Effects Of Hematopoietic Stem Cells Research Articles

Heme oxygenase-1 protects cells from replication stress

Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress.Experiments were conducted in control and HMOX1-deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1, the effect that was further enhanced in response to δ-aminolevulinic acid (ALA), a substrate in heme synthesis. This was associated with replication stress, as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1-deficient patient. Interestingly, in the absence of HO-1, the speed of fork progression was higher, and the response to DNA conformational hindrance less stringent, indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead, we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53, an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin, which in turn induces strong lipid peroxidation.Our results indicate that protection against replication stress is a universal feature of HO-1, presumably contributing to its widely recognized cytoprotective activity.

Hematopoietic stem cell transplantation ameliorates maternal diabetes-mediated gastrointestinal symptoms and autism-like behavior in mouse offspring.

Epidemiological studies have shown that maternal diabetes is associated with autism spectrum disorder development, although the detailed mechanism remains unclear. We have previously found that maternal diabetes induces persistent epigenetic changes and gene suppression in neurons, subsequently triggering autism‐like behavior (ALB). In this study, we investigated the potential role and effect of hematopoietic stem cells (HSCs) on maternal diabetes–mediated gastrointestinal (GI) dysfunction and ALB in a mouse model. We show in vitro that transient hyperglycemia induced persistent epigenetic changes and gene suppression of tight junction proteins. In vivo, maternal diabetes–mediated oxidative stress induced gene suppression and inflammation in both peripheral blood mononuclear cells and intestine epithelial cells, subsequently triggering GI dysfunction with increased intestinal permeability and altered microbiota compositions, as well as suppressed gene expression in neurons and subsequent ALB in offspring; HSC transplantation (HSCT) ameliorates this effect by systematically reversing maternal diabetes–mediated oxidative stress. We conclude that HSCT can ameliorate maternal diabetes–mediated GI symptoms and autism‐like behavior in mouse offspring.

Mechanisms of stem cells action: reality and hypotheses

Despite the long history of stem cells studying, the mechanisms of their action are still not fully understood. A classic example is the described non-hematological effect of hematopoietic stem cells (HSC), presumably due to the ability to transdifferentiation and cell fusion. The focus of clinical application in the field of regenerative medicine is mesenchymal stromal cells (MSC), and the understanding of the ways of implementfnion of their regenerative potential has evolved significantly. MSC were discovered and described as a structural unit of the HSC niche responsible for the repair of connective tissue through differentiation. However, it later turned out that they are a regulator of various processes in the body as a whole. These processes include anti-inflammatory, antifibrotic, and immunomodulatory effects, which are realized in various ways. Among these pathways, the paracrine mechanism has already been identified – the release of various growth factors, exosomes and microvesicles, the mechanism of direct transfer of mitochondria and other cellular organelles from cell to cell using tunneling nanotubes, the mechanism of efferocytosis. One of the latest discoveries in this field was the immunomodulatory effect of apoptotic MSC.In general, the study of the stem cells mechanisms of action is a living, developing field of science in which the last word will not be said soon.

Crohn’s Disease: Hematopoietic and Mesenchymal Stem Cell Therapy as Potential Treatment

Purpose: The purpose of this work was to comprehensively review literature to determine the safety and efficacy of stem cells for the treatment of Crohn’s disease and compare the effects of hematopoietic stem cells (HSCs) to that of mesenchymal stem cells (MSCs). Results: The data search included 5 animal models and clinical trials found on PubMed. The 5 studies included for HSCs and the 15 trails for MSCs found them to be a safe and effective as a treatment option (allogeneic and autologous bone marrow and adipose derived) for CD except for one multicenter randomized trial for HSC therapy. Most trials investigate the impact of stem cells specifically on perianal fistula, a common occurrence in patients with CD. Conclusion: While both HSCs and MSCs proved their safety and efficacy, MSC studies showed a greater therapeutic effect over HSCs. Adipose MSCs and Bone Marrow Derived MSCs reached similar clinical healing rates, with studies backing support for both sides. Across the board, autologous stem cell transplants proved to be safer and more effective compared to their allogeneic counterparts.

Co-transplantation of Human Fetal Mesenchymal and Hematopoietic Stem Cells in Type 1 Diabetic Mice Model.

