Defined sets of transcriptional factors can reprogram human somatic cells to induced pluripotent stem (iPS) cells. However, many types of human cells are not easily accessible to minimally invasive procedures. Here we evaluated dental pulp cells (DPCs) as an optimal source of iPS cells, since they are easily obtained from extracted teeth and can be expanded under simple culture conditions. From all 6 DPC lines tested with the conventional 3 or 4 reprogramming factors, iPS cells were effectively established from 5 DPC lines. Furthermore, determination of the HLA types of 107 DPC lines revealed 2 lines homozygous for all 3 HLA loci and showed that if an iPS bank is established from these initial pools, the bank will cover approximately 20% of the Japanese population with a perfect match. Analysis of these data demonstrates the promising potential of DPC collections as a source of iPS cell banks for use in regenerative medicine.

SUMMARY Induced pluripotent stem cell (iPSC)-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8ab+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs). iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by defined transcription factors has been a well-established technique and will provide an invaluable resource for regenerative medicine. However, the low reprogramming efficiency of human iPSC is still a limitation for clinical application. Here we showed that the reprogramming potential of human dental pulp cells (DPCs) obtained from immature teeth is much higher than those of mature teeth DPCs. Furthermore, immature teeth DPCs can be reprogrammed by OCT3/4 and SOX2, conversely these two factors are insufficient to convert mature teeth DPCs to pluripotent states. Using a gene expression profiles between these two DPC groups, we identified a new transcript factor, distal-less homeobox 4 (DLX4), which was highly expressed in immature teeth DPCs and significantly promoted human iPSC generation in combination with OCT3/4, SOX2, and KLF4. We further show that activation of TGF-β signaling suppresses the expression of DLX4 in DPCs and impairs the iPSC generation of DPCs. Our findings indicate that DLX4 can functionally replace c-MYC and supports efficient reprogramming of immature teeth DPCs.

Human dental pulp cells (hDPCs) are a promising resource for regenerative medicine and tissue engineering and can be used for derivation of induced pluripotent stem cells (iPSCs). However, current protocols use reagents of animal origin (mainly fetal bovine serum, FBS) that carry the potential risk of infectious diseases and unwanted immunogenicity. Here, we report a chemically defined protocol to isolate and maintain the growth and differentiation potential of hDPCs. hDPCs cultured under these conditions showed significantly less primary colony formation than those with FBS. Cell culture under stringently defined conditions revealed a donor-dependent growth capacity; however, once established, the differentiation capabilities of the hDPCs were comparable to those observed with FBS. DNA array analyses indicated that the culture conditions robustly altered hDPC gene expression patterns but, more importantly, had little effect on neither pluripotent gene expression nor the efficiency of iPSC induction. The chemically defined culture conditions described herein are not perfect serum replacements, but can be used for the safe establishment of iPSCs and will find utility in applications for cell-based regenerative medicine.

Hypoxia enhances the reprogramming efficiency of human dermal fibroblasts to become induced pluripotent stem cells (iPSCs). Because we showed previously that hypoxia facilitates the isolation and maintenance of human dental pulp cells (DPCs), we examined here whether it promotes the reprogramming of DPCs to become iPSCs. Unlike dermal fibroblasts, early and transient hypoxia (3% O2) induced the transition of DPCs to iPSCs by 3.3- to 5.1-fold compared with normoxia (21% O2). The resulting iPSCs closely resembled embryonic stem cells as well as iPSCs generated in normoxia, as judged by morphology and expression of stem cell markers. However, sustained hypoxia strongly inhibited the appearance of iPSC colonies and altered their morphology, and anti-oxidants failed to suppress this effect. Transient hypoxia increased the expression levels of NANOG and CDH1 and modulated the expression of numerous genes, including those encoding chemokines and their receptors. Therefore, we conclude that hypoxia, when optimized for cell type, is a simple and useful tool to enhance the reprogramming of somatic cells to become iPSCs.