Ectomesenchymal Stem Cells Research Articles

Immediate or Delayed Transplantation of a Vein Conduit Filled with Nasal Olfactory Stem Cells Improves Locomotion and Axogenesis in Rats after a Peroneal Nerve Loss of Substance.

Over the recent years, several methods have been experienced to repair injured peripheral nerves. Among investigated strategies, the use of natural or synthetic conduits was validated for clinical application. In this study, we assessed the therapeutic potential of vein guides, transplanted immediately or two weeks after a peroneal nerve injury and filled with olfactory ecto-mesenchymal stem cells (OEMSC). Rats were randomly allocated to five groups. A3 mm peroneal nerve loss was bridged, acutely or chronically, with a 1 cm long femoral vein and with/without OEMSCs. These four groups were compared to unoperated rats (Control group). OEMSCs were purified from male olfactory mucosae and grafted into female hosts. Three months after surgery, nerve repair was analyzed by measuring locomotor function, mechanical muscle properties, muscle mass, axon number, and myelination. We observed that stem cells significantly (i) increased locomotor recovery, (ii) partially maintained the contractile phenotype of the target muscle, and (iii) augmented the number of growing axons. OEMSCs remained in the nerve and did not migrate in other organs. These results open the way for a phase I/IIa clinical trial based on the autologous engraftment of OEMSCs in patients with a nerve injury, especially those with neglected wounds.

P75NTR-/- mice exhibit an alveolar bone loss phenotype and inhibited PI3K/Akt/β-catenin pathway.

ObjectivesThe aim of this study was to investigate the role of p75 neurotrophin receptor (p75NTR) in regulating the mouse alveolar bone development and the mineralization potential of murine ectomesenchymal stem cells (EMSCs). Moreover, we tried to explore the underlying mechanisms associated with the PI3K/Akt/β‐catenin pathway.Materials and methodsp75NTR knockout (p75NTR−/−) mice and wild‐type (WT) littermates were used. E12.5d p75NTR−/− and WT EMSCs were isolated in the same pregnant p75NTR‐/+ mice from embryonic maxillofacial processes separately. Mouse alveolar bone mass was evaluated using micro‐CT. Differential osteogenic differentiation pathways between p75NTR−/− and WT EMSCs were analysed by RNA‐sequencing. The PI3K inhibitor LY294002 and PI3K agonist 740Y‐P were used to regulate the PI3K/Akt pathway in EMSCs. p75NTR overexpression lentiviruses, p75NTR knock‐down lentiviruses and recombined mouse NGF were used to transfect cells.ResultsThe alveolar bone mass was found reduced in the p75NTR knockout mouse comparing to the WT mouse. During mineralization induction, p75NTR−/− EMSCs displayed decreased osteogenic capacity and downregulated PI3K/Akt/β‐catenin signalling. The PI3K/Akt/β‐catenin pathway positively regulates the potential of differential mineralization in EMSCs. The promotive effect of p75NTR overexpression can be attenuated by LY294002, while the inhibitory effect of p75NTR knock‐down on Runx2 and Col1 expression can be reversed by 740Y‐P.ConclusionDeletion of p75NTR reduced alveolar bone mass in mice. P75NTR positively regulated the osteogenic differentiation of EMSCs via enhancing the PI3K/Akt/β‐catenin pathway.

Hesperidin promotes peripheral nerve regeneration based on tissue engineering strategy using alginate/chitosan hydrogel: in vitro and in vivo study

