Stem cell-secreted factor therapy regenerates the ovarian niche and rescues follicles.

BACKGROUND: Ovarian senescence is a normal age-associated phenomenon, but increasingly younger women are affected by diminished ovarian reserves or premature ovarian insufficiency. There is an urgent need for developing therapies to improve ovarian function in these patients. In this context, previous studies suggest that stem cell-secreted factors could have regenerative properties in the ovaries. OBJECTIVE: This study aimed to test the ability of various human plasma sources, enriched in stem cell-secreted factors, and the mechanisms behind their regenerative properties, to repair ovarian damage and to promote follicular development. STUDY DESIGN: In the first phase, the effects of human plasma enriched in bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization, umbilical cord blood plasma, and their activated forms on ovarian niche, follicle development, and breeding performance were assessed in mouse models of chemotherapy-induced ovarian damage (n=7 per group). In addition, the proteomic profile of each plasma was analyzed to find putative proteins and mechanism involved in their regenerative properties in ovarian tissue. In the second phase, the most effective plasma treatment was validated in human ovarian cortex xenografted in immunodeficient mice (n=4 per group). RESULTS: Infusion of human plasma enriched bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization or of umbilical cord blood plasma-induced varying degrees of microvessel formation and cell proliferation and reduced apoptosis in ovarian tissue to rescue follicular development and fertility in mouse models of ovarian damage. Plasma activation enhanced these effects. Activated granulocyte colony-stimulating factor plasma was the most potent inducing ovarian rescue in both mice and human ovaries, and proteomic analysis indicated that its effects may be mediated by soluble factors related to cell cycle and apoptosis, gene expression, signal transduction, cell communication, response to stress, and DNA repair of double-strand breaks, the most common form of age-induced damage in oocytes. CONCLUSION: Our findings suggested that stem cell-secreted factors present in both granulocyte colony-stimulating factor-mobilized and umbilical cord blood plasma could be an effective treatment for increasing the reproductive outcomes in women with impaired ovarian function owing to several causes. The activated granulocyte colony-stimulating factor plasma, which is already enriched in both stem cell-secreted factors and platelet-enclosed growth factors, seems to be the most promising treatment because of its most potent restorative effects on the ovary together with the autologous source.

Dnmt3a-null hematopoietic stem and progenitor cells expand after busulfan treatment.

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) comprise the majority of mutations found in clonal hematopoiesis (CH), an age-related condition that was recently found to affect outcomes in patients undergoing hematopoietic stem cell transplant (HSCT). Recent studies have indicated that patients with CH have worse prognoses after HSCT, suggesting stress imposed by HSCT preconditioning agents may impact hematopoietic stem cell (HSC) dynamics in transplant recipients. In this study, we used a competitive transplantation mouse model to investigate how treatment with the common preconditioning agents 5-fluorouracil (5-FU) and busulfan (BU) affect the prevalence of Dnmt3a-/- HSCs and progenitor cells in competition with wild-type cells. We found that, though sufficient to deplete peripheral blood counts, 5-FU preconditioning did not significantly alter the frequency of Dnmt3a-null hematopoietic stem and progenitor cells (HSPCs) in mosaic mice. In contrast, mice treated with BU had a sevenfold decline in total bone marrow cells and an increase in Dnmt3a-null HSPCs that was detectable in peripheral blood. Indeed, even though all mosaic mice had a starting engraftment of ∼10%-40%, 85%-100% of HSPCs were Dnmt3a-null in four of seven mice after BU treatment, indicating these cells expand dramatically during recovery. Overall, these results suggest that individual preconditioning regimens have different effects on the expansion of Dnmt3a-mutant cells in patients with pre-existing CH. Thus, the presence of CH-associated mutants should be evaluated prior to selecting preconditioning regimens for HSCT.

[Modalities of mobilization and hematopoietic stem cells objectives in autologous transplantation: Guidelines from the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)]. / Modalités de mobilisation des cellules souches hématopoïétiques autologues et objectifs cellulaires en cellules CD34+ : recommandations de la Société francophone de greffe de mœlle et de thérapie cellulaire (SFGM-TC).

