Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands

It has been postulated that the stem cells of somatic tissues protect themselves from mutation and cancer risk by selective segregation of their template DNA strands. Self-renewing mammary epithelial stem cells that were originated during allometric growth of the mammary ducts in pubertal females were labeled using [3H]-thymidine (3HTdR). After a prolonged chase during which much of the branching duct morphogenesis was completed, 3HTdR-label retaining epithelial cells (LREC) were detected among the epithelium of the maturing glands. Labeling newly synthesized DNA in these glands with a different marker, 5-bromodeoxyuridine (5BrdU), resulted in the appearance of doubly labeled nuclei in a large percentage of the LREC. By contrast, label-retaining cells within the stroma did not incorporate 5BrdU during the pulse, indicating that they were not traversing the cell cycle. Upon chase, the second label (5BrdU) was distributed from the double-labeled LREC to unlabeled mammary cells while 3HTdR was retained. These results demonstrate that mammary LREC selectively retain their 3HTdR-labeled template DNA strands and pass newly synthesized 5BrdU-labeled DNA to their progeny during asymmetric divisions. Similar results were obtained in mammary transplants containing self-renewing, lacZ-positive epithelial cells suggesting that cells capable of expansive self-renewal may repopulate new mammary stem cell niches during the allometric growth of new mammary ducts.     

The Olfactory Bulb in Newborn Piglet Is a Reservoir of Neural Stem and Progenitor Cells

The olfactory bulb (OB) periventricular zone is an extension of the forebrain subventricular zone (SVZ) and thus is a source of neuroprogenitor cells and neural stem cells. While considerable information is available on the SVZ-OB neural stem cell (NSC)/neuroprogenitor cell (NPC) niche in rodents, less work has been done on this system in large animals. The newborn piglet is used as a preclinical translational model of neonatal hypoxic-ischemic brain damage, but information about the endogenous sources of NSCs/NPCs in piglet is needed to implement endogenous or autologous cell-based therapies in this model. We characterized NSC/NPC niches in piglet forebrain and OB-SVZ using western blotting, histological, and cell culture methods. Immunoblotting revealed nestin, a NSC/NPC marker, in forebrain-SVZ and OB-SVZ in newborn piglet. Several progenitor or newborn neuron markers, including Dlx2, musashi, doublecortin, and polysialated neural cell adhesion molecule were also detected in OB-SVZ by immunoblotting. Immunohistochemistry confirmed the presence of nestin, musashi, and doublecortin in forebrain-SVZ and OB-SVZ. Bromodeoxyuridine (BrdU) labeling showed that the forebrain-SVZ and OB-SVZ accumulate newly replicated cells. BrdU-positive cells were immunolabeled for astroglial, oligodendroglial, and neuronal markers. A lateral migratory pathway for newly born neuron migration to primary olfactory cortex was revealed by BrdU labeling and co-labeling for doublecortin and class III β tubulin. Isolated and cultured forebrain-SVZ and OB-SVZ cells from newborn piglet had the capacity to generate numerous neurospheres. Single cell clonal analysis of neurospheres revealed the capacity for self-renewal and multipotency. Neurosphere-derived cells differentiated into neurons, astrocytes, and oligodendrocytes and were amenable to permanent genetic tagging with lentivirus encoding green fluorescent protein. We conclude that the piglet OB-SVZ is a reservoir of NSCs and NPCs suitable to use in autologous cell therapy in preclinical models of neonatal/pediatric brain injury.     

Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules

Mechanisms that regulate partitioning of the endoplasmic reticulum (ER) during cell division are largely unknown. Previous studies have mostly addressed ER partitioning in cultured cells, which may not recapitulate physiological processes that are critical in developing, intact tissues. We have addressed this by analysing ER partitioning in asymmetrically dividing stem cells, in which precise segregation of cellular components is essential for proper development and tissue architecture. We show that in Drosophila neural stem cells, called neuroblasts, the ER asymmetrically partitioned to centrosomes early in mitosis. This correlated closely with the asymmetric nucleation of astral microtubules (MTs) by centrosomes, suggesting that astral MT association may be required for ER partitioning by centrosomes. Consistent with this, the ER also associated with astral MTs in meiotic Drosophila spermatocytes and during syncytial embryonic divisions. Disruption of centrosomes in each of these cell types led to improper ER partitioning, demonstrating the critical role for centrosomes and associated astral MTs in this process. Importantly, we show that the ER also associated with astral MTs in cultured human cells, suggesting that this centrosome/astral MT-based partitioning mechanism is conserved across animal species.     