Introduction: Cell therapy can overcome the limitation of conventional treatments (including different medications and β cell replacement) for type 1 diabetes. Based- on several studies human fetal mesenchymal and hematopoietic stem cells are ideal candidates for stem cell therapy. On the other hand, co-transplantation of them can improve their effects. Accordingly, the aim of this research is co-transplantation of human fetal mesenchymal and hematopoietic stem cells in type 1 diabetes.Materials and Methods: The liver of legally aborted fetus was harvested. Then, mononuclear cells were isolated and extracted mesenchymal stromal cells and CD34+ hematopoietic stem cells were cultured. Expression of pluripotency markers were evaluated. For molecular imaging, mesenchymal stromal cells were labeled using GFP- vector. BALB/c inbred male mice were modeled by injection a single dose of Streptozotocin. Diabetic animals were received stem cells. After stem cell transplantation, in vivo imaging was performed and blood glucose levels were measured weekly.Results: Fetal mesenchymal stromal cells were demonstrated differentiation potential. Expression of pluripotency markers were positive. The mean of blood glucose levels were reduced in mixed mesenchymal and hematopoietic stem cells transplantation. A lot of GFP-labeled mesenchymal stem cells were engrafted in the pancreas of animal models that received a mixed suspension of hematopoietic and mesenchymal stromal cells.Conclusions: Human fetal stem cells are valuable source for cell therapy and co-transplantation of mesenchymal stromal cells can improve therapeutic effects of hematopoietic stem cells.

Effect of Hematopoietic Stem Cells and Platelet-Rich Plasma on the Healing of Experimental Skin Burned Tissues: A Comparative Study in Adult Male Mice

Background: Unfortunately burns are a common problem, leading to scarring or death. Platelet rich plasma (PRP) is a well known method that could be used as topical application for burn either second or third degree; it harbors many growth factors that can accelerate the healing. This Study Aimed: To analyze the use of PRP in deep second-degree burn, in comparison with the hematopoietic stem cells (CD34+) in mice. Materials and Methods: Seventy male adult mice were divided into four groups (control group, burn only, burn treated with CD34+ cells (injected once intradermal with 0.3–0.4 × 106 /kg and burn treated with PRP (1 ml injected once intradermal) at the edge of burn after 24hs from its induction. All burned groups were exposed to thermal burn. On day 9 and 19, the animals were sacrificed and skin biopsies were taken for H&E and Masson's trichrome staining. Assessment of angiogenesis and quantification study for matrix metalloproteinase 13 (MMP13) was done. Results: Wounds treated with PRP revealed fast wound closure in comparison with burned only group. Significant decrease in collagen fibers and increase in MMP13 deposition, as well as angiogenic markers (Angio1 and 2 and VEGF) expression was observed. However CD34+ cells treated group showed highly significant improvement in previously mentioned markers. Histopathological changes of burned skin improved in all treated groups after 19 days particularly in CD34 + cells treated group. Conclusion: These results indicated that CD34+ cells treatment could exert beneficial effects on healing process more than that of PRP in second degree burns.

Transplantation of Hematopoietic Stem Cells Promotes Functional Improvement Associated with NT-3-MEK-1 Activation in Spinal Cord-Transected Rats.

Transected spinal cord injury (SCT) is a devastating clinical disease that strongly affects a patient’s daily life and remains a great challenge for clinicians. Stem-cell therapy has been proposed as a potential therapeutic modality for SCT. To investigate the effects of hematopoietic stem cells (HSCs) on the recovery of structure and function in SCT rats and to explore the mechanisms associated with recovery, 57 adult Sprague-Dawley rats were randomly divided into sham (n = 15), SCT (n = 24), and HSC transplantation groups (n = 15). HSCs (passage 3) labeled by Hoechst 33342, were transplanted intraspinally into the rostral, scar and caudal sites of the transected lesion at 14 days post-operation. Both in vitro and in vivo, HSCs exhibited a capacity for cell proliferation and differentiation. Following HSC transplantation, the animals’ Basso, Beattie, and Bresnahan (BBB). locomotion scale scores increased significantly between weeks 4 and 24 post-SCT, which corresponded to an increased number of 5-hydroxytryptamine (5-HT) fibers and oligodendrocytes. The amount of astrogliosis indicated by immunohistochemical staining, was markedly decreased. Moreover, the decreased expression of neurotrophin- 3 (NT-3) and mitogen-activated protein kinase kinase-1 (MEK-1) after SCT was effectively restored by HSC transplantation. The data from the current study indicate that intraspinally administered HSCs in the chronic phase of SCT results in an improvement in neurological function. Further, the results indicate that intraspinally administered HSCs benefit the underlying mechanisms involved in the enhancement of 5-HT-positive fibers and oligogenesis, the suppression of excessive astrogliosis and the upregulation of NT3-regulated MEK-1 activation in the spinal cord. These crucial findings reveal not only the mechanism of cell therapy, but may also contribute to a novel therapeutic target for the treatment of spinal cord injury (SCI).

