Culturing and Neuronal Differentiation of Human Dental Pulp Stem Cells.

A major issue in studying human neurogenetic disorders, especially rare syndromes affecting the nervous system, is the ability to grow neuronal cultures that accurately represent these disorders for analysis. Although there has been some success in generating induced pluripotent stem cells (iPSC) from both skin and blood, there are still limitations to the collection, production and use of iPSC derived neurons. We have had significant success in collecting and growing human dental pulp stem cells (DPSC) from exfoliated teeth sent directly to our laboratory by the parents of children with a variety of rare neurogenetic syndromes. This protocol outlines our current methods for the growth and expansion of DPSC from exfoliated (baby) teeth. These DPSC can be differentiated into a variety of cell types including osteoblasts, chondrocytes, and mixed neuron and glial cultures. Here we provide our protocol for the differentiation of early passage DPSC cultures into neurons for molecular and cellular studies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Collection and transportation of exfoliated teeth Basic Protocol 2: Dental pulp extraction Basic Protocol 3: Passage, freezing, and thawing of DPSC cultures Basic Protocol 4: Differentiation of DPSC into mixed neuronal cultures.

Significant transcriptional changes in 15q duplication but not Angelman syndrome deletion stem cell-derived neurons.

Background: The inability to analyze gene expression in living neurons from Angelman (AS) and Duplication 15q (Dup15q) syndrome subjects has limited our understanding of these disorders at the molecular level. Method: Here, we use dental pulp stem cells (DPSC) from AS deletion, 15q Duplication, and neurotypical control subjects for whole transcriptome analysis. We identified 20 genes unique to AS neurons, 120 genes unique to 15q duplication, and 3 shared transcripts that were differentially expressed in DPSC neurons vs controls. Results: Copy number correlated with gene expression for most genes across the 15q11.2-q13.1 critical region. Two thirds of the genes differentially expressed in 15q duplication neurons were downregulated compared to controls including several transcription factors, while in AS differential expression was restricted primarily to the 15q region. Here, we show significant downregulation of the transcription factors FOXO1 and HAND2 in neurons from 15q duplication, but not AS deletion subjects suggesting that disruptions in transcriptional regulation may be a driving factor in the autism phenotype in Dup15q syndrome. Downstream analysis revealed downregulation of the ASD associated genes EHPB2 and RORA, both genes with FOXO1 binding sites. Genes upregulated in either Dup15q cortex or idiopathic ASD cortex both overlapped significantly with the most upregulated genes in Dup15q DPSC-derived neurons. Conclusions: Finding a significant increase in both HERC2 and UBE3A in Dup15q neurons and significant decrease in these two genes in AS deletion neurons may explain differences between AS deletion class and UBE3A specific classes of AS mutation where HERC2 is expressed at normal levels. Also, we identified an enrichment for FOXO1-regulated transcripts in Dup15q neurons including ASD-associated genes EHPB2 and RORA indicating a possible connection between this syndromic form of ASD and idiopathic cases.

Culturing and Neuronal Differentiation of Human Dental Pulp Stem Cells.

A major issue in studying human neurogenetic disorders, especially rare syndromes affecting the nervous system, is the ability to grow neuronal cultures that accurately represent these disorders for analysis. Although there has been some success in generating induced pluripotent stem (iPS) cells from both skin and blood, there are still limitations to the collection and production of iPS cells from these biospecimens. We have had significant success in collecting and growing human dental pulp stem (DPS) cells from exfoliated teeth sent to our laboratory by the parents of children with a variety of rare neurogenetic syndromes. This protocol outlines our current methods for the growth and expansion of DPS cells from exfoliated (baby) teeth. These DPS cells can be differentiated into a variety of cell types including osteoblasts, chondrocytes, and mixed neuron and glial cultures. Here we provide our protocol for the differentiation of early passage DPS cell cultures into neurons for molecular studies. © 2017 by John Wiley & Sons, Inc.

Dental Pulp Stem Cells Model Early Life and Imprinted DNA Methylation Patterns.