About 2.8% of trauma patients suffer from peripheral nerve injury and it becomes a serious challenge in the world. Therefore, various methods and materials have been used for regenerating peripheral nerve damages. The aim of this study is to evaluate the effect of different dosages of Hesperidin on peripheral nerve regeneration. For this purpose, different concentration (0.1%, 1%, and 10% (w/v)) of Hesperidin was loaded into cross-linked alginate/chitosan hydrogel and its characters such as morphology, swelling properties, weight loss, hemo-, and cytocompatibility were evaluated. For functional analysis, the fabricated hydrogels without and with different amounts of hesperidin were administrated after creating sciatic nerve injury in a rat model, and the various experiments like walking-foot-print analysis, Hot plate latency test, gastrocnemius muscle wet weight loss, and histopathological examination were used. Our results indicated that prepared hydrogels contained the porosity of 90%, with the interconnected pores. Biodegradability of fabricated hydrogels was confirmed with weight loss assessment and the weight loss percentage was about 80% after 14 days. Moreover, hydrogels had good blood compatibility and antibacterial properties. The MTT assay indicated that the developed hydrogels do not have toxicity effect and they have ability to increase the proliferation rate of Olfactory Ecto-mesenchymal stem cells (OE-MSCs). The results of in vivo study showed that hesperidin specially the group contained 1% of hesperidin improve sciatic nerve regeneration and hesperidin could be considered as a potential material for peripheral nerve regeneration.

The role and potential mechanism of p75NTR in mineralization via in vivo p75NTR knockout mice and in vitro ectomesenchymal stem cells.

ObjectiveThe aim of this study is to investigate the role and potential mechanism of p75NTR in mineralization in vivo using p75NTR‐knockout mice and in vitro using ectomesenchymal stem cells (EMSCs).Materials and methodsFemur bone mass and daily incisor mineralization speed were assessed in an in vivo p75NTR‐knockout mouse model. The molecular signatures alkaline phosphatase (ALP), collagen type 1 (Col1), melanoma‐associated antigen (Mage)‐D1, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), distal‐less homeobox 1 (Dlx1) and Msh homeobox 1 (Msx1) were examined in vitro in EMSCs isolated from p75NTR+/+ and p75NTRExIII−/− mice.Resultsp75NTR‐knockout mice were smaller in body size than heterozygous and wild‐type mice. Micro‐computed tomography and structural quantification showed that the osteogenic ability of p75NTRExIII‐knockout mice was significantly decreased compared with that of wild‐type mice (P < .05). Weaker ALP and alizarin red staining and reduced expression of ALP, Col1, Runx2, BSP, OCN and OPN were also observed in p75NTRExIII−/− EMSCs. Moreover, the distance between calcein fluorescence bands in p75NTRExIII‐knockout mice was significantly smaller than that in wild type and heterozygous mice (P < .05), indicating the lower daily mineralization speed of incisors in p75NTRExIII‐knockout mice. Further investigation revealed a positive correlation between p75NTR and Mage‐D1, Dlx1, and Msx1.Conclusionp75NTR not only promotes osteogenic differentiation and tissue mineralization, but also shows a possible relationship with the circadian rhythm of dental hard tissue formation.

Priming TLR3 and TLR4 in human adipose- and olfactory mucosa-derived mesenchymal stromal cells and comparison of their cytokine secretions.

Mesenchymal stromal cells (MSCs) have potent immunomodulatory abilities to regulate most of the immune cells. Not only the tissue origin of MSCs can affect their functions, but also their microenvironment can strongly influence their biology, particularly through toll-like receptors (TLR)/ligands interaction. In the present study, we compared MSCs derived from two different sources, i.e. human olfactory ecto-mesenchymal stem cells (OE-MSCs) and adipose tissue (AT-MSCs), in terms of their immunosuppressive effects before and after TLR3 and TLR4 stimulation through low-level and short-term TLR-priming protocol. After isolation and characterization of OE-MSCs and AT-MSCs, flow cytometry analyses were used to assess the expression of TLR3, TLR4 by MSCs. Secretion and expression levels of immune-related genes were analyzed using ELISA and RT-qPCR techniques. Based on the results, the proliferation potential of OE-MSCs was significantly higher than that of AT-MSCs. The gene expression and also protein levels of both TLR3 and TLR4 were significantly higher in OE-MSCs, compared to AT-MSCs. Among the examined cytokines and chemokines, OE-MSCs exhibited significantly higher levels of CCL5, IL-8, and TGF-β production, in comparison with AT-MSCs. However, IL-6 secretion by AT-MSCs was considerably more than that by OE-MSCs. OE-MSCs were only affected by the TLR4 ligand, and IL-8 and IL-6 production levels increased after LPS treatment. However, only IL-8 significantly increased after adding LPS or Poly (I:C) to the AT-MSC media. According to the obtained data, OE-MSCs exhibited a higher proliferative potential and greater expression levels of TLR3 and TLR4 genes, compared to AT-MSCs. However, unlike AT-MSCs, the expression of TLR3 by OE-MSCs was nonfunctional. Finally, based on our findings, OE-MSCs have a stronger secretion of immunosuppressive cytokines both before and after LPS or PIC treatment, compared to AT-MSCs.

