Carl Trolle

Delivery of Differentiation Factors by Mesoporous Silica Particles Assists Advanced Differentiation of Transplanted Murine Embryonic Stem Cells

Stem cell transplantation holds great hope for the replacement of damaged cells in the nervous system. However, poor long‐term survival after transplantation and insufficiently robust differentiation of stem cells into specialized cell types in vivo remain major obstacles for clinical application. Here, we report the development of a novel technological approach for the local delivery of exogenous trophic factor mimetics to transplanted cells using specifically designed silica nanoporous particles. We demonstrated that delivering Cintrofin and Gliafin, established peptide mimetics of the ciliary neurotrophic factor and glial cell line‐derived neurotrophic factor, respectively, with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also their long‐term survival in vivo. We propose that the delivery of growth factors by mesoporous nanoparticles is a potentially versatile and widely applicable strategy for efficient differentiation and functional integration of stem cell derivatives upon transplantation.

Boundary cap neural crest stem cells homotopically implanted to the injured dorsal root transitional zone give rise to different types of neurons and glia in adult rodents

BackgroundThe boundary cap is a transient group of neural crest-derived cells located at the presumptive dorsal root transitional zone (DRTZ) when sensory axons enter the spinal cord during development. Later, these cells migrate to dorsal root ganglia and differentiate into subtypes of sensory neurons and glia. After birth when the DRTZ is established, sensory axons are no longer able to enter the spinal cord. Here we explored the fate of mouse boundary cap neural crest stem cells (bNCSCs) implanted to the injured DRTZ after dorsal root avulsion for their potential to assist sensory axon regeneration.ResultsGrafted cells showed extensive survival and differentiation after transplantation to the avulsed DRTZ. Transplanted cells located outside the spinal cord organized elongated tubes of Sox2/GFAP expressing cells closely associated with regenerating sensory axons or appeared as small clusters on the surface of the spinal cord. Other cells, migrating into the host spinal cord as single cells, differentiated to spinal cord neurons with different neurotransmitter characteristics, extensive fiber organization, and in some cases surrounded by glutamatergic terminal-like profiles.ConclusionsThese findings demonstrate that bNCSCs implanted at the site of dorsal root avulsion injury display remarkable differentiation plasticity inside the spinal cord and in the peripheral compartment where they organize tubes associated with regenerating sensory fibers. These properties offer a basis for exploring the ability of bNCSCs to assist regeneration of sensory axons into the spinal cord and replace lost neurons in the injured spinal cord.

Murine neural crest stem cells and embryonic stem cell-derived neuron precursors survive and differentiate after transplantation in a model of dorsal root avulsion

Spinal root avulsion results in paralysis and sensory loss, and is commonly associated with chronic pain. In addition to the failure of avulsed dorsal root axons to regenerate into the spinal cord, avulsion injury leads to extensive neuroinflammation and degeneration of second‐order neurons in the dorsal horn. The ultimate objective in the treatment of this condition is to counteract degeneration of spinal cord neurons and to achieve functionally useful regeneration/reconnection of sensory neurons with spinal cord neurons. Here we compare survival and migration of murine boundary cap neural crest stem cells (bNCSCs) and embryonic stem cells (ESCs)‐derived, predifferentiated neuron precursors after their implantation acutely at the junction between avulsed dorsal roots L3–L6 and the spinal cord. Both types of cells survived transplantation, but showed distinctly different modes of migration. Thus, bNCSCs migrated into the spinal cord, expressed glial markers and formed elongated tubes in the peripheral nervous system (PNS) compartment of the avulsed dorsal root transitional zone (DRTZ) area. In contrast, the ESC transplants remained at the site of implantation and differentiated to motor neurons and interneurons. These data show that both stem cell types successfully survived implantation to the acutely injured spinal cord and maintained their differentiation and migration potential. These data suggest that, depending on the source of neural stem cells, they can play different beneficial roles for recovery after dorsal root avulsion. Copyright

Human Embryonic Stem Cell-Derived Progenitors Assist Functional Sensory Axon Regeneration after Dorsal Root Avulsion Injury

Dorsal root avulsion results in permanent impairment of sensory functions due to disconnection between the peripheral and central nervous system. Improved strategies are therefore needed to reconnect injured sensory neurons with their spinal cord targets in order to achieve functional repair after brachial and lumbosacral plexus avulsion injuries. Here, we show that sensory functions can be restored in the adult mouse if avulsed sensory fibers are bridged with the spinal cord by human neural progenitor (hNP) transplants. Responses to peripheral mechanical sensory stimulation were significantly improved in transplanted animals. Transganglionic tracing showed host sensory axons only in the spinal cord dorsal horn of treated animals. Immunohistochemical analysis confirmed that sensory fibers had grown through the bridge and showed robust survival and differentiation of the transplants. Section of the repaired dorsal roots distal to the transplant completely abolished the behavioral improvement. This demonstrates that hNP transplants promote recovery of sensorimotor functions after dorsal root avulsion, and that these effects are mediated by spinal ingrowth of host sensory axons. These results provide a rationale for the development of novel stem cell-based strategies for functionally useful bridging of the peripheral and central nervous system.

