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
The global DNA methylome profiles (right) identified from human dental pulp stem cells (DPSCs) derived from exfoliated baby teeth are more similar to those of early life tissues derived from the embryo and placenta than induced pluripotent stem cells (iPSCs) derived from skin. For investigation of human diseases with altered DNA methylation patterns, such as Dup15q syndrome, DPSCs are a relevant cell culture model.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
This study demonstrated that immortalized dental pulp stem cells (DPSCs) do not form tumors in animals and that immortalized DPSCs can be differentiated into neurons in culture. These results lend ...support to the use of primary and immortalized DPSCs for future therapeutic approaches to treatment of neurobiological diseases.
Dental pulp stem cells (DPSCs) provide an exciting new avenue to study neurogenetic disorders. DPSCs are neural crest‐derived cells with the ability to differentiate into numerous tissues including neurons. The therapeutic potential of stem cell‐derived lines exposed to culturing ex vivo before reintroduction into patients could be limited if the cultured cells acquired tumorigenic potential. We tested whether DPSCs that spontaneously immortalized in culture acquired features of transformed cells. We analyzed immortalized DPSCs for anchorage‐independent growth, genomic instability, and ability to differentiate into neurons. Finally, we tested both spontaneously immortalized and human telomerase reverse transcriptase (hTERT)‐immortalized DPSC lines for the ability to form tumors in immunocompromised animals. Although we observed increased colony‐forming potential in soft agar for the spontaneously immortalized and hTERT‐immortalized DPSC lines relative to low‐passage DPSC, no tumors were detected from any of the DPSC lines tested. We noticed some genomic instability in hTERT‐immortalized DPSCs but not in the spontaneously immortalized lines tested. We determined that immortalized DPSC lines generated in our laboratory, whether spontaneously or induced, have not acquired the potential to form tumors in mice. These data suggest cultured DPSC lines that can be differentiated into neurons may be safe for future in vivo therapy for neurobiological diseases.
Significance
This study demonstrated that immortalized dental pulp stem cells (DPSCs) do not form tumors in animals and that immortalized DPSCs can be differentiated into neurons in culture. These results lend support to the use of primary and immortalized DPSCs for future therapeutic approaches to treatment of neurobiological diseases.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Duplication of chromosome 15q11-q13 (dup15q) accounts for approximately 3% of autism cases. Chromosome 15q11-q13 contains imprinted genes necessary for normal mammalian neurodevelopment controlled by ...a differentially methylated imprinting center (imprinting center of the Prader-Willi locus, PWS-IC). Maternal dup15q occurs as both interstitial duplications and isodicentric chromosome 15. Overexpression of the maternally expressed gene UBE3A is predicted to be the primary cause of the autistic features associated with dup15q. Previous analysis of two postmortem dup15q frontal cortical samples showed heterogeneity between the two cases, with one showing levels of the GABAA receptor genes, UBE3A and SNRPN in a manner not predicted by copy number or parental imprint.
Postmortem human brain tissue (Brodmann area 19, extrastriate visual cortex) was obtained from 8 dup15q, 10 idiopathic autism and 21 typical control tissue samples. Quantitative PCR was used to confirm duplication status. Quantitative RT-PCR and Western blot analyses were performed to measure 15q11-q13 transcript and protein levels, respectively. Methylation-sensitive high-resolution melting-curve analysis was performed on brain genomic DNA to identify the maternal:paternal ratio of methylation at PWS-IC.
Dup15q brain samples showed a higher level of PWS-IC methylation than control or autism samples, indicating that dup15q was maternal in origin. UBE3A transcript and protein levels were significantly higher than control and autism in dup15q, as expected, although levels were variable and lower than expected based on copy number in some samples. In contrast, this increase in copy number did not result in consistently increased GABRB3 transcript or protein levels for dup15q samples. Furthermore, SNRPN was expected to be unchanged in expression in dup15q because it is expressed from the single unmethylated paternal allele, yet SNRPN levels were significantly reduced in dup15q samples compared to controls. PWS-IC methylation positively correlated with UBE3A and GABRB3 levels but negatively correlated with SNRPN levels. Idiopathic autism samples exhibited significantly lower GABRB3 and significantly more variable SNRPN levels compared to controls.
Although these results show that increased UBE3A/UBE3A is a consistent feature of dup15q syndrome, they also suggest that gene expression within 15q11-q13 is not based entirely on copy number but can be influenced by epigenetic mechanisms in brain.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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 ≥72h, 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.
•Shed teeth can be stored at RT for days in media and still produce viable DPSCs.•Neurons can be made from hTERT immortalized DPSC as well as primary DPSCs.•Differentiation of DPSCs changes 1305 transcripts.•94 transcripts are affected by immortalization and differentiation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
...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.
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
and
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
and
, 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.
Finding a significant increase in both
and
in Dup15q neurons and significant decrease in these two genes in AS deletion neurons may explain differences between AS deletion class and
specific classes of AS mutation where
is expressed at normal levels. Also, we identified an enrichment for FOXO1-regulated transcripts in Dup15q neurons including ASD-associated genes
and
indicating a possible connection between this syndromic form of ASD and idiopathic cases.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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