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Dental pulp-derived stem cells (DPSCs) are considered to be of great promise for use in tissue repair and regenerative medicine. DPSCs can easily be collected from discarded teeth with little ethical concerns and harvested in a minimally invasive and safe manner. However, unfractionated clonogenic DPSCs are heterogenous and have variations in their phenotype. In this review paper, we summarize further isolation methods of DPSC subpopulations including immunoselection methods and a granulocyte colony-stimulating factor (G-CSF) gradient mobilization method for therapeutic clinical applications. The fractionated DPSC subpopulations exhibit stem cell properties in vitro: (i) high expression of pluripotency markers, Oct3/4, Nanog, and Sox2; (ii) high stability in long-term expansion; (iii) multi-lineage differentiation capacity; (iv) high migratory activity; and (v) high expression of trophic factors to enhance proliferation, migration, and anti-apoptotic and immunomodulatory effects as well as angiogenesis and neurite extension. DPSC subpopulations have higher angiogenic, neurogenic, and regenerative potential compared with bone marrow stem cells and adipose stem cells, presenting an alternate versatile stem cell source for cellular therapies. Preclinical efficacy of DPSC subpopulations has also been investigated in various tissue/organ disease models including pulpitis, and currently a few clinical trials are underway to determine their safety and efficacy. Therefore, the major aim of this review is to highlight the recent progress in DPSC biology, trends in preclinical regenerative studies, and future perspectives.