High-Throughput Assay for the Identification of Compounds Regulating Osteogenic Differentiation of Human Mesenchymal Stromal Cells

Abstract

Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. They present multilineage differentiation potential and trophic and immunosuppressive abilities, making them the best candidate for clinical applications. Several molecules have been described to increase bone formation and were mainly discovered by candidate approaches towards known signaling pathways controlling osteogenesis. However, their bone forming potential is still limited, making the search for novel molecules a necessity. High-throughput screening (HTS) not only allows the screening of a large number of diverse chemical compounds, but also allows the discovery of unexpected signaling pathways and molecular mechanisms for a certain application, even without the prior knowledge of the full molecular pathway. Typically HTS is performed in cell lines, however, in this manuscript we have performed a phenotypical screen on more clinically relevant human mesenchymal stromal cells, as a proof of principle that HTS can be performed in those cells and can be used to find small molecules that impact stem cell fate. From a library of pharmacologically active small molecules, we were able to identify novel compounds with increased osteogenic activity. These compounds allowed achieving levels of bone-specific alkaline phosphatase higher than any other combination previously known. By combining biochemical techniques, we were able to demonstrate that a medium to high-throughput phenotypic assay can be performed in academic research laboratories allowing the discovery of novel molecules able to enhance stem cell differentiation.

High-throughput identification of small molecules that affect human embryonic vascular development

“…2A). The library was screened at a single concentration (4.5 μM) according to previous studies using ECs (21)(22)(23)(24) and other cells (25,26), in a volume of 200 μL per well of EGM-2 medium containing 0.25% DMSO (vol/vol). To identify compounds that selectively target ECs, we screened the same library against human anterior cruciate ligament (ACL) cells.…”

Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the Layer-by-Layer technique

“…For comparison, cells were also cultured in the top of the plastic surface of culture dishes or over the electrospun fiber samples in the presence of dexamethasone (positive control of osteogenesis) which is known to be an osteogenesis inductive factor. 110 Experiments were further done without dexamethasone and without pDNA using hMSCs cultured in the top of the 51 plastic surface of the cell culture dishes or over electrospun fiber mats (these were considered negative controls of the osteogenesis process).…”

Small molecule delivery through nanofibrous scaffolds for musculoskeletal regenerative engineering

“…However, the utilization of small molecules for engineering musculoskeletal tissues has been largely overlooked. This trend is very likely to be reversed as advanced screening technologies have recently generated a large number of novel regenerative small molecule drugs, and advanced tissue engineering scaffolds continue to provide promise for controllable drug delivery to target tissues [8, 22, 28–30]. …”

“…Thanks to the advances in high throughput screening technologies, over a hundred of osteogenic small molecules have been discovered in the past decade [8, 21, 25, 28, 29, 49, 50]. Of these osteogenic small molecules, a number of them might represent promising candidates for next-generation bone regenerative medicines because their osteogenic activities have been observed in various preclinical animal models [8, 21, 51–54].…”