The modalities of mobilization of hematopoietic stem cells in autologous transplantation have evolved in recent years. The Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC) organized the 9th hematopoietic stem cell transplantation clinical practices harmonization workshop series in September 2018 in Lille, France, to conduct a review of current practices of the society centers and of international recommendations. The cell dose objectives have been revised. The modalities of mobilization including the use of plerixafor have been specified allowing reaching the objectives of collection while limiting the number of apheresis. Collections failures have become exceptional.

Neuroprotective effects of erythropoietin on rat retinas subjected to oligemia.

OBJECTIVES: Erythropoietin may have neuroprotective potential after ischemia of the central nervous system. Here, we conducted a study to characterize the protective effects of erythropoietin on retinal ganglion cells and gliotic reactions in an experimentally induced oligemia model. METHODS: Rats were subjected to global oligemia by bilateral common carotid artery occlusion and then received either vehicle or erythropoietin via intravitreal injection after 48 h; they were euthanized one week after the injection. The densities of retinal ganglion cells and contents of glial fibrillary acidic protein (astrocytes/Müller cells) and cluster of differentiation 68 clone ED1 (microglia/macrophages), assessed by fluorescence intensity, were evaluated in frozen retinal sections by immunofluorescence and epifluorescence microscopy. RESULTS: Retinal ganglion cells were nearly undetectable one week after oligemia compared with the sham controls; however, these cells were partially preserved in erythropoietin-treated retinas. The contents of glial fibrillary acidic protein and cluster of differentiation 68 clone ED1, markers for reactive gliosis, were significantly higher in retinas after bilateral common carotid artery occlusion than those in both sham and erythropoietin-treated retinas. CONCLUSIONS: The number of partially preserved retinal ganglion cells in the erythropoietin-treated group suggests that erythropoietin exerts a neuroprotective effect on oligemic/ischemic retinas. This effect could be related to the down-modulation of glial reactivity, usually observed in hypoxic conditions, clinically observed during glaucoma or retinal artery occlusion conditions. Therefore, glial reactivity may enhance neurodegeneration in hypoxic conditions, like normal-tension glaucoma and retinal ischemia, and erythropoietin is thus a candidate to be clinically applied after the detection of decreased retinal blood flow.

Neuroprotective effects of erythropoietin on rat retinas subjected to oligemia

OBJECTIVES: Erythropoietin may have neuroprotective potential after ischemia of the central nervous system. Here, we conducted a study to characterize the protective effects of erythropoietin on retinal ganglion cells and gliotic reactions in an experimentally induced oligemia model. METHODS: Rats were subjected to global oligemia by bilateral common carotid artery occlusion and then received either vehicle or erythropoietin via intravitreal injection after 48 h; they were euthanized one week after the injection. The densities of retinal ganglion cells and contents of glial fibrillary acidic protein (astrocytes/Müller cells) and cluster of differentiation 68 clone ED1 (microglia/macrophages), assessed by fluorescence intensity, were evaluated in frozen retinal sections by immunofluorescence and epifluorescence microscopy. RESULTS: Retinal ganglion cells were nearly undetectable one week after oligemia compared with the sham controls; however, these cells were partially preserved in erythropoietin-treated retinas. The contents of glial fibrillary acidic protein and cluster of differentiation 68 clone ED1, markers for reactive gliosis, were significantly higher in retinas after bilateral common carotid artery occlusion than those in both sham and erythropoietin-treated retinas. CONCLUSIONS: The number of partially preserved retinal ganglion cells in the erythropoietin-treated group suggests that erythropoietin exerts a neuroprotective effect on oligemic/ischemic retinas. This effect could be related to the down-modulation of glial reactivity, usually observed in hypoxic conditions, clinically observed during glaucoma or retinal artery occlusion conditions. Therefore, glial reactivity may enhance neurodegeneration in hypoxic conditions, like normal-tension glaucoma and retinal ischemia, and erythropoietin is thus a candidate to be clinically applied after the detection of decreased retinal blood flow.

Erythropoietin-regulated oxidative stress negatively affects enucleation during terminal erythropoiesis.

