Ex vivo reprogramming of human hematopoietic stem cells is accompanied by increased transcripts of genes regulating metabolic integrity.

The regenerative potential of human hematopoietic stem cells (HSCs) is functionally defined by their ability to provide life-long blood cell production and to repopulate myeloablated allogeneic transplant recipients. The expansion of HSC numbers is dependent not only on HSC divisions but also on a coordinated adaptation of HSCs to metabolic stress. These variables are especially critical during the ex vivo culture of HSCs with cytokine combinations, which frequently results in HSC exhaustion.

Ex vivo expansion of hematopoietic stem cells: Finally transitioning from the lab to the clinic.

Hematopoietic stem cells (HSCs) have been used for therapeutic purposes for decades in the form of autologous and allogeneic transplantation and are currently emerging as an attractive target for gene therapy. A low stem cell dose is a major barrier to the application of HSC therapy in several situations, primarily umbilical cord blood transplantation and gene modification. Strategies that promote ex vivo expansion of the numbers of functional HSCs could overcome this barrier, hence have been the subject of intense and prolonged research.

Evaluation of a clinical-grade, cryopreserved, ex vivo-expanded stem cell product from cryopreserved primary umbilical cord blood demonstrates multilineage hematopoietic engraftment in mouse xenografts.

Background Aims: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34 cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs.

Pleckstrin-2 is essential for erythropoiesis in β-thalassemic mice, reducing apoptosis and enhancing enucleation.

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking.

Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of Expanded Human Hematopoietic Stem Cells.

expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Within the heterogeneous pool of expanded HSCs, the precise phenotypic, transcriptomic and metabolic profile and thus, the identity of HSCs that confer LT repopulation potential remains poorly described.

Ex Vivo Expansion of Adult Hematopoietic Stem and Progenitor Cells with Valproic Acid.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic stem cell transplantation but lack a matched donor. However, the limited number of HSCs within each UCB unit remains a major challenge for their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of human HSCs in ex vivo cultures initiated with CD34 cells isolated from UCBs can overcome this limitation.

Expansion and preservation of the functional activity of adult hematopoietic stem cells cultured ex vivo with a histone deacetylase inhibitor.

Attempts to expand ex vivo the numbers of human hematopoietic stem cells (HSCs) without compromising their marrow repopulating capacity and their ability to establish multilineage hematopoiesis has been the subject of intense investigation. Although most such efforts have focused on cord blood HSCs, few have been applied to adult HSCs, a more clinically relevant HSC source for gene modification. To date, the strategies that have been used to expand adult HSCs have resulted in modest effects or HSCs with lineage bias and a limited ability to generate T cells in vivo.

Ex vivo HSC expansion challenges the paradigm of unidirectional human hematopoiesis.

Understanding mechanisms that determine the behavior of human hematopoietic stem cells (HSCs) is essential for developing novel strategies to expand ex vivo the number of fully functional HSCs. In this review, we focus on the complex interplay between intrinsic mechanisms regulated by transcriptional and mitochondrial networks and extrinsic signals imposed by the bone marrow microenvironment, which in concert regulate the balance between HSC self-renewal and differentiation. Such integrated signaling mechanisms that dictate the fate of HSCs in vivo must be recapitulated ex vivo to achieve successful expansion of clinically relevant HSCs.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34 cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA).

Mitochondrial Role in Stemness and Differentiation of Hematopoietic Stem Cells.

Quiescent and self-renewing hematopoietic stem cells (HSCs) rely on glycolysis rather than on mitochondrial oxidative phosphorylation (OxPHOS) for energy production. HSC reliance on glycolysis is considered an adaptation to the hypoxic environment of the bone marrow (BM) and reflects the low energetic demands of HSCs. Metabolic rewiring from glycolysis to mitochondrial-based energy generation accompanies HSC differentiation and lineage commitment.

Ex vivo human HSC expansion requires coordination of cellular reprogramming with mitochondrial remodeling and p53 activation.

