Abstract
BackgroundExpression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette.ResultsTwo or more colored cells (~ 10), after staining with a chromogen such a 3,3′-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells.ConclusionThese findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.
Highlights
Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining
Compared with other Dental pulp stem cells (DPSCs), SHEDs exhibit a greater rate of proliferation [4, 8, 9], and express the mesenchymal stem cells (MSCs) surface markers (CD44, CD73, and CD90), osteoblast markers [alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and collagen type I α2 chain (COL1A2)], cartilage cell markers [collagen type X α1 Chain (COL10A1) and aggrecan (ACAN)], adipose cell markers [peroxisome proliferator-activated receptor-gamma2 (PPAR-γ2) and lipoprotein lipase (LPL)], and neuronal stem cell marker, nestin [10]
It has been demonstrated that 1) expression levels of stemness factors [e.g., octamerbinding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and ALP differ among the donors tested, 2) HDDPCs with high ALP activity and enriched with OCT3/4 and SOX2 tend to be reprogrammed into induced pluripotent stem cells when they are transfected with vectors carrying Yamanaka’s reprogramming factors, and 3) these ALP-enriched cells have the ability to differentiate into osteogenic or adipose cells when they are induced to differentiate into an osteoblastic or adipogenic lineage [13, 14]. These findings suggested that ALP-positive cells in HDDPCs could serve as a useful source in regenerative medicine
Summary
Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Compared with other DPSCs, SHEDs exhibit a greater rate of proliferation [4, 8, 9], and express the MSC surface markers (CD44, CD73, and CD90), osteoblast markers [alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and collagen type I α2 chain (COL1A2)], cartilage cell markers [collagen type X α1 Chain (COL10A1) and aggrecan (ACAN)], adipose cell markers [peroxisome proliferator-activated receptor-gamma (PPAR-γ2) and lipoprotein lipase (LPL)], and neuronal stem cell marker, nestin [10]. Little is known about SSCs within the dental pulp, which have been hereinafter called “human deciduous tooth-derived dental pulp cells (HDDPCs).”
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