Neuronal Differentiation and Extensive Migration of Human Neural Precursor Cells following Co-Culture with Rat Auditory Brainstem Slices

Congenital or acquired hearing loss is often associated with a progressive degeneration of the auditory nerve (AN) in the inner ear. The AN is composed of processes and axons of the bipolar spiral ganglion neurons (SGN), forming the connection between the hair cells in the inner ear cochlea and the cochlear nuclei (CN) in the brainstem (BS). Therefore, replacement of SGNs for restoring the AN to improve hearing function in patients who receive a cochlear implantation or have severe AN malfunctions is an attractive idea. A human neural precursor cell (HNPC) is an appropriate donor cell to investigate, as it can be isolated and expanded in vitro with maintained potential to form neurons and glia. We recently developed a post-natal rodent in vitro auditory BS slice culture model including the CN and the central part of the AN for initial studies of candidate cells. Here we characterized the survival, distribution, phenotypic differentiation, and integration capacity of HNPCs into the auditory circuitry in vitro. HNPC aggregates (spheres) were deposited adjacent to or on top of the BS slices or as a monoculture (control). The results demonstrate that co-cultured HNPCs compared to monocultures (1) survive better, (2) distribute over a larger area, (3) to a larger extent and in a shorter time-frame form mature neuronal and glial phenotypes. HNPC showed the ability to extend neurites into host tissue. Our findings suggest that the HNPC-BS slice co-culture is appropriate for further investigations on the integration capacity of HNPCs into the auditory circuitry.Funding Agencies|Swedish Research Council|2008-2822|Marianne and Marcus Wallenbergs Foundation||Petrus and Augusta Hedlunds Foundation||Swedish Association of Hard of Hearing People||Acta Otolaryngologicas Foundation||Foundation Tysta Skolan||Ollie and Elof Ericssons Foundation for Medical Research||Karolinska Institutet Foundations||Medical faculty||Lund University||</p

Exogenous BDNF and Chondroitinase ABC Consisted Biomimetic Microenvironment Regulates Survival, Migration and Differentiation of Human Neural Progenitor Cells Transplanted into a Rat Auditory Nerve

Current putative regeneration oriented studies express possible role of stem cell based implantation strategy in the restoration of fundamental perception of hearing. The present work utilizes a rat auditory nerve (AN) directed transplantation of human neural progenitor cells (HNPCs) as a cell replacement therapy for impaired auditory function. Groups of b-bungarotoxin induced auditory function compromised female rats were used to transplant HNPCs in the nerve trunk. In the treatment groups, brain derived neurotrophic factor (BDNF), peptide amphiphile nanofiber bioactive gel (Bgel) and Chondroitinase ABC (ChABC), a digestive enzyme that cleaves the core of chondroitin sulphate proteoglycans, were added along with HNPCs while the control groups were with PA inert gel (Igel) and devoid of ChABC. Six weeks post transplantation survival, migration, and differentiation of HNPCs were studied and compared. The groups treated with BDNF and Bgel showed improved survival and differentiation of transplanted HNPCs while the ChABC treated group showed significant migration of HNPCs along the AN and elongation of neuronal fibers along the nerve towards the cochlear nucleus (CN) which was characterized by immunocytochemical markers for human Nuclei (HuN), human mitochondria (HuM) and neuronal β-tubulin (Tuj1). These findings show that addition of BDNF and ChABC consisted Bgel environment facilitated HNPC survival, migration and differentiation along the transplanted rat AN towards the CN. This transplantation strategy provides unique experimental validation for futuristic role of cell based biomaterial consisted neurotrophic factor application in clinically transferable treatment of sensorineural hearing loss (SNHL) along with cochlear implants (CI)

Brain stem slice conditioned medium contains endogenous BDNF and GDNF that affect neural crest boundary cap cells in co-culture

Conditioned medium (CM), made by collecting medium after a few days in cell culture and then re-using it to further stimulate other cells, is a known experimental concept since the 1950s. Our group has explored this technique to stimulate the performance of cells in culture in general, and to evaluate stem- and progenitor cell aptitude for auditory nerve repair enhancement in particular. As compared to other mediums, all primary endpoints in our published experimental settings have weighed in favor of conditioned culture medium, where we have shown that conditioned culture medium has a stimulatory effect on cell survival. In order to explore the reasons for this improved survival we set out to analyze the conditioned culture medium. We utilized ELISA kits to investigate whether brain stem (BS) slice CM contains any significant amounts of brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF). We further looked for a donor cell with progenitor characteristics that would be receptive to BDNF and GDNF. We chose the well-documented boundary cap (BC) progenitor cells to be tested in our in vitro co-culture setting together with cochlear nucleus (CN) of the BS. The results show that BS CM contains BDNF and GDNF and that survival of BC cells, as well as BC cell differentiation into neurons, were enhanced when BS CM were used. Altogether, we conclude that BC cells transplanted into a BDNF and GDNF rich environment could be suitable for treatment of a traumatized or degenerated auditory nerve.Fil: Kaiser, Andreas. Karolinska Huddinge Hospital; SueciaFil: Kale, Ajay. Karolinska Huddinge Hospital; SueciaFil: Novozhilova, Ekaterina. Karolinska Huddinge Hospital; SueciaFil: Siratirakun, Piyaporn. Karolinska Huddinge Hospital; SueciaFil: Aquino, Jorge Benjamin. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Thonabulsombat, Charoensri. Mahidol University. Faculty of Science. Department of Anatomy; TailandiaFil: Ernfors, Patrik. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Olivius, Petri. Linköping University. Faculty of Health Sciences. Department of Clinical and Experimental Medicine; Sueci

Synapse-formation in co-cultured HNPCs (adjacent deposit).