The limited number of hematopoietic stem cells (HSCs) in umbilical cord blood (UCB) units restricts their use for stem cell transplantation. Ex vivo treatment of UCB-CD34 cells with valproic acid (VPA) increases the number of transplantable HSCs. In this study, we demonstrate that HSC expansion is not merely a result of proliferation of the existing stem cells but, rather, a result of a rapid reprogramming of CD34CD90 cells into CD34CD90 cells, which is accompanied by limited numbers of cell divisions.

SOD1, an unexpected novel target for cancer therapy.

Cancer cells have elevated levels of reactive oxygen species (ROS), which are generated in majority by the mitochondria. In the mitochondrial matrix, the manganese dismutase SOD2 acts as a major anti-oxidant enzyme. The deacetylase SIRT3 regulates the activity of SOD2.

SOD2 to SOD1 switch in breast cancer.

Cancer cells are characterized by elevated levels of reactive oxygen species, which are produced mainly by the mitochondria. The dismutase SOD2 localizes in the matrix and is a major antioxidant. The activity of SOD2 is regulated by the deacetylase SIRT3.

SirT3 regulates the mitochondrial unfolded protein response.

The mitochondria of cancer cells are characterized by elevated oxidative stress caused by reactive oxygen species (ROS). Such an elevation in ROS levels contributes to mitochondrial reprogramming and malignant transformation. However, high levels of ROS can cause irreversible damage to proteins, leading to their misfolding, mitochondrial stress, and ultimately cell death.

KLF6-SV1 drives breast cancer metastasis and is associated with poor survival.

Metastasis is the major cause of cancer mortality. A more thorough understanding of the mechanisms driving this complex multistep process will aid in the identification and characterization of therapeutically targetable genetic drivers of disease progression. We demonstrate that KLF6-SV1, an oncogenic splice variant of the KLF6 tumor suppressor gene, is associated with increased metastatic potential and poor survival in a cohort of 671 lymph node-negative breast cancer patients.

Estrogen receptor mediates a distinct mitochondrial unfolded protein response.

Unfolded protein responses (UPRs) of the endoplasmic reticulum and mitochondrial matrix have been described. Here, we show that the accumulation of proteins in the inter-membrane space (IMS) of mitochondria in the breast cancer cell line MCF-7 activates a distinct UPR. Upon IMS stress, overproduction of reactive oxygen species (ROS) and phosphorylation of AKT triggers estrogen receptor (ER) activity, which further upregulates the transcription of the mitochondrial regulator NRF1 and the IMS protease OMI (officially known as HTRA2).

Bortezomib enhances the efficacy of fulvestrant by amplifying the aggregation of the estrogen receptor, which leads to a proapoptotic unfolded protein response.

Purpose: Fulvestrant is known to promote the degradation of the estrogen receptor (ER) in the nucleus. However, fulvestrant also promotes the aggregation of the newly synthesized ER in the cytoplasm. Accumulation of protein aggregates leads to cell death but this effect is limited as a result of their elimination by the proteasome.

Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition.

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time.

The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells.

Identifying prosurvival mechanisms in stressed neuronal cells would provide protective strategies to hinder neurodegeneration. Recent evidence shows that vascular endothelial growth factor (VEGF), a well-established mitogen in endothelial cells, can mediate neuroprotection against damaging insults through the activation of its cognate receptor VEGFR2. In addition, growth factor receptor signaling pathways have been shown to crosstalk with cAMP-dependent Protein Kinase A (PKA) to protect neuronal cells from harmful stimuli.

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death.

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 microM) and potent (5.

Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium.

We reported previously that cadmium, an oxidative stressor, induced cyclooxygenase-2 (COX-2) upregulation in mouse neuronal cells that culminated in cell death. Herein, we show that cadmium induces reactive oxygen species (ROS) that activate c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) and their substrates, activating transcription factor 2 (ATF-2), CRE-binding protein (CREB) and c-Jun. This response is accompanied by induction of heme-oxygenase-1 (HO-1), poly(ADP-ribose) polymerase cleavage and a caspase-independent cell death.