Early embryonic stages of pluripotency are modeled for epigenomic studies primarily with human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSCs). For analysis of DNA methylation however, ESCs and iPSCs do not accurately reflect the DNA methylation levels found in preimplantation embryos. Whole genome bisulfite sequencing (WGBS) approaches have revealed the presence of large partially methylated domains (PMDs) covering 30%-40% of the genome in oocytes, preimplantation embryos, and placenta. In contrast, ESCs and iPSCs show abnormally high levels of DNA methylation compared to inner cell mass (ICM) or placenta. Here we show that dental pulp stem cells (DPSCs), derived from baby teeth and cultured in serum-containing media, have PMDs and mimic the ICM and placental methylome more closely than iPSCs and ESCs. By principal component analysis, DPSC methylation patterns were more similar to two other neural stem cell types of human derivation (EPI-NCSC and LUHMES) and placenta than were iPSCs, ESCs or other human cell lines (SH-SY5Y, B lymphoblast, IMR90). To test the suitability of DPSCs in modeling epigenetic differences associated with disease, we compared methylation patterns of DPSCs derived from children with chromosome 15q11.2-q13.3 maternal duplication (Dup15q) to controls. Differential methylation region (DMR) analyses revealed the expected Dup15q hypermethylation at the imprinting control region, as well as hypomethylation over SNORD116, and novel DMRs over 147 genes, including several autism candidate genes. Together these data suggest that DPSCs are a useful model for epigenomic and functional studies of human neurodevelopmental disorders. Stem Cells 2017;35:981-988.

Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells.

These data relate to the differentiation of human dental pulp stem cells (DPSC) and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT) through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells). The data augment another study to characterize immortalized DPSC for the study of neurogenetic "Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders" [1]. Two copies of one typical control cell line (technical replicates) were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown.

Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders.

A major challenge to the study and treatment of neurogenetic syndromes is accessing live neurons for study from affected individuals. Although several sources of stem cells are currently available, acquiring these involve invasive procedures, may be difficult or expensive to generate and are limited in number. Dental pulp stem cells (DPSCs) are multipotent stem cells that reside deep the pulp of shed teeth. To investigate the characteristics of DPSCs that make them a valuable resource for translational research, we performed a set of viability, senescence, immortalization and gene expression studies on control DPSC and derived neurons. We investigated the basic transport conditions and maximum passage number for primary DPSCs. We immortalized control DPSCs using human telomerase reverse transcriptase (hTERT) and evaluated neuronal differentiation potential and global gene expression changes by RNA-seq. We show that neurons from immortalized DPSCs share morphological and electrophysiological properties with non-immortalized DPSCs. We also show that differentiation of DPSCs into neurons significantly alters gene expression for 1305 transcripts. Here we show that these changes in gene expression are concurrent with changes in protein levels of the transcriptional repressor REST/NRSF, which is known to be involved in neuronal differentiation. Immortalization significantly altered the expression of 183 genes after neuronal differentiation, 94 of which also changed during differentiation. Our studies indicate that viable DPSCs can be obtained from teeth stored for ≥72 h, these can then be immortalized and still produce functional neurons for in vitro studies, but that constitutive hTERT immortalization is not be the best approach for long term use of patient derived DPSCs for the study of disease.

Acetaminophen: antipyretic or hypothermic in mice? In either case, PGHS-1b (COX-3) is irrelevant.

Acetaminophen (AC) reduces the core temperatures (T(c)) of febrile and non-febrile mice alike. Evidence has been adduced that the selectively AC-sensitive PGHS isoform, PGHS-1b (COX-3), mediates these effects. PGHS-1b, however, has no catalytic potency in mice. To resolve this contradiction, AC was injected intravenously (i.v.) into conscious PGHS-1 gene-sufficient (wild-type (WT)) and -deficient (PGHS-1(-/-)) mice 60 min before or after pyrogen-free saline (PFS) or E. coli LPS (10 microg/kg) i.v. T(c) was monitored continuously; brain and plasma PGE(2) levels were determined hourly. AC at <160 mg/kg did not affect T(c) when given before PFS or LPS; at 160 mg/kg, it caused a approximately 2.5 degrees C T(c) fall in 60 min. LPS given after AC (all doses) induced a approximately 1 degrees C fever, not different from that in AC-untreated mice. But this rise was insufficient to overcome the hypothermia of the 160 mg/kg-treated mice; their T(c) culminated 1 degrees C below baseline. LPS given before AC similarly elevated T(c) approximately 1 degrees C. This rise was reduced to baseline in 30 min by 80 mg AC/kg; T(c) rebounded to its febrile level over the next 30 min. At 160 mg/kg, AC reduced T(c) to 4 degrees C below baseline in 60 min, where it remained until the end of the experiment. WT and PGHS-1(-/-) mice responded similarly to all the treatments. The basal brain and plasma PGE(2) levels of PFS mice and the elevated plasma levels of LPS mice were unchanged by AC at 160 mg/kg; but the latter's brain levels were reduced at 1h, then recovered. Thus, AC could exert an anti-PGHS-2 effect when this enzyme is upregulated in the brain of febrile mice. The hypothermia it induces in non-febrile mice, therefore, is due to another mechanism. PGHS-1b is not involved in either case.