Differentiating erythroblasts are exposed to an oxidative environment. The dynamics of oxidative status during terminal erythropoiesis and how they affect cell differentiation in response to erythropoietin (Epo) are unclear. Here, we show that Epo induces reactive oxygen species (ROS) production in the early stages of terminal erythropoiesis. The levels of ROS correlate with CD71 surface expression and the uptake of iron and transferrin. ROS decreases in the late stages of terminal erythropoiesis, when the cells are preparing for enucleation. Consistently, treatment of erythroblasts with a low dose (5 mM) of N-acetyl-cysteine (NAC), a ROS scavenger, promotes enucleation. However, a high dose (20 mM) of NAC leads to significant cell death. Our study reveals an important function of Epo in regulating the dynamics of oxidative status and enucleation.

Early production of human neutrophils and platelets posttransplant is severely compromised by growth factor exposure.

The critical human cells that produce neutrophils and platelets within 3 weeks in recipients of hematopoietic transplants are thought to produce these mature blood cells with the same kinetics in sublethally irradiated immunodeficient mice. Quantification of their numbers indicates their relative underrepresentation in cord blood (CB), likely explaining the clinical inadequacy of single CB units in rescuing hematopoiesis in myelosuppressed adult patients. We here describe that exposure of CD34(+) CB cells ex vivo to growth factors that markedly expand their numbers and colony-forming cell content also rapidly (within 24 hours) produce a significant and sustained net loss of their original short-term repopulating activity. This loss of short-term in vivo repopulating activity affects early platelet production faster than early neutrophil output, consistent with their origin from distinct input populations. Moreover, this growth factor-mediated loss is not abrogated by published strategies to increase progenitor homing despite evidence that the effect on rapid neutrophil production is paralleled in time and amount by a loss of the homing of their committed clonogenic precursors to the bone marrow. These results highlight the inability of in vitro or phenotype assessments to reliably predict clinical engraftment kinetics of cultured CB cells.

Comparative study of neural differentiation of bone marrow mesenchymal stem cells by different induction methods.

Neurogenic differentiation of bone marrow (BM) mesenchymal stem cells (MSCs) offers a new hope for patients with many neurological disorders. Several chemical inducers are employed to induce BMMSCs differentiation into nerve cells. In the present study, we compared different inducers [2-mercaptoethanol (BME), tretinoin (ATRA), dimethyl sulfoxide/butylated hydroxyanisole (DMSO/BHA), and indomethacin/3-isobutyl-1-methylxanthine (indomethacin/IBMX)] on the neurogenic differentiation of BMMSCs and aimed to identify a more efficient and safer method. The MSCs were first identified by their ability to adhere to plastic and by the expression of positive (CD44, CD90, and CD105) and negative (CD34) markers assessed by flow cytometry. The efficiency of the neurogenic differentiation was determined by assessing the mRNA and protein expression of nestin, microtubule-associated protein-2 (MAP2), neuron specific enolase (NSE), and glial fibrillary acidic protein (GFAP) by reverse transcription-polymerase chain reaction and western-blot, respectively. The effect of these inducers on cell viability was also evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. This comprehensive study shows that indomethacin/IBMX is better than BME, DMSO/BHA, and ATRA both in terms of efficiency and safety, while BME suppressed the growth and proliferation of MSCs.

Concentración de interleuquina-6 en el vítreo de pacientes con desprendimiento de retina / Interleukin-6 concentrations in the vitreous body of patients with retinal detachment

OBJETIVO: Determinar los niveles de interleuquina-6 (IL-6) en el vítreo de pacientes con desprendimiento de retina (DdR). MATERIAL Y MÉTODO: Mediante vitrectomía vía pars plana, se recogieron muestras no diluidas de vítreo de 40 pacientes sin antecedentes de cirugía vítrea o intraocular previa, que fueron divididos en 2 grupos: A (n = 20) pacientes con DdR y B (n = 20) pacientes con membrana premacular y agujero macular. La concentración de IL-6 se determinó mediante radioinmunoensayo. RESULTADOS: La concentración vítrea de IL-6 en el grupo A fue 122,4 + -16 pg/mL (rango 91,5-620) y en el grupo B fue 46 +/- 23 pg/mL (rango 3-150) (p < 0,001). CONCLUSIONES: Estos resultados demuestran que la concentración vítrea de IL-6 está más elevada en los pacientes con DdR en comparación con el grupo control

