Xinxin Huang

SIRT1 Positively Regulates Autophagy and Mitochondria Function in Embryonic Stem Cells Under Oxidative Stress

SIRT1, an NAD‐dependent deacetylase, plays a role in regulation of autophagy. SIRT1 increases mitochondrial function and reduces oxidative stress, and has been linked to age‐related reactive oxygen species (ROS) generation, which is highly dependent on mitochondrial metabolism. H2O2 induces oxidative stress and autophagic cell death through interference with Beclin 1 and the mTOR signaling pathways. We evaluated connections between SIRT1 activity and induction of autophagy in murine (m) and human (h) embryonic stem cells (ESCs) upon ROS challenge. Exogenous H2O2 (1 mM) induced apoptosis and autophagy in wild‐type (WT) and Sirt1−/− mESCs. High concentrations of H2O2 (1 mM) induced more apoptosis in Sirt1−/−, than in WT mESCs. However, addition of 3‐methyladenine, a widely used autophagy inhibitor, in combination with H2O2 induced more cell death in WT than in Sirt1−/− mESCs. Decreased induction of autophagy in Sirt1−/− mESCs was demonstrated by decreased conversion of LC3‐I to LC3‐II, lowered expression of Beclin‐1, and decreased LC3 punctae and LysoTracker staining. H2O2 induced autophagy with loss of mitochondrial membrane potential and disruption of mitochondrial dynamics in Sirt1−/− mESCs. Increased phosphorylation of P70/85‐S6 kinase and ribosomal S6 was noted in Sirt1−/− mESCs, suggesting that SIRT1 regulates the mTOR pathway. Consistent with effects in mESCs, inhibition of SIRT1 using Lentivirus‐mediated SIRT1 shRNA in hESCs demonstrated that knockdown of SIRT1 decreased H2O2‐induced autophagy. This suggests a role for SIRT1 in regulating autophagy and mitochondria function in ESCs upon oxidative stress, effects mediated at least in part by the class III PI3K/Beclin 1 and mTOR pathways. Stem Cells 2014;32:1183–1194

Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

The importance of hypoxia and extra physiologic oxygen shock/stress for collection and processing of stem and progenitor cells to understand true physiology/pathology of these cells ex vivo.

Purpose of reviewHematopoietic stem (HSCs) and progenitor (HPCs) cells reside in a hypoxic (lowered oxygen tension) environment, in vivo. We review literature on growth of HSCs and HPCs under hypoxic and normoxic (ambient air) conditions with a focus on our recent work demonstrating the detrimental effects of collecting and processing cells in ambient air through a phenomenon termed extra physiologic oxygen shock/stress (EPHOSS), and we describe means to counteract EPHOSS for enhanced collection of HSCs. Recent findingsCollection and processing of bone marrow and cord blood cells in ambient air cause rapid differentiation and loss of HSCs, with increases in HPCs. This apparently irreversible EPHOSS phenomenon results from increased mitochondrial reactive oxygen species, mediated by a p53-cyclophilin D–mitochondrial permeability transition pore axis, and involves hypoxia inducing factor-1&agr; and micro-RNA 210. EPHOSS can be mitigated by collecting and processing cells in lowered (3%) oxygen, or in ambient air in the presence of, cyclosporine A which effects the mitochondrial permeability transition pore, resulting in increased HSC collections. SummaryOur recent findings may be advantageous for HSC collection for hematopoietic cell transplantation, and likely for enhanced collection of other stem cell types. EPHOSS should be considered when ex-vivo cell analysis is utilized for personalized medicine, as metabolism of cells and their response to targeted drug treatment ex vivo may not mimic what occurs in vivo.