LPS-activated complement, not LPS per se, triggers the early release of PGE2 by Kupffer cells.

The intravenous injection of LPS rapidly evokes fever. We have hypothesized that its onset is mediated by prostaglandin (PG)E(2) quickly released by Kupffer cells (Kc). LPS, however, does not stimulate PGE(2) production by Kc as rapidly as it induces fever; but complement (C) activated by LPS could be the exciting agent. To test this hypothesis, we injected LPS (2 or 8 microg/kg) or cobra venom factor (CVF, an immediate activator of the C cascade that depletes its substrate, ultimately causing hypocomplementemia; 25 U/animal) into the portal vein of anesthetized guinea pigs and measured the appearance of PGE(2), TNF-alpha, IL-1beta, and IL-6 in the inferior vena cava (IVC) over the following 60 min. LPS (at both doses) and CVF induced similar rises in PGE(2) within the first 5 min after treatment; the rises in PGE(2) due to CVF returned to control in 15 min, whereas PGE(2) rises due to LPS increased further, then stabilized. LPS given 3 h after CVF to the same animals also elevated PGE(2), but after a 30- to 45-min delay. CVF per se did not alter basal PGE(2) and cytokine levels and their responses to LPS. These in vivo effects were substantiated by the in vitro responses of primary Kc from guinea pigs to C (0.116 U/ml) and LPS (200 ng/ml). These results indicate that LPS-activated C rather than LPS itself triggers the early release of PGE(2) by Kc.

Nociception and the differential expression of cyclooxygenase-1 (COX-1), the COX-1 variant retaining intron-1 (COX-1v), and COX-2 in mouse dorsal root ganglia (DRG).

Prostaglandins (PGs) formed via the cyclooxygenase (COX) pathway mediate hyperalgesia in sensory nerve endings. To investigate the role of the COX isoforms in pain transmission we recently studied nociception in COX-isozyme-deficient mice using models of "sharp" rapidly transmitted pain (hot-plate) and slowly developing, diffuse pain (writhing) [Ballou L, Botting RM, Goorha S, Zhang J, Vane JR. Nociception in cyclooxygenase isozyme-deficient mice. Proc Natl Acad Sci USA 2000;97:10272]. Our results demonstrated that COX-1 (and not COX-2) was the primary isoform involved in nociception in both model systems. Given the importance of dorsal root ganglia (DRG) in pain transmission we examined the expression patterns of COX-1, -2 and the recently described variant of COX-1 retaining intron-1, originally referred to as "COX-3" but hereafter referred to as COX-1 variant (COX-1v), in mouse L4 or L5 DRG taken from normal and COX-isozyme-deficient mice. Messenger RNA and protein for COX isoforms from DRG, spinal cord as well as, heart, brain, kidney, spleen and skin of adult mice were isolated and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Patterns of COX-isoform expression were determined using immunohistochemical techniques. We found that COX-1 and COX-1v were both expressed in neurons while COX-2 expression was completely undetectable in the DRG. Immunohistochemical analysis of COX expression in DRG of mice exhibiting the chronic pain and inflammation associated with collagen-induced arthritis (CIA) expressed COX-1 and COX-1v while no COX-2 could be detected. For purposes of comparison, COX-1v mRNA was also expressed in heart, brain, spinal cord, kidney, spleen and skin. Together, these data support a role for COX-1 and perhaps COX-1v, not COX-2, as the primary producers of PGs in mouse DRG in normal and in mice subject to chronic pain and inflammation. These data also suggest potential alternative analgesic mechanisms of action for the newly developed, COX-2 selective inhibitors and the nonsteroidal anti-inflammatory drugs (NSAIDs) in pain transmission in the peripheral nervous system.