OBJECTIVE: To measure interleuquin-6 (IL-6) levels in the vitreous body of patients with retinal detachment (RD). PATIENTS AND METHODS: Undiluted vitreous samples were obtained from 40 patients with no history of prior vitreous or intraocular surgery. Patients were divided into two groups: A (n = 20) patients with RD and B (n = 20) patients with pre-retinal macular membranes and macular holes. IL-6 was determined using radioimmunoassay. RESULTS: IL-6 vitreous concentration in group A was 122.4 + -16 pg/mL (range 91.5-620) and in group B was 46 +/- 23 pg/mL (range 3-150) (p < .001). CONCLUSIONS: These results show that the concentration of IL-6 in the vitreous body was significantly higher in patients with RD than in the control group

Flow cytometric detection of altered signaling in myelodysplastic syndrome and cytopenia.

Multiparameter flow cytometric analysis allows for precise evaluation of growth factor stimulated intracellular signaling in distinct immunophenotype defined hematopoetic populations. Our analysis of intracellular phosphoprotein in response to major hematopoietic growth factors or cytokines showed several interesting findings. Although there was no characteristic signaling abnormality that was diagnostic for MDS, MDS cases were often associated with more signaling aberrancies involving more cellular populations. Higher than average response in the CD34(+)CD117(+) progenitor cells to Flt3 ligand and stem cell factor stimulation was frequently associated with high risk features or disease progression in MDS. Although preliminary results hint an adverse prognostic role of dysregulated FLT3 pathway in MDS cases, whether this observation adds independent prognostic value to the existing prognostic system needs to be further explored in future prospective studies.

Potential pharmacological interventions against hematotoxicity: an overview.

Various treatment regimens, including chemotherapy, are known to induce heavy oxidative stress on the system, which in turn leads to adverse effects on healthy tissues. Blood being prone to oxidative stress is affected the most. At this juncture, it might not be prudent to anticipate having chemotherapeutic agents with no hematotoxicity; the best way forward is to look for potential anti-hematotoxic compounds, which could be supplemented to exposed patients, thus reducing the toxic burden on blood cells. We mined existing literature for reviewing possible interventions against hematotoxicity and figured that there is a great lacuna in this field in terms of not having such useful information at one place. This review presents the possible entities based on their antioxidant potentials, including their mechanistic pathways.

Generation and characterization of a JAK2V617F-containing erythroleukemia cell line.

The JAK2V617F mutation is found in the majority of patients with myeloproliferative neoplasms (MPNs). Transgenic expression of the mutant gene causes MPN-like phenotypes in mice. We have produced JAK2V617F mice with p53 null background. Some of these mice developed acute erythroleukemia. From one of these mice, we derived a cell line designated J53Z1. J53Z1 cells were stained positive for surface markers CD71 and CD117 but negative for Sca-1, TER-119, CD11b, Gr-1, F4/80, CD11c, CD317, CD4, CD8a, CD3e, B220, CD19, CD41, CD42d, NK-1.1, and FceR1. Real time PCR analyses demonstrated expressions of erythropoietin receptor EpoR, GATA1, and GATA2 in these cells. J53Z1 cells grew rapidly in suspension culture containing fetal bovine serum with a doubling time of ∼18 hours. When transplanted into C57Bl/6 mice, J53Z1 cells induced acute erythroleukemia with massive infiltration of tumor cells in the spleen and liver. J53Z1 cells were responsive to stimulation with erythropoietin and stem cell factor and were selectively inhibited by JAK2 inhibitors which induced apoptosis of the cells. Together, J53Z1 cells belong to the erythroid lineage, and they may be useful for studying the role of JAK2V617F in proliferation and differentiation of erythroid cells and for identifying potential therapeutic drugs targeting JAK2.