Effect of hematopoietic stem cell on tumor necrosis factor-毩expression, spiral artery remodeling and placental apoptosis in lead-exposed pregnant mice

Objective: To evaluate the effectiveness of hematopoietic stem cell (HSC) on TNF-α expression, spiral artery remodeling, and placental apoptosis. Methods: The study used pregnant mice divided to five groups of treatment: Group 1, without treatment; Group 2, 1/12 LD50 of lead and Aquadest; Group 3, 1/16 LD50 of lead and Aquadest, Group 4, 1/12 LD50 of lead and 1 × 107 HSC; and group 5, 1/16 LD50 of lead and 1 × 107 HSC. Histopathology examination was performed to identify spiral artery remodeling. Immunohistochemistry analysis was conducted to characterize TNF-α expression and the apoptosis index. The data from all five groups were compared using the ANOVA test. Results: TNF-α expression differed significantly among the groups that were not exposed to lead, exposed to lead and exposed to lead after HSC treatment (P 0.05). Conclusions: In lead-exposed pregnant mice, the administration of hematopoietic stem cell may decrease TNF-α expression, spiral artery remodeling, and placental apoptosis index but in the group of 1/16 LD50 of lead and HSC treatment, apoptosis index has no differences with those of control group.

Effectiveness of Search for an Unrelated Donor of Hematopoietic Stem Cells using Russian Date Base "Bone Marrow Donor Search": Experience of Raisa Gorbacheva Memorial Institute for Children Oncology, Hematology and Transplantation

Background & Aims. The key condition for allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the presence of HLA-compatible related or unrelated donor. If related donor is not found, further search is carried out in the Bone Marrow Donor Worldwide (BMDW) international data base, which is not effective enough (about 80-85 %), because of genotype specificity of Russian Federation residents. The recruitment procedure using BMDW takes a lot of time and is expensive. Therefore, there are good reasons to develop an alternative Russian data base, Bone Marrow Donor Search (BMDS), which includes data from Russian bone marrow donor registries and has a good potential. The aim is to evaluate the effectiveness of hematopoietic stem cell (HSC) donor search and transplant quality using the BMDS search system. Methods. 34 allo-HSCT recipients with malignancies and hematological diseases were enrolled in the study in RM Gorbacheva Scientific Research Institute of Pediatric Hematology and Transplantation from November, 2012, to March, 2016. A HLA-compatible donor was found for each patient in the BMDS (www.bmds.info), which includes data from 13 Russian registries of HSC donors. Results. 34 allo-HSCTs were performed from unrelated donors recruited using Russian registries: 1 in 2012; 3 in 2013; 5 in 2014; 21 in 2015; and 4 in the 1<sup>st</sup> quarter of 2016. The greatest effectiveness of the BMDS search was in 2015 (14 %, n = 17). In 30 cases (88.2 %) a complete 10/10 compatibility for 5 HLA-gene loci was observed; in 4 cases (11.8 %) there was an incomplete compatibility (9/10). AB0 compatibility was only in 7 cases (20.6 %). In 15 cases (44.1 %) bone marrow was used for transplant harvesting; in 19 cases (55.9 %) peripheral blood stem cells were harvested by means of cytapheresis. The CD34+ count in the transplant was 1.2-12.0 х 10<sup>6</sup> CD34+ cell/kg (median: 5.0 * 10<sup>6</sup> CD34+ cell/kg). Engraftment was observed in 79.4 % of cases (п = 27), graft failure in 17.7 % of cases (n = 6), and early posttransplant mortality in 2.9 % of cases (n = 1). Conclusion. There was an increasing efficiency of search for a HLA-compatible unrelated HSC donor using a Russian BMDS search system for Russian residents with a graft quality similar to the one found in the international BMDW database.

