Effect of Octamer-Binding Transcription Factor 4 Overexpression on the Neural Induction of Human Dental Pulp Stem Cells.

Stem cell-based therapy is a potential alternative strategy for brain repair, with neural stem cells (NSC) presenting as the most promising candidates. Obtaining sufficient quantities of NSC for clinical applications is challenging, therefore alternative cell types, such as neural crest-derived dental pulp stem cells (DPSC), may be considered. Human DPSC possess neurogenic potential, exerting positive effects in the damaged brain through paracrine effects. However, a method for conversion of DPSC into NSC has yet to be developed. Here, overexpression of octamer-binding transcription factor 4 (OCT4) in combination with neural inductive conditions was used to reprogram human DPSC along the neural lineage. The reprogrammed DPSC demonstrated a neuronal-like phenotype, with increased expression levels of neural markers, limited capacity for sphere formation, and enhanced neuronal but not glial differentiation. Transcriptomic analysis further highlighted the expression of genes associated with neural and neuronal functions. In vivo analysis using a developmental avian model showed that implanted DPSC survived in the developing central nervous system and respond to endogenous signals, displaying neuronal phenotypes. Therefore, OCT4 enhances the neural potential of DPSC, which exhibited characteristics aligning with neuronal progenitors. This method can be used to standardise DPSC neural induction and provide an alternative source of neural cell types.

A senescence stress secretome is a hallmark of therapy-related myeloid neoplasm stromal tissue occurring soon after cytotoxic exposure.

Therapy-related myeloid neoplasm (tMN) is considered a direct consequence of DNA damage in hematopoietic stem cells. Despite increasing recognition that altered stroma can also drive leukemogenesis, the functional biology of the tMN microenvironment remains unknown. We performed multiomic (transcriptome, DNA damage response, cytokine secretome and functional profiling) characterization of bone marrow stromal cells from tMN patients. Critically, we also compared (i) patients with myeloid neoplasm and another cancer but without cytotoxic exposure, (ii) typical primary myeloid neoplasm, and (iii) age-matched controls to decipher the microenvironmental changes induced by cytotoxics vs. neoplasia. Strikingly, tMN exhibited a profoundly senescent phenotype with induction of CDKN1A and ß-Galactosidase, defective phenotype, and proliferation. Moreover, tMN stroma showed delayed DNA repair and defective adipogenesis. Despite their dormant state, tMN stromal cells were metabolically highly active with a switch toward glycolysis and secreted multiple pro-inflammatory cytokines indicative of a senescent-secretory phenotype that inhibited adipogenesis. Critically, senolytics not only eliminated dormant cells, but also restored adipogenesis. Finally, sequential patient sampling showed senescence phenotypes are induced within months of cytotoxic exposure, well prior to the onset of secondary cancer. Our data underscores a role of senescence in the pathogenesis of tMN and provide a valuable resource for future therapeutics.

Feasibility, acceptability, and appropriateness of a mobile health stroke intervention among Ghanaian health workers.

BACKGROUND: mHealth interventions can improve care delivery in settings with limited resources. The Phone-based Interventions under Nurse Guidance after Stroke (PINGS) is a nurse-led, mHealth-centered approach to blood pressure (BP) control among recent stroke survivors with hypertension in Ghana. It has 4 key components: (1) home blood pressure monitoring, (2) nurse-coordinated mhealth consults, (3) phone alerts as medication reminders, and (4) patient motivational messages delivered as interactive voice recordings. OBJECTIVE: To assess the feasibility, acceptability, and appropriateness for scale up of the PINGS intervention in Ghana, from the perspective of health workers. METHODS: Between July and August 2021, we deployed an online questionnaire describing the components of PINGS to a cross-section of health workers in Ghana. The questionnaire used an adaptation of psychometrically validated Likert scale measures to elicit agreement or disagreement with attributes of the intervention. The questionnaire was distributed online to approximately 4000 healthcare workers via email and social media platforms. A summary of descriptive statistics was obtained; summed composite scores were then calculated, dichotomized, and binary logistic regression performed using R programming software. RESULTS: Of 653 health workers who completed the survey, 57.2% were male; 73.2% clinicians; median age was 33 years (IQR 29, 37). Respondents' primary workplaces were public (64.4%), quasi-government (9.4%), and private, including mission-based (26.2%) facilities. PINGS was deemed feasible, acceptable, and appropriate by 93.9%, 94.8%, and 95.1% of respondents respectively. Clinical staff had higher odds of finding PINGS feasible (OR 4.10; C.I. 2.15, 8.0; p < 0.001), acceptable (OR 3.76, C.I. 1.87, 7.69; p < 0.001), or appropriate (OR 2.91, C.I. 1.41, 5.95; p = 0.004) compared to non-clinical staff. There was no statistically significant difference in the rating of each measure when analyzed by age, sex, years of health work experience, geographic location, type, or level of health facility. CONCLUSION: An overwhelming majority of health workers (particularly clinical staff) considered PINGS to be a feasible, acceptable, and appropriate for BP control among stroke survivors in Ghana.

