Ken-ichi Tezuka

A more efficient method to generate integration-free human iPS cells

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ∼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.

Dental Pulp Cells for Induced Pluripotent Stem Cell Banking

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.

Characterization of Dental Pulp Stem Cells of Human Tooth Germs

In previous studies, human dental pulp stem cells (hDPSCs) were mainly isolated from adults. In this present study, we characterized hDPSCs isolated from an earlier developmental stage to evaluate the potential usage of these cells for tissue-regenerative therapy. hDPSCs isolated at the crown-completed stage showed a higher proliferation rate than those isolated at a later stage. When the cells from either group were cultured in medium promoting differentiation toward cells of the osteo/odontoblastic lineage, both became alkaline-phosphatase-positive, produced calcified matrix, and were also capable of forming dentin-like matrix on scaffolds in vivo. However, during long-term passage, these cells underwent a change in morphology and lost their differentiation ability. The results of a DNA array experiment showed that the expression of several genes, such as WNT16, was markedly changed with an increasing number of passages, which might have caused the loss of their characteristics as hDPSCs.

Suppression of differentiation and proliferation of early chondrogenic cells by Notch.

Notch is a transmembrane protein involved in cell fate determination. In the present study, we observed temporally and spatially restricted expression of Notch1 in developing cartilage. Notch1 was localized starting from the mesenchymal condensation stage of embryonic mouse forelimbs. Interestingly, although localization could not be detected in the proliferating chondrocytes, obvious immunoreactivity indicating its expression was retained in the perichondrial region. Next, we investigated the expression of Notch1 and related molecules in a chondrogenic cell line, ATDC5 cells. Notch1, Delta-like (Dll)1, Deltex2, and Deltex3 were coexpressed after 6-day insulin treatment. Expression of Hairy and Enhancer of split homologue (HES)-1 followed thereafter. These results suggest that Notch may have a role in the early stage of chondrogenesis. To assess the effect of Notch activation, we cultured ATDC5 cells with a myeloma clone constitutively expressing Dll1, a ligand of Notch. We also used an adenovirus vector to express the constitutively active Notch1 intracellular domain (NIC). Activating either the endogenous or exogenous Notch receptor dramatically inhibited chondrogenic cell differentiation of ATDC5 cells, as assessed by Alcian blue staining of the cells and chondrocyte differentiation markers. Last, we investigated the effect of NIC on the proliferation of the ATDC5 cells. Expression of NIC by the adenovirus strongly suppressed thymidine incorporation. These results indicate that Notch is expressed in the initial stage of chondrogenic cell differentiation and has a strong inhibitory effect on both differentiation and proliferation of the cells when activated. The expression of Notch decreases as chondrogenic differentiation proceeds; however, a population of the cells with sustained expression of Notch1 become perichondrial cells. Considering that the perichondrium acts as a stem cell source of osteoblasts and chondrocytes, Notch1 may have a role in the formation of these cells by suppressing both differentiation and proliferation.

Computer-simulated bone architecture in a simple bone-remodeling model based on a reaction-diffusion system

Bone is a complex system with functions including those of adaptation and repair. To understand how bone cells can create a structure adapted to the mechanical environment, we propose a simple bone remodeling model based on a reaction-diffusion system influenced by mechanical stress. Two-dimensional bone models were created and subjected to mechanical loads. The conventional finite element method (FEM) was used to calculate stress distribution. A stress-reactive reaction-diffusion model was constructed and used to simulate bone remodeling under mechanical loads. When an external mechanical stress was applied, stimulated bone formation and subsequent activation of bone resorption produced an efficient adaptation of the internal shape of the model bone to a given stress, and demonstrated major structures of trabecular bone seen in the human femoral neck. The degree of adaptation could be controlled by modulating the diffusion constants of hypothetical local factors. We also tried to demonstrate the deformation of bone structure during osteoporosis by the modulation of a parameter affecting the balance between formation and resorption. This simple model gives us an insight into how bone cells can create an architecture adapted to environmental stress, and will serve as a useful tool to understand both physiological and pathological states of bone based on structural information.

TGF-β suppresses POEM expression through ERK1/2 and JNK in osteoblasts

POEM, also called nephronectin, is an extracellular matrix protein that is considered to play a critical role as an adhesion molecule in the development and functioning of various tissues, such as kidneys and bones. In the present study, we examined the molecular mechanism of POEM gene expression, and found that transforming growth factor‐β (TGF‐β) strongly inhibited POEM expression in the mouse osteoblastic cell line, MC3T3‐E1. TGF‐β‐induced decrease of POEM expression occurred in both time‐ and dose‐dependent manners through the activation of TGF‐β receptor I and extracellular signal‐regulated kinase/c‐Jun N‐terminal kinase pathways.

Derivation of iPSCs after culture of human dental pulp cells under defined conditions.

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-enhanced Derivation of iPSCs from Human Dental Pulp Cells

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.

The homeobox gene DLX4 promotes generation of human induced pluripotent stem cells

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.

iBone: A Reaction Diffusion Based Shape Optimization Method

Bone is a highly specialized form of connective tissue consisting of org anic and inorganic materials. Bone is continuously remodeled by bone forming osteoblasts and resorbing ost eoclast . In terrestrial vertebrates, these two activities are strictl y ba anced and adapt the shape of bone to the local stress with limited calcium intake from foods. However, the cell based-adaptation mechanism underlying this system have not been well considered. Recently, we have found that human mesenchymal stem cells strongly stimulated towards osteoblasti c lineage form a condensation pattern similar to Turing patterns observed in reaction-diffusion models. We c onstructed a hypothetical model of bone remodeling (iBone), by coupling the bone forming and resorbing ac t vities based on the reaction-diffusion model weighed by local stress. When an externa l mechanical stress was applied to a sample model, stimulated bone formation and subsequent activation of bone resorption efficiently adapted the shape of it to the given stress, and created flat stress distribution. iB one could also repair fractures which caused uneven stress distribution. The efficacy of i Bone proposes a principal model how bone cells can form a cooperative system that adapt the microst ru ture of bone to the voluntary mechanical loads; and, suggests that element based-parallel computi ng system, such as reaction-diffusion system, can be applied for designing efficient stress adapta tion models.