Cerebrospinal Fluid and Photobiomodulation Effects on Neural Gene Expression in Dental Pulp Stem Cells.

Introduction: Dental pulp cells, a unique source of ectomesenchymal pluripotent stem cells, are originated from the skull neural crest. They are considered as one ideal source of cells for the regenerative medicine applications. Cerebrospinal fluid (CSF), a transparent fluid found in the brain and spinal cord, is enriched with electrolytes, proteins, and growth factors such as EGF, bFGF, BDNF, GDNF, and neuropeptides and can be utilized as a trigger in order to induce the neural differentiation. On the other hand, photobiomodulation (PBM), with the ability to prevent cell apoptosis, can induce cell proliferation by means of increasing the ATP synthesis in mitochondria and facilitating the secretion of the growth factors. In this research, we first aimed to isolate and culture the dental pulp stem cells (DPSCs) and subsequently to investigate their potential for neural differentiation. Methods: Human dental pulp stem cells (hDPSCs) were isolated from the pulp tissues using an outgrowth method and subsequently cultured. In order to access the cells' differentiation potential, cells were firstly classified into four groups which were treated with CSF, gallium aluminum arsenide diode laser irradiation (808 nm; 30 mW power output) and a combination of both, while the fourth group was considered as the control. MTT assay was then used to examine the viability of cells following the treatments. After 4, 7, and 14 days the cell morphology in the treated groups was evaluated while RT-PCR was used in order to evaluate the Nestin and β-tubulinIII neural gene marker expressions. Results: It was shown that PBM has the ability to elevate the proliferation of DPSCs. Also, the differentiated morphology was obvious in the CSF treated group, especially on day 14 with the formation of three-dimensional (3D) structures. The results of gene expression analysis showed that on the fourth day of post-treatment, Nestin and β-tubulinIII gene expressions were reduced in all groups while a rising trend in their expression was observed subsequently on days 7 and 14. Conclusion: In accordance with previous studies, including functional and protein base researches, it has been demonstrated that CSF has a direct role in neural induction. Although past works have been significant, none of them shows a 3D structure. In this article, we investigated the dual effect of PBM and CSF. Initial results confirmed the upregulation of neural-related transcription factors. The 3D organization of the formed tissue could imply the initiation of organogenesis which has not been reported before. In sum, the dual effect of CSF and PBM has been shown to have the potential for contributing to the initiation of neurogenesis and organogenesis.

