Gunnar Hargus

Differentiation plasticity of chondrocytes derived from mouse embryonic stem cells.

Evidence exists that cells of mesenchymal origin show a differentiation plasticity that depends on their differentiation state. We used in vitro differentiation of embryonic stem cells through embryoid bodies as a model to analyze chondrogenic and osteogenic differentiation because embryonic stem cells recapitulate early embryonic developmental phases during in vitro differentiation. Here, we show that embryonic stem cells differentiate into chondrocytes, which progressively develop into hypertrophic and calcifying cells. At a terminal differentiation stage, cells expressing an osteoblast-like phenotype appeared either by transdifferentiation from hypertrophic chondrocytes or directly from osteoblast precursor cells. Chondrocytes isolated from embryoid bodies initially dedifferentiated in culture but later re-expressed characteristics of mature chondrocytes. The process of redifferentiation was completely inhibited by transforming growth factor β3. In clonal cultures of chondrocytes isolated from embryoid bodies, additional mesenchymal cell types expressing adipogenic properties were observed, which suggests that the subcultured chondrocytes indeed exhibit a certain differentiation plasticity. The clonal analysis confirmed that the chondrogenic cells change their developmental fate at least into the adipogenic lineage. In conclusion, we show that chondrocytic cells are able to transdifferentiate into other mesenchymal cells such as osteogenic and adipogenic cell types. These findings further strengthen the view that standardized selection strategies will be necessary to obtain defined cell populations for therapeutic applications.

Neural Cell Adhesion Molecule L1-Transfected Embryonic Stem Cells Promote Functional Recovery after Excitotoxic Lesion of the Mouse Striatum

We have generated a murine embryonic stem cell line constitutively expressing L1 at all stages of neural differentiation to investigate the effects of L1 overexpression on stem cell proliferation, migration, differentiation, cell death, and ability to influence drug-induced rotation behavior in an animal model of Huntingtons disease. L1-transfected cells showed decreased cell proliferation in vitro, enhanced neuronal differentiation in vitro and in vivo, and decreased astrocytic differentiation in vivo without influencing cell death compared with nontransfected cells. L1 overexpression also resulted in an increased yield of GABAergic neurons and enhanced migration of embryonic stem cell-derived neural precursor cells into the lesioned striatum. Mice grafted with L1-transfected cells showed recovery in rotation behavior 1 and 4 weeks, but not 8 weeks, after transplantation compared with mice that had received nontransfected cells, thus demonstrating for the first time that a recognition molecule is capable of improving functional recovery during the initial phase in a syngeneic transplantation paradigm.

Embryonic Stem Cell-Derived L1 Overexpressing Neural Aggregates Enhance Recovery after Spinal Cord Injury in Mice

An obstacle to early stem cell transplantation into the acutely injured spinal cord is poor survival of transplanted cells. Transplantation of embryonic stem cells as substrate adherent embryonic stem cell-derived neural aggregates (SENAs) consisting mainly of neurons and radial glial cells has been shown to enhance survival of grafted cells in the injured mouse brain. In the attempt to promote the beneficial function of these SENAs, murine embryonic stem cells constitutively overexpressing the neural cell adhesion molecule L1 which favors axonal growth and survival of grafted and imperiled cells in the inhibitory environment of the adult mammalian central nervous system were differentiated into SENAs and transplanted into the spinal cord three days after compression lesion. Mice transplanted with L1 overexpressing SENAs showed improved locomotor function when compared to mice injected with wild-type SENAs. L1 overexpressing SENAs showed an increased number of surviving cells, enhanced neuronal differentiation and reduced glial differentiation after transplantation when compared to SENAs not engineered to overexpress L1. Furthermore, L1 overexpressing SENAs rescued imperiled host motoneurons and parvalbumin-positive interneurons and increased numbers of catecholaminergic nerve fibers distal to the lesion. In addition to encouraging the use of embryonic stem cells for early therapy after spinal cord injury L1 overexpression in the microenvironment of the lesioned spinal cord is a novel finding in its functions that would make it more attractive for pre-clinical studies in spinal cord regeneration and most likely other diseases of the nervous system.