Glucocorticoid hormone-induced chromatin remodeling enhances human hematopoietic stem cell homing and engraftment

Efficient hematopoietic stem cell (HSC) homing is important for hematopoietic cell transplantation (HCT), especially when HSC numbers are limited, as in the use of cord blood (CB). In a screen of small-molecule compounds, we identified glucocorticoid (GC) hormone signaling as an activator of CXCR4 expression in human CB HSCs and hematopoietic progenitor cells (HPCs). Short-term GC pretreatment of human CB HSCs and HPCs promoted SDF-1–CXCR4-axis-mediated chemotaxis, homing, and long-term engraftment when these cells were transplanted into primary- and secondary-recipient NSG mice. Mechanistically, activated glucocorticoid receptor binds directly to a glucocorticoid response element in the CXCR4 promoter and recruits the SRC-1–p300 complex to promote H4K5 and H4K16 histone acetylation, facilitating transcription of CXCR4. These results suggest a new and readily available means to enhance the clinical efficacy of CB HCT.

Cutting Edge: Progesterone Directly Upregulates Vitamin D Receptor Gene Expression for Efficient Regulation of T Cells by Calcitriol

The two nuclear hormone receptor ligands progesterone and vitamin D (vit.D) play important roles in regulating T cells. The mechanism that connects these two hormones in regulating T cells has not been established. In this study, we report that progesterone is a novel inducer of vit.D receptor (VDR) in T cells and makes T cells highly sensitive to calcitriol. At the molecular level, the induction by progesterone is mediated by two progesterone receptor-binding elements in the intron region after the first noncoding exon of the human VDR gene. Increased expression of VDR by progesterone allows highly sensitive regulation of T cells by vit.D even when vit.D levels are suboptimal. This novel regulatory pathway allows enhanced induction of regulatory T cells but suppression of Th1 and Th17 cells by the two nuclear hormones. The results have significant ramifications in effective regulation of T cells to prevent adverse immune responses during pregnancy.

Antagonism of PPAR-γ signaling expands human hematopoietic stem and progenitor cells by enhancing glycolysis

Hematopoietic stem cells (HSCs) quiescently reside in bone marrow niches and have the capacity to self-renew or differentiate to form all of the blood cells throughout the lifespan of an animal. Allogeneic HSC transplantation is a life-saving treatment for malignant and nonmalignant disorders. HSCs isolated from umbilical cord blood (CB) are used for hematopoietic cell transplantation (HCT), but due to the limited numbers of HSCs in single units of umbilical CB, a number of methods have been proposed for ex vivo expansion of human HSCs. We show here that antagonism of peroxisome proliferator-activated receptor (PPAR)-γ promotes ex vivo expansion of phenotypically and functionally defined subsets of human CB HSCs and hematopoietic progenitor cells (HSPCs). PPAR-γ antagonism in CB HSPCs strongly downregulated expression of several differentiation-associated genes, as well as fructose-bisphosphatase 1 (FBP1; which encodes a negative regulator of glycolysis), and enhanced glycolysis without compromising mitochondrial metabolism. The expansion of CB HSPCs by PPAR-γ antagonism was completely suppressed by removal of glucose or inhibition of glycolysis. Moreover, knockdown of FBP1 expression promoted glycolysis and ex vivo expansion of long-term repopulating CB HSPCs, whereas overexpression of FBP1 suppressed the expansion of CB HSPCs that was induced by PPAR-γ antagonism. Our study suggests the possibility for a new and simple means for metabolic reprogramming of CB HSPCs to improve the efficacy of HCT.

Combinations of antioxidants and/or of epigenetic enzyme inhibitors allow for enhanced collection of mouse bone marrow hematopoietic stem cells in ambient air

Hematopoietic cell transplantation (HCT) is a treatment for malignant and non-malignant disorders. However, sometimes the numbers of donor hematopoietic stem cells (HSC) are limiting, which can compromise the success of HCT. We recently published that collection and processing of mouse bone marrow (BM) and human cord blood cells in a hypoxic atmosphere of 3% O2 or in ambient air (~21% O2) in the presence of cyclosporine A yields increased numbers of HSC. We now show that collection and processing of mouse BM cells in ambient air in the presence of specific combinations of anti-oxidants and/or inhibitors of epigenetic enzymes can also enhance the collection of HSC, information of potential relevance for enhanced efficacy of HCT.

