We aimed to immortalize primarily isolated human deciduous tooth-derived dental pulp cells (HDDPCs) by transfection with piggyBac (PB)-based transposon vectors carrying E7 from human papilloma virus 16 or complementary DNA (cDNA) encoding human telomerase reverse transcriptase (hTERT). HDDPCs were co-transfected with pTrans (conferring PB transposase expression) + pT-pac (conferring puromycin acetyltransferase expression) + pT-tdTomato (conferring tdTomato cDNA expression) and pT-E7 (conferring E7 expression) or pTrans + pT-pac + pT-EGFP (conferring enhanced green fluorescent protein cDNA expression) + pT-hTERT (conferring hTERT expression). After six days, these cells were selected in medium containing 5 μg/mL puromycin for one day, and then cultured in normal medium allowing cell survival. All resultant colonies were harvested and propagated as a pool. Stemness and tumorigenic properties of the established cell lines (“MT_E7” for E7 and “MT_hTERT” for hTERT) with untransfected parental cells (MT) were examined. Both lines exhibited proliferation similar to that of MT, with alkaline phosphatase activity and stemness-specific factor expression. They displayed differentiation potential into multi-lineage cells with no tumorigenic property. Overall, we successfully obtained HDDPC-derived immortalized cell lines using a PB-based transfection system. The resultant and parental cells were indistinguishable. Thus, E7 and hTERT could immortalize HDDPCs without causing cancer-associated changes or altering phenotypic properties.

Alkaline phosphatase (ALP) is a ubiquitous membrane-bound glycoprotein capable of providing inorganic phosphate by catalyzing the hydrolysis of organic phosphate esters, or removing inorganic pyrophosphate that inhibits calcification. In humans, four forms of ALP cDNA have been cloned, among which tissue-nonspecific ALP (TNSALP) (TNSALP) is widely distributed in the liver, bone, and kidney, making it an important marker in clinical and basic research. Interestingly, TNSALP is highly expressed in juvenile cells, such as pluripotent stem cells (i.e., embryonic stem cells and induced pluripotent stem cells (iPSCs)) and somatic stem cells (i.e., neuronal stem cells and bone marrow mesenchymal stem cells). Hypophosphatasia is a genetic disorder causing defects in bone and tooth development as well as neurogenesis. Mutations in the gene coding for TNSALP are thought to be responsible for the abnormalities, suggesting the essential role of TNSALP in these events. Moreover, a reverse-genetics-based study using mice revealed that TNSALP is important in bone and tooth development as well as neurogenesis. However, little is known about the role of TNSALP in the maintenance and differentiation of juvenile cells. Recently, it was reported that cells enriched with TNSALP are more easily reprogrammed into iPSCs than those with less TNSALP. Furthermore, in bone marrow stem cells, ALP could function as a “signal regulator” deciding the fate of these cells. In this review, we summarize the properties of ALP and the background of ALP gene analysis and its manipulation, with a special focus on the potential role of TNSALP in the generation (and possibly maintenance) of juvenile cells.

Pluripotent stem cells are classified as naïve and primed cells, based on their in vitro growth characteristics and potential to differentiate into various types of cells. Human-induced pluripotent stem cells (iPSCs, also known as epiblast stem cells [EpiSCs]) have limited capacity to differentiate and are slightly more differentiated than naïve stem cells (NSCs). Although there are several in vitro protocols that allow iPSCs to differentiate into pancreatic lineage, data concerning generation of β-cells from these iPSCs are limited. Based on the pluripotentiality of NSCs, it was hypothesized that NSCs can differentiate into pancreatic β-cells when placed under an appropriate differentiation induction condition. We examined whether NSCs can be efficiently induced to form potentially pancreatic β cells after being subjected to an in vitro protocol. Several colonies resembling in vitro-produced β-cell foci, with β-cell-specific marker expression, were observed when NSC-derived embryoid bodies (EBs) were induced to differentiate into β-cell lineage. Conversely, EpiSC-derived EBs failed to form such foci in vitro. Intrapancreatic grafting of the in vitro-formed β-cell foci into nude mice (BALB/c-nu/nu) generated a cell mass containing insulin-producing cells (IPCs), without noticeable tumorigenesis. These NSCs can be used as a promising resource for curing type 1 diabetes.

Background 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. 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. Results Two 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. Conclusion These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.

Tissue-specific stem cells exist in tissues and organs, such as skin and bone marrow. However, their pluripotency is limited compared to embryonic stem cells. Culturing primary cells on plastic tissue culture dishes can result in the loss of multipotency, because of the inability of tissue-specific stem cells to survive in feeder-less dishes. Recent findings suggest that culturing primary cells in medium containing feeder cells, particularly genetically modified feeder cells expressing growth factors, may be beneficial for their survival and proliferation. Therefore, the aim of this study was to elucidate the role of genetically modified human feeder cells expressing growth factors in maintaining the integrity of primary cultured human deciduous dental pulp cells. Feeder cells expressing leukemia inhibitory factor, bone morphogenetic protein 4, and basic fibroblast growth factor were successfully engineered, as evidenced by PCR. Co-culturing with mitomycin-C-treated feeder cells enhanced the proliferation of newly isolated human deciduous dental pulp cells, promoted their differentiation into adipocytes and neurons, and maintained their stemness properties. Our findings suggest that genetically modified human feeder cells may be used to maintain the integrity of primary cultured human deciduous dental pulp cells.