Embryonic stem cell-derived L1 overexpressing neural aggregates enhance recovery in Parkinsonian mice

Parkinsons disease is the second most common neurodegenerative disease, after Alzheimers disease, and the most common movement disorder. Drug treatment and deep brain stimulation can ameliorate symptoms, but the progressive degeneration of dopaminergic neurons in the substantia nigra eventually leads to severe motor dysfunction. The transplantation of stem cells has emerged as a promising approach to replace lost neurons in order to restore dopamine levels in the striatum and reactivate functional circuits. We have generated substrate-adherent embryonic stem cell-derived neural aggregates overexpressing the neural cell adhesion molecule L1, because it has shown beneficial functions after central nervous system injury. L1 enhances neurite outgrowth and neuronal migration, differentiation and survival as well as myelination. In a previous study, L1 was shown to enhance functional recovery in a mouse model of Huntingtons disease. In another study, a new differentiation protocol for murine embryonic stem cells was established allowing the transplantation of stem cell-derived neural aggregates consisting of differentiated neurons and radial glial cells into the lesioned brain. In the present study, this embryonic stem cell line was engineered to overexpress L1 constitutively at all stages of differentiation and used to generate stem cell-derived neural aggregates. These were monitored in their effects on stem cell survival and differentiation, rescue of endogenous dopaminergic neurons and ability to influence functional recovery after transplantation in an animal model of Parkinsons disease. Female C57BL/6J mice (2 months old) were treated with the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intraperitoneally to deplete dopaminergic neurons selectively, followed by unilateral transplantation of stem cell-derived neural aggregates into the striatum. Mice grafted with L1 overexpressing stem cell-derived neural aggregates showed better functional recovery when compared to mice transplanted with wild-type stem cell-derived neural aggregates and vehicle-injected mice. Morphological analysis revealed increased numbers and migration of surviving transplanted cells, as well as increased numbers of dopaminergic neurons, leading to enhanced levels of dopamine in the striatum ipsilateral to the grafted side in L1 overexpressing stem cell-derived neural aggregates, when compared to wild-type stem cell-derived neural aggregates. The striatal levels of gamma-aminobutyric acid were not affected by L1 overexpressing stem cell-derived neural aggregates. Furthermore, L1 overexpressing, but not wild-type stem cell-derived neural aggregates, enhanced survival of endogenous host dopaminergic neurons after transplantation adjacent to the substantia nigra pars compacta. Thus, L1 overexpressing stem cell-derived neural aggregates enhance survival and migration of transplanted cells, differentiation into dopaminergic neurons, survival of endogenous dopaminergic neurons, and functional recovery after syngeneic transplantation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinsons disease.

Close homologue of adhesion molecule L1 promotes survival of Purkinje and granule cells and granule cell migration during murine cerebellar development.

Several L1‐related adhesion molecules, expressed in a well‐coordinated temporospatial pattern during development, are important for fine tuning of specific cerebellar circuitries. We tested the hypothesis that CHL1, the close homologue of L1, abundantly expressed in the developing and adult cerebellum, is also required for normal cerebellar histogenesis. We found that constitutive ablation of CHL1 in mice caused significant loss (20–23%) of Purkinje and granule cells in the mature 2‐month‐old cerebellum. The ratio of stellate/basket interneurons to Purkinje cells was abnormally high (+38%) in CHL1‐deficient (CHL1−/−) mice compared with wild‐type (CHL1+/+) littermates, but the γ‐aminobutyric acid (GABA)ergic synaptic inputs to Purkinje cell bodies and dendrites were normal, as were numbers of Golgi interneurons, microglia, astrocytes, and Bergmann glia. Purkinje cell loss occurred before the first postnatal week and was associated with enhanced apoptosis, presumably as a consequence of CHL1 deficiency in afferent axons. In contrast, generation of granule cells, as indicated by in vivo analyses of cell proliferation and death, was unaffected in 1‐week‐old CHL1−/− mice, but numbers of migrating granule cells in the molecular layer were increased. This increase was likely related to retarded cell migration because CHL1−/− granule cells migrated more slowly than CHL1+/+ cells in vitro, and Bergmann glial processes guiding migration in vivo expressed CHL1 in wild‐type mice. Granule cell deficiency in adult CHL1−/− mice appeared to result from decreased precursor cell proliferation after the first postnatal week. Our results indicate that CHL1 promotes Purkinje and granule cell survival and granule cell migration during cerebellar development. J. Comp. Neurol. 513:496–510, 2009.

Loss of Sox9 function results in defective chondrocyte differentiation of mouse embryonic stem cells "in vitro"

The transcription factor Sox9 plays an important role during chondrogenesis. After early conditional inactivation of Sox9 in mesenchymal limb bud cells of mice, mesenchymal condensations as well as cartilage and bone are completely absent in the developing limbs. We analyzed chondrogenic differentiation of Sox9-/- mouse embryonic stem cells in vitro, using two clones with different targeted mutations. We found that the development of mature and hypertrophic chondrocytes is completely inhibited in the absence of Sox9 confirming that Sox9 is required for the formation of cartilage. In contrast, Sox9+/- mouse embryonic stem cells showed continuous but reduced differentiation into mature chondrocytes. Interestingly, the formation of early chondrogenic condensations expressing characteristic marker genes such as scleraxis, Sox5 and Sox6 was not inhibited in the absence of Sox9 in vitro. Thus, we propose that the earliest step of chondrogenesis could be regulated by a non cell-autonomous function of Sox9.