Plant-derived soybean peroxidase stimulates osteoblast collagen biosynthesis, matrix mineralization, and accelerates bone regeneration in a sheep model.

Bone defects arising from fractures or disease represent a significant problem for surgeons to manage and are a substantial economic burden on the healthcare economy. Recent advances in the development of biomaterial substitutes provides an attractive alternative to the current "gold standard" autologous bone grafting. Despite on-going research, we are yet to identify cost effective biocompatible, osteo-inductive factors that stimulate controlled, accelerated bone regeneration.We have recently reported that enzymes with peroxidase activity possess previously unrecognised roles in extracellular matrix biosynthesis, angiogenesis and osteoclastogenesis, which are essential processes in bone remodelling and repair. Here, we report for the first time, that plant-derived soybean peroxidase (SBP) possesses pro-osteogenic ability by promoting collagen I biosynthesis and matrix mineralization of human osteoblasts in vitro. Mechanistically, SBP regulates osteogenic genes responsible for inflammation, extracellular matrix remodelling and ossification, which are necessary for normal bone healing. Furthermore, SBP was shown to have osteo-inductive properties, that when combined with commercially available biphasic calcium phosphate (BCP) granules can accelerate bone repair in a critical size long bone defect ovine model. Micro-CT analysis showed that SBP when combined with commercially available biphasic calcium phosphate (BCP) granules significantly increased bone formation within the defects as early as 4 weeks compared to BCP alone. Histomorphometric assessment demonstrated accelerated bone formation prominent at the defect margins and surrounding individual BCP granules, with evidence of intramembranous ossification. These results highlight the capacity of SBP to be an effective regulator of osteoblastic function and may be beneficial as a new and cost effective osteo-inductive agent to accelerate repair of large bone defects.

Phone-Based Intervention under Nurse Guidance after Stroke (PINGS II) Study: Protocol for a Phase III Randomized Clinical Trial.

OBJECTIVES: The Sub-Saharan African (SSA) region now has the highest estimated effect size of hypertension for stroke causation worldwide. An urgent priority for countries in SSA is to develop and test self-management interventions to control hypertension among those at highest risk of adverse outcomes. Thus the overall objective of the Phone-based Intervention under Nurse Guidance after Stroke II study (PINGS-2) is to deploy a hybrid study design to assess the efficacy of a theoretical-model-based, mHealth technology-centered, nurse-led, multi-level integrated approach to improve longer term blood pressure (BP) control among stroke survivors. MATERIALS AND METHODS: A phase III randomized controlled trial involving 500 recent stroke survivors to be enrolled across 10 Ghanaian hospitals. Using a computer-generated sequence, patients will be randomly assigned 1:1 into the intervention or usual care arms. The intervention comprises of (i) home BP monitoring at least once weekly with nurse navigation for high domiciliary BP readings; (2) medication reminders using mobile phone alerts and (3) education on hypertension and stroke delivered once weekly via audio messages in preferred local dialects. The intervention will last for 12 months. The control group will receive usual care as determined by local guidelines. The primary outcome is the proportion of patients with systolic BP <140 mm Hg at 12 months. Secondary outcomes will include medication adherence, self-management of hypertension, major adverse cardiovascular events, health related quality of life and implementation outcomes. CONCLUSION: An effective PINGS intervention can potentially be scaled up and disseminated across healthcare systems in low-and-middle income countries challenged with resource constraints to reduce poor outcomes among stroke survivors.

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.

EphB and Ephrin-B interactions mediate human mesenchymal stem cell suppression of activated T-cells.

