Jana Hlučilová

Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs.

In recent studies using a rat aortic balloon occlusion model, we have demonstrated that spinal grafting of rat or human neuronal precursors or human postmitotic hNT neurons leads to progressive amelioration of spasticity and rigidity and corresponding improvement in ambulatory function. In the present study, we characterized the optimal dosing regimen and safety profile of human spinal stem cells (HSSC) when grafted into the lumbar spinal cord segments of naive immunosuppressed minipigs. Gottingen-Minnesota minipigs (18–23 kg) were anesthetized with halothane, mounted into a spine-immobilization apparatus, and received five bilateral injections of HSSC delivered in 2, 4, 6, 8, or 10 μl of media targeted into L2-L5 central gray matter (lamina VII). The total number of delivered cells ranged between 2,500 and 100,000 per injection. Animals were immunosuppressed with Prograf® for the duration of study. After cell grafting, ambulatory function was monitored daily using a Tarlovs score. Sensory functions were assessed by mechanically evoked skin twitch test. Animals survived for 6–7 weeks. Three days before sacrifice animals received daily injections of bromodeoxyuridine (100 mg/kg; IV) and were then transcardially perfused with 4% paraformaldehyde. Th12-L6 spinal column was then dissected; the spinal cord was removed and scanned with MRI. Lumbar transverse spinal cord sections were then cut and stained with a combination of human-specific (hNUMA, hMOC, hNSE, hSYN) or nonspecific (DCX, MAP2, GABA, CHAT) antibodies. The total number of surviving cells was estimated using stereological quantification. During the first 12–24 h after cell grafting, a modest motor weakness was observed in three of eight animals but was no longer present at 4 days to 7 weeks. No sensory dysfunction was seen at any time point. Postmortem MRI scans revealed the presence of the individual grafts in the targeted spinal cord areas. Histological examination of spinal cord sections revealed the presence of hNUMA-immunoreactive grafted cells distributed between the base of the dorsal horn and the ventral horn. In all grafts intense hMOC, DCX, and hSYN immunoreactivity in grafted cells was seen. In addition, a rich axodendritic network of DCX-positive processes was identified extending 300–700 μm from the grafts. On average, 45% of hNUMA-positive neurons were GABA immunoreactive. Stereological analysis of hNUMA-positive cells showed an average of 2.5- to 3-fold increase in number of surviving cells compared with the number of injected cells. Analysis of spinal structural morphology showed that in animals injected with more than 50,000 cells/injection or volumes of injectate higher than 6 μl/injection there was tissue expansion and disruption of the local axodendritic network. Based on these data the safe total number of injected cells and volume of injectate were determined to be 30,000 cells delivered in ≤6 μl of media. These data demonstrate that highly reproducible delivery of a potential cell therapeutic candidate into spinal parenchyma can be achieved across a wide range of cell doses by direct intraspinal injections. The resulting grafts uniformly showed robust cell survival and progressive neuronal maturation.

Allogeneic and autogenous transplantations of MSCs in treatment of the physeal bone bridge in rabbits

BackgroundThe aim of this experimental study on New Zealands white rabbits was to find differences in the results of treating the distal physeal femoral defect by the transplantation of autologous or allogeneic mesenchymal stem cells (MSCs). After the excision of a created bone bridge in the distal physis of the right femur, modified composite scaffold with MSCs was transplanted into the defect. In animal Group A (n = 11) autogenous MSCs were implanted; in animal Group B (n = 15) allogeneic MSCs were implanted. An iatrogenic physeal defect of the left femur of each animal not treated by MSCs transplantation served as control. The rabbits were euthanized four months after the transplantation. The treatment results were evaluated morphometrically (femoral length and valgus deformity measurement) and histologically (character and quality of the new cartilage).ResultsFour months after the transplantation, the right femurs of the animals in Group A were on average longer by 0.50 ± 0.04 cm (p = 0.018) than their left femurs, the right femurs of rabbits in Group B were on average longer by 0.43 ± 0.01 cm (p = 0.028) than their left femurs.4 months after the therapeutic transplantation of MSCs valgus deformity of the distal part of the right femur of animals in Group A was significantly lower (by 4.45 ± 1.86°) than that of their left femur (p = 0.028), in Group B as well (by 3.66 ± 0.95° than that of their left femur p = 0.001). However, no significant difference was found between rabbits with transplanted autogenous MSCs (Group A) and rabbits with transplanted allogeneic MSCs (Group B) either in the femur length (p = 0.495), or in its valgus deformity (p = 0.1597). After the MSCs transplantation the presence of a newly formed hyaline cartilage was demonstrated histologically in all the animals (both groups). The ability of transplanted MSCs to survive in the damaged physis was demonstrated in vivo by magnetic resonance, in vitro by Perls reaction and immunofluorescence.ConclusionThe transplantation of both autogenous and allogeneic MSCs into a defect of the growth plate appears as an effective method of surgical treatment of physeal cartilage injury. However, the Findings point to the conclusion that there is no clear difference in the final effect of the transplantation procedure used.

Synthetic poly(amino acid) hydrogels with incorporated cell-adhesion peptides for tissue engineering

Preparation of soft poly(amino acid) hydrogels containing biomimetic cell‐adhesive peptides was investigated. Covalently crosslinked gels were formed by radical co‐polymerization of methacryloylated macromonomer poly[N5‐(2‐hydroxyethyl)‐L‐glutamine‐stat‐L‐alanine‐stat‐methacryloyllysine] with 2‐hydroxyethyl methacrylate (HEMA) as minor co‐monomer. Hydrogels carrying biomimetic peptides were prepared by using methacryloylated peptides, such as methacryloyl–GGGRGDSG–OH and methacryloyl–GGGYIGSR–OH, as additional monomers in the polymerization mixture. Mechanical stability and swelling in water of the hydrogels obtained for different solid:water and polypeptide:HEMA ratios were evaluated. The microporosity of gels (5–20 µm), dependent on the polyHEMA phase separation in water, was followed by low‐vacuum SEM. The effect of biomimetic modification of hydrogels with RGDS and YIGSR peptides on the seeding efficiency of porcine mesenchymal stem cells (MSCs) was studied in vitro. While unmodified hydrogels showed very low cell adhesion, due to their highly hydrophilic nature, the incorporation of adhesive peptides significantly improved the adhesion and viability of seeded cells. Copyright