Stromal Cell-derived Inducing Activity Method Research Articles

Impaired Neural Differentiation of Induced Pluripotent Stem Cells Generated from a Mouse Model of Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.

Transplantation of telencephalic neural progenitors induced from embryonic stem cells into subacute phase of focal cerebral ischemia

Cerebral ischemia causes neuronal death and disruption of neural circuits in the central nervous system. Various neurological disorders caused by cerebral infarction can severely impair quality of life and are potentially fatal. Functional recovery in the chronic stage mainly depends on physical treatment and rehabilitation. We aim to establish cell therapy for cerebral ischemia using embryonic stem (ES) cells, which have self-renewing and pluripotent capacities. We previously reported that the transplanted monkey and mouse ES cell-derived neural progenitors, by stromal cell-derived inducing activity method, could survive and differentiate into various types of neurons and glial cells, and form the neuronal network in basal ganglia. In this report, we induced the differentiation of the neural progenitors from mouse ES cells using the serum-free suspension culture method and confirmed the expression of various basal ganglial neuronal markers and neurotransmitter-related markers both in vitro and in vivo, which was thought to be suitable for replacing damaged striatum after middle cerebral artery occlusion. This is the first report that used selectively induced telencephalic neural progenitors into ischemia model. Furthermore, we purified the progenitors expressing the neural progenitor marker Sox1 by fluorescence-activated cell sorting and Sox1-positive neural progenitors prevented tumor formation in ischemic brain for 2 months. We also analyzed survival and differentiation of transplanted cells and functional recovery from ischemic damage.

Development of effective isolation method of ES cells for analysis of differentiation

Neuroectoderm development is a milestone of vertebrate neurogenesis. However, the molecular mechanism underlying the differentiation of neuroectoderm is still unclear, especially in mammals. ES cells co-cultured with PA6 cells can differentiate to neuroectoderm by the stromal cell-derived inducing activity method (SDIA method), but contamination of PA6 cells is an obstacle to the analysis of molecular mechanisms of differentiation. Here we describe a novel method by which differentiated ES cells are easily isolated from PA6 cells. We attempted to induce the differentiation of ES cells using paraformaldehyde-fixed PA6 cells. RT-PCR and DNA microarray analysis revealed that the background noise derived from contaminated PA6 cells disappeared when fixed PA6 cells were used. Furthermore, genes up-regulated during the differentiation of ES cells were expressed in a developing mouse embryo. Thus, our newly developed method will be very useful for identifying novel genes associated with mouse neuroectoderm development in vitro and in vivo.

Differentiation of dopaminergic neurons from human embryonic stem cells: Modulation of differentiation by FGF-20

Derivation of midbrain dopaminergic (DA) neurons from human embryonic stem (hES) cells has been of particular interest because of the clinical potential for DA neuron transplantation in patients with Parkinson's disease (PD). Several protocols for DA neuron differentiation from mouse embryonic stem cells and hES cells have been reported: however, protocols involving hES cells have yet to be improved. Here, we used a slightly modified stromal cell-derived inducing activity method, consisting four different culture stages, to show that KhES-1 cells differentiate into tyrosine hydroxylase (TH)-positive DA neurons. Quantitative real-time PCR analysis showed a marked induction of the DA neuron marker genes NURR1, paired-like homeodomain transcription factor 3 (PITX3), LIM homeobox transcription- factor 1, beta (LMX1B), engrailed-1 (EN1), dopamine transporter (DAT), and aromatic amino acid decarboxylase (AADC) during differentiation. Treatment with fibroblast growth factor (FGF)-20 and FGF-2 at the final differentiation stage induced the increase of DA neuron development-related transcription factors such as NURR1, PITX3, LMX1B, and EN1. FGF-20 and FGF-2 enhanced DA neuron differentiation from hES cell-derived neural progenitor cells directly without any soluble factors from PA6 cells. These results provide valuable information that will assist in efficient DA neuron differentiation from hES cells and for future transplant application.