Olfactory Ecto-Mesenchymal Stem Cells in Laryngeal Nerve Regeneration in Rats

Selective intralaryngeal reinnervation has been shown to be effective in experimental models. This consists of independently reinnerving the adductor and abductor of laryngeal muscles of the larynx, in order to prevent any misalignment of the axonal regrowth, improve the functional recovery and tend toward reduction of synkinesis. The surgical technique remains complex. Current research focuses on simplifying and improving this technique. Olfactory ectomesenchymal stem cells (OEMSC) represent an interesting candidate for cell therapy and could be obtained from olfactory mucosa. Recent reports suggest a neuroregenerative potential in various animal models of central and peripheral nervous systems injuries. The aim of this study was dual: to develop a simple surgical model of selective reinnervation applicable in humans and to evaluate the relevance of OEMSC-based cell therapy for improving axonal guidance. Eight Fisher syngeneic rats were used to carry out the OEMSCs culture. Thirty-four Fisher syngeneic rats were operated on, divided into three groups depending on the transplanting. For all the rats, we have performed a side to end anastomosis of the vagal nerve with the inferior laryngeal nerve by interposition of a nerve graft from the left femoral nerve. Then, the first group didn't have any injection, the second group has an injection of thrombin and the third group has an injection of one million EOMSCs. Three months after surgery, laryngeal muscle activity was evaluated by videolaryngoscopy and electromyography recordings. In order to illustrate the quality of axonal regrowth, a fluorescent tracer was injected into the right posterior crico-arytenoid muscle (PCA) to reveal the cellular bodies of the motoneurons responsible for reinnervation of the PCA in the central nervous system. In our study, no improvement was found during the videolaryngological functional evaluation or with regard to the electrical activity of the PCA muscle. The cells colabelled in retrograde tracing were numerous in all groups, reflecting abnormal axonal regeneration. The interposition of a nerve graft, as side to end anastomosis between the vagus nerve and the inferior laryngeal nerve, filled with OEMSCs, does not provide better reinnervation of a hemilarynx.

Motor and sensitive recovery after injection of a physically cross-linked PNIPAAm-g-PEG hydrogel in rat hemisectioned spinal cord.

In line with experiments showing that implanted hydrogels are promising tools, we designed and injected, after a C2 spinal cord hemisection, a thermoresponsive and thermoreversible physically cross-linked poly(N-isopropylacrylamide)-poly(ethylene glycol) copolymer in order to reduce functional deficits and provide a favorable environment to axotomized axons. Nasal olfactory ecto-mesenchymal stem cells were cultured on the hydrogel in order to verify its biocompatibility. Then, inflammatory reaction (Interleukin-1β and 6, Tumor Necrosis Factor-α) was examined 15 days post-hydrogel injection. Functional recovery (postural and locomotor activities, muscle strength and tactile sensitivity) was assessed once a week, during 12 weeks. Finally, at 12 weeks post-injection, spinal reflexivity and ventilatory adjustments were measured, and the presence of glial cells and regenerated axons were determined in the injured area. Our results indicate that cells survived and proliferated on the hydrogel which, itself, did not induce an enhanced inflammation. Furthermore, we observed significant motor and sensitive improvements in hydrogel-injected animals. Hydrogel also induced H-reflex recovery close to control animals but no improved ventilatory adjustment to electrically-evoked isometric contractions. Finally, regrowing axons were visualized within the hydrogel with no glial cells colonization. Our results emphasize the effectiveness of our copolymer and its high therapeutic potential to repair the spinal cord after injury.

Conductive hydrogels based on agarose/alginate/chitosan for neural disorder therapy

Electroactive scaffolds derived from carbohydrate hydrogels were synthesized, resulting in a large shift in the conductivity of chitosan (CS) from 10−6 S/cm to about 10-3 S/cm, assigned to CS-oligoaniline. Several analyses including UV–vis spectroscopy and cyclic voltammetry were performed, before examining the carbohydrate-based scaffolds for their ability to recapitulate the neural tissue microenvironment. Good conductivity and resemblance of the modulus to soft tissue of the optimized hydrogels led to appropriate cellular activity and neural regeneration. The loss of dopaminergic neurons as the prominent source of dopamine in the central nervous system results in the deterioration of multiple brain functions such as voluntary movement and behavioral processes. To overcome this, olfactory ecto-mesenchymal stem cells (OE-MSCs) were induced to differentiate into dopaminergic neuron-like cells on hydrogels through a monolayer arrangement cell culture by using cocktail neurotrophic factors including sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF). The differentiation capacity of a series of OE-MSCs on the conductive hydrogel was evaluated by real-time PCR, immunocytochemistry and flow cytometry, and the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) neural and dopaminergic markers. The results of this study represent the first steps in designing and implementing advanced platforms based on conductive polysaccharide hydrogels for neural disorder therapies, such as the treatment of Parkinson’s disease.