Acetaminophen-induced hypothermia in mice is mediated by a prostaglandin endoperoxide synthase 1 gene-derived protein.

Acetaminophen is a widely used antipyretic analgesic, reducing fever caused by bacterial and viral infections and by clinical trauma such as cancer or stroke. In rare cases in humans, e.g., in febrile children or HIV or stroke patients, acetaminophen causes hypothermia while therapeutic blood levels of the drug are maintained. In C57/BL6 mice, acetaminophen caused hypothermia that was dose related and maximum (>2 degrees C below normal) with a dose of 300 mg/kg. The reduction and recovery of body temperature was paralleled by a fall of >90% and a subsequent rise of prostaglandin (PG)E(2) concentrations in the brain. In cyclooxygenase (COX)-2(-/-) mice, acetaminophen (300 mg/kg) produced hypothermia accompanied by a reduction in brain PGE(2) levels, whereas in COX-1(-/-) mice, the hypothermia to this dose of acetaminophen was attenuated. The brains of COX-1(-/-) mice had approximately 70% lower levels of PGE(2) than those of WT animals, and these levels were not reduced further by acetaminophen. The putative selective COX-3 inhibitors antipyrine and aminopyrine also reduced basal body temperature and brain PGE(2) levels in normal mice. We propose that acetaminophen is a selective inhibitor of a COX-1 variant and this enzyme is involved in the continual synthesis of PGE(2) that maintains a normal body temperature. Thus, acetaminophen reduces basal body temperature below normal in mice most likely by inhibiting COX-3.

Intracerebroventricular interleukin-6, macrophage inflammatory protein-1 beta and IL-18: pyrogenic and PGE(2)-mediated?

This study was undertaken to determine whether cyclooxygenase (COX)-2, the critical enzyme in the production of febrigenic prostaglandin (PG) E(2), may be involved centrally in the fever induced in mice by homologous interleukin (IL)-6, macrophage inflammatory protein (MIP)-1 beta, and interleukin (IL)-18, a member of the pyrogenic IL-1 beta family. To this end, the core temperatures (Tc) of COX-1 and COX-2 gene-ablated mice and of their normal wild-type (WT) counterparts were recorded after intracerebroventricular (i.c.v.) challenge with recombinant murine (rm) IL-6 (10 ng/mouse), rmMIP-1 beta (20 pg/mouse), rmIL-18 (0.01-1 microgram/mouse), rmIL-1 beta (positive control; 0.1 microgram/mouse), or their vehicle (0.1% bovine serum albumin [BSA] in sterile phosphate-buffered saline [PBS]; 5 microl/mouse). rmIL-6 caused a approximately 1 degrees C T(c) rise in WT mice that peaked at approximately 120 min and gradually recovered over the next 3 h; COX-1(-/-) mice exhibited a relatively faster (peak at 45 min) and shorter (recovery at 150 min) febrile course, whereas COX-2(-/-) mice did not develop fever. rmMIP-1 beta induced a 1 degrees C fever (peak at 60 min) with a long time course (recovery incomplete at 300 min) in both WT and COX-2(-/-) mice; COX-1(-/-) mice displayed a quick-onset (peak at 40 min) and shorter (recovery at approximately 240 min) fever. rmIL-18 did not cause any thermal response at any dose whether administered intraperitoneally (i.p.) or i.c.v. in WT mice; COX gene-ablated mice, therefore, were not tested. These data indicate that COX-2-dependent PGE(2) is critical for the febrile response to IL-6, but not to MIP-1 beta. IL-18 i.p. or i.c.v. is not pyrogenic.

Both constitutive and inducible prostaglandin H synthase affect dermal wound healing in mice.