Noncanonical NF-κB pathway controls the production of type I interferons in antiviral innate immunity.

Production of type I interferons (IFN-I) is a crucial innate immune mechanism against viral infections. IFN-I induction is subject to negative regulation by both viral and cellular factors, but the underlying mechanism remains unclear. We report that the noncanonical NF-κB pathway was stimulated along with innate immune cell differentiation and viral infections and had a vital role in negatively regulating IFN-I induction. Genetic deficiencies in major components of the noncanonical NF-κB pathway caused IFN-I hyperinduction and rendered cells and mice substantially more resistant to viral infection. Noncanonical NF-κB suppressed signal-induced histone modifications at the Ifnb promoter, an action that involved attenuated recruitment of the transcription factor RelA and a histone demethylase, JMJD2A. These findings reveal an unexpected function of the noncanonical NF-κB pathway and highlight an important mechanism regulating antiviral innate immunity.

Effects of agitation speed on the ex vivo expansion of cord blood hematopoietic stem/progenitor cells in stirred suspension culture.

The mononuclear cells were cultivated in stirred flasks at different agitation speeds of 30 rpm, 45 rpm, 60 rpm and 80 rpm. At the agitation speed of 30 rpm, total cells achieved higher expansion folds and the CFC density increased. When at higher agitation speed of 60 rpm or 80 rpm, the number of cells dropped rapidly and characteristics of hematopoietic stem/progenitor cells (HSPCs) were not maintained. Moreover, the culture duration of 6-9 days was better for HSPCs ex vivo expansion. These data indicated that HSPCs should be cultured at relatively low agitation speed and for a short-term period when cultured in stirred suspension system.

[The comparative characteristics of antibacterial properties of the peptides of the active site of GM-CSF, and substances delivered from supernatants of hematopoietic progenitor CD34+45- cells].

The antibacterial activity of synthetic peptides of the active site of GM-CSF and supernatants of CD34+45- hematopoietic progenitor cells has been investigated GM-CSF peptides and cell supernatants were found to possess pronounced antibacterial activity, at that a combination of these substances has a more pronounced activity in comparison with the single substances. Possible mechanisms of the identified effects of synthetic peptides and substances from the supernatants of CD34+5- cells are discussed.

Production of functional classical brown adipocytes from human pluripotent stem cells using specific hemopoietin cocktail without gene transfer.

Brown adipose tissue is attracting much attention due to its antiobestic effects; however, its development and involvement in metabolic improvement remain elusive. Here we established a method for a high-efficiency (>90%) differentiation of human pluripotent stem cells (hPSCs) into functional classical brown adipocytes (BAs) using specific hemopoietin cocktail (HC) without exogenous gene transfer. BAs were not generated without HC, and lack of a component of HC induced white adipocyte (WA) marker expressions. hPSC-derived BA (hPSCdBA) showed respiratory and thermogenic activation by ß-adrenergic receptor (AdrRß) stimuli and augmented lipid and glucose tolerance, whereas human multipotent stromal cell-derived WA (hMSCdWA) improved lipid but inhibited glucose metabolism. Cotransplantation of hPSCdBA normalized hMSCdWA-induced glucose intolerance. Surprisingly, hPSCdBAs expressed various hemopoietin genes, serving as stroma for myeloid progenitors. Moreover, AdrRß stimuli enhanced recovery from chemotherapy-induced myelosuppression. Our study enhances our understanding of BA, identifying roles in metabolic and hemogenic regulation.

HUVEC co-culture and haematopoietic growth factors modulate human proliferative monocyte activity.

Monocytes and macrophages are often claimed to have limited potential for proliferation in vivo and in vitro although a human monocyte subset with increased potential to proliferate in culture, termed the proliferative monocyte (PM), has previously been identified. The response of the putatively less mature PM to conditions conducive to haematopoietic stem cell culture was determined. Co-culture of monocytes on a HUVEC monolayer induced up to four cell divisions in a 9 day period. The PM response to haematopoietic growth factors (Flt3L, SCF, IL-6, IL-3 and M-CSF) was determined. M-CSF induced the greatest proliferative response in PM; IL-3 and Flt3L reduced basal and M-CSF-induced proliferation. The inhibition of M-CSFR kinase activity by GW2580 indicated that the ligand(s) for this receptor was a potent inducer of proliferation of this subset; inhibitors of intracellular signalling pathways also reduced PM proliferation.