Abstract B34: Single cell RNA sequencing identifies the NRASG12V-mediated AML self-renewal signature.

Abstract Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Using a murine model of AML that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V), we have found that NRASG12V enforced leukemia self-renewal gene expression signatures and was required to maintain an MLL-AF9 and Myb-dependent leukemia self-renewal gene expression program. In functional assays, NRASG12V was required for leukemia self-renewal independently of its effects on growth and survival (Sachs et al. in revisions). Similarly, recent work has shown that NRASG12V has bimodal, mutually exclusive effects in hematopoietic stem cells (HSCs) providing a proliferation advantage in some HSCs and a self-renewal advantage in others (Li et al. Nature 2013). These data show that mutant NRAS enforces different behaviors in similar cells and suggest that these discrepant effects may be mediated by different NRAS-activated pathways and gene expression pathways. In order to define the mechanisms by which NRASG12V directs self-renewal in AML, we investigated the NRASG12V-mediated transcriptional profile of self-renewing leukemic subgroups and single cells from our AML model. We sorted our primary murine AML into immunophenotypic subgroups and performed bulk cell RNA sequencing on these groups. The Mac-1High group displayed poor self-renewal capacity in colony forming assays and in vivo leukemia transplant assays. In contrast, the Mac-1Low/c-Kit+/Sca-1+ (MLowKS) subgroup was enriched for self-renewal capacity and displayed significantly increased colony forming ability and enhanced in vivo leukemia transplantability. Using principal component analysis and unsupervised hierarchical clustering, we found that each of our immunophenotypic subpopulations were transcriptionally distinct. Using this dataset, we identified the NRASG12V-directed leukemia self-renewal gene expression signature. Gene Set Enrichment Analysis (GSEA, et al. 2005) of this dataset showed that the MLowKS group preferentially expressed previously published transcriptional signatures of leukemia self-renewal. These data also show that NRASG12V enforces distinct gene expression signatures among leukemic subpopulations. Since others have shown that NRASG12V can enforce bimodal behaviors among cells within the same hematopoietic compartment (HSCs, Li et al. Nature 2013), we speculated that the NRASG12V–mediated transcriptional signature critical for leukemia self-renewal may be variable among individual cells. In order to identify that signature and to assess the cell-to-cell heterogeneity of NRASG12V-mediated transcription, we performed single cell RNA sequencing of unsorted and sorted leukemia cells from our murine AML model. In unsupervised hierarchical clustering algorithms, our single cells clustered into five distinct transcriptional signatures, demonstrating that individual leukemia cells are transcriptionally distinct and identifying the NRASG12V-mediated leukemia self renewal signature. Currently, we employ ultra-high parameter flow cytometry (mass cytometry, CyTOF) to identify the cell type specific signaling pathways associated with these discrepant transcriptional signatures. These data identify the NRASG12V-mediated self-renewal gene expression signature at the single cell level and will allow us to identify the specific signaling pathways that activate this signature. Citation Format: Zohar Sachs, Rebecca S. LaRue, Klara Noble, Susan K. Rathe, Aaron L. Sarver, Ngoc A. Ha, David A. Largaespada. Single cell RNA sequencing identifies the NRASG12V-mediated AML self-renewal signature. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B34.

Abstract B15: NRASG12V oncogene mediates self-renewal in a murine model of acute myelogenous leukemia

Abstract Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V). We performed gene expression microarray and RNA sequencing of our AML cells in the presence and absence of NRASG12V. By employing computational approaches to explore NRASG12V-responsive genes in our model, we found that NRASG12V enforced the leukemia self-renewal gene expression signature and was required to maintain an MLL-AF9 and Myb-dependent leukemia self-renewal gene expression program. In functional assays, NRASG12V was required for leukemia self-renewal independently of its effects on growth and survival. We used CyTOF (mass cytometry) for a multiplexed analysis of RAS-dependent signaling intermediates, and found that Mac-1Low cells, which harbor leukemia stem cells, were preferentially sensitive to NRASG12V withdrawal. Using RAS-pathway inhibitors, we found NRASG12V maintained leukemia self-renewal through mTor and Mek pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction. Recent work has shown that NRASG12V has bimodal effects in hematopoietic stem cells (Li et al. Nature 2013). To understand the mechanism of these bimodal effects, we have performed single cell RNA sequencing on our AML model. We expect that these analyses will reveal the cell-type specific NRASG12V –mediated mechanisms of leukemia self renewal. Citation Format: Zohar Sachs, Rebecca S. LaRue, Hanh T. Nguyen, Karen Sachs, Nurul Azyan Mohd Hassan, Ernesto Diaz-Flores, Susan K. Rathe, Aaron L. Sarver, Sean C. Bendall, Ngoc A. Ha, Miechaleen D. Diers, Garry P. Nolan, Kevin M. Shannon, David A. Largaespada. NRASG12V oncogene mediates self-renewal in a murine model of acute myelogenous leukemia. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B15. doi: 10.1158/1557-3125.RASONC14-B15