Mouse embryonic palatal mesenchymal cells maintain stemness through the PTEN-Akt-mTOR autophagic pathway

BackgroundBoth genetic and environmental factors are implicated in the pathogenesis of cleft palate. However, the molecular and cellular mechanisms that regulate the development of palatal shelves, which are composed of mesenchymal cells, have not yet been fully elucidated. This study aimed to determine the stemness and multilineage differentiation potential of mouse embryonic palatal mesenchyme (MEPM) cells in palatal shelves and to explore the underlying regulatory mechanism associated with cleft palate formation.MethodsPalatal shelves excised from mice models were cultured in vitro to ascertain whether MEPM are stem cells through immunofluorescence and flow cytometry. The osteogenic, adipogenic, and chondrogenic differentiation potential of MEPM cells were also determined to characterize MEPM stemness. In addition, the role of the PTEN-Akt-mTOR autophagic pathway was investigated using quantitative RT-PCR, Western blotting, and transmission electron microscopy.ResultsMEPM cells in culture exhibited cell surface marker expression profiles similar to that of mouse bone marrow stem cells and exhibited positive staining for vimentin (mesodermal marker), nestin (ectodermal marker), PDGFRα, Efnb1, Osr2, and Meox2 (MEPM cells markers). In addition, exposure to PDGFA stimulated chemotaxis of MEPM cells. MEPM cells exhibited stronger potential for osteogenic differentiation as compared to that for adipogenic and chondrogenic differentiation. Undifferentiated MEPM cells displayed a high concentration of autophagosomes, which disappeared after differentiation (at passage four), indicating the involvement of PTEN-Akt-mTOR signaling.ConclusionsOur findings suggest that MEPM cells are ectomesenchymal stem cells with a strong osteogenic differentiation potential and that maintenance of their stemness via PTEN/AKT/mTOR autophagic signaling prevents cleft palate development.

Overexpression of TG2 enhances the differentiation of ectomesenchymal stem cells into neuron‑like cells and promotes functional recovery in adult rats following spinal cord injury.

Ectomesenchymal stem cells (EMSCs) represent a type of adult stem cells derived from the cranial neural crest. These cells are capable of self-renewal and have the potential for multidirectional differentiation. Tissue transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family of Ca2+-dependent crosslinking enzymes. However, the effect of TG2 on neural differentiation and proliferation of EMSCs remains unknown. To determine whether TG2 improves EMSC proliferation and neurogenesis, a stable TG2-overexpressing EMSC cell line (TG2-EMSCs) was established by using an adenovirus system. Immunofluorescence staining and western blot analyses demonstrated that TG2 overexpression had beneficial effects on the rate of EMSC neurogenesis, and that the proliferative capacity of TG2-EMSCs was higher than that of controls. Furthermore, the results of western blotting revealed that extracellular matrix (ECM) and neurotrophic factors were upregulated during the differentiation of TG2-EMSCs. Notably, TG2-EMSC transplantation in an animal model of spinal cord injury (SCI), TG2-EMSCs differentiated into neuron-like cells and enhanced the repair of SCI. Taken together, these results demonstrated that TG2 gene transfection may offer a novel strategy to enhance EMSC proliferation and neurogenesis in vivo and in vitro, which may ultimately facilitate EMSC-based transplantation therapy in patients with SCI.

Olfactory mucosa stem cells: An available candidate for the treatment of the Parkinson's disease.

Olfactory ectomesenchymal stem cells (OE-MSCs) possess the immunosuppressive activity and regeneration capacity and hold a lot of promises for neurodegenerative disorders treatment. This study aimed to determine OE-MSCs which are able to augment and differentiate into functional neurons and regenerate the CNS and also examine whether the implantation of OE-MSCs in the pars compacta of the substantia nigra (SNpc) can improve Parkinson's symptoms in a rat model-induced with 6-hydroxydopamine. We isolated OE-MSCs from lamina propria in olfactory mucosa and characterized them using flow cytometry and immunocytochemistry. The therapeutic potential of OE-MSCs was evaluated by the transplantation of isolated cells using a rat model of acute SN injury as a Parkinson's disease. Significant behavioral improvement in Parkinsonian rats was elicited by the OE-MSCs. The results demonstrate that the expression of PAX2, PAX5, PITX3, dopamine transporter, and tyrosine hydroxylase was increased by OE-MSCs compared to the control group which is analyzed with real-time polymerase chain reaction technique and immunohistochemical staining. In the outcome, the transplantation of 1,1'-dioctadecyl-3,3,3'3'-tetramethyl indocarbocyanine perchlorate labeled OE-MSCs that were fully differentiated to dopaminergic neurons contribute to a substantial improvement in patients withParkinson's. Together, our results provide that using OE-MSCs in neurodegenerative disorders might lead to better neural regeneration.