Neuroprotection for ischaemic stroke: translation from the bench to the bedside.

Neuroprotection seeks to restrict injury to the brain parenchyma following an ischaemic insult by preventing salvageable neurons from dying. The concept of neuroprotection has shown promise in experimental studies, but has failed to translate into clinical success. Many reasons exist for this including the heterogeneity of human stroke and the lack of methodological agreement between preclinical and clinical studies. Even with the proposed Stroke Therapy Academic Industry Roundtable criteria for preclinical development of neuroprotective agents for stroke, we have still seen limited success in the clinic, an example being NXY-059, which fulfilled nearly all the Stroke Therapy Academic Industry Roundtable criteria. There are currently a number of ongoing trials for neuroprotective strategies including hypothermia and albumin, but the outcome of these approaches remains to be seen. Combination therapies with thrombolysis also need to be fully investigated, as restoration of oxygen and glucose will always be the best therapy to protect against cell death from stroke. There are also a number of promising neuroprotectants in preclinical development including haematopoietic growth factors, and inhibitors of the nicotinamide adenine dinucleotide phosphate oxidases, a source of free radical production which is a key step in the pathophysiology of acute ischaemic stroke. For these neuroprotectants to succeed, essential quality standards need to be adhered to; however, these must remain realistic as the evidence that standardization of procedures improves translational success remains absent for stroke.

Involvement of intracellular reactive oxygen species and mitochondria in the radiosensitivity of human hematopoietic stem cells.

Reactive oxygen species (ROS) can cause significant biological damage and are produced from low linear energy transfer-ionizing radiation, such as X-rays. Although hematopoietic stem cells (HSCs) are known to be particularly sensitive to ionizing radiation, little is known about the roles of mitochondria and ROS production in determining the radiosensitivity of HSCs. The clonogenic survival of CD34(+) HSCs, intracellular mitochondrial content, and intracellular ROS production after irradiation were investigated to elucidate the involvement of mitochondria and ROS in the individual radiosensitivity of HSCs detected in human placental/umbilical cord blood. The results showed that large individual differences exist in the initial numbers of each progenitor cell type, as well as in the surviving fraction of cells. When supplemented with an appropriate cytokine combination, a statistically significant increase in ROS production was observed at 3 h after 2 or 4 Gy of irradiation (P < 0.05), with nearly a return to initial levels by 6 h. In contrast, no significant difference was observed under cytokine-free conditions. At this stage, no significant correlations were observed between ROS production, intracellular mitochondrial content, and the surviving fractions of each HSC progenitor. These results suggest that the kinetics of ROS generation during the 6 h after ionizing radiation have little effect on the different radiation sensitivity of HSCs.

Dampened ERK signaling in hematopoietic progenitor cells in rheumatoid arthritis.

In rheumatoid arthritis (RA), hematopoietic progenitor cells (HPC) have age-inappropriate telomeric shortening suggesting premature senescence and possible restriction of proliferative capacity. In response to hematopoietic growth factors RA-derived CD34(+) HPC expanded significantly less than age-matched controls. Cell surface receptors for stem cell factor (SCF), Flt 3-Ligand, IL-3 and IL-6 were intact in RA HPC but the cells had lower transcript levels of cell cycle genes, compatible with insufficient signal strength in the ERK pathway. Cytokine-induced phosphorylation of ERK1/2 was diminished in RA HPC whereas phosphorylated STAT3 and STAT5 molecules accumulated to a similar extent as in controls. Confocal microscopy demonstrated that the membrane-proximal colocalization of K-Ras and B-Raf was less efficient in RA-derived CD34(+) cells. Thus, hyporesponsiveness of RA HPC to growth factors results from dampening of the ERK signaling pathways; with a defect localized in the very early steps of the ERK signaling cascade.