Self-Assembly of Tooth Root Organoid from Postnatal Human Dental Stem Cells.

Challenges remain in simultaneously regenerating the multiple diverse tissues of the tooth root in a spatially organized manner. Previously, our research group has established that scaffold-free tissue engineering approaches enable dental pulp stem/progenitor cells (DPSCs) and periodontal ligament (PDL) stem/progenitor cells (PDLSCs) to self-assemble into dentin-pulp and PDL-cementum organoids, respectively. In this study, we leveraged the innate self-organizing capacity of DPSCs and PDLSCs to now engineer organoids that resemble the full tooth root. Scaffold-free engineered tissues were generated using a heterogeneous mixture of human DPSCs and PDLSCs. Within 2 days of construct formation, PDLSCs and DPSCs became spatially restricted to the periphery and center of the constructs, respectively, emulating their anatomical positions in the tooth root. Histological and microcomputed tomography analyses showed that organoids exhibited a striated mineral pattern with a central unmineralized core, surrounded by a mineralized tissue structure, enclosed within a second peripheral unmineralized tissue, similar to the natural tooth root. Interestingly, DPSCs gave rise to the central unmineralized tissue and the inner portion of the mineralized tissue, and PDLSCs generated the outer portion of the mineralized tissue and the peripheral soft tissue. Quantitative image analysis of immunofluorescent staining revealed increased dentin sialophosphoprotein expression in the region of mineralized tissue associated with DPSCs and increased cementum protein-1 expression in the portion formed by PDLSCs, demonstrating that tooth root organoids comprise two biochemically distinct mineralized tissues characteristic of dentin-like and cementum-like structures, respectively. In addition, PDL-associated protein-1 expression was localized to the peripheral soft tissue, suggesting the formation of a rudimentary PDL-like structure. This study demonstrates that DPSCs and PDLSCs have an inherent ability to orchestrate the formation of a full tooth root-like structure. These organoids present a biomimetic model system to study cellular dynamics driving dental tissue repair or could be utilized therapeutically as biological dental implants.

Differential Effects of Escherichia coli- Versus Porphyromonas gingivalis-derived Lipopolysaccharides on Dental Pulp Stem Cell Differentiation in Scaffold-free Engineered Tissues.

INTRODUCTION: To leverage the therapeutic capabilities of dental pulp stem cells (DPSCs) for regenerative endodontic applications, a better understanding of their innate defense and reparative processes is needed. Lipopolysaccharide (LPS) is a major virulent factor of gram-negative bacteria and contributor to endodontic infections. We have developed 3-dimensional scaffold-free DPSC tissues that self-organize into dentin-pulp organoids comprising a mineralized dentin-like tissue on the periphery and an unmineralized pulp-like core. In this study, scaffold-free DPSC constructs were used as controllable experimental models to study the DPSC response to bacterial challenge. METHODS: Scaffold-free constructs were engineered using DPSCs isolated from human third molars. To simulate bacterial exposure, DPSC constructs were exposed to either Porphyromonas gingivalis-derived LPS or Escherichia coli-derived LPS. The effects of LPS on DPSC differentiation, proliferation, and apoptosis were evaluated. RESULTS: Engineered tissues lacking LPS treatment self-organized into dentin-pulp organoids. LPS treatment did not negatively affect DPSC proliferation or apoptosis in the engineered tissues. Both E. coli LPS and P. gingivalis LPS inhibited the up-regulation of RUNX2 messenger RNA expression and reduced the expression of the odontoblast-associated proteins (P < .05), suggesting that LPS is inhibiting odontoblastic differentiation. However, only E. coli LPS treatment significantly reduced mineral deposition in the DPSC (P < .05) constructs, indicating that E. coli LPS but not P. gingivalis LPS reduced functional differentiation of DPSCs and prevented DPSCs from self-organizing into a dentin-pulp complex-like structure. CONCLUSIONS: This study establishes scaffold-free DPSC constructs as models of oral disease. Furthermore, it emphasizes the diversity of LPS derived from different bacterial species and highlights the necessity of using LPS derived from clinically relevant bacteria in basic science investigations.

Promoting and Orienting Axon Extension Using Scaffold-Free Dental Pulp Stem Cell Sheets.