Primate Embryonic Stem Cell-Derived Neuronal Progenitors Transplanted into Ischemic Brain

Transplantation of stem cells has the possibility of restoring neural functions after stroke damage. Therefore, we transplanted neuronal progenitors generated from monkey embryonic stem (ES) cells into the ischemic mouse brain to test this possibility. Monkey ES cells were caused to differentiate into neuronal progenitors by the stromal cell-derived inducing activity method. Focal cerebral ischemia was induced by occluding the middle cerebral artery by the intraluminal filament technique. The donor cells were transplanted into the ischemic lateral striatum at 24 h after the start of reperfusion. The cells transplanted into the ischemic brain became located widely around the ischemic area, and, moreover, the transplanted cells differentiated into various types of neurons and glial cells. Furthermore, at 28 days after the transplantation, over 10 times more cells in the graft were labeled with Fluorogold (FG) by stereotactic focal injection of FG into the anterior thalamus and substantia nigra on the grafted side when compared with the number at 14 days. From these results we confirmed the survival and differentiation of, as well as network formation by, monkey ES-cell-derived neuronal progenitors transplanted into the ischemic mouse brain.

Survival and differentiation of neural progenitor cells derived from embryonic stem cells and transplanted into ischemic brain

Cell replacement therapy including the use of embryonic stem cells (ESCs) may represent a novel treatment for damage from stroke. In this study, the authors transplanted neural progenitor cells (NPCs) derived from ESCs into ischemic brain and analyzed their survival and differentiation. Multipotential NPCs were generated from ESCs by using the stromal cell-derived inducing activity method. These cells could differentiate in vitro into neurons, glia, and oligodendrocytes, thus revealing them to be neural stem cells. The NPCs were then transplanted into ischemic brain. At 2 weeks postischemia, the transplanted cells occupied 18.8 +/-2.5% of the hemispheric area; by 4 weeks postischemia, 26.5 +/- 4% of the hemisphere. At 4 weeks after transplantation, green fluorescent protein (GFP)-positive transplanted cells showed mature neuronal morphological features. The authors also investigated the expression of differentiation markers and various neurotransmitters. Transplanted cells were immunopositive for neuronal nuclei, beta-tubulin-III, and glial fibrillary acidic protein. Of the GFP-positive cells, 33.3 +/-11.5% were positive for glutamate decarboxylase, 13.3 +/- 5.8% for glutamate, 2.1 +/- 2.5% for tyrosine hydroxylase, 1.8 +/- 2% for serotonin, and 0.4 +/- 0.2% for choline acetyltransferase. The authors confirmed the survival and differentiation of ESC-derived NPCs transplanted into the ischemic brain. Surviving transplanted cells expressed several neural markers and neurotransmitters. These findings indicate that these cells can function in the brain.

Efficient induction of dopaminergic neurons from embryonic stem cells for application to Parkinson's disease.

For cell replacement therapy of neurodegenerative diseases such as Parkinson's disease (PD), methods for efficiently generating midbrain dopaminergic (DA) neurons from embryonic stem (ES) cells have been investigated. Two aspects of DA neuron generation are considered: genetic modification and manipulation of culture conditions. A transcription factor known as critical for development of DA neurons, Nurr1, was introduced into ES cells to see how they facilitate the generation of DA neurons from ES cells. Also, two culture procedures, the 5-stage method and stromal cell-derived inducing activity (SDIA) method, were used for ES cell differentiation. Using the 5-stage method, we and others previously demonstrated that Nurr1-overexpressing ES cells, under treatment of signaling molecules such as SHH and FGF8 followed by treatment of ascorbic acid, can differentiate into DA neurons with a high efficiency (> 60% of TH+/Tuj1+ neurons). Furthermore, using the SDIA method with treatment of signaling molecules, we found that Nurr1-overexpressing ES cells can differentiate to DA neurons with the highest efficiency ever reported (approximately 90% of TH+/Tuj1+ neurons). Importantly, our semi-quantitative and real-time PCR analyses demonstrate that all known DA marker genes (e.g., TH, AADC and DAT) were up-regulated in Nurr1- overexpressing ES cells when compared to the na ve ES cells. These cells produced increased dopamine compared to na ve D3 cells after differentiation. In the in vivo context after transplantation, the genetically modified ES cells also showed the highly increased dopaminergic neuronal phenotypes. Thus, the combination of genetic engineering and appropriate culture conditions provides a useful tool to generate a good cell source from ES cells for cell replacement therapy of degenerative diseases such as PD.

Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity.

We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells, including midbrain tyrosine hydroxylase-positive (TH(+)) dopaminergic neurons, from mouse embryonic stem cells. We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells. Induced neurons contain TH(+) neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH(+) neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (approximately 5 weeks). In addition, 8% of the colonies contain large patches of Pax6(+)-pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinson's disease and retinitis pigmentosa.