Evidence for Heterogeneity of Astrocyte De-Differentiation in vitro: Astrocytes Transform into Intermediate Precursor Cells Following Induction of ACM from Scratch-Insulted Astrocytes

Our previous study definitely demonstrated that the mature astrocytes could undergo a de-differentiation process and further transform into pluripotential neural stem cells (NSCs), which might well arise from the effect of diffusible factors released from scratch-insulted astrocytes. However, these neurospheres passaged from one neurosphere-derived from de-differentiated astrocytes possessed a completely distinct characteristic in the differentiation behavior, namely heterogeneity of differentiation. The heterogeneity in cell differentiation has become a crucial but elusive issue. In this study, we show that purified astrocytes could de-differentiate into intermediate precursor cells (IPCs) with addition of scratch-insulted astrocyte-conditioned medium (ACM) to the culture, which can express NG2 and A2B5, the IPCs markers. Apart from the number of NG2⁺ and A2B5⁺ cells, the percentage of proliferative cells as labeled with BrdU progressively increased with prolonged culture period ranging from 1 to 10 days. Meanwhile, the protein level of A2B5 in cells also increased significantly. These results revealed that not all astrocytes could de-differentiate fully into NSCs directly when induced by ACM, rather they generated intermediate or more restricted precursor cells that might undergo progressive de-differentiation to generate NSCs.     

Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro

We recently reported that neural stem cells (NSCs) become senescent and commit to astrocytic differentiation upon X-ray irradiation. Surprisingly, under self-renewing culture conditions, some of these senescent cells undergo p53-independent apoptosis, which can be suppressed by caspase inhibition and BCL2 overexpression. Inhibition of apoptosis proved beneficial for astroglial differentiation efficiency; hence the toxicity of DNA damage on NSCs was specifically tested in context of the culture conditions. In this regard, self-renewal-promoting culture conditions proved incompatible with terminal astrocyte differentiation and impacted negatively on the viability of NSCs following DNA damage-induced cell cycle exit. On the contrary, a switch to differentiation-supporting conditions ablated apoptosis and conveyed tolerance to DNA damage. Thus, stem cell death has likely not originated from DNA break toxicity, while the potentially confounding effect of stem cell niche should always be taken in consideration in stem cell irradiation experiments.     

Studies on BrdU labeling of hematopoietic cells: stem cells and cell lines

Studies using chronic in vivo BrdU exposure, isolating primitive stem cells, and determining BrdU labeling, indicate that stem cells cycle. BrdU is also incorporated into DNA during damage/repair. DNA, which has incorporated BrdU due to cycle transit is heavier than normal, while the density of DNA with damage/repair incorporation is intermediate. DNA density of purified lineage-rhodamine low (rho(low)) Hoechst low (Ho(low)) stem cells or FDC-P1 cell line cells-was assessed in vitro, after exposure to cytokines and BrdU (cycling model) or cytokines and BrdU with bleomycin to induce strand breaks and hydroxyurea to halt cycle progression (damage/repair model). We determined DNA density using cesium chloride (CsCl) gradients and either fluorometry or dot blot chemiluminesence. DNA from BrdU labeled cycling Lin-rho(lo)Ho(lo) or FDC-P1 cells was heavier than normal DNA, while damage repair DNA had an intermediate density. We then assessed BrdU labeling of Lin-rho(lo)Ho(lo) cells in vivo. We found that 70.9% of lin-rho(lo)Ho(lo) cells labeled at 5 weeks. DNA density of these cells was low, in the damage/repair range, but similar results were obtained with stem cells, which had proliferated in vivo. Dilution of BrdU in in vitro culture of proliferating FDC-P1 cells also resulted in damage/repair density. We conclude that in vitro BrdU labeling models can distinguish between proliferation and damage/repair, but that we cannot obtain high enough in vivo levels to address this issue. All together, while we cannot absolutely exclude damage/repair as contributing to stem cell BrdU labeling, the data indicate that primitive bone marrow stem cells are probably a cycling population.     

The role of DNA damage in neural stem cells ageing

Neural stem cells (NSCs) are pluripotent stem cells with the potential to differentiate into a variety of nerve cells. NSCs are susceptible to both intracellular and extracellular insults, thus causing DNA damage. Extracellular insults include ultraviolet, ionizing radiation, base analogs, modifiers, alkyl agents and others, while intracellular factors include Reactive oxygen species (ROS) radicals produced by mitochondria, mismatches that occur during DNA replication, deamination of bases, loss of bases, and more. When encountered with DNA damage, cells typically employ three coping strategies: DNA repair, damage tolerance, and apoptosis. NSCs, like many other stem cells, have the ability to divide, differentiate, and repair DNA damage to prevent mutations from being passed down to the next generation. However, when DNA damage accumulates over time, it will lead to a series of alterations in the metabolism of cells, which will cause cellular ageing. The ageing and exhaustion of neural stem cell will have serious effects on the body, such as neurodegenerative diseases. The purpose of this review is to examine the processes by which DNA damage leads to NSCs ageing and the mechanisms of DNA repair in NSCs.     