Current treatments of facial nerve injury result in poor functional outcomes due to slow and inefficient axon regeneration and aberrant reinnervation. To address these clinical challenges, bioactive scaffold-free cell sheets were engineered using neurotrophic dental pulp stem/progenitor cells (DPCs) and their aligned extracellular matrix (ECM). DPCs endogenously supply high levels of neurotrophic factors (NTFs), growth factors capable of stimulating axonal regeneration, and an aligned ECM provides guidance cues to direct axon extension. Human DPCs were grown on a substrate comprising parallel microgrooves, inducing the cells to align and deposit a linearly aligned, collagenous ECM. The resulting cell sheets were robust and could be easily removed from the underlying substrate. DPC sheets produced NTFs at levels previously shown capable of promoting axon regeneration, and, moreover, inducing DPC alignment increased the expression of select NTFs relative to unaligned controls. Furthermore, the aligned DPC sheets were able to stimulate functional neuritogenic effects in neuron-like cells in vitro. Neuronally differentiated neuroblastoma SH-SY5Y cells produced neurites that were significantly more oriented and less branched when cultured on aligned cell sheets relative to unaligned sheets. These data demonstrate that the linearly aligned DPC sheets can biomechanically support axon regeneration and improve axonal guidance which, when applied to a facial nerve injury, will result in more accurate reinnervation. The aligned DPC sheets generated here could be used in combination with commercially available nerve conduits to enhance their bioactivity or be formed into stand-alone scaffold-free nerve conduits capable of facilitating improved facial nerve recovery.

Dental Pulp Cell Sheets Enhance Facial Nerve Regeneration via Local Neurotrophic Factor Delivery.

An effective strategy for sustained neurotrophic factor (NTF) delivery to sites of peripheral nerve injury (PNI) would accelerate healing and enhance functional recovery, addressing the major clinical challenges associated with the current standard of care. In this study, scaffold-free cell sheets were generated using human dental pulp stem/progenitor cells, that endogenously express high levels of NTFs, for use as bioactive NTF delivery systems. Additionally, the effect of fibroblast growth factor 2 (FGF2) on NTF expression by dental pulp cell (DPC) sheets was evaluated. In vitro analysis confirmed that DPC sheets express high levels of NTF messenger RNA (mRNA) and proteins, and the addition of FGF2 to DPC sheet culture increased total NTF production by significantly increasing the cellularity of sheets. Furthermore, the DPC sheet secretome stimulated neurite formation and extension in cultured neuronal cells, and these functional effects were further enhanced when DPC sheets were cultured with FGF2. These neuritogenic results were reversed by NTF inhibition substantiating that DPC sheets have a positive effect on neuronal cell activity through the production of NTFs. Further evaluation of DPC sheets in a rat facial nerve crush injury model in vivo established that in comparison with untreated controls, nerves treated with DPC sheets had greater axon regeneration through the injury site and superior functional recovery as quantitatively assessed by compound muscle action potential measurements. This study demonstrates the use of DPC sheets as vehicles for NTF delivery that could augment the current methods for treating PNIs to accelerate regeneration and enhance the functional outcome. Impact statement The major challenges associated with current treatments of peripheral nerve injuries (PNIs) are prolonged repair times and insufficient functional recovery. Dental pulp stem/progenitor cells (DPCs) are known to endogenously express high levels of neurotrophic factors (NTFs), growth factors that enhance axon regeneration. In this study, we demonstrate that scaffold-free DPC sheets can act as effective carrier systems to facilitate the delivery and retention of NTF-producing DPCs to sites of PNIs and improve functional nerve regeneration. DPC sheets have high translational feasibility and could augment the current standard of care to enhance the quality of life for patients dealing with PNIs.

Self-Assembly of an Organized Cementum-Periodontal Ligament-Like Complex Using Scaffold-Free Tissue Engineering.

A major challenge in regenerating periodontal tissues is emulating its complex structure containing both mineralized and soft tissues. In this study, scaffold-free tissue constructs engineered using periodontal ligament cells (PDLCs), which contain a population of adult stem/progenitor cells, self-assembled into an organized multi-tissue structure comprising a mineralized cementum-like core enclosed within a periodontal ligament (PDL)-like tissue. Scaffold-free engineered constructs were formed by culturing human PDLCs to form a cell sheet on six-well dishes containing two minutien pins placed 7 mm apart. The cell sheet was contracted by the cells to roll into the pins forming a cylindrical construct anchored on either end by the pins. These tissues were approximately 1 mm in diameter and 7 mm long and contained only the cells and their endogenous matrix. These scaffold-free engineered constructs exhibited two structurally distinct tissues, one in the center of the construct and another on the periphery. The center tissue was mineralized and expressed alkaline phosphatase and bone sialoprotein, similar to cementum. The peripheral tissue was not calcified and expressed periodontal ligament-associated protein-1 and periostin, which is characteristic of the periodontal ligament. This tissue organization was seen after in vitro culture and maintained in vivo following subcutaneous implantation in immunocompromised mice. These data demonstrate that scaffold-free tissue engineering facilitates PDLCs to self-assemble into an organized cementum-PDL-like complex. These engineered tissues could be used as implantable grafts to regenerate damaged periodontal tissues or as model systems to study PDLC biology and mechanisms driving organized tissue assembly within the periodontium.