In an attempt to define the roles of prostaglandin H synthase 1 (PGHS-1, cyclooxygenase-1, COX-1) and prostaglandin H synthase 2 (PGHS-2, cyclooxygenase-2, COX-2) in wound healing, we investigated the healing of incisional dermal wounds in wild-type, PGHS-1 null, and PGHS-2 null mice. We measured tensile strength of the wounds, levels of PGHS-1 and PGHS-2 mRNA in the wound site, and histologic markers for the inflammatory, proliferative, and remodeling phases of wound healing. Although no gross visible differences were noted among healed wounds of the different mouse types, measurement of tensile strength showed that both PGHS-1 and PGHS-2 null wounds were weaker (75% and 70%, respectively) than wild-type wounds at 12 days after incision. At Day 8 the endothelial staining was 70% greater in the wounds of PGHS-2 null mice compared with their wild-type counterparts. In contrast at Day 12, staining for macrophages and myofibroblasts was less in PGHS-1 null wounds compared with wild-type and PGHS-2 null tissue. Compensatory expression of the alternate PGHS mRNA could be demonstrated by RT-PCR in the wounds of PGHS null mice on Days 1 and 4. We conclude that both PGHS-1 and PGHS-2 genes play distinct roles in the process of dermal wound healing.

The tissue-specific, compensatory expression of cyclooxygenase-1 and -2 in transgenic mice.

Prostaglandins are essential regulators of tissue homeostasis, reproduction and inflammation. We have recently shown that cells derived from cyclooxygenase (COX)-deficient mice express higher, compensatory levels of the remaining COX isozyme [Kirtikara et al., J. Exp. Med., 187, 517 (1998)]. To assess this compensatory expression phenomenon in vivo, we quantified COX-1 and COX-2 mRNA levels in various organs of COX-1- and COX-2-ablated mice using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. We found that COX-1 and COX-2 mRNAs in the brains of COX-ablated mice were elevated > 2-fold compared with wild-type (WT) animals. COX-2 mRNA was enhanced approximately 2-fold in the kidneys and stomachs of COX-1-deficient mice while COX-1 expression remained unchanged. Conversely, the livers of COX-2-deficient mice expressed 15-fold higher COX-1 mRNA levels, while hepatic COX-2 mRNA levels were not significantly altered in the COX-1-ablated mice. Steady state levels of COX-1 and COX-2 mRNAs in the hearts, lungs and spleens of WT, COX-1- and COX-2-deficient mice were indistinguishable from each other. Peritoneal macrophages isolated from COX-1- and COX-2-ablated mice also expressed significantly higher steady-state levels of cytoplasmic phospholipase A2 and 5-lipooxygenase mRNAs suggesting a global upregulation of eicosanoid biosynthetic pathways in COX-deficient mice. These data suggest that expression of both COX-1 and COX-2 can be re-programmed to compensate for the lack of both alleles of the alternate COX gene in transgenic mice.

The involvement of NF-kappaB in the constitutive overexpression of cyclooxygenase-2 in cyclooxygenase-1 null cells.

We recently reported that there was enhanced cyclooxygenase (COX)-2 expression and prostaglandin E(2) biosynthesis in COX-1 deficient (COX-1(-/-)) cells. We also observed that the growth of COX-1(-/-) cells was significantly retarded compared to wild-type (WT) and COX-2 deficient (COX-2(-/-)) cells. In this study, COX-2 expression and its promoter activity were compared in immortalized, nontransformed fibroblasts from WT, COX-1(-/-) or COX-2(-/-) mice in the context of the role of COX-2 as a growth regulator. When compared with WT cells expressing both COX isoenzymes, constitutive COX-2 protein and promoter activity were significantly higher in COX-1(-/-) cells as determined by Western blotting and luciferase assays using a 5'-flanking promoter construct of the murine COX-2 gene. The luciferase assay using a series of luciferase-linked COX-2 promoter deletions transfected into COX-1(-/-) cells indicated that a region involving NF-kappaB plays a significant role in regulating constitutive COX-2 expression. Data from electrophoretic mobility shift assays showed that COX-1(-/-) cells contained higher levels of activated NF-kappaB than either WT or COX-2(-/-) cells. Furthermore, COX-2 promoter activity was significantly inhibited by the oligonucleotides (ODNs) containing the NF-kappaB element (NF-kappaB decoy ODNs) but not by the scrambled control ODNs, as examined by the luciferase assay. These findings indicate that constitutive COX-2 promoter activity and protein expression are enhanced in COX-1(-/-) fibroblasts and that signaling via the NF-kappaB pathway is involved in the transcriptional control of constitutive COX-2 expression.