and modulating hematopoiesis. Toll-like receptors (TLRs) function as receptors for different conserved pathogen associated products as well as certain host derived molecules. TLRs are expressed in several hematopoietic and non-hematopoietic cells, and their activation plays a key role in innate and adaptive immune responses to infectious agents, as well as in the development of pathologic conditions like autoimmune disease. In inflammatory conditions like sepsis, cytokines like G-CSF are elevated in serum, CD34+ cells increase in circulation, and bone marrow myelopoiesis is enhanced. We hypothesized that MSCs express TLRs and are capable to respond to TLR agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis according to respective needs. In vitro cultured bone marrow MSCs were analyzed for mRNA expression of TLRs. TLR 1, 3, 4, 5, 6, and 9 expression, but not TLR 2, 7, 8, and 10 expression was detectable. Compared to human conventional and plasmacytoid blood dendritic cells, MSCs expressed TLR-3 and TLR-4 at levels up to 2 log higher than did conventional DCs, while TLR 9 expression was low. Upon in vitro stimulation with TLR-4 agonists, mRNA transcripts of flt3-ligand and thrombopoietin increased, and MSCs secreted high amounts of G-CSF, GM-CSF, and M-CSF, whereas stimulation with TLR-9 agonists did not lead to changes in cytokine levels measured in supernatants. TLR-4 stimulated MSC conditioned media enhanced myeloid colony formation from human CD34+ cells about 2.5× fold compared to non-stimulated MSC conditioned media. Furthermore, in co-culture assays of MSCs with CD34+ cells, TLR-4 stimulated MSCs were capable to retain 8 fold more CD34+ cells in divisions 0–3 and maintained overall 2 fold higher numbers of CD34+ cells. In secondary colony formation assays, CD34+ cells from TLR-4 stimulated MSC co-cultures produced about 2.5 fold more myeloid colonies, with some being CFU-GEMM. Moreover, hematopoietic cells taken from TLR-4 stimulated MSC co-cultures were able to engraft conditioned newborn Rag 2−/− gc−/− mice and differentiated into B, T, and myeloid cells, while CD34+ cells from co-cultures of non-stimulated MSCs did not engraft.

These findings thus suggest a regulatory mechanism how, e.g. in gram-negative infection, inflammatory signals are translated via stromal cells into sustained early hematopoiesis and myeloid differentiation to efficiently meet the requests of an ongoing innate immune response.