Phenotypic screening reveals topoisomerase I as a breast cancer stem cell therapeutic target.

Cancer stem cells (CSCs) are a subpopulation generally thought to be responsible for cancer initiation and progression. Because CSCs are often rare in the total tumor cell population and differentiate rapidly when grown in culture, it has been challenging to uncover compounds that selectively target CSCs. We previously described CSC-emulating cells derived from breast cancer cell lines that maintained a stable undifferentiated state. We optimized a phenotypic assay with these cells and screened 1,280-bioactive compounds, identifying five that preferentially inhibited CSC-like cell proliferation. Using a compound-guided target identification approach, we found high topoisomerase I (Topo I) expression levels in breast CSC-like cells and primary breast CSCs. Structurally unrelated small molecules targeting Topo I preferentially inhibited CSC-like cells. These results illustrate the substantial power of this CSC phenotypic screening platform and promote Topo I as a potential molecular therapeutic target for therapies aimed at expunging CSCs.

Mitochondrial structure, function and dynamics are temporally controlled by c-Myc.

Although the c-Myc (Myc) oncoprotein controls mitochondrial biogenesis and multiple enzymes involved in oxidative phosphorylation (OXPHOS), the coordination of these events and the mechanistic underpinnings of their regulation remain largely unexplored. We show here that re-expression of Myc in myc-/- fibroblasts is accompanied by a gradual accumulation of mitochondrial biomass and by increases in membrane polarization and mitochondrial fusion. A correction of OXPHOS deficiency is also seen, although structural abnormalities in electron transport chain complexes (ETC) are not entirely normalized. Conversely, the down-regulation of Myc leads to a gradual decrease in mitochondrial mass and a more rapid loss of fusion and membrane potential. Increases in the levels of proteins specifically involved in mitochondrial fission and fusion support the idea that Myc affects mitochondrial mass by influencing both of these processes, albeit favoring the latter. The ETC defects that persist following Myc restoration may represent metabolic adaptations, as mitochondrial function is re-directed away from producing ATP to providing a source of metabolic precursors demanded by the transformed cell.

Point mutations in c-Myc uncouple neoplastic transformation from multiple other phenotypes in rat fibroblasts.

Deregulation of c-Myc (Myc) occurs in many cancers. In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism. Although Myc is wild type in most cancers (wtMyc), it occasionally acquires point mutations in certain lymphomas. Some of these mutations confer a survival advantage despite partially attenuating proliferation and transformation. Here, we have evaluated four naturally-occurring or synthetic point mutations of Myc for their ability to affect these phenotypes, as well as to promote genomic instability, to generate reactive oxygen species and to up-regulate aerobic glycolysis and oxidative phosphorylation. Our findings indicate that many of these phenotypes are genetically and functionally independent of one another and are not necessary for transformation. Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation. One mutation (Q131R) was greatly impaired for nearly all of the studied Myc phenotypes, yet was able to retain some ability to transform. These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state.

Permanently blocked stem cells derived from breast cancer cell lines.

Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. However, an unanticipated result was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immunocompromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs.

Regulation of reactive oxygen species homeostasis by peroxiredoxins and c-Myc.

Peroxiredoxins (Prxs) are highly conserved proteins found in most organisms, where they function primarily to scavenge reactive oxygen species (ROS). Loss of the most ubiquitous member of the family, Prx1, is associated with the accumulation of oxidatively damaged DNA and a tumor-prone phenotype. Prx1 interacts with the transcriptional regulatory domain of the c-Myc oncoprotein and suppresses its transforming activity. The DNA damage in tissues of prx1-/- mice is associated in some cases with only modest increases in total ROS levels. However, these cells show dramatic increases in nuclear ROS and reduced levels of cytoplasmic ROS, which explains their mutational susceptibility. In the current work, we have investigated whether changes in other ROS scavengers might account for the observed ROS redistribution pattern in prx1-/- cells. We show approximately 5-fold increases in Prx5 levels in prx1-/- embryo fibroblasts relative to prx1+/+ cells. Moreover, Prx5 levels normalize when Prx1 expression is restored. Prx5 levels also appear to be highly dependent on c-Myc, and chromatin immunoprecipitation experiments showed differential occupancy of c-Myc and Prx1 complexes at E-box elements in the prx5 gene proximal promoter. This study represents a heretofore unreported mechanism for the c-Myc-dependent regulation of one Prx family member by another and identifies a novel means by which cells reestablish ROS homeostasis when one of these family members is compromised.

