Studies at the Department are aimed at exploring the possibity of cell therapy to repair and improve the biological functioning of defective nervous tissue. This aim can be achieved through the transplantation of isolated and well-characterized cells into the injured CNS in sufficient numbers and quality so that they can induce the recovery of function. Embryonic or mesenchymal stem cells may serve as one of the cell sources for transplantation. Axons are capable of regeneration even in the adult mammalian CNS; however, in larger lesions, especially after spinal cord injury, the axons are unable to overcome the tissue defect. Hence, one of the top priorities of our research is to develop a technique for the regeneration and repair of CNS injury. Biocompatible heterogenous hydrogels have been found to be capable of promoting regeneration of damaged tissue and raise the potential for regeneration of axonal ingrowth.
      Research at the Department of Tissue Culture and Stem Cells focuses on these main topics:
- Labeling and in vitro differentiation of bone marrow mesenchymal stem cells (MSC)
- Transplantation of embryonic stem cells (ESC) and MSC into the injured CNS
- Bridging spinal cord lesions by using polymer hydrogels

      In the majority of transplantation studies, donor cells are labeled before transplantation (retroviral labeling, bromodeoxyuridine, red fluorescent "cell tracker" PKH 26, green fluorescent protein [GFP]). After a certain time, the host organism is sacrificed and the evaluation of the location of the transplanted cells is carried out by microscopic analysis. This method, however, does not give us data about the dynamics of the process or about the migration of the transplanted stem cells in the host organism and requires sacrifing the host organism.
      Initial findings confirmed that nanoparticles based on microcrystals of iron oxides in brain cells could be observed by MR imaging. The nanoparticle contrast agents are introduced into the cells during cell culture incubation and later transplanted into the tissue. The position and migration of the transplanted cells can be tracked by MR imaging techniques due to the fact that the presence of superparamagnetic iron-oxide particles in the cells increases the contrast in the MR image. For labeling we have chosen commercially available contrast agents based on dextran-coated iron-oxide nanoparticles. The cells were co-labeled, e.g., with BrdU or Prussian blue.

Fig 1.
A: Bone marrow mesenchymal stem cells grown in culture labeled with the contrast agent, stained with Prussian blue and colabeled with BrdU.
B: Transmission electron microphotograph showing a cluster of iron particles surrounded by a cell membrane, confirming the presence of iron inside the cell.

      The iron particles in cells in culture were observed as dark dots using an optical microscope with phase contrast. Iron inside the cells was visualised by Prussian blue staining. Transmission electron microscopy confirmed that the iron particles were inside the cells. The iron particles were observed as membrane-bound clusters within the cell cytoplasm. The membranous vesicles containing iron particles indicated an endocytotic process of iron uptake. We have used in vivo MR imaging to observe the fate of magnetic iron-oxide-labeled rat MSC transplanted into adult rats with cortical photochemical lesions.

      Recently, we have concentrated on studies of ESC and MSC as carriers for gene and cell therapy in the injured CNS. MSC are pluripotent progenitor cells that have the capacity to migrate and exhibit site-dependent differentiation in response to environmental signals. Intracerebral implantation or intravenous infusion of MSC in an animal model of cortical or spinal cord photochemical lesion resulted in a preferential migration of cells into the lesion. We observed the migration of magnetically labeled cells implanted either intracerebrally into the contralateral hemisphere or injected intravenously into the femoral vein. The presence of the cells, visible on MR images after 1 week as a hypointense signal in the lesion was confirmed by subsequent Prussian blue and anti-BrdU staining. We showed that some commercially available contrast agents can be used as cell markers in noninvasive MR tracking. MSC labeled with iron-oxide nanoparticles migrate into an injured site and therefore can be used to track implanted cells in the brain.

Fig. 2. Intracerebral injection of MSCs into brains with photochemical lesions.
A: Prussian blue staining of an injection site in the contralateral hemisphere and a photochemical lesion, four weeks after grafting. Higher magnification microphotograph of antiBrdU staining showing BrdU-positive MSCs in the lesion (B) and prussian blue-stained MSCs in the left edge of the photochemical lesion (C). Implanted rat MSCs at the border of the lesion four weeks after grafting, double-stained for BrdU (red; D) and the neuronal marker NeuN (green; E). Co-expression of both markers appears as yellow (F).

      The implantation of non-resorbable biocompatible polymer hydrogels into defects in the central nervous system (CNS) can reduce glial scar formation, bridge the lesion and lead to tissue regeneration within the hydrogel. We implanted hydrogels based on crosslinked poly hydroxyethyl-methacrylate (pHEMA) and poly N-(2-hydroxypropyl)-methacrylamide (pHPMA) into the rat cortex and spinal cord and evaluated the cellular invasion into the hydrogels by means of immunohistochemical methods and tetramethylammonium diffusion measurements. Our hydrogels are a class of materials based on poly-HEMA that form a three-dimensional polymer network on which is adsorbed a large amount of water (60-98%). They are in many ways similar to the environment in developing nervous tissue; they are capable of providing mechanical support to ingrowing cells and axons, and their chemical and physical properties can be tailored to a specific use.

Fig. 3. T2 weighted images of cortical photochemical lesion and MSC labeled with iron-oxide nanoparticles implanted into the contralateral hemisphere (A) and intravenously (B). The cell implant was sharply bounded and clearly visible as a hypointense area in the MR images. Seven days after transplantation (A) or intravenous injection (B), a hypointense signal was observed in the lesion that persisted for the next 5 weeks.

      In the pHPMA hydrogels, we found a massive ingrowth of connective tissue elements. We found mainly astrocytes in the pHEMA hydrogels. These changes were accompanied by a decrease in the extracellular space volume fraction and a tortuosity increase, showing cell invasion and the development of new diffusion barriers that hinder the diffusion of TMA+ in the hydrogel. In the near future we will employ cell-polymer constructs in order to facilitate the regeneration of injured spinal cord. The scaffold of the cell-polymer constructs is made of non-resorbable biocompatible macroporous hydrogels with communicating pores. This scaffold will be seeded with pre-labeled ESC or MSC. The properties of the cell-polymer constructs will be characterized in vitro. We will examine the morphological properties of the cells, their viability, their membrane properties and the accumulation of ECM molecules. Preliminary results show that the cell populations can inhabit the hydrogels, modify the microenvironment by producing extracellular matrix (ECM) molecules, and differentiate into neurons and glial cells.

Fig: 4: Immunostaining with anti-neurofilament antibodies shows intensely stained axons that have crossed the interface and penetrated the pHPMA hydrogel matrix. At the lower right is a selected area at higher magnification (B). GFAP immunocytochemistry shows glial cells infiltrating the porous structure of the hydrogel and sending processes into the implant (C). Capillary sprouts were observed within the hydrogel. On this fluorescent microphotograph showing immunostaining for chondroitin sulphate proteoglycans (CS56), some of these sprouts are marked with an asterisk (A).

      The diffusion parameters of the cell-polymer constructs will be determined by the TMA+ ionophoretic method. The cell-polymer constructs will then be implanted into injured rat spinal cords in order to observe the extent of tissue ingrowth. Simultaneously, systemic infusion of ESC or MSC will help to re-populate the lesion edges. The properties of the scaffold will be modified, preferably by coating with various proteins (chondroitinase, collagen) or their important epitopes (RGD peptides of laminin), in order to selectively support axonal ingrowth into the implants.