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.

SDF-1/CXCL12 modulates mitochondrial respiration of immature blood cells in a bi-phasic manner.

SDF-1/CXCL12 is a potent chemokine required for the homing and engraftment of hematopoietic stem and progenitor cells. Previous data from our group has shown that in an SDF-1/CXCL12 transgenic mouse model, lineage(-) Sca-1(+) c-Kit(+) (LSK) bone marrow cells have reduced mitochondrial membrane potential versus wild-type. These results suggested that SDF-1/CXCL12 may function to keep mitochondrial respiration low in immature blood cells in the bone marrow. Low mitochondrial metabolism helps to maintain low levels of reactive oxygen species (ROS), which can influence differentiation. To test whether SDF-1/CXCL12 regulates mitochondrial metabolism, we employed the human leukemia cell line HL-60, that expresses high levels of the SDF-1/CXCL12 receptor, CXCR4, as a model of hematopoietic progenitor cells in vitro. We treated HL-60 cells with SDF-1/CXCL12 for 2 and 24h. Oxygen consumption rates (OCR), mitochondrial-associated ATP production, mitochondrial mass, and mitochondrial membrane potential of HL-60 cells were significantly reduced at 2h and increased at 24h as compared to untreated control cells. These biphasic effects of SDF-1/CXCL12 were reproduced with lineage negative primary mouse bone marrow cells, suggesting a novel function of SDF-1/CXCL12 in modulating mitochondrial respiration by regulating mitochondrial oxidative phosphorylation, ATP production and mitochondrial content.

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 H2 O2 (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.

Mouse hematopoietic cell-targeted STAT3 deletion: stem/progenitor cell defects, mitochondrial dysfunction, ROS overproduction, and a rapid aging-like phenotype.

Nuclear transcription factor Stat3 is important for proper regulation of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) proliferation, survival, and cytokine signaling responses. A new, noncanonical role for Stat3 in mitochondrial function has been discovered recently. However, there is little information on the role(s) of mitochondrial Stat3 in HSC/HPC function, especially potential effects of Stat3/mitochondrial dysregulation in human diseases. We investigated hematopoietic cell-targeted deletion of the STAT3 gene in HSCs/HPCs with a focus on mitochondrial function. We found that STAT3(-/-) mice, which have a very shortened lifespan, dysfunctional/dysregulated mitochondrial function and excessive reactive oxygen species production in HSCs/HPCs that coincides with pronounced defects in function. These animals have a blood phenotype with similarities to premature aging and to human diseases of myelodysplastic syndrome and myeloproliferative neoplasms such as erythroid dysplasia, anemia, excessive myeloproliferation, and lymphomyeloid ratio shifts. We show herein that the lifespan of STAT3(-/-) animals is lengthened by treatment with a reactive oxygen species scavenger, which lessened the severity of the blood phenotype. These data suggest a need for more detailed studies of role(s) of Stat3 in HSC/HPC mitochondrial function in human diseases and raise the idea that mitochondrial Stat3 could be used as a potential therapeutic target.

Superoxide flashes, reactive oxygen species, and the mitochondrial permeability transition pore: potential implications for hematopoietic stem cell function.

PURPOSE OF REVIEW: Reactive oxygen species (ROS) have an important function in blood cell homeostasis and hematopoietic diseases. Recent discoveries concerning how ROS are generated and regulated in mitochondria via the mitochondrial permeability transition pore (mPTP) and the new phenomenon, superoxide flashes, and ROS-induced ROS release, have not been investigated in hematopoietic stem and progenitor cells, but likely have important implications for their regulation and survival. Here we relate our opinions about these potential implications. RECENT FINDINGS: The mPTP has been recently implicated in ROS generation via binding of Stat3 transcription factor to a central component of the pore. SUMMARY: The implications of this new information for hematopoiesis regulation and transplantation methodologies could prove to be important, especially as they relate to myeloid neoplasm oncogenesis and potentially new therapeutic targets. New details about ROS production suggest that techniques for bone marrow and umbilical cord blood harvest may benefit from means to downmodulate ROS.

Modelling the sitagliptin effect on dipeptidyl peptidase-4 activity in adults with haematological malignancies after umbilical cord blood haematopoietic cell transplantation.

BACKGROUND AND OBJECTIVES: Dipeptidyl peptidase-4 (DPP4) inhibition is a potential strategy to increase the engraftment rate of haematopoietic stem/progenitor cells. A recent clinical trial using sitagliptin, a DPP4 inhibitor approved for type 2 diabetes mellitus, has been shown to be a promising approach in adults with haematological malignancies after umbilical cord blood (UCB) haematopoietic cell transplantation (HCT). On the basis of data from this clinical trial, a semi-mechanistic model was developed to simultaneously describe DPP4 activity after multiple doses of sitagliptin in subjects with haematological malignancies after a single-unit UCB HCT. METHODS: The clinical study included 24 patients who received myeloablative conditioning followed by oral sitagliptin with single-unit UCB HCT. Using a nonlinear mixed-effects approach, a semi-mechanistic pharmacokinetic-pharmacodynamic model was developed to describe DPP4 activity from these trial data, using NONMEM version 7.2 software. The model was used to drive Monte Carlo simulations to probe the various dosage schedules and the attendant DPP4 response. RESULTS: The disposition of sitagliptin in plasma was best described by a two-compartment model. The relationship between sitagliptin concentrations and DPP4 activity was best described by an indirect response model with a negative feedback loop. Simulations showed that twice daily or three times daily dosage schedules were superior to a once daily schedule for maximal DPP4 inhibition at the lowest sitagliptin exposure. CONCLUSION: This study provides the first pharmacokinetic-pharmacodynamic model of sitagliptin in the context of HCT, and provides a valuable tool for exploration of optimal dosing regimens, which are critical for improving the time to engraftment in patients after UCB HCT.