Conductive hydrogel based on chitosan-aniline pentamer/gelatin/agarose significantly promoted motor neuron-like cells differentiation of human olfactory ecto-mesenchymal stem cells

Developing a simple produces for efficient derivation of motor neurons (MNs) is essential for neural tissue engineering studies. Stem cells with high capacity for neural differentiation and scaffolds with the potential to promote motor neurons differentiation are promising candidates for neural tissue engineering. Recently, human olfactory ecto-mesenchymal stem cells (OE-MSCs), which are isolated easily from the olfactory mucosa, are considered a new hope for neuronal replacement due to their neural crest origin. Herein, we synthesized conducting hydrogels using different concentration of chitosan-g-aniline pentamer, gelatin, and agarose. The chemical structures, swelling and deswelling ratio, ionic conductivity and thermal properties of the hydrogel were characterized. Scaffolds with 10% chitosan-g-aniline pentamer/gelatin (S10) were chosen for further investigation and the potential of OE-MSCs as a new source for programming to motor neuron-like cells investigated on tissue culture plate (TCP) and conductive hydrogels. Cell differentiation was evaluated at the level of mRNA and protein synthesis and indicated that conductive hydrogels significantly increased the markers related to motor neurons including Hb-9, Islet-1 and ChAT compared to TCP. Taken together, the results suggest that OE-MSCs would be successfully differentiated into motor neuron-like cells on conductive hydrogels and would have a promising potential for treating motor neuron-related diseases.

Alginate/chitosan hydrogel containing olfactory ectomesenchymal stem cells for sciatic nerve tissue engineering.

Regeneration and functional recovery after peripheral nerve damage still remain a significant clinical problem. In this study, alginate/chitosan (alg/chit) hydrogel was used for the transplantation of olfactory ectomesenchymal stem cells (OE-MSCs) to promote peripheral nerve regeneration. The OE-MSCs were isolated from olfactory mucosa biopsies and evaluated by different cell surface markers and differentiation capacity. After creating sciatic nerve injury in a rat model, OE-MSCs were transplanted to the injured area with alg/chit hydrogel which was prepared and well-characterized. The prepared hydrogel had the porosity of 91.3 ± 1.27%, the swelling ratio of 379% after 240 min, weight loss percentages of 80 ± 5.56% after 14 days, and good blood compatibility. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 4',6-diamidino-2-phenylindole, and LIVE/DEAD staining were done to assay the behavior of OE-MSCs on alg/chit hydrogel and the results confirmed that the hydrogel can provide a suitable substrate for cell survival. For functional analysis, alg/chit hydrogel with and without OE- MSCs was injected into a 3-mm sciatic nerve defect of Wistar rats. The results of the sciatic functional index, hot plate latency, electrophysiological assessment, weight-loss percentage of wet gastrocnemius muscle, and histopathological examination using hematoxylin-eosin and Luxol fast blue staining showed that utilizing alg/chit hydrogel with OE-MSCs to the sciatic nerve defect enhance regeneration compared to the control group and hydrogel without cells.