The window and mechanisms of major age-related decline in the production of new neurons within the dentate gyrus of the hippocampus

While it is well known that production of new neurons from neural stem/progenitor cells (NSC) in the dentate gyrus (DG) diminishes greatly by middle age, the phases and mechanisms of major age-related decline in DG neurogenesis are largely unknown. To address these issues, we first assessed DG neurogenesis in multiple age groups of Fischer 344 rats via quantification of doublecortin-immunopositive (DCX+) neurons and then measured the production, neuronal differentiation and initial survival of new cells in the subgranular zone (SGZ) of 4-, 12- and 24-month-old rats using four injections (one every sixth hour) of 5'-bromodeoxyuridine (BrdU), and BrdU-DCX dual immunostaining. Furthermore, we quantified the numbers of proliferating cells in the SGZ of these rats using Ki67 immunostaining. Numbers of DCX+ neurons were stable at 4-7.5 months of age but decreased progressively at 7.5-9 months (41% decline), 9-10.5 months (39% decline), and 10.5-12 months (34% decline) of age. Analyses of BrdU(+) cells at 6 h after the last BrdU injection revealed a 71-78% decline in the production of new cells per day between 4-month-old rats and 12- or 24-month-old rats. Numbers of proliferating Ki67+ cells (putative NSCs) in the SGZ also exhibited similar (72-85%) decline during this period. However, the extent of both neuronal differentiation (75-81%) and initial 12-day survival (67-74%) of newly born cells was similar in all age groups. Additional analyses of dendritic growth of 12-day-old neurons revealed that newly born neurons in the aging DG exhibit diminished dendritic growth compared with their age-matched counterparts in the young DG. Thus, major decreases in DG neurogenesis occur at 7.5-12 months of age in Fischer 344 rats. Decreased production of new cells due to proliferation of far fewer NSCs in the SGZ mainly underlies this decline.     

Impact of the interplay between stemness features,p53 and pol iota on replication pathway choices

Using human embryonic, adult and cancer stem cells/stem cell-like cells (SCs), we demonstrate that DNA replication speed differs in SCs and their differentiated counterparts. While SCs decelerate DNA replication, differentiated cells synthesize DNA faster and accumulate DNA damage. Notably, both replication phenotypes depend on p53 and polymerase iota (POLι). By exploring protein interactions and newly synthesized DNA, we show that SCs promote complex formation of p53 and POLι at replication sites. Intriguingly, in SCs the translocase ZRANB3 is recruited to POLι and required for slow-down of DNA replication. The known role of ZRANB3 in fork reversal suggests that the p53–POLι complex mediates slow but safe bypass of replication barriers in SCs. In differentiated cells, POLι localizes more transiently to sites of DNA synthesis and no longer interacts with p53 facilitating fast POLι-dependent DNA replication. In this alternative scenario, POLι associates with the p53 target p21, which antagonizes PCNA poly-ubiquitination and, thereby potentially disfavors the recruitment of translocases. Altogether, we provide evidence for diametrically opposed DNA replication phenotypes in SCs and their differentiated counterparts putting DNA replication-based strategies in the spotlight for the creation of therapeutic opportunities targeting SCs.     

Genome-wide screen for differential DNA methylation associated with neural cell differentiation in mouse

Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in in-vitro induced neural stem cells (NSCs) and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score>1.96) enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.     

De-differentiation Response of Cultured Astrocytes to Injury Induced by Scratch or Conditioned Culture Medium of Scratch-Insulted Astrocytes

Our previous reports indicated that astrocytes (ASTs) in injured adult rat spinal cord underwent a process of de-differentiation, and may acquire the potential of neural stem cells (NSCs). However, the AST de-differentiation and transitional rejuvenation process following injury is still largely unclear. The aim of the present study was to determine whether injured in vitro ASTs can re-enter the multipotential-like stem cell pool and regain NSC characteristics, and to further understand the mechanism of AST de-differentiation. We used an in vitro scratch-wound model to evoke astrocytic response to mechanical injury. GFAP and nestin double-labeled indirect immunofluorescence were carried out to characterize these scratched cells at various periods. Western-blot analysis was used to determine the changes of GFAP and nestin expression following injury. Furthermore, the rate of proliferation was determined by immunocytochemical detection of BrdU incorporating cells. These scratch-wound ASTs were cultured with stem cells medium to explore their ability to generate neurospheres and examine the self-renewal and multi-potency of such neurospheres. Moreover, scratched AST culture supernatant as conditioned cultured medium (ACM) was used to investigate if some diffusible factors derived from injured ASTs could induce de-differentiation of AST. The results showed: (1) the nestin positivity first appeared in GFAP-positive cells at the edge of the scratch, subsequently, disseminated into un-insulted zone. The expression of nestin in AST was increased with longer culture, while that of GFAP was decreased. Furthermore, these nestin-immunoreactive ASTs could generate neurospheres, which showed self-renewal and could be differentiated into neurons, ASTs and oligodendrocytes. (2) Scratched ASTs culture supernatant can induce astrocytic proliferation and de-differentiation. These results reveal that the in vitro injured ASTs can de-differentiate into nestin-positive stem/precursor cells, the process of de-differentiation may arise from direct injury or some diffusible factors released from injured ASTs.     

Effects of Oxygen Concentration on the Proliferation and Differentiation of Mouse Neural Stem Cells In Vitro

Background and purpose Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro. Methods NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively. Results The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions. Conclusions These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation.     

Properties of a fetal multipotent neural stem cell (NEP cell)

Multipotent neural stem cells (NSCs) present in the developing neural tube (E10.5, neuroepithelial cells; NEP) were examined for the expression of candidate stem cell markers, and the expression of these markers was compared with later appearing precursor cells (E14.5) that can be distinguished by the expression of embryonic neural cell adhesion molecule (E-NCAM) and A2B5. NEP cells possess gap junctions, express connexins, and appear to lack long cilia. Most candidate markers, including Nestin, Presenilin, Notch, and Numb, were expressed by both NEP cells as well as other cell populations. Fibroblast growth factor receptor 4 (FGFR4), Frizzled 9 (Fz9), and SRY box-containing gene 2 (Sox2) as assessed by immunocytochemistry and in situ hybridization are markers that appear to distinguish NSCs from other precursor cells. Neither Hoechst 33342 nor rhodamine-123 staining, telomerase (Tert) expression, telomerase activity, or breakpoint cluster region protein 1 (Bcrp1) transporter expression could be used to distinguish NEP stem cells from other dividing cells. NEP cells, however, lacked expression of several lineage markers that are expressed by later appearing cells. These included absence of expression of CD44, E-NCAM, A2B5, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor-alpha (PDGFR alpha), suggesting that negative selection using cell surface epitopes could be used to isolate stem cell populations from mixed cultures of cells. Using mixed cultures of cells isolated from E14.5 stage embryos, we show that NEP cells can be enriched by depleting differentiating cells that express E-NCAM or A2B5 immunoreactivity. Overall, our results show that a spectrum of markers used in combination can reliably distinguish multipotent NSCs from other precursor cells as well as differentiated cells present in the CNS.     

HMGB1 enhances embryonic neural stem cell proliferation by activating the MAPK signaling pathway

Neural stem cells (NSCs) are involved in neural tube formation. As the high-mobility group box 1 (HMGB1) protein is involved in neurulation and is present at elevated levels in neural tube defects (NTDs) induced by hyperthermia, we have now investigated the effects of HMGB1 on proliferation, differentiation, and MAPK signaling pathways of NSCs in vitro. We constructed a lentivirus vector with HMGB1 siRNA and used it to infect NSCs. Down-regulation of HMGB1 expression was confirmed. Proliferation of NSCs was determined by MTS and nestin/BrdU double-labeling. Differentiation of NSCs was assessed using β-tubulinIII and GFAP. Knockdown of HMGB1 significantly suppressed NSC proliferation but hardly affected differentiation, which was regulated by decreased expression of MAPK signaling pathways. Thus, HMGB1 has beneficial effects on neurulation and may serve as a new target for the prevention of NTDs.     

Visualization of DNA in pachytene by monoclonal antibodies against BrdU reveals synaptonemal complex-like structures.

Most of the techniques used to visualize structures of the synaptonemal complex (SC) are based on specific staining properties or immunocytochemical detection of proteinaceous SC components. The SC is therefore considered to be mostly protein. We have now accomplished visualization of the DNA within the SC by use of the BrdU antibody technique, following BrdU substitution during the last premeiotic S phase. Preparations of mouse meiotic chromosomes were obtained by spreading on a water surface. The DNA content in the SCs, which appeared as light threads, was clearly lower than the DNA content in the surrounding chromatin. At higher magnification, dark, longitudinal structures appeared in these threads. These structures are made up of DNA, which forms the inner part of the lateral elements of the SCs. Within the SCs, DNA is confined mostly to these threads. Thus, DNA staining reveals the same structures already known from protein staining of SCs. The DNA mass surrounding the SCs is often nonhomogeneously distributed along the chromosome axis. The more dense parts appear to be chromomeres. The DNA staining technique described in this paper may therefore be a useful complement to standard protein staining techniques for pachytene chromosomes.     

The fate of parental nucleosomes during SV40 DNA replication.

The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.