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Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cellderived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell-derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms. IntroductionParkinson disease (PD) is a neurodegenerative disorder characterized by the loss of midbrain dopaminergic (DA) neurons, with subsequent reductions in striatal dopamine levels. While initial pharmacological treatment with L-dihydroxyphenylalanin (L-DOPA) can attenuate symptoms, the efficacy of this treatment gradually decreases over time. The development of motor complications then requires additional treatments, including deep brain stimulation and fetal DA neuron transplantation (1-3). Both studies of animal models and clinical investigations have shown that transplantation of fetal DA neurons can produce symptomatic relief (4-8). The technical and ethical difficulties in obtaining sufficient and appropriate donor fetal brain tissue, however, have limited the application of this therapy.ES cells are self-renewing, pluripotent cells derived from the inner cell mass of the preimplantation blastocyst. These cells have many of the characteristics required of a cell source for cell-replacement therapy, including proliferation and differentiation capacities (9). We previously discovered that a strong neuralizing activity, which we called stromal cell-derived inducing activity (SDIA), is present

Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell–derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell–derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms

Summary Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.

The process of palate fusion was examined in 13- and 14-day-old mouse fetuses by using in situ staining for nuclear DNA fragmentation (TUNEL method) and immunofluorescent staining for keratin, with special reference to the disruption of the midline epithelial seam. TUNEL-positive cells were found in the disappearing midline seam and the oral and nasal epithelial triangles at some late stages of palate fusion, but not in the palatal shelves prior to contact or in the intact midline epithelial seam. It seems that DNA fragmentation or apoptosis is required for the midline epithelial seam to disrupt, but may not be necessary for initial contact of palatal shelves or for the epithelial fusion of opposing palatal shelves. A similar sign of apoptotic cell death was observed in the disappearing epithelial seam between the fusing nasal septum and dorsal palate. We have demonstrated that apoptotic programmed cell death does occur at some stages of palate fusion, although the present results do not exclude the possibility of epithelial-mesenchymal transformation and the oral and nasal migration of midline epithelial cells.

Recent studies have revealed that neural precursor cells can be expanded not only from the subventricular zone and hippocampus but also from other regions of the human embryonic brain. To determine the regional differences of these precursor cells, we divided the brain of a 9-week-old human embryo into four parts, i.e., telencephalon, diencephalon, mesencephalon, and rhombencephalon. All cultures of the tissues yielded neurospheres, and these spheres gave rise to neurons, astrocytes, and oligodendrocytes. An analysis of clonal populations revealed that these precursor cells were multipotent, and two region-specific differences in neural precursor cells were revealed: 1) The precursor cells from the rostral part of the brain tended to proliferate faster than those from the caudal part, and 2) the precursor cells from the diencephalon and mesencephalon gave rise to more tyrosine hydoxylase (TH)-positive neurons than those from the telencephalon and rhombencephalon. When 50-day-cultured spheres were caused to differentiate, the percentage of TH-positive cells per total cell population was 1.2% for diencephalic and mesencephalic precursors, whereas it was 0.4% for telencephalic and rhombencephalic ones. Furthermore, the TH-positive cells from diencephalic and mesencephalic precursors were large, multipolar, and gamma-aminobutyric acid (GABA)-negative, which suggested that these cells were midbrain dopaminergic neurons. In contrast, TH-positive cells from telencephalic and rhombencephalic precursors were small, bipolar, and GABA-positive. These results suggest that human neural precursor cells might have the potential to differentiate into a variety of cells but retain regional specificity.

R‐loops, which consist of DNA : RNA hybrids and displaced single‐strand DNA, are a major threat to genome stability. We have previously reported that a key Fanconi anemia protein, FANCD2, accumulates on large fragile genes during mild replication stress in a manner depending on R‐loops. In this study, we found that FANCD2 suppresses R‐loop levels. Furthermore, we identified FANCD2 interactions with RNA processing factors, including hnRNP U and DDX47. Our data suggest that FANCD2, which accumulates with R‐loops in chromatin, recruits these factors and thereby promotes efficient processing of long RNA transcripts. This may lead to a reduction in transcription–replication collisions, as detected by PLA between PCNA and RNA Polymerase II, and hence, lowered R‐loop levels. We propose that this mechanism might contribute to maintenance of genome stability during mild replication stress.