EMSCs Build an All‐in‐One Niche via Cell–Cell Lipid Raft Assembly for Promoted Neuronal but Suppressed Astroglial Differentiation of Neural Stem Cells

The therapeutic efficiency of allogenic/intrinsic neural stem cells (NSCs) after spinal cord injury is severely compromised because the hostile niche at the lesion site incurs massive astroglial but not neuronal differentiation of NSCs. Although many attempts are made to reconstruct a permissive niche for nerve regeneration, solely using a living cell material to build an all-in-one, multifunctional, permissive niche for promoting neuronal while inhibiting astroglial differentiation of NSCs is not reported. Here, ectomesenchymal stem cells (EMSCs) are reported to serve as a living, smart material that creates a permissive, all-in-one niche which provides neurotrophic factors, extracellular matrix molecules, cell-cell contact, and favorable substrate stiffness for directing NSC differentiation. Interestingly, in this all-in-one niche, a corresponding all-in-one signal-sensing platform is assembled through recruiting various niche signaling molecules into lipid rafts for promoting neuronal differentiation of NSCs, and meanwhile, inhibiting astrocyte overproliferation through the connexin43/YAP/14-3-3θ pathway. In vivo studies confirm that EMSCs can promote intrinsic NSC neuronal differentiation and domesticating astrocyte behaviors for nerve regeneration. Collectively, this study represents an all-in-one niche created by a single-cell material-EMSCs for directing NSC differentiation.

Differentiation of human mesenchymal stem cells (MSC) to dopaminergic neurons: A comparison between Wharton’s Jelly and olfactory mucosa as sources of MSCs

The generation of dopaminergic neurons from stem cells is a potential therapeutic approach to treat neurodegenerative disorders, such as Parkinson’s disease. The current study aims to investigate the potential of two different types of mesenchymal stem cells derived from human Wharton’s jelly and nasal cavity for differentiation into dopaminergic neurons. The differentiation capacities of both cell types were evaluated using real-time PCR, immunocytochemistry, flow cytometry and HPLC. Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) are noted for their capability to differentiate into mesodermal and non-mesodermal cells, including neurons. However, it was demonstrated that having the same neuroectodermal origin as the nervous system, the olfactory ectomesenchymal stem cells (OE-MSCs) expressed the neural marker MAP2 as well as dopaminergic markers such as tyrosine hydroxylase (TH), dopamine transporter (DAT) and PITX3 to a greater extent than the WJ-MSCs both at the level of mRNA and protein. Furthermore, quantitative flow cytometric evaluation of these markers at 12 days post-induction supported the above-mentioned results. Finally, the assessment of the functionality of differentiated cells and their ability to synthesize dopamine measured by HPLC revealed that the OE-MSC-derived dopaminergic cells released almost the same amount of dopamine as that secreted by WJ-MSC-derived cells. Thus it showed the difference in their functionality to be negligible. Overall, it may be concluded that higher proliferation and differentiation capacity of OE-MSCs, along with their easier harvestability and autologous transplantability compared with WJ-MSCs, makes them a better cell source for stem cell therapy of neurodegenerative disorders which are caused by degeneration of dopaminergic neurons.

Human olfactory stem cells: As a promising source of dopaminergic neuron-like cells for treatment of Parkinson's disease

The production of dopaminergic (DA) neurons from stem cells holds a great promise for future clinical treatment of neurodegenerative diseases, such as Parkinson’s disease (PD). Olfactory ecto-mesenchymal stem cells (OE-MSCs) derived from the adult human olfactory mucosa can be easily isolated and expanded in culture while maintaining their immense plasticity. In addition to reduced ethical concerns, OE-MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. The goal of this study was to define the potentiality of human olfactory mucosa-derived MSCs aimed at differentiation into DA neuron-like cells. OE-MSCs were induced to differentiate to DA neuron-like cells in vitro by using sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), basic fibroblast growth factor (bFGF), Glial cell line-derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF). Then the differentiated neurons were characterized for expression of DA neuron markers by Real-time PCR, immunocytochemistry and flow cytometry. Our findings showed that differentiated OE-MSCs could significantly express DA neuron markers at mRNA and protein levels along with dopamine release 12 days post-differentiation. These results support the viability and feasibility of using OE-MSCs as a source of in vitro generated DA neuron-like cells for treatment of DA disorders namely PD.

