Generation of Neural Crest-Like Cells From Human Periodontal Ligament Cell-Derived Induced Pluripotent Stem Cells.

Neural crest cells (NCC) hold great promise for tissue engineering, however the inability to easily obtain large numbers of NCC is a major factor limiting their use in studies of regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC. In the present study, we examined the potential of neural crest tissue-derived periodontal ligament (PDL) iPSC to differentiate into neural crest-like cells (NCLC) relative to iPSC generated from a non-neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with NCC characteristics. We isolated PDL iPSC- and FF iPSC-derived NCLC, which highly expressed HNK1. A high proportion of the HNK1-positive cell populations generated, expressed the MSC markers, whilst very few cells expressed the pluripotency markers or the hematopoietic markers. The PDL and FF HNK1-positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell-associated markers than the PDL and FF HNK1-negative populations. Interestingly, the HNK1-positive cells derived from the PDL-iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1-positive cells derived from the FF-iPSC. Our work suggests that HNK1-enriched NCLC from neural crest tissue-derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1-low population and non-neural crest iPSC-derived NCLC. J. Cell. Physiol. 232: 402-416, 2017. © 2016 Wiley Periodicals, Inc.

Immunomodulatory Properties of Induced Pluripotent Stem Cell-Derived Mesenchymal Cells.

MSC-like populations derived from induced pluripotent stem cells (iPSC-MSC) serve as an alternative stem cell source due to their high proliferative capacity. In this study, we assessed the immunomodulatory potential of iPSC-MSC generated from periodontal ligament (PDL) and gingival (GF) tissue. The iPSC-MSC lines exhibited a similar level of suppression of mitogen-stimulated peripheral blood mononuclear cells (PBMNC) proliferation compared to their respective parental fibroblast populations in vitro. Moreover, iPSC-MSC demonstrated the ability to suppress T-cells effector cells, Th1/Th2/Th17 populations, and increase levels of Treg cells. In order to investigate the mechanisms involved, expression of common MSC-derived soluble factors known to supress lymphocyte proliferation were assessed in iPSC-MSC cultured with PBMNC with direct cell-cell contact or separated in transwells. Real-time PCR analysis of factors known to be involved in MSC mediated immune regulation, found a general trend of elevated IDO1 and IL6 transcript levels in iPSC-MSC lines and their respective primary cells co-cultured with activated PBMNC, with a wide range of gene expression levels between the different mesenchymal cell types. The results suggest that different iPSC-MSC may be useful as a potential alternative source of cells for future clinical use in therapeutic applications because of their potent immunosuppressive properties. J. Cell. Biochem. 117: 2844-2853, 2016. © 2016 Wiley Periodicals, Inc.

Immunomodulatory properties of mesenchymal stem cell in experimental arthritis in rat and mouse models: A systematic review.

BACKGROUND: Despite recent advances in the treatment of arthritis with the development of disease-modifying antirheumatic drugs, 30% of patients still fail to respond to treatment. Given the potent anti-inflammatory and immunomodulatory properties of mesenchymal stem cells (MSC) and their ability to repair damaged cartilage, bone, and tendons, it has been proposed that MSC could be ideal for cell-based treatment of arthritis. OBJECTIVE: This systematic review investigates evidence from studies on the therapeutic efficacy of MSC in rodent models of arthritis. METHODS: PubMed, Embase, MEDLINE, and Wed of Science were searched to June 2015 for quantitative studies examining the outcome of treating animal models of arthritis with MSC. Inclusion criteria were as follows: administration of mesenchymal stem as a treatment approach for arthritis; animal models only; and published in English. We followed the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. RESULTS: The literature search identified 30 studies which met the inclusion criteria. A range of MSC populations were assessed in various rodent models of arthritis. Of these, 19 demonstrated positive outcomes while 11 studies failed to demonstrate positive effects. Owing to the extensive variation in the experimental design, cells investigated and the outcome measures described in the manuscripts, no meta-analysis was possible. Furthermore, the numerical values for the primary outcome measure of clinical paw score were frequently not published in the manuscripts analyzed, as they were only illustrated in graphical form. CONCLUSIONS: Numerous studies have investigated the utility of a range of MSC populations in the treatment of experimental arthritis. The results obtained from these studies have been highly inconsistent, with multiple studies identifying a statistically significant improvement in arthritis scores after treatment with MSC, while other studies identified a statistically significant deterioration in arthritis scores and thirdly some studies showed no effect. Further studies using standardized protocols and outcome measures are needed to determine fully the potential of MSC populations in the treatment of experimental arthritis.

Differentiation of iPSC to Mesenchymal Stem-Like Cells and Their Characterization.

Mesenchymal stem cells (MSC) are a unique population of adult stem cells that have the capacity to differentiate into numerous cell types as well as the ability to modulate the immune system. As such, MSC represent a promising stem cell population for use in the clinical treatment of a range of disorders involving tissue regeneration as well as the immune system. The lack of accessibility to MSC is currently limiting the use of MSC in mainstream clinical treatment strategies. It is therefore imperative for the future success of stem cell-based treatment approaches that are more reliable, and accessible sources of MSC are identified. The present chapter describes a method for generating MSC-like cells from induced pluripotent stem cells (iPSC), with equivalent growth and functional properties to parental MSC populations.

Mutations of protocadherin 19 in female epilepsy (PCDH19-FE) lead to allopregnanolone deficiency.

Protocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE; also known as epilepsy and mental retardation limited to females, EFMR; MIM300088) is an infantile onset epilepsy syndrome with or without intellectual disability (ID) and autism. We investigated transcriptomes of PCDH19-FE female and control primary skin fibroblasts, which are endowed to metabolize neurosteroid hormones. We identified a set of 94 significantly dysregulated genes in PCDH19-FE females. Intriguingly, 43 of the 94 genes (45.7%) showed gender-biased expression; enrichment of such genes was highly significant (P = 2.51E-47, two-tailed Fisher exact test). We further investigated the AKR1C1-3 genes, which encode crucial steroid hormone-metabolizing enzymes whose key products include allopregnanolone and estradiol. Both mRNA and protein levels of AKR1C3 were significantly decreased in PCDH19-FE patients. In agreement with this, the blood levels of allopregnanolone were also (P < 0.01) reduced. In conclusion, we show that the deficiency of neurosteroid allopregnanolone, one of the most potent GABA receptor modulators, may contribute to PCDH19-FE. Overall our findings provide evidence for a role of neurosteroids in epilepsy, ID and autism and create realistic opportunities for targeted therapeutic interventions.

Generation of functional mesenchymal stem cells from different induced pluripotent stem cell lines.

The therapeutic potential of mesenchymal stem cells (MSC) has highlighted the need for identifying easily accessible and reliable sources of these cells. An alternative source for obtaining large populations of MSC is through the controlled differentiation of induced pluripotent stem cells (iPSC). In the present study, colonies of iPSC were cultured in MSC culture media for 2 weeks. Serial passaging then selected for fast growing MSC-like cells with a typical fibroblastic morphology and the capacity to proliferate on standard culture flasks without feeder cells. MSC-like cells were developed from iPSC lines arising from three different somatic tissues: gingiva, periodontal ligament (PDL), and lung. The iPSC-MSC like cells expressed key MSC-associated markers (CD73, CD90, CD105, CD146, and CD166) and lacked expression of pluripotent markers (TRA160, TRA181, and alkaline phosphatase) and hematopoietic markers (CD14, CD34, and CD45). In vitro iPSC-MSC-like cells displayed the capacity to differentiate into osteoblasts, adipocytes, and chondrocytes. In vivo subcutaneous implantation of the iPSC-MSC-like cells into NOD/SCID mice demonstrated that only the PDL-derived iPSC-MSC-like cells exhibited the capacity to form mature mineralized structures which were histologically similar to mature bone. These findings demonstrate that controlled induction of iPSC into fibroblastic-like cells that phenotypically and functionally resemble adult MSC is an attractive approach to obtain a readily available source of progenitor cells for orthopedic and dental-related tissue-engineering applications. However, a detailed characterization of the iPSC-MSC-like cells will be important, as MSC-like cells derived from different iPSC lines exhibit variability in their differentiation capacity.

Clinical utility of stem cells for periodontal regeneration.

The aim of this review is to discuss the clinical utility of stem cells in periodontal regeneration by reviewing relevant literature that assesses the periodontal-regenerative potential of stem cells. We considered and described the main stem cell populations that have been utilized with regard to periodontal regeneration, including bone marrow-derived mesenchymal stem cells and the main dental-derived mesenchymal stem cell populations: periodontal ligament stem cells, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, stem cells from apical papilla and dental follicle precursor cells. Research into the use of stem cells for tissue regeneration has the potential to significantly influence periodontal treatment strategies in the future.

Epilepsy and mental retardation limited to females with PCDH19 mutations can present de novo or in single generation families.

BACKGROUND: Epilepsy and mental retardation limited to females (EFMR) is an intriguing X-linked disorder affecting heterozygous females and sparing hemizygous males. Mutations in the protocadherin 19 (PCDH19) gene have been identified in seven unrelated families with EFMR. METHODS AND RESULTS: Here, we assessed the frequency of PCDH19 mutations in individuals with clinical features which overlap those of EFMR. We analysed 185 females from three cohorts: 42 with Rett syndrome who were negative for MECP2 and CDKL5 mutations, 57 with autism spectrum disorders, and 86 with epilepsy with or without intellectual disability. No mutations were identified in the Rett syndrome and autism spectrum disorders cohorts suggesting that despite sharing similar clinical characteristics with EFMR, PCDH19 mutations are not generally associated with these disorders. Among the 86 females with epilepsy (of whom 51 had seizure onset before 3 years), with or without intellectual disability, we identified two (2.3%) missense changes. One (c.1671C-->G, p.N557K), reported previously without clinical data, was found in two affected sisters, the first EFMR family without a multigenerational family history of affected females. The second, reported here, is a novel de novo missense change identified in a sporadic female. The change, p.S276P, is predicted to result in functional disturbance of PCDH19 as it affects a highly conserved residue adjacent to the adhesion interface of EC3 of PCDH19. CONCLUSIONS: This de novo PCDH19 mutation in a sporadic female highlights that mutational analysis should be considered in isolated instances of girls with infantile onset seizures and developmental delay, in addition to those with the characteristic family history of EFMR.

X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment.

Epilepsy and mental retardation limited to females (EFMR) is a disorder with an X-linked mode of inheritance and an unusual expression pattern. Disorders arising from mutations on the X chromosome are typically characterized by affected males and unaffected carrier females. In contrast, EFMR spares transmitting males and affects only carrier females. Aided by systematic resequencing of 737 X chromosome genes, we identified different protocadherin 19 (PCDH19) gene mutations in seven families with EFMR. Five mutations resulted in the introduction of a premature termination codon. Study of two of these demonstrated nonsense-mediated decay of PCDH19 mRNA. The two missense mutations were predicted to affect adhesiveness of PCDH19 through impaired calcium binding. PCDH19 is expressed in developing brains of human and mouse and is the first member of the cadherin superfamily to be directly implicated in epilepsy or mental retardation.