Isolation and characterization of lymphoid enhancer factor-1-positive deciduous dental pulp stem-like cells after transfection with a piggyBac vector containing LEF1 promoter-driven selection markers.

OBJECTIVE: Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. LEF1 is also an important member of the Wnt/ß-catenin signaling pathway that plays important roles in the self-renewal and differentiation of embryonic stem cells. We speculated that LEF1 might function in the stem cells from human exfoliated deciduous teeth (SHED). In this study, we attempted to isolate such LEF1-positive cells from human deciduous dental pulp cells (HDDPCs) by genetic engineering technology, using the human LEF1 promoter. DESIGN: A piggyBac transposon plasmid (pTA-LEN) was introduced into HDDPCs, using the Neon® transfection system. After G418 selection, the emerging colonies were assessed for EGFP-derived fluorescence by fluorescence microscopy. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using RNA isolated from these colonies to examine stem cell-specific transcript expression. Osteoblastic or neuronal differentiation was induced by cultivating the LEF1-positive cells with differentiation-inducing medium. RESULTS: RT-PCR analysis confirmed the expression of several stem cell markers, including OCT3/4, SOX2, REX1, and NANOG, in LEF1-positive HDDPCs, which could be differentiated into osteoblasts and neuronal cells. CONCLUSIONS: The isolated LEF1-positive HDDPCs exhibited the properties of stem cells, suggesting that LEF1 might serve as a marker for SHED.

Alkaline phosphatase and OCT-3/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells.

AIM: The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells. METHODS: We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells. RESULTS: Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3/4 expression was also prominent in those two lines. Furthermore, these two lines proliferated faster than the other three lines. The transfection of HDDPC with Yamanaka factors resulted in the generation of iPS cells from HDDPC-1 and -5. CONCLUSION: The number of cells with the stemness property of HDDPC differs among individuals, which suggests that HDDPC showing an increased expression of both ALP and OCT-3/4 can be more easily reprogrammed to generate iPS cells after the forced expression of reprogramming factors.