Long non-coding RNA H19 promotes the osteogenic differentiation of rat ectomesenchymal stem cells via Wnt/β-catenin signaling pathway.

To investigate the molecular mechanism of long noncoding RNA (lncRNA) H19 that promotes osteogenic differentiation in rat ectomesenchymal stem cells (EMSCs). EMSCs were isolated from rat fetal facial processes by flow cytometry. Osteogenic markers CD29, CD90, CD44, CD57, Nestin and sox10 were detected by fluorescent immunoassay. β-catenin and Wnt pathway target genes were detected by Real-time polymerase chain reaction (RT-PCR) and Western blot after the construction of transient interference H19, a stable expression of H19 EMSCs cell line and the induction of osteogenic differentiation of EMSCs cells. EMSCs of H19 overexpression were treated with Wnt/beta-catenin signaling pathway inhibitor Wnt-C59, and the expressions of beta-catenin and osteogenic markers were detected by RT-PCR and Western blot. Furthermore, the mechanism of H19 regulating Wnt/beta-catenin signaling pathway was explored by transfecting miR-22 and miR-141 mimics and luciferase reporter assays. EMSCs were successfully isolated and identified, osteogenic markers CD29, CD90, CD44, CD57, Nestin and sox10 were significantly overexpressed. Osteogenesis-induced solution significantly increased the expression of H19 and osteogenic markers ALP, Runx2, BMP and OCN in EMSCs (p<0.05). Interference with H19 significantly inhibited the expressions of osteogenic markers, beta-catenin and target genes of Wnt/beta-catenin signaling pathway (p<0.05), while upregulation of H19 significantly promoted the expressions of these markers and genes in EMSCs (p<0.05). Wnt-C59 inhibitors treatment inhibited the Wnt/beta-catenin signaling pathway and osteogenic differentiation in EMSCs with H19 overexpression (p<0.05). Furthermore, H19 could block the inhibitory effect of miR-22 and miR-141 on β-catenin and activate the Wnt/beta-catenin signaling pathway after transfecting miR-22 mimics and miR-141 mimics in EMSCs (p<0.05). LncRNA H19 can promote the osteogenic differentiation of rat EMSCs by activating Wnt/beta-catenin signal, providing a theoretical basis for the application of EMSCs in tooth tissue engineering regeneration and repair.

The spatiotemporal expression and mineralization regulation of p75 neurotrophin receptor in the early tooth development.

The aim of this study was to investigate the spatiotemporal expression and potential role of p75NTR in tooth morphogenesis and tissue mineralization. The dynamic morphology of the four stages (from the beginning of E12.5 d, then E13.5 d and E15.5 d, to the end of E18.5 d) was observed, and the expressions of p75NTR and Runx2 were traced. The ectomesenchymal stem cells (EMSCs) were harvested in vitro, and the biological characteristics were observed. Moreover, the mineralization capability of EMSCs was evaluated. The relations between p75NTR and ALP, Col-1 and Runx2 were investigated. The morphologic results showed that the dental lamina appeared at E12.5 d, the bud stage at E13.5 d, the cap stage at E15.5 d and the bell stage at E18.5 d. p75NTR and Runx2 showed the similar expression pattern. EMSCs from the four stages showed no significant difference in proliferation. But the positive rate of p75NTR in the E12.5 d cells was significantly lower than that in the other three stages (P<0.05). Moreover, the higher positive rate of p75NTR the cells were, the stronger mineralization capability they showed. p75NTR was well positively correlated with the mineralization-related markers ALP, Col-1 and Runx2, which increased gradually with the mature of dental germs. p75NTR might play an important role in the regulation of tooth morphogenesis, especially dental hard tissue formation.

The Effects of Acellular Spinal Cord Scaffolds-Genipin-Sonic Hedgehog on Proliferation and Differentiation of Ectomesenchymal Stem Cells

The Effects of Acellular Spinal Cord Scaffolds-Genipin-Sonic Hedgehog on Proliferation and Differentiation of Ectomesenchymal Stem Cells