Onzin, a c-Myc-repressed target, promotes survival and transformation by modulating the Akt-Mdm2-p53 pathway.

The c-Myc oncoprotein is a general transcription factor whose target genes dictate the c-Myc phenotype. One such target of c-Myc, 'onzin', is normally expressed at high levels in myeloid cells and is dramatically downregulated in response to c-Myc overexpression. We show here that short hairpin interfering RNA-mediated knockdown of endogenous onzin results in a reduced growth rate and a proapoptotic phenotype. In contrast, onzin overexpression in fibroblasts is associated with an increased growth rate, resistance to apoptotic stimuli, loss of the G2/M checkpoint, and tumorigenic conversion. Onzin-overexpressing cells fail to induce p53 in response to apoptotic stimuli and contain higher levels of the active, phosphorylated forms of Akt1 and, more strikingly, of Mdm2. Using yeast two-hybrid and coimmunoprecipitation assays, we show that onzin directly interacts with both proteins. Green fluorescent protein tagging also confirms directly that Akt1 and Mdm2 colocalize with onzin, although the precise subcellular distribution of each protein is dependent on its relative abundance. Collectively, our results identify onzin as a novel regulator of several p53-dependent aspects of the c-Myc phenotype via its dramatic effect on Mdm2. This is reminiscent of the c-Myc --> p19(ARF)--mid R: Mdm2 pathway and might function as a complementary arm to ensure the proper cellular response to oncogenic and/or apoptotic stimuli.

Regulation of reactive oxygen species, DNA damage, and c-Myc function by peroxiredoxin 1.

Overexpression of c-Myc results in transformation and multiple other phenotypes, and is accompanied by the deregulation of a large number of target genes. We previously demonstrated that peroxiredoxin 1 (Prdx1), a scavenger of reactive oxygen species (ROS), interacts with a region of the c-Myc transcriptional regulatory domain that is essential for transformation. This results either in the suppression or enhancement of some c-Myc functions and in the altered expression of select target genes. Most notably, c-Myc-mediated transformation is inhibited, implying a tumor suppressor role for Prdx1. Consistent with this, prdx1-/- mice develop age-dependent hemolytic anemias and/or malignancies. We now show that erythrocytes and embryonic fibroblasts from these animals contain higher levels of ROS, and that the latter cells show evidence of c-Myc activation, including the ability to be transformed by a ras oncogene alone. In contrast, other primary cells from prdx1-/- mice do not have elevated ROS, but nonetheless show increased oxidative DNA damage. This apparent paradox can be explained by the fact that ROS localize primarily to the cytoplasm of prdx1+/+ cells, whereas in prdx1-/- cells, much higher levels of nuclear ROS are seen. We suggest that increased DNA damage and tumor susceptibility in prdx1-/- animals results from this shift in intracellular ROS. prdx1-/- mice should be useful in studying the role of oxidative DNA damage in the causation of cancer and its prevention by antioxidants. They should also help in studying the relationship between oncogenes such as c-Myc and DNA damage.

Identification of the presenilins in hematopoietic cells with localization of presenilin 1 to neutrophil and platelet granules.

Most cases of familial Alzheimer disease (AD) are caused by mutations in presenilin 1 (PS1) and presenilin 2 (PS2). Presenilins are required for the proteolytic processing of the beta amyloid precursor protein, which yields beta amyloid peptide, the major component of extracellular amyloid plaques. In addition, presenilins are essential for proteolytic processing of other membrane proteins, including Notch, TrkB, and APLP2. Notch directs neural and hematopoietic development. Here we show mRNA and protein expression of PS1 in both lymphoid and myeloid cells, while PS2 mRNA is present only in lymphocytes. Expression of PS1 was found throughout myeloid development from CD34+ stem cells to platelets and neutrophils. PS1 expression was found in avian as well as mammalian blood cells. In neutrophils, PS1 colocalized with myeloperoxidase and CD63 within the azurophil granules as demonstrated by subcellular fractionation and double labeling immunogold electron microscopy. In platelets, PS1 colocalized with glucose transporter (GLUT-3) in the membrane of alpha granules, as evidenced by immunogold electron microscopy. The colocalization of PS1 and amyloid precursor protein in cell-specific granules suggests a conserved function across different tissues. These studies indicate that PS1 may play multiple roles in blood cell physiology and that blood tissue may provide a model to study PS1 interactions with other proteins.