A Canadian perception of the nursing practice, research, and theoretical implications associated with Autologous Cell Transplantation

Heart failure is a progressive disorder. An estimated 400,000 Canadians are diagnosed annually with heart failure, and a quarter experience severe heart failure that is unresponsive to medical therapy. Autologous cell transplantation (ACT) has been proposed as a new approach for cardiac repair, and holds enormous potential for the regeneration of injured myocardium cells. Currently, ACT is under investigation in Canada. The use of ACT as a treatment alternative for heart failure patients has been established over the past 5 years across Europe and the United States. This paper will present a Canadian perception of the nursing practice, research, and theoretical implications associated with this new and innovative therapy.

A Canadian perception of the nursing practice, research, and theoretical implications associated with Autologous Cell Transplantation

Heart failure is a progressive disorder. An estimated 400,000 Canadians are diagnosed annually with heart failure, and a quarter experience severe heart failure that is unresponsive to medical therapy. Autologous cell transplantation (ACT) has been proposed as a new approach for cardiac repair, and holds enormous potential for the regeneration of injured myocardium cells. Currently, ACT is under investigation in Canada. The use of ACT as a treatment alternative for heart failure patients has been established over the past 5 years across Europe and the United States. This paper will present a Canadian perception of the nursing practice, research, and theoretical implications associated with this new and innovative therapy.

A Canadian perception of the nursing practice, research, and theoretical implications associated with Autologous Cell Transplantation

Heart failure is a progressive disorder. An estimated 400,000 Canadians are diagnosed annually with heart failure, and a quarter experience severe heart failure that is unresponsive to medical therapy. Autologous cell transplantation (ACT) has been proposed as a new approach for cardiac repair, and holds enormous potential for the regeneration of injured myocardium cells. Currently, ACT is under investigation in Canada. The use of ACT as a treatment alternative for heart failure patients has been established over the past 5 years across Europe and the United States. This paper will present a Canadian perception of the nursing practice, research, and theoretical implications associated with this new and innovative therapy.

Surgical Injury and Ischemia Prime the Adipose Stromal Vascular Fraction and Increase Angiogenic Capacity in a Mouse Limb Ischemia Model

The adipose-derived stromal vascular fraction (SVF) is an effective source for autologous cell transplantation. However, the quality and quantity of SVFs vary depending on the patient’s age, complications, and other factors. In this study, we developed a method to reproducibly increase the cell number and improve the quality of adipose-derived SVFs by surgical procedures, which we term “wound repair priming.” Subcutaneous fat from the inguinal region of BALB/c mice was surgically processed (primed) by mincing adipose parenchyma (injury) and ligating the subcutaneous fat-feeding artery (ischemia). SVFs were isolated on day 0, 1, 3, 5, or 7 after the priming procedures. Gene expression levels of the primed SVFs were measured via microarray and pathway analyses which were performed for differentially expressed genes. Changes in cellular compositions of primed SVFs were analyzed by flow cytometry. SVFs were transplanted into syngeneic ischemic hindlimbs to measure their angiogenic and regeneration potential. Hindlimb blood flow was measured using a laser Doppler blood perfusion imager, and capillary density was quantified by CD31 staining of ischemic tissues. Stabilization of HIF-1 alpha and VEGF-A synthesis in the SVFs were measured by fluorescent immunostaining and Western blotting, respectively. As a result, the number of SVFs per fat weight was increased significantly on day 7 after priming. Among the differentially expressed genes were innate immunity-related signals on both days 1 and 3 after priming. In primed SVFs, the CD45-positive blood mononuclear cell fraction decreased, and the CD31-CD45-double negative mesenchymal cell fraction increased on day 7. The F4/80-positive macrophage fraction was increased on days 1 and 7 after priming. There was a serial decrease in the mesenchymal-gated CD34-positive adipose progenitor fraction and mesenchymal-gated CD140A-positive/CD9-positive preadipocyte fraction on days 1 and 3. Transplantation of primed SVFs resulted in increased capillary density and augmented blood flow, improving regeneration of the ischemic limbs. HIF-1 alpha was stabilized in the primed cutaneous fat in situ, and VEGF-A synthesis of the primed SVFs was on a peak on 5 days after priming. Wound repair priming thus resulted in SVFs with increased number and augmented angiogenic potential.

Recent Progress in Gene Therapy and Other Targeted Therapeutic Approaches for Beta Thalassemia

Beta-thalassemia is a genetic disorder characterized by the impaired synthesis of the betaglobin chain of adult hemoglobin. The disorder has a complex pathophysiology that affects multiple organ systems. The main complications of beta thalassemia are ineffective erythropoiesis, chronic hemolytic anemia and hemosiderosis-induced organ dysfunction. Regular blood transfusions are the main therapy for beta thalassemia major; however, this treatment can cause cardiac and hepatic hemosiderosis – the most common cause of death in these patients. This review focuses on unique future therapeutic interventions for thalassemia that reverse splenomegaly, reduce transfusion frequency, decrease iron toxicity in organs, and correct chronic anemia. The targeted effective protocols include hemoglobin fetal inducers, ineffective erythropoiesis correctors, antioxidants, vitamins, and natural products. Resveratrol is a new herbal therapeutic approach which serves as fetal Hb inducer in beta thalassemia. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for beta thalassemia major and is preferred over iron chelation and blood transfusion for ensuring long life in these patients. Meanwhile, several molecular therapies, such as ActRIIB/IgG1 Fc recombinant protein, have emerged to address complications of beta thalassemia or the adverse effects of current drugs. Regarding gene correction strategies, a phase III trial called HGB-207 (Northstar-2; NCT02906202) is evaluating the efficacy and safety of autologous cell transplantation with LentiGlobin. Advanced gene-editing approaches aim