Mesenchymal stromal/stem cells (MSC) express the contact-dependent erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinase family and their cognate ephrin ligands, which are known to regulate thymocyte maturation and selection, T-cell transendothelial migration, activation, co-stimulation, and proliferation. However, the contribution of Eph/ephrin molecules in mediating human MSC suppression of activated T-cells remains to be determined. In the present study, we showed that EphB2 and ephrin-B2 are expressed by ex vivo expanded MSC, while the corresponding ligands, ephrin-B1 and EphB4, respectively, are highly expressed by T-cells. Initial studies demonstrated that EphB2-Fc and ephrin-B2-Fc molecules suppressed T-cell proliferation in allogeneic mixed lymphocyte reaction (MLR) assays compared with human IgG-treated controls. While the addition of a third-party MSC population demonstrated dramatic suppression of T-cell proliferation responses in the MLR, blocking the function of EphB2 or EphB4 receptors using inhibitor binding peptides significantly increased T-cell proliferation. Consistent with these observations, shRNA EphB2 or ephrin-B2 knockdown expression in MSC reduced their ability to inhibit T-cell proliferation. Importantly, the expression of immunosuppressive factors, indoleamine 2, 3-dioxygenase, transforming growth factor-ß1, and inducible nitric oxide synthase expressed by MSC, was up-regulated after stimulation with EphB4 and ephrin-B1 in the presence of interferon (IFN)-γ, compared with untreated controls. Conversely, key factors involved in T-cell activation and proliferation, such as interleukin (IL)-2, IFN-γ, tumor necrosis factor-α, and IL-17, were down-regulated by T-cells treated with EphB2 or ephrin-B2 compared with untreated controls. Studies utilizing signaling inhibitors revealed that inhibition of T-cell proliferation is partly mediated through EphB2-induced ephrin-B1 reverse signaling or ephrin-B2-mediated EphB4 forward signaling by activating Src, PI3Kinase, Abl, and JNK kinase pathways, activated by tyrosine phosphorylation. Taken together, these observations suggest that EphB/ephrin-B interactions play an important role in mediating human MSC inhibition of activated T cells.

Human adult dental pulp stem cells enhance poststroke functional recovery through non-neural replacement mechanisms.

Human adult dental pulp stem cells (DPSCs), derived from third molar teeth, are multipotent and have the capacity to differentiate into neurons under inductive conditions both in vitro and following transplantation into the avian embryo. In this study, we demonstrate that the intracerebral transplantation of human DPSCs 24 hours following focal cerebral ischemia in a rodent model resulted in significant improvement in forelimb sensorimotor function at 4 weeks post-treatment. At this time, 2.3 ± 0.7% of engrafted cells had survived in the poststroke brain and demonstrated targeted migration toward the stroke lesion. In the peri-infarct striatum, transplanted DPSCs differentiated into astrocytes in preference to neurons. Our data suggest that the dominant mechanism of action underlying DPSC treatment that resulted in enhanced functional recovery is unlikely to be due to neural replacement. Functional improvement is more likely to be mediated through DPSC-dependent paracrine effects. This study provides preclinical evidence for the future use of human DPSCs in cell therapy to improve outcome in stroke patients.

Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues.

Human adult dental pulp stem cells (DPSCs) reside within the perivascular niche of dental pulp and are thought to originate from migrating cranial neural crest (CNC) cells. During embryonic development, CNC cells differentiate into a wide variety of cell types, including neurons of the peripheral nervous system. Previously, we have demonstrated that DPSCs derived from adult human third molar teeth differentiate into cell types reminiscent of CNC embryonic ontology. We hypothesized that DPSCs exposed to the appropriate environmental cues would differentiate into functionally active neurons. The data demonstrated that ex vivo-expanded human adult DPSCs responded to neuronal inductive conditions both in vitro and in vivo. Human adult DPSCs, but not human foreskin fibroblasts (HFFs), acquired a neuronal morphology, and expressed neuronal-specific markers at both the gene and protein levels. Culture-expanded DPSCs also exhibited the capacity to produce a sodium current consistent with functional neuronal cells when exposed to neuronal inductive media. Furthermore, the response of human DPSCs and HFFs to endogenous neuronal environmental cues was determined in vivo using an avian xenotransplantation assay. DPSCs expressed neuronal markers and acquired a neuronal morphology following transplantation into the mesencephalon of embryonic day-2 chicken embryo, whereas HFFs maintained a thin spindle fibroblastic morphology. We propose that adult human DPSCs provide a readily accessible source of exogenous stem/precursor cells that have the potential for use in cell-therapeutic paradigms to treat neurological disease.