Hypoxia Signaling Pathway in Stem Cell Regulation: Good and Evil

Purpose of ReviewThis review summarizes the role of hypoxia and hypoxia-inducible factors (HIFs) in the regulation of stem cell biology, specifically focusing on maintenance, differentiation, and stress responses in the context of several stem cell systems. Stem cells for different lineages/tissues reside in distinct niches, and are exposed to diverse oxygen concentrations. Recent studies have revealed the importance of the hypoxia signaling pathway for stem cell functions.Recent FindingsHypoxia and HIFs contribute to maintenance of embryonic stem cells, generation of induced pluripotent stem cells, functionality of hematopoietic stem cells, and survival of leukemia stem cells. Harvest and collection of mouse bone marrow and human cord blood cells in ambient air results in fewer hematopoietic stem cells recovered due to the phenomenon of Extra PHysiologic Oxygen Shock/Stress (EPHOSS).SummaryOxygen is an important factor in the stem cell microenvironment. Hypoxia signaling and HIFs play important roles in modeling cellular metabolism in both stem cells and niches to regulate stem cell biology, and represent an additional dimension that allows stem cells to maintain an undifferentiated status and multilineage differentiation potential.

Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment

Enhancement of hematopoietic stem cell (HSC) homing and engraftment is clinically critical, especially for cord blood (CB) hematopoietic cell transplantation. Here we report that specific HDAC5 inhibition highly upregulates CXCR4 surface expression in human CB HSCs and progenitor cells (HPCs). This results in enhanced SDF-1/CXCR4-mediated chemotaxis and increased homing to the bone marrow environment, with elevated SCID-repopulating cell (SRC) frequency and enhanced long-term and secondary engraftment in NSG mice. HDAC5 inhibition increases acetylated p65 levels in the nucleus, which is important for CXCR4 transcription. Inhibition of nuclear factor-κB (NF-κB) signaling suppresses HDAC5-mediated CXCR4 upregulation, enhanced HSC homing, and engraftment. Furthermore, activation of the NF-κB signaling pathway via TNFα also results in significantly increased CXCR4 surface expression, enhanced HSC homing, and engraftment. These results demonstrate a previously unknown negative epigenetic regulation of HSC homing and engraftment by HDAC5, and allow for a new and simple translational strategy to enhance HSC transplantation.Enhancement of haematopoietic stem cell (HSC) homing and engraftment is critical for haematopoietic cell transplantation. Here, the authors find that HDAC5 inhibition enhances HSC homing and engraftment by increasing p65 acetylation and enhancing NF-kB mediated CXCR4 transcription.

Enhancing human cord blood hematopoietic stem cell engraftment by targeting nuclear hormone receptors

Purpose of review Allogeneic hematopoietic cell transplantation (HCT) is a life-saving therapy for hematological and nonhematological diseases. Cord blood is a source of transplantable hematopoietic stem cells (HSCs), but limited numbers of HSCs in single cord blood units, which may cause delayed neutrophil, platelet, and immune cell reconstitution, is a major problem for efficient transplantation. Ex-vivo expansion and enhanced homing of cord blood HSC may overcome this disadvantage and improve its long-term engraftment. Here, we discuss the role of nuclear hormone receptors signaling in human cord blood HSC engraftment. Recent findings Antagonizing retinoid acid receptor (RAR) signaling promotes human HSC expansion and increases myeloid cell production. Cord blood CD34+ cells expanded by SR1 promotes efficient myeloid recovery after transplantation compared with control groups, and leads to successful engraftment. Short-term treatment of glucocorticoids enhances homing and long-term engraftment of human HSCs and HPCs in NSG mice. Peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) antagonism expands human HSCs and HPCs by preventing differentiation and enhancing glucose metabolism. These findings demonstrate that nuclear hormone receptor signaling components might be promising targets for improving human cord blood HCT. Summary Better understanding of molecular mechanisms underlying human HSC expansion and homing mediated by nuclear hormone receptor signaling pathways will facilitate enhanced HCT efficacy.