<p><b>A</b>. SV2A (white)- expression pattern was confined largely to the core of the cell mass. SV2A+ HNPC expressing GFAP (red) indicated maturing neuronal progenitors (arrow). It may be suggested that cells with a more mature cellular profile expressing GFP and SV2A but negative for GFAP were of a neuronal phenotype (arrowheads). <b>B</b>. High magnification of occasional SV2A (white)/GFP+ HNPCs (arrows). <b>C</b>. Notably, GFP/SV2A (white)- expressing processes (arrowheads) indicated a capacity for functional interaction of donor-host cells. BS = brain stem slice. Scale bar equals 100 µm in A and 50 µm in B, C.</p

Distribution and migration of HNPCs cultured adjacent to BS slices.

<p><b>A</b>. At 2 weeks migration of h nuc (red) and GFP (green)+ HNPCs was found directed both opposite and towards the BS. Note the large fraction of Tuj1 (white)+ HNPCs located at the sphere periphery. <b>B</b>. At 4 weeks the distribution of h nuc (red)/GFP (green) positive cells was more extensive cf to 2 weeks, with densely packed HNPCs found at the graft/host border. <b>C</b>. A stream of migrating h nuc/GFP/GFAP+ cells toward the host was seen. Only at 4 weeks a visible rim (arrowheads) at the donor-host border of cells was found, including very extensive GFAP+ processes (arrows) <b>D</b>. At the sphere core at both time-points densely packed HNPCs nuclei were found (h nuc, red). <b>E</b>. Tuj1 (white)/h nuc (red)-positive cells exhibited immature (arrow) and migrating cell (arrowhead) profiles in the periphery. <b>F</b>. H nuc (red)/Tuj1 (white)+ migrating cells were found at the periphery (arrowheads). <b>G</b>. H nuc (red)/GFAP (white)- expressing migrating HNPCs (arrowheads) were observed with an increasing toward the host. <b>H</b>. GFP/DCX (red)/hTau (white)+ cells were found migrating in parallel in a chain-like formation perpendicular to the BS slice border. <b>I</b>. Neuronal HNPCs, h nuc (red)/Tuj1 (white)+, found within the host tissue. BS = brain stem slice. Scale bar equals 200 µm in A, B; 100 µm in C, 25 µm in D, 50 µm in E–H, 100 µm in I.</p

Experimental groups, including size of seeded spheres, sphere placement with regard to the BS slice, survival times and rates.

<p>The survival rate was measured as number of specimens displaying ≥40 human nuclei-positive cells/the total numbers of spheres seeded in the respective group.</p

HNPCs cultured on top of BS slices form honeycomb-like networks and primarily neurons.

<p><b>A</b>. Distribution of HNPCs detected by h nuc (red)- or GFP (green)- expression was uniform throughout the entire surface of the BS slice. GFAP (white)- expression was mainly seen in the center of the human cell mass. <b>B</b>. GFP (green)/GFAP (white)-labelling revealed large numbers of HNPC processes expressing GFAP. <b>C</b>. Neuronal differentiation was confirmed by Tuj1 (white)-staining co-localizing with h nuc (red) and GFP (green). Arrowhead in insert shows GFP/Tuj1+ process. <b>D</b>. Neuronal differentiation was also confirmed by DCX (red)/GFP+ processes (arrowheads in insert). <b>E</b>. A honeycomb-like pattern was formed by GFP+ HNPCs network on the surface of the BS slice. <b>F</b>. The vast majority of GFP-labeled cells had morphologies of migrating/differentiating neurons (arrowheads). Scale bar equals 200 µm in A, 100 µm in B; 200 µm in C, D; 50 µm in E, 100 µm in F.</p

Increased neuronal differentiation in adjacent co-cultured HNPCs.

<p><b>A</b>. DCX-staining (white) revealed an obvious preference of neuronal differentiation of HNPCs closer to the BS slice. In agreement, a significant decrease in numbers of nestin-expressing HNPC occurred from 2 to 4 weeks of co-culture. <b>B</b>. At 4 weeks a pronounced neuronal differentiation had occurred and many GFP/DCX (red)/hTau (white) double-labelled HNPCs were found. <b>C</b>. At 2 weeks a significant number of HNPCs were found in a transitional differentiation stage judged by the co-expression of nestin (red) and DCX (white, arrows). But, here also DCX+ cells with neuron-like profiles were found (arrowhead). <b>D</b>. Several “neurogenic” clusters of approximately 10–30 h nuc (red)/Tuj1 (white) were found at 2 weeks (arrowhead). <b>E</b>. Similar clusters expressing DCX (red) were also identified. <b>F</b>. At 4 weeks a large number of DCX (red)/hTau (white)+ HNPCs displayed complex morphologies with multi-branched extensions (arrowheads). <b>G</b>. GFP (green)/DCX (red)/Tau (white)+ HNPCs observed close to the BS tissue at 4 weeks. These cells most often exhibited profiles of migrating cells or early/late differentiated neurons (arrowheads). <b>H</b>. Near and within the BS slice GFP (green)/DCX (red)/Tau (white)+ HNPCs were both migrating (arrowheads) and maturing (arrows). Scale bar equals 200 µm in A; 100 µm in B; 50 µm in C–F, 25 µm in F, G and 40 µm in H.</p

Distribution area of HNPCs after monoculture or co-culture.

<p>The graph shows the mean distribution area in mm² for HNPCs after monoculture of co-culture after adjacent placement to an organoypic cultured brainstem slice. The area was estimated for all groups including 0.3 mm and 1.0 mm sized spheres, at 2 or 4 weeks of culture. All samples with surviving cells were included in the measurements (n = 2–9). Bars represent mean±SD, and white bars represent monocultures and black bars co-cultures. m = monoculture; c = co-culture.</p