In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and ...support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d‐hDPSCs), which promoted survival and neurite outgrowth in 2‐dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d‐hDPSCs in a 3‐dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d‐hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell‐based therapies as treatment for peripheral nerve injury.—Martens, W., Sanen, K., Georgiou, M., Struys, T., Bronckaers, A., Ameloot, M., Phillips, J., Lambrichts, I. Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue‐engineered collagen construct in vitro. FASEB J. 28, 1634–1643 (2014). www.fasebj.org
Abstract Adipose-derived stem cells were isolated from rats and differentiated to a Schwann cell-like phenotype in vitro . The differentiated cells (dADSCs) underwent self-alignment in a tethered ...type-1 collagen gel, followed by stabilisation to generate engineered neural tissue (EngNT-dADSC). The pro-regenerative phenotype of dADSCs was enhanced by this process, and the columns of aligned dADSCs in the aligned collagen matrix supported and guided neurite extension in vitro . EngNT-dADSC sheets were rolled to form peripheral nerve repair constructs that were implanted within NeuraWrap conduits to bridge a 15 mm gap in rat sciatic nerve. After 8 weeks regeneration was assessed using immunofluorescence imaging and transmission electron microscopy and compared to empty conduit and nerve graft controls. The proportion of axons detected in the distal stump was 3.5 fold greater in constructs containing EngNT-dADSC than empty tube controls. Our novel combination of technologies that can organise autologous therapeutic cells within an artificial tissue construct provides a promising new cellular biomaterial for peripheral nerve repair.
Abstract A new combination of tissue engineering techniques provides a simple and effective method for building aligned cellular biomaterials. Self-alignment of Schwann cells within a tethered type-1 ...collagen matrix, followed by removal of interstitial fluid produces a stable tissue-like biomaterial that recreates the aligned cellular and extracellular matrix architecture associated with nerve grafts. Sheets of this engineered neural tissue supported and directed neuronal growth in a co-culture model, and initial in vivo tests showed that a device containing rods of rolled-up sheets could support neuronal growth during rat sciatic nerve repair (5 mm gap). Further testing of this device for repair of a critical-sized 15 mm gap showed that, at 8 weeks, engineered neural tissue had supported robust neuronal regeneration across the gap. This is, therefore, a useful new approach for generating anisotropic engineered tissues, and it can be used with Schwann cells to fabricate artificial neural tissue for peripheral nerve repair.
Olfactory ensheathing cells (OECs) are a promising potential cell therapy to aid regeneration. However, there are significant challenges in isolating and characterizing them. In the current study, we ...have explored methods to enhance the recovery of cells expressing OEC marker p75NTR from rat mucosa. With the addition of a 24-hour differential adhesion step, the expression of p75NTR was significantly increased to 73 ± 5% and 46 ± 18% on PDL and laminin matrices respectively. Additionally, the introduction of neurotrophic factor NT-3 and the decrease in serum concentration to 2% FBS resulted in enrichment of OECs, with p75NTR at nearly 100% (100 ± 0% and 98 ± 2% on PDL and laminin respectively), and candidate fibroblast marker Thy1.1 decreased to zero. Culturing OECs at physiologically relevant oxygen tension (2-8%) had a negative impact on p75NTR expression and overall cell survival. Regarding cell potency, co-culture of OECs with NG108-15 neurons resulted in more neuronal growth and potential migration at atmospheric oxygen. Moreover, OECs behaved similarly to a Schwann cell line positive control. In conclusion, this work identified key bioprocessing fundamentals that will underpin future development of OEC-based cell therapies for potential use in spinal cord injury repair. However, there is still much work to do to create optimized isolation methods.
Abstract
Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, ...inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive “off-the-shelf” alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycER
TAM
gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycER
TAM
-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75
NTR
, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycER
TAM
-derived PA5 hOMCs have potential as a regenerative therapy for neural cells.
Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, inter-patient ...variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive "off-the-shelf" alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycER
gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycER
-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75
, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycER
-derived PA5 hOMCs have potential as a regenerative therapy for neural cells.
Peripheral nerve injury as a result of trauma affects approximately 1 million people in Europe and America annually. The current clinical gold standard treatment for repairing long gaps is the nerve ...autograft, in which only -50% of cases result in satisfactory functional recovery. Tissue-engineered cellular bridging devices for surgical implantation into peripheral nerve injury sites could provide an attractive alternative to the autograft. This project reports the development of a robust, anisotropic biomaterial with highly aligned cells that can fonn the basis of a peripheral nerve repair device. Engineered neural tissue (EngNT), which is formed from columns of Schwann cells or stem cells within a 3D aligned collagen matrix, can promote directed neurite outgrowth in vitro. This study demonstrates that sheets of EngNT can be arranged to form the 'endoneurium' of a peripheral nerve repair device within a NeuraWrap™ outer tube, and can be used for the repair of critical sized defects in rat. Schwann cells are the preferred cell type for peripheral nerve repair because of their ability to enhance axon migration and secrete factors that further increase regeneration. However the use of autologous Schwann cells has a number of disadvantages, including the sacrifice of host nerve tissue for their extraction and slow expansion times in vitro. Various therapeutic cell types and a bovine collagen source that can potentially be used to make EngNT to form the device core were investigated. EngNT devices containing Schwann cell-like cells from adipose-derived stem cells (dADSC) or human neural progenitor cells differentiated to glial cells (dCX) were tested in a critical sized gap in the rat sciatic nerve model. The in vivo experiments demonstrated that there is potential, for the dADSCs to be used for peripheral nerve repair. The results from the dCX repairs were less clear. The technology reported here offers a simple, rapid and effective method for the manufacture of an aligned cellular biomaterial, and could be applied to a range of tissue engineering applications. This study demonstrates that there is potential for EngNT to be used in the construction of nerve repair conduits.
This study aimed to develop a 3D culture model to test the extent to which transplanted stem cells modulate astrocyte reactivity, where exacerbated glial cell activation could be detrimental to CNS ...repair success.
The reactivity of rat astrocytes to bone marrow mesenchymal stem cells, neural crest stem cells (NCSCs) and differentiated adipose-derived stem cells was assessed after 5 days. Schwann cells were used as a positive control.
NCSCs and differentiated Schwann cell-like adipose-derived stem cells did not increase astrocyte reactivity. Highly reactive responses to bone marrow mesenchymal stem cells and Schwann cells were equivalent.
This approach can screen therapeutic cells prior to in vivo testing, allowing cells likely to trigger a substantial astrocyte response to be identified at an early stage. NCSCs and differentiated Schwann cell-like adipose-derived stem cells may be useful in treating CNS damage without increasing astrogliosis.
Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like ...domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.
Display omitted
•Autism-disrupted eIF4G microexons mediate activity-dependent responses•eIF4G microexons suppress the expression of critical synaptic proteins•eIF4G microexon-deficient mice display social behavior and memory deficits•eIF4G microexons coalesce with neuronal granule components and stall ribosomes
Gonatopoulos-Pournatzis et al. demonstrate that conserved, autism-disrupted microexons in eIF4G translation initiation factors regulate the neuronal proteome and control higher order cognitive functions. The microexons function as a translational brake and elicit ribosome stalling on transcripts encoding synaptic proteins through their propensity to coalesce with FMRP and other neuronal granule components.
PDF
