Establishing a Workflow Towards Understanding Osteogenic Lineage Commitment of Enriched Homogeneous Populations of Human Dental Pulp Stem Cells

Immunoheterogeneity is a potential hindrance to the maximum applicability of mesenchymal stromal cells (MSCs) in biotherapeutics and regenerative medicine. The primary causes of this heterogeneity are donor variation, diversity of tissue sources, and the culture conditions under which MSCs clonally propagate. Immunoheterogeneity could manifest as functional heterogeneity within these stem cells, affecting their ability to differentiate into adult tissue lineages. The impacted healthy human third molar tooth is a rich source of MSCs, which are isolatable both from the dental pulp and the dental follicle. Dental pulp stem cells (DPSCs), though widely characterized for their multipotential lineage differentiation, demonstrate considerable immunoheterogeneity, which demands the enrichment of these stem cells post-isolation. The aim of this study is to enrich and understand the functional differences in sub-populations of MSCs towards a specific lineage. In this protocol, we have highlighted detailed, stepwise methods for sorting and culturing post-sort and optimized methods for quantifying the differentiation of sorted DPSCs towards the osteogenic lineage. Our workflow is designed with additional considerations for cell sorter availability, maintenance of cell viability during long-distance transport to a state-of-the-art facility, and optimization of post-sorting cell culture and downstream experiments. This protocol can be universally followed to understand functional differences within MSCs isolated from different tissue sources and their implications for multi-lineage differentiation. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Assessing multiparametric expression of MSC surface markers and single cell sorting for enrichment of stem cells Support Protocol 1: Antibody panel design Support Protocol 2: MSC culture, preparation of cell suspension, and sample staining Support Protocol 3: Setting up controls Support Protocol 4: Analysis and data interpretation Basic Protocol 2: Quantitative and qualitative assessment of osteogenic differentiation of post-sorted DPSCs Support Protocol 5: Differentiation of MSCS (pre-sort and post-sort considerations) Support Protocol 6: Cytochemical staining and quantification Support Protocol 7: Immunofluorescence staining Support Protocol 8: RNA isolation and qPCR-based characterization.