In vivo DPP-4 inhibition to enhance engraftment of single-unit cord blood transplants in adults with hematological malignancies.

Delayed engraftment is a significant limitation of umbilical cord blood (UCB) transplantation due to low stem cell numbers. Inhibition of dipeptidyl peptidase (DPP)-4 enhanced engraftment in murine transplants. We evaluated the feasibility of systemic DPP-4 inhibition using sitagliptin to enhance engraftment of single-unit UCB grafts in adults with hematological malignancies. Twenty-four patients (21-58 years) received myeloablative conditioning, followed by sitagliptin 600 mg orally days -1 to +2, and single UCB grafts day 0. Seventeen receiving red cell-depleted (RCD) grafts, matched at 4 (n=10) or 5 (n=7) of 6 human leucocyte antigen (HLA) loci with median nucleated cell dose 3.6 (2.5-5.2)×10(7)/kg, engrafted at median of 21 (range, 13-50) days with cumulative incidence of 94% (95% confidence interval, 84%-100%) at 50 days. Plasma DDP-4 activity was reduced to 23%±7% within 2 h. Area under DPP-4 activity-time curve (AUCA) correlated with engraftment; 9 of 11 with AUCA <6,000 activity·h engrafted within ≤21 days, while all 6 with higher AUCA engrafted later (P=0.002). Seven patients receiving red cell replete grafts had 10-fold lower colony forming units after thawing compared with RCD grafts, with poor engraftment. Systemic DPP-4 inhibition was well tolerated and may enhance engraftment. Optimizing sitagliptin dosing to achieve more sustained DPP-4 inhibition may further improve outcome.

Optical imaging of progenitor cell homing to patient-derived tumors.

Capitalizing on cellular homing to cancer is a promising strategy for targeting malignant cells for diagnostic, monitoring and therapeutic purposes. Murine C17.2 neural progenitor cells (NPC) demonstrate a tropism for cell line-derived tumors, but their affinity for patient-derived tumors is unknown. We tested the hypothesis that NPC accumulate in patient-derived tumors at levels detectable by optical imaging. Mice bearing solid tumors after transplantation with patient-derived leukemia cells and untransplanted controls received 10(6) fluorescent DiR-labeled NPC daily for 1-4 days, were imaged, then sacrificed. Tissues were analyzed by immunofluorescence and flow cytometry to detect tumor cell engraftment (CD45) and NPC (FITC-ß galactosidase or DiR). Tumors consisted primarily of CD45-positive cells and demonstrated mild fluorescence, corresponding to frequent clusters of FITC-ß gal-positive cells. Both transplanted and control mice demonstrated the highest fluorescent signal in the spleens and other tissues of the reticuloendothelial activating system. However, only rare FITC-ß gal-positive cells were detected in the mildly engrafted transplanted spleens and none in the control spleens, suggesting that their high DiR signal reflects the sequestration of DiR-positive debris. The mildly engrafted transplanted kidneys demonstrated low fluorescent signal and rare FITC-ß gal-positive cells whereas control kidneys were negative. Results indicate that NPC accumulate in tissues containing patient-derived tumor cells in a manner that is detectable by ex vivo optical imaging and proportional to the level of tumor engraftment, suggesting a capacity to home to micrometastatic disease. As such, NPC could have significant clinical applications for the targeted diagnosis and treatment of cancer.

Dipeptidylpeptidase 4 negatively regulates colony-stimulating factor activity and stress hematopoiesis.

Enhancement of hematopoietic recovery after radiation, chemotherapy, or hematopoietic stem cell (HSC) transplantation is clinically relevant. Dipeptidylpeptidase (DPP4) cleaves a wide variety of substrates, including the chemokine stromal cell-derived factor-1 (SDF-1). In the course of experiments showing that inhibition of DPP4 enhances SDF-1-mediated progenitor cell survival, ex vivo cytokine expansion and replating frequency, we unexpectedly found that DPP4 has a more general role in regulating colony-stimulating factor (CSF) activity. DPP4 cleaved within the N-termini of the CSFs granulocyte-macrophage (GM)-CSF, G-CSF, interleukin-3 (IL-3) and erythropoietin and decreased their activity. Dpp4 knockout or DPP4 inhibition enhanced CSF activities both in vitro and in vivo. The reduced activity of DPP4-truncated versus full-length human GM-CSF was mechanistically linked to effects on receptor-binding affinity, induction of GM-CSF receptor oligomerization and signaling capacity. Hematopoiesis in mice after radiation or chemotherapy was enhanced in Dpp4(-/-) mice or mice receiving an orally active DPP4 inhibitor. DPP4 inhibition enhanced engraftment in mice without compromising HSC function, suggesting the potential clinical utility of this approach.