Forced Runx1 expression in human neural stem/progenitor cells transplanted to the rat dorsal root ganglion cavity results in extensive axonal growth specifically from spinal cord-derived neurospheres.

Cell replacement therapy holds great promise for treating a wide range of human disorders. However, ensuring the predictable differentiation of transplanted stem cells, eliminating their risk of tumor formation, and generating fully functional cells after transplantation remain major challenges in regenerative medicine. Here, we explore the potential of human neural stem/progenitor cells isolated from the embryonic forebrain (hfNSPCs) or the spinal cord (hscNSPCs) to differentiate to projection neurons when transplanted into the dorsal root ganglion cavity of adult recipient rats. To stimulate axonal growth, we transfected hfNSPC- and hscNSPC-derived neurospheres, prior to their transplantation, with a Tet-Off Runx1-overexpressing plasmid to maintain Runx1 expression in vivo after transplantation. Although pronounced cell differentiation was found in the Runx1-expressing transplants from both cell sources, we observed extensive, long-distance growth of axons exclusively from hscNSPC-derived transplants. These axons ultimately reached the dorsal root transitional zone, the boundary separating peripheral and central nervous systems. Our data show that hscNSPCs have the potential to differentiate to projection neurons with long-distance axonal outgrowth and that Runx1 overexpression is a useful approach to induce such outgrowth in specific sources of NSPCs.

Neural crest stem cells from hair follicles and boundary cap have different effects on pancreatic islets in vitro

Purpose: Neural crest stem cells derived from the boundary cap (bNCSCs), markedly promote survival, proliferation and function of insulin producing β-cells in vitro and in vivo after coculture/transplantation with pancreatic islets [1, 2]. Recently, we have shown that beneficial effects on β-cells require cadherin contacts between bNCSCs and β-cells [3, 4]. Here we investigated whether hair follicle (HF) NCSCs, a potential source for human allogeneic transplantation, exert similar positive effects on β-cells. Materials and Methods: We established cocultures of HF-NCSCs or bNCSCs from mice expressing enhanced green fluorescent protein together with pancreatic islets from DxRed expressing mice or NMRI mice and compared their migration towards islet cells and effect on proliferation of β-cells as well as intracellular relations between NCSCs and islets using qRT-PCR analysis and immunohistochemistry. Results: Whereas both types of NCSCs migrated extensively in the presence of islets, only bNCSCs demonstrated directed migration toward islets, induced β-cell proliferation and increased the presence of cadherin at the junctions between bNCSCs and β-cells. Even in direct contact between β-cells and HF-NCSCs, no cadherin expression was detected. Conclusions: These observations indicate that HF-NCSCs do not confer the same positive effect on β-cells as demonstrated for bNCSCs. Furthermore, these data suggest that induction of cadherin expression by HF-NCSCs may be useful for their ability to support β-cells in coculture and after transplantation.

In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles

AIM Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous nanoparticles could be effective for stem cell differentiation in vitro. MATERIALS & METHODS We used a mouse embryonic stem cell line expressing green fluorescent protein under the promoter for the MN-specific gene Hb9 to visualize the level of MN differentiation. The differentiation of stem cells was evaluated by expression of MN-specific transcription factors monitored by quantitative real-time PCR reactions and immunocytochemistry. RESULTS Mesoporous nanoparticles have strong affiliation to the embryoid bodies, penetrate inside the embryoid bodies and come in contact with differentiating cells. CONCLUSION Repeated administration of soluble factors into a culture medium can be avoided due to a sustained release effect using mesoporous silica.

Boundary cap neural crest stem cell transplants contribute Mts1/S100A4-expressing cells in the glial scar

AIM During development, boundary cap neural crest stem cells (bNCSCs) assist sensory axon growth into the spinal cord. Here we repositioned them to test if they assist regeneration of sensory axons in adult mice after dorsal root avulsion injury. MATERIALS & METHODS Avulsed mice received bNCSC or human neural progenitor (hNP) cell transplants and their contributions to glial scar formation and sensory axon regeneration were analyzed with immunohistochemistry and transganglionic tracing. RESULTS hNPs and bNCSCs form similar gaps in the glial scar, but unlike hNPs, bNCSCs contribute Mts1/S100A4 (calcium-binding protein) expression to the scar and do not assist sensory axon regeneration. CONCLUSION bNCSC transplants contribute nonpermissive Mts1/S100A4-expressing cells to the glial scar after dorsal root avulsion.