Potential Therapeutic Effect of Hematopoietic Stem Cells on Cerebellar Ataxia in Adult Female Rats Subjected to Cerebellar Damage by Monosodium Glutamate

Background: Research evidence has indicated that monosodium glutamate (MSG) consumption produces certain deleterious effects on the cerebellum of adult rats at high doses which can consequently affect cerebellar function. The use of stem cells in nervous system disorders is a growing field, which in numerous reports has shown promising results in the restoration of neurological function. Aim: To compare the effect of injection of human umbilical cord blood CD34+ stem cells versus CD34- fraction in a rat model of cerebellar damage induced by monosodium glutamate. Methods: Forty adult female albino rats were equally randomized into 4 groups: group I served as control, group II received MSG, group III received MSG followed by CD34+ stem cell separated from umbilical cord blood of human male fetuses, group IV received MSG followed by the CD34- fraction. At the end of the experiment, all rats were subjected to assessment of motor function, histological and immunohistochemical techniques as well as a polymerase chain reaction analysis of male-specific Sry gene. Results: Group II showed a significant decrease in the mean number of Purkinje cells and cells of the molecular layer. Nissl’s granules and length of dendrites of Purkinje cells were markedly decreased. Marked increase of GFAP immunoexpression in astrocytes was also detected. Group III stem cells showed improvement in motor function after 4 weeks of treatment. The CD34- group (IV) showed more increase in the number of cells in the molecular, granular and Purkinje cell layers as well as an increase in Nissl’s granules and Purkinje cell dendrite length compared to CD34+ stem cell group (III). There was also a significant decrease in optical density of GFAP immunoexpression of the CD 34- group compared to both MSG and CD34+ groups. The Sry gene was not detected in either of the CD34+ and CD34- groups implying that the improvement happened without homing of stem cells in the cerebellum. Conclusion: Both CD34 -ve and CD34+ve stem cells improved cerebellar structure and function against damage induced by monosodium glutamate; however CD34- stem cells showed more improvement than CD34+ stem cells.

Fbxw7-dependent Degradation of Notch Is Required for Control of “Stemness” and Neuronal-Glial Differentiation in Neural Stem Cells

Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7(F/F) mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stem cells was sustained in association with the accumulation of Notch1 and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stem cells in the brain.

The effects of hematopoietic stem cells on mice vascular endothelial cells of diabetic retinopathy

The purpose of the study was to determine whether hematopoietic stem cell (HSCs) mobilization can regulate early diabetic retinopathy in mice. Mice were divided into four groups: control group, normal mice HSC-mobilized group, diabetic mice control group and diabetic mice HSC-mobilized group. Murine stem cell growth factor (SCF) and recombined human granulocyte colony stimulating factor (rhG-csf) were administered to the mice with diabetes and without diabetes for continuous 5 days to induce autologous HSCs mobilization, and subcutaneous injection of physiological saline was used as control. The changes associated with autologous HSCs mobilization were characterized using flow cytometry, Immunohistochemistry and semiquantitative RT-PCR. Evans blue quantitative test was used to measure the breakdown level of blood-retina barrier. HSCs were marked by CD34-/low and Sca1+ in this study. The acceleration of endothelial cell regeneration was observed. A decrease of VEGF expression due to autologous stem cell mobilization was found. HSCs could increase the content of VEGFR-2 in mouse retina and significantly downregulated the expression of VEGF and ang-2 in diabetic mice. The experiment suggest that autologous HSCs mobilization can be approach of therapeutic vascular reconstruction and functional restoration of blood-retina barrier in mice.