Mouse ES cell lines show a variable degree of chondrogenic differentiation in vitro

Pluripotent mouse embryonic stem (ES) cells differentiate in vitro spontaneously into cell types of all three primary germ layers when cultivated as cell aggregates, so‐called ‘embryoid bodies’. Many reports have shown that this system recapitulates cellular developmental processes and gene expression patterns of early embryogenesis. During ES cell differentiation, efficient and directed differentiation into a specific cell type is influenced by many parameters, for example, the batch of the serum used or the application of growth factors and signalling molecules. Because all ES cell lines are considered to be pluripotent, one should not expect remarkable differences regarding their spontaneous differentiation efficiencies. However, here we show that different ES cell lines exhibit a variable degree of spontaneous chondrogenic differentiation indicating that lines with a specific differentiation capacity could be selected. This is an important aspect if ES cells are applied for tissue regeneration.

Tenascin-R Promotes Neuronal Differentiation of Embryonic Stem Cells and Recruitment of Host-Derived Neural Precursor Cells After Excitotoxic Lesion of the Mouse Striatum

Loss of GABAergic projection neurons under excitotoxic conditions in the striatum is associated with a disturbance of motor and cognitive functions as seen, for instance, in Huntingtons disease. Since current treatments cannot replace degenerated neurons, research on alternative therapeutic approaches needs to be pursued. In this context, the transplantation of genetically modified stem cells into lesioned brain areas of patients is a possible alternative. In this study, green fluorescent protein‐labeled murine embryonic stem cells (ESCs) were stably transfected to overexpress the extracellular matrix molecule tenascin‐R (TNR), which is expressed by striatal GABAergic neurons. TNR‐overexpressing ESCs were analyzed in comparison with their parental cells regarding neural differentiation and migration in vitro, and after transplantation into the striatum of quinolinic acid‐treated mice, which serve as a model for Huntingtons disease. In comparison with sham‐transfected control cells, TNR‐overexpressing ESCs showed enhanced differentiation into neurons in vitro, reduced migration in vitro and in vivo, and increased generation of GABAergic neurons and decreased numbers of astrocytes 1 month and 2 months after transplantation, but without significant effects on locomotor functions. Interestingly, TNR‐overexpressing ESCs transplanted into the striatum attracted host‐derived neuroblasts from the rostral migratory stream and promoted stem cell‐mediated recruitment of host‐derived newborn neurons within the grafted area. Thus, we show for the first time that overexpression of an extracellular matrix molecule by in vitro predifferentiated ESCs exerts beneficial effects on tissue regeneration in a mouse model of neurodegenerative disease.

Transplanted L1 Expressing Radial Glia and Astrocytes Enhance Recovery after Spinal Cord Injury

A major obstacle for the transplantation of neural stem cells (NSCs) into the lesioned spinal cord is their predominant astrocytic differentiation after transplantation. We took advantage of this predominant astrocytic differentiation of NSCs and expressed the paradigmatic beneficial neural cell adhesion molecule L1 in radial glial cells and reactive and nonreactive astrocytes as novel cellular vehicles to express L1 under the control of the promoter for the human glial fibrillary acidic protein (GFAP-L1 NSCs). Behavioral analysis and electrophysiological H-reflex recordings revealed that mice transplanted with GFAP-L1 NSCs showed enhanced locomotor recovery in comparison to mice injected with wild type (WT) NSCs or control mice injected with phosphate-buffered saline (PBS). This functional recovery was further accelerated in mice transplanted with L1-expressing radial glial cells that had been immunoisolated from GFAP-L1 NSCs (GFAP-L1-i cells). Morphological analysis revealed that mice grafted with GFAP-L1 NSCs exhibited increased neuronal differentiation and migration of transplanted cells, as well as increased soma size and cholinergic synaptic coverage of host motoneurons and increased numbers of endogenous catecholaminergic nerve fibers caudal to the lesion site. These findings show that L1-expressing astrocytes and radial glial cells isolated from GFAP-L1 NSC cultures represent a novel strategy for improving functional recovery after spinal cord injury, encouraging the use of the human GFAP promoter to target beneficial transgene expression in transplanted stem cells.

Ultrastructural analysis of mouse embryonic stem cell-derived chondrocytes.

Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue.