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1.  Fancd2 and p21 function independently in maintaining the size of hematopoietic stem and progenitor cell pool in mice 
Stem cell research  2013;11(2):687-692.
Fanconi anemia patients suffer from progressive bone marrow failure. An overactive p53 response to DNA damage contributes to the progressive elimination of Fanconi anemia hematopoietic stem and progenitor cells (HSPC), and hence presents a potential target for therapeutic intervention. To investigate whether the cell cycle regulatory protein p21 is the primary mediator of the p53-dependent stem cell loss, p21/Fancd2 double-knockout mice were generated. Surprisingly double mutant mice displayed even more severe loss of HSPCs than Fancd2−/− single mutants. p21 deletion did not rescue the abnormal cell cycle profile and had no impact on the long-term repopulating potential of Fancd2−/− bone marrow cells. Collectively, our data indicate that p21 has an indispensable role in maintaining a normal HSPC pool and suggest that other p53-targeted factors, not p21, mediate the progressive elimination of HSPC in Fanconi anemia.
doi:10.1016/j.scr.2013.04.010
PMCID: PMC3737285  PMID: 23721813
Bone Marrow Failure; Fanconi Anemia; Hematopoietic Stem Cell; p21
2.  Oxymetholone Therapy of Fanconi Anemia Suppresses Osteopontin Transcription and Induces Hematopoietic Stem Cell Cycling 
Stem Cell Reports  2014;4(1):90-102.
Summary
Androgens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2−/− mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2−/− mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure.
Graphical Abstract
Highlights
•OXM treatment causes loss of quiescence and stem cell exhaustion•RNaseq analysis reveals transcriptional changes in key pathways in Fancd2−/− HSPC•OXM suppresses transcription of osteopontin, a known stem cell quiescence promoter•Unlike previous notions, OXM has no effect on telomerase expression or EPO signaling
In this article, Zhang and colleagues show that 18-month-old Fancd2−/− mice recapitulated key human Fanconi anemia phenotypes, including peripheral pancytopenia and macrocytosis. Chronic oxymetholone treatment improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells, but eventually resulted in stem cell exhaustion. RNaseq analysis implicated downregulation of osteopontin as an important mechanism for the drug’s action.
doi:10.1016/j.stemcr.2014.10.014
PMCID: PMC4297866  PMID: 25434823
3.  Bone marrow failure in Fanconi anemia is triggered by an exacerbated p53/p21 DNA damage response that impairs hematopoietic stem and progenitor cells 
Cell Stem Cell  2012;11(1):36-49.
SUMMARY
Fanconi anemia (FA) is an inherited DNA repair deficiency syndrome. FA patients undergo progressive bone marrow failure (BMF) during childhood, which frequently requires allogeneic hematopoietic stem cell transplantation. The pathogenesis of this BMF has been elusive to date. Here we found that FA patients exhibit a profound defect in hematopoietic stem and progenitor cells (HSPCs) that is present before the onset of clinical BMF. In response to replicative stress and unresolved DNA damage, p53 is hyperactivated in FA cells and triggers a late p21Cdkn1a-dependent G0/G1 cell-cycle arrest. Knockdown of p53 rescued the HSPC defects observed in several in vitro and in vivo models, including human FA or FA-like cells. Taken together, our results identify an exacerbated p53/p21 “physiological” response to cellular stress and DNA damage accumulation as a central mechanism for progressive HSPC elimination in FA patients, and have implications for clinical care.
doi:10.1016/j.stem.2012.05.013
PMCID: PMC3392433  PMID: 22683204
4.  Non-Invasive Stem Cell Therapy in a Rat Model for Retinal Degeneration and Vascular Pathology 
PLoS ONE  2010;5(2):e9200.
Background
Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP.
Methodology/Principal Findings
Animals received syngeneic MSCs (1×106 cells) by tail vein at an age before major photoreceptor loss. Principal results: both rod and cone photoreceptors were preserved (5–6 cells thick) at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs.
Conclusions/Significance
These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal degeneration and other ocular vascular diseases.
doi:10.1371/journal.pone.0009200
PMCID: PMC2821411  PMID: 20169166
5.  Identification of Synaptic Targets of Drosophila Pumilio 
PLoS Computational Biology  2008;4(2):e1000026.
Drosophila Pumilio (Pum) protein is a translational regulator involved in embryonic patterning and germline development. Recent findings demonstrate that Pum also plays an important role in the nervous system, both at the neuromuscular junction (NMJ) and in long-term memory formation. In neurons, Pum appears to play a role in homeostatic control of excitability via down regulation of para, a voltage gated sodium channel, and may more generally modulate local protein synthesis in neurons via translational repression of eIF-4E. Aside from these, the biologically relevant targets of Pum in the nervous system remain largely unknown. We hypothesized that Pum might play a role in regulating the local translation underlying synapse-specific modifications during memory formation. To identify relevant translational targets, we used an informatics approach to predict Pum targets among mRNAs whose products have synaptic localization. We then used both in vitro binding and two in vivo assays to functionally confirm the fidelity of this informatics screening method. We find that Pum strongly and specifically binds to RNA sequences in the 3′UTR of four of the predicted target genes, demonstrating the validity of our method. We then demonstrate that one of these predicted target sequences, in the 3′UTR of discs large (dlg1), the Drosophila PSD95 ortholog, can functionally substitute for a canonical NRE (Nanos response element) in vivo in a heterologous functional assay. Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB), which is an anatomical site of memory storage.
Author Summary
The Drosophila Pumilio (Pum) protein was originally identified as a translational control factor for embryo patterning. Subsequent studies have identified Pum's role in multiple biological processes, including the maintenance of germline stem cell, the proliferation and migration of primordial germ cells, olfactory leaning and memory, and synaptic plasticity. Pum is highly conserved across phyla, i.e., from worm to human; however, the mRNA targets of Pum within each tissue and organism are largely unknown. On the other hand, the prediction of RNA binding sites remains a hard question in the computational field. We were interested in finding Pum targets in the nervous system using fruit flies as a model organism. To accomplish this, we used the few Pum binding sequences that had previously been shown in vivo as “training sequences” to construct bioinformatic models of the Pum binding site. We then predicted a few Pum mRNA targets among the genes known to function in neuronal synapses. We then used a combination of “golden standards” to verify these predictions: a biochemical assay called gel shifts, and in vivo functional assays both in embryo and neurons. With these approaches, we successfully confirmed one of the targets as Dlg, which is the Drosophila ortholog of human PSD95. Therefore, we present a complete story from computational study to real biological functions.
doi:10.1371/journal.pcbi.1000026
PMCID: PMC2265480  PMID: 18463699

Results 1-5 (5)