Radiation-Induced Senescence in p16+/LUC Mouse Lung Compared to Bone Marrow Multilineage Hematopoietic Progenitor Cells.

We defined the time course of ionizing radiation-induced senescence in lung compared to bone marrow of p16+/LUC mice in which the senescence-induced biomarker (p16) is linked to a luciferase reporter gene. Periodic in situ imaging revealed increased luciferase activity in the lungs of 20 Gy thoracic irradiated, but not 8 Gy total-body irradiated (TBI) mice beginning at day 75 and increasing to day 170. In serial sections of explanted lungs, senescent cells appeared in the same areas as did fibrosis in the 20 Gy thoracic irradiated, but not the 8 Gy TBI group. Lungs from 8 Gy TBI mice at one year did show increased RNA levels for p16, p21, p19 and TGF-ß. Individual senescent cells in 20 Gy irradiated mouse lung included those with epithelial, endothelial, fibroblast and hematopoietic cell biomarkers. Rare senescent cells in the lungs of 8 Gy TBI mice at one year were of endothelial phenotype. Long-term bone marrow cultures (LTBMCs) were established at either day 60 or one year after 8 Gy TBI. In freshly removed marrow at both times after irradiation, there were increased senescent cells. In LTBMCs, there were increased senescent cells in both weekly harvested single cells and in colonies of multilineage hematopoietic progenitor cells producing CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, mega-karyocyte) that were formed in secondary cultures when these single cells were plated in semisolid media. LTBMCs from TBI mice produced fewer CFU-GEMM; however, the relative percentage of senescent cell-containing colonies was increased as measured by both p16-luciferase and ß-galactosidase. Therefore, 20 Gy thoracic radiation, as well as 8 Gy TBI, induces senescent cells in the lungs. With bone marrow, 8 Gy TBI induced senescence in both hematopoietic cells and in colony-forming progenitors. The p16+/LUC mouse strain provides a valuable system in which to compare the kinetics of radiation-induced senescence between organs in vivo, and to evaluate the potential role of senescent cells in irradiation pulmonary fibrosis.

Quantifying Senescence-Associated Phenotypes in Primary Multipotent Mesenchymal Stromal Cell Cultures.

Cellular senescence is a tumor suppressor mechanism that removes potentially neoplastic cells from the proliferative pool. Senescent cells naturally accumulate with advancing age; however, excessive/aberrant accumulation of senescent cells can disrupt normal tissue function. Multipotent mesenchymal stromal cells (MSCs), which are actively evaluated as cell-based therapy, can undergo replicative senescence or stress-induced premature senescence. The molecular characterization of MSCs senescence can be useful not only for understanding the clinical correlations between MSCs biology and human age or age-related diseases but also for identifying competent MSCs for therapeutic applications. Because MSCs are involved in regulating the hematopoietic stem cell niche, and MSCs dysfunction has been implicated in age-related diseases, the identification and selective removal of senescent MSC may represent a potential therapeutic target. Cellular senescence is generally defined by senescence-associated (SA) permanent proliferation arrest (SAPA) accompanied by persistent DNA damage response (DDR) signaling emanating from persistent DNA lesions including damaged telomeres. Alongside SA cell cycle arrest and DDR signaling, a plethora of phenotypic hallmarks help define the overall senescent phenotype including a potent SA secretory phenotype (SASP) with many microenvironmental functions. Due to the complexity of the senescence phenotype, no single hallmark is alone capable of identifying senescent MSCs. This protocol highlights strategies to validate MSCs senescence through the measurements of several key SA hallmarks including lysosomal SA Beta-galactosidase activity (SA-ßgal), cell cycle arrest, persistent DDR signaling, and the inflammatory SASP.

Long-Term Engraftment of Primary Bone Marrow Stromal Cells Repairs Niche Damage and Improves Hematopoietic Stem Cell Transplantation.

Hematopoietic stem cell (HSC) transplantation represents a curative treatment for various hematological disorders. However, delayed reconstitution of innate and adaptive immunity often causes fatal complications. HSC maintenance and lineage differentiation are supported by stromal niches, and we now find that bone marrow stroma cells (BMSCs) are severely and permanently damaged by the pre-conditioning irradiation required for efficient HSC transplantation. Using mouse models, we show that stromal insufficiency limits the number of donor-derived HSCs and B lymphopoiesis. Intra-bone transplantation of primary, but not cultured, BMSCs quantitatively reconstitutes stroma function in vivo, which is mediated by a multipotent NT5E+ (CD73)+ ENG- (CD105)- LY6A+ (SCA1)+ BMSC subpopulation. BMSC co-transplantation doubles the number of functional, donor-derived HSCs and significantly reduces clinically relevant side effects associated with HSC transplantation including neutropenia and humoral immunodeficiency. These data demonstrate the potential of stroma recovery to improve HSC transplantation.

Contrasting roles for C/EBPα and Notch in irradiation-induced multipotent hematopoietic progenitor cell defects.

Ionizing radiation (IR) is associated with reduced hematopoietic function and increased risk of hematopoietic malignancies, although the mechanisms behind these relationships remain poorly understood. Both effects of IR have been commonly attributed to the direct induction of DNA mutations, but evidence supporting these hypotheses is largely lacking. Here we demonstrate that IR causes long-term, somatically heritable, cell-intrinsic reductions in hematopoietic stem cell (HSC) and multipotent hematopoietic progenitor cell (mHPC) self-renewal that are mediated by C/EBPα and reversed by Notch. mHPC from previously irradiated (>9 weeks prior), homeostatically restored mice exhibit gene expression profiles consistent with their precocious differentiation phenotype, including decreased expression of HSC-specific genes and increased expression of myeloid program genes (including C/EBPα). These gene expression changes are reversed by ligand-mediated activation of Notch. Loss of C/EBPα expression is selected for within previously irradiated HSC and mHPC pools and is associated with reversal of IR-dependent precocious differentiation and restoration of self-renewal. Remarkably, restoration of mHPC self-renewal by ligand-mediated activation of Notch prevents selection for C/EBPα loss of function in previously irradiated mHPC pools. We propose that environmental insults prompt HSC to initiate a program limiting their self-renewal, leading to loss of the damaged HSC from the pool while allowing this HSC to temporarily contribute to differentiated cell pools. This "programmed mediocrity" is advantageous for the sporadic genotoxic insults animals have evolved to deal with but becomes tumor promoting when the entire HSC compartment is damaged, such as during total body irradiation, by increasing selective pressure for adaptive oncogenic mutations.

Differentiation of human adipose-derived stem cells into neuron-like cells which are compatible with photocurable three-dimensional scaffolds.

Multipotent human adipose-derived stromal/stem cells (hADSCs) hold a great promise for cell-based therapy for many devastating human diseases, such as spinal cord injury and stroke. If exogenous hADSCs can be cultured in a three-dimensional (3D) scaffold with effective proliferation and differentiation capacity, it will better mimic the in vivo environment, which will have profound impact on the therapeutic application of hADSCs. In this study, a group of elastic-dominant, porous bioscaffolds from photocurable chitosan and gelatin were fabricated and proven to be biocompatible with both hADSCs and hADSC-derived neuron-like cells (hADSC-NLCs) in vitro. The identity of harvested hADSCs was confirmed by their positive immunostaining of mesenchymal stem cell surface markers, CD29, CD44, and CD105, and also positive expression of stem markers, Sox-2, Oct-4, c-Myc, Nanog, and Klf4. Their multipotency was further confirmed by trilineage differentiation of hADSCs toward adipocyte, osteoblast, and chondrocyte. It was found that hADSCs could be conditioned to differentiate into neurons in vitro as determined by immunostaining the markers of Tuj1, MAP2, NeuN, and Synapsin. The hADSCs and hADSC-NLCs were proven to be biocompatible with 3D scaffold, which actually facilitated the proliferation and differentiation of hADSCs in vitro, by MTT assay and their neuronal gene expression profiling. Moreover, hADSC-NLCs, which were mixed with 3D scaffold and transplanted into traumatic brain injury mouse model, survived in vivo and led to the better repair of the damaged brain area. The immunohistochemical studies revealed that 3D scaffold indeed improved the viability of transplanted cells, their ability to incorporate into the in vivo neural circuit, and their capacity for tissue repair. This study indicates that hADSCs would have great therapeutic application potential as seeding cells for in vivo transplantation to treat various neurological diseases when co-applied with porous chitosan/gelatin bioscaffolds.

Basic fibroblast growth factor protects C17.2 cells from radiation-induced injury through ERK1/2.

AIMS: To establish a radiation-induced neural injury model using C17.2 neural stem cells (NSCs) and to investigate whether basic fibroblast growth factor (bFGF) can protect the radiation-induced injury of C17.2 NSCs. Furthermore, we aim to identify the possible mechanisms involved in this model. METHODS: C17.2 NSCs received a single exposure (3, 6, and 9 Gy, respectively) at a dose rate of 300 cGy/min with a control group receiving 0 Gy. Different concentrations of bFGF were added for 24 h, 5 min postirradiation. The MTS assay and flow cytometry were used to detect cytotoxicity and apoptosis. Expression of FGFR1, ERK1/2, and p-ERK1/2 proteins was detected with or without U0126 was pretreated prior to C17.2 NSCs receiving irradiation. RESULTS: C17.2 NSCs showed a dose-dependent cell death as the dose of radiation was increased. Additionally, the rate of apoptosis in the C17.2 NSCs reached 31.2 ± 1.23% in the 6 Gy irradiation group, which was the most significant when compared to the other irradiation treated groups. bFGF showed protective effect on cell apoptosis in a dose-dependent manner. The mean percentage of apoptotic cells decreased to 7.83 ± 1.75% when 100 ng/mL bFGF was given. Furthermore, U0126 could block the protective effect of bFGF by inhibiting the phosphorylation of ERK1/2. CONCLUSIONS: An in vitro cellular model of radiation-induced apoptosis of NSCs, in C17.2 NSCs, was developed successfully. Additionally, bFGF can protect neurons from radiation injury in vitro via the ERK1/2 signal transduction pathway.

Bmi1 confers resistance to oxidative stress on hematopoietic stem cells.

BACKGROUND: The polycomb-group (PcG) proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC) 1, maintained self-renewing hematopoietic stem cells (HSCs) during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26Stop(FL)Bmi1). Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26Stop(FL)Bmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS). CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it.

Differential effect of ionizing radiation exposure on multipotent and differentiation-restricted bone marrow mesenchymal stem cells.

Debilitating effects of bone marrow from ionizing radiation exposure has been well established for hematopoietic stem cells; however, radiation toxicity of mesenchymal stem cells (MSCs) has been controversial. The present study addressed if ionizing radiation exposure differently affected bone marrow MSCs with various differentiation commitments. Mouse bone-marrow-derived MSCs, D1 cells of early passages (≤ 5 passages; p5) maintained the complete characteristics of multipotent MSCs, whereas, after ≥ 45 passages (p45) the differentiation capability of D1 cells became partially restricted. Both p5 and p45 D1 cells were subjected to single dose irradiation by radioactive isotope (137)Cs. Radiation treatment impaired cell renewal and differentiation activities of p5 D1 cells; however, p45 D1 cells were less affected. Radiation treatment upregulated both pro- and anti-apoptotic genes of p5 D1 cells in a dose-dependent manner, potentially resulting in the various apoptosis thresholds. It was found that constitutive as well as radiation-induced phosphorylation levels of histone H2AX was significantly higher in p45 D1 cells than in p5 D1 cells. The increased repair activity of DNA double-strand breakage may play a role for p45 D1 cells to exhibit the relative radioresistance. In conclusion, the radiation toxicity predominantly affecting multipotent MSCs may occur at unexpectedly low doses, which may, in part, contribute to the catabolic pathology of bone tissue.

Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow.

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and can give rise to multiple mesenchymal lineages. Because MSCs have only been isolated from tissue in culture, the equivalent cells have not been identified in vivo and little is known about their physiological roles or even their exact tissue location. In this study, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of MSCs (PDGFRalpha+Sca-1+CD45-TER119-) from adult mouse bone marrow. Individual MSCs generated colonies at a high frequency and could differentiate into hematopoietic niche cells, osteoblasts, and adipocytes after in vivo transplantation. Naive MSCs resided in the perivascular region in a quiescent state. This study provides the useful method needed to identify MSCs as defined in vivo entities.

Time-dependent neurosphere-forming ability of adult rat spinal cord after irradiation.

To determine whether there was evidence for long-term time-dependent changes in neurosphere-forming ability of rat spinal cord after irradiation, a 15-mm length of spinal cord (C2-T2) of 10-week-old female rats was irradiated with a single dose of 2, 5, 10 or 19 Gy. Cells were isolated from the central 10-mm segment of the irradiated spinal cord immediately or at 0.5, 1, 2 or 5 months to form neurospheres. The number and sizes of neurospheres were determined at day 10, 12, 14 and 16 in vitro. The multipotential properties of neurosphere cells were assessed by immunocytochemistry using lineage-specific markers for neurons and glia. In nonirradiated controls, the number and size of the neurospheres decreased with increasing age of the animals. Regardless of the time after irradiation, there was a dose-dependent decrease in the number and size of neurospheres obtained from the irradiated cord compared to age-matched controls. Using three-way ANOVA, the number of neurospheres was dependent on radiation dose (P < 0.0001), time after irradiation (P < 0.0001), and day of counting in vitro (P < 0.0001). Compared to cells cultured immediately after irradiation, there was an increase in the relative plating efficiency of neurospheres cultured 1 month after irradiation. However, no further increase was apparent up to 5 months after irradiation. The multipotential properties of neurosphere cells in vitro remained unchanged with increasing time after irradiation. These results may suggest a time-dependent recovery of radiation damage using neurosphere-forming ability as the end point and agree with data that show time-dependent recovery of radiation damage in spinal cord using histological or functional end points.

Survival of transplanted neural progenitor cells enhanced by brain irradiation.

OBJECT: Authors of previous studies have reported that adult transplanted neural progenitor cells (NPCs) are suitable for brain cell replacement or gene delivery. In this study, the authors evaluated survival and integration of adult rat-derived NPCs after transplantation and explored the potential impact on transplant survival of various mechanical and biological factors of clinical importance. METHODS: Adult female Fischer 344 rats were used both as a source and recipient of transplanted NPCs. Both 9L and RG2 rat glioma cells were used to generate in vivo brain tumor models. On the 5th day after tumor implantation, NPCs expressing green fluorescent protein (GFP) were administered either intravenously (3.5 x 10(7) cells) or by stereotactic injection (1 x 10(4)-1 x 10(6) cells) into normal or tumor-bearing brain. The authors evaluated the effect of delivery method (sharp compared with blunt needles, normal compared with zero-volume needles, phosphate-buffered saline compared with medium as vehicle), delivery sites (intravenous compared with intratumoral compared with intraparenchymal), and pretreatment with an immunosuppressive agent (cyclosporin) or brain irradiation (20-40 Gy) on survival and integration of transplanted NPCs. RESULTS: Very few cells survived when less than 10(5) cells were transplanted. When 10(5) cells or more were transplanted, only previously administered brain irradiation significantly affected survival and integration of NPCs. Although GFP-containing NPCs could be readily detected 1 day after injection, few cells survived 4 days to 1 week unless preceded by whole-brain radiation (20 or 40 Gy in a single fraction), which increased the number of GFP-containing NPCs within the tissue more than fivefold. CONCLUSIONS: The authors' findings indicate that most NPCs, including those from a syngeneic autologous source, do not survive at the site of implantation, but that brain irradiation can facilitate subsequent survival in both normal and tumor-bearing brain. An understanding of the mechanisms of this effect could lead to improved survival and clinical utility of transplanted NPCs.

Altered growth and radiosensitivity in neural precursor cells subjected to oxidative stress.

PURPOSE: To determine whether changes in oxidative stress could enhance the sensitivity of neural precursor cells to ionizing radiation. MATERIALS AND METHODS: Two strategies were used whereby oxidative stress was modulated endogenously, through manipulation cell culture density, or exogenously, through direct addition of hydrogen peroxide. RESULTS: Cells subjected to increased endogenous oxidative stress through low-density growth routinely exhibited an inhibition of growth following irradiation. However, cells subjected to chronic exogenous oxidative treatments showed increased sensitivity to proton and gamma-irradiation compared to untreated controls. Reduced survival of irradiated cultures subjected to oxidizing conditions was corroborated using enzymatic viability assays, and was observed over a range of doses (1 - 5 Gy) and post-irradiation re-seeding densities (20 - 200 K/plate). CONCLUSIONS: Collectively our results provide further support for the importance of redox state in the regulation of neural precursor cell function, and suggest that oxidative stress can inhibit the proliferative potential of cells through different mechanisms. This is likely to compromise survival and under conditions where excess exogenous oxidants might predominate, sensitivity to irradiation may be enhanced.

Selective irradiation of the vascular endothelium has no effect on the survival of murine intestinal crypt stem cells.

The possible role of vascular endothelial cell damage in the loss of intestinal crypt stem cells and the subsequent development of the gastrointestinal (GI) syndrome is addressed. Mice received whole-body epithermal neutron irradiation at a dose rate of 0.57 +/- 0.04 Gy x min(-1). An additional dose was selectively targeted to endothelial cells from the short-ranged (5-9 microm) particles released from neutron capture reactions in 10B confined to the blood by incorporation into liposomes 70-90 nm in diameter. Different liposome formulations produced 45 +/- 7 or 118 +/- 12 microg/g 10B in the blood at the time of neutron irradiation, which resulted in total absorbed dose rates in the endothelial cells of 1.08 +/- 0.09 or 1.90 +/- 0.16 Gy x min(-1), respectively. At 3.5 d after irradiation, the intestinal crypt microcolony assay showed that the 2- to 3-fold increased doses to the microvasculature, relative to the nonspecific whole-body neutron beam doses, caused no additional crypt stem cell loss beyond that produced by the neutron beam alone. The threshold dose for death from the GI syndrome after neutron-beam-only irradiation was 9.0 +/- 0.6 Gy. There were no deaths from the GI syndrome, despite calculated absorbed doses to endothelial cells as high as 27.7 Gy, in the groups that received neutron beam doses of <9.0 Gy with boronated liposomes in the blood. These data indicate that endothelial cell damage is not causative in the loss of intestinal crypt stem cells and the eventual development of the GI syndrome.

Loss of p53 induces changes in the behavior of subventricular zone cells: implication for the genesis of glial tumors.

The role of multipotential progenitors and neural stem cells in the adult subventricular zone (SVZ) as cell-of-origin of glioblastoma has been suggested by studies on human tumors and transgenic mice. However, it is still unknown whether glial tumors are generated by all of the heterogeneous SVZ cell types or only by specific subpopulations of cells. It has been proposed that transformation could result from lack of apoptosis and increased self-renewal, but the definition of the properties leading to neoplastic transformation of SVZ cells are still elusive. This study addresses these questions in mice carrying the deletion of p53, a tumor-suppressor gene expressed in the SVZ. We show here that, although loss of p53 by itself is not sufficient for tumor formation, it provides a proliferative advantage to the slow- and fast-proliferating subventricular zone (SVZ) populations associated with their rapid differentiation. This results in areas of increased cell density that are distributed along the walls of the lateral ventricles and often associated with increased p53-independent apoptosis. Transformation occurs when loss of p53 is associated with a mutagenic stimulus and is characterized by dramatic changes in the properties of the quiescent adult SVZ cells, including enhanced self-renewal, recruitment to the fast-proliferating compartment, and impaired differentiation. Together, these findings provide a cellular mechanism for how the slow-proliferating SVZ cells can give rise to glial tumors in the adult brain.

Ionizing radiation enhances the engraftment of transplanted in vitro-derived multipotent astrocytic stem cells.

The subependymal zone (SEZ) is a region of persistent neurogenesis in the adult mammalian brain containing a neural stem cell (NSC) pool that continuously generates migratory neuroblasts that travel in chains through the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate and functionally integrate into existing neural circuitry. NSCs can be isolated from the SEZ and cultured to generate either neurospheres (NSs) or multipotent astrocytic stem cells (MASCs), with both possessing the stem cell characteristics of multipotency and self-renewal. NSs and MASCs home to the SEZ after transplantation into the lateral ventricle (LV) and contribute to neuroblast migration, with minimal engraftment into the OB observed in the adult mouse. Recent studies have compared the relatively uncharacterized NSC with the more established hematopoietic stem cell (HSC) in an effort to determine the level of stemness possessed by the NSC. Depletion of native HSCs in the bone marrow by lethal irradiation (LI) is necessary to maximize functional engraftment of donor HSCs. Our data show that the NSC pool and neuroblasts in the SEZ can be significantly and permanently depleted by exposure to LI. Attenuation of donor-derived migratory neuroblast engraftment into the OB is observed after transplantation of gfp+ MASCs into the LV of LI animals, whereas engraftment is significantly enhanced after transplantation into animals exposed to sublethal levels of ionizing radiation. By increasing receptiveness of the NSC niche through depletion of indigenous cells, the adult SEZ-RMS-OB can be used as a model to further characterize the NSC.

Enhanced recovery of radiation-induced translocations from spermatogonial stem cells of p53 null mice.

X-ray-induced (4Gy) chromosomal translocations were studied in mouse spermatogonial stem cells with different p53 status by meiotic analysis at the spermatocyte stage, many cell generations after the moment irradiation. The results show enhanced recovery of translocations from p53 -/- mice relative to +/- and +/+ littermates. The enhanced recovery is probably due to an altered cell cycle distribution of the stem cells in the -/- mice leading to less radioresistant G(0)-G(1) transition cells, rather than differences in apoptotic response. Experiments with the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide (3-AB) indicate that, in contrast to the situation in +/+ mice, no sensitization in the p53-deficient mice occurred for both testis weight loss and the recovery of induced translocations. This result also points to the presence of less radioresistant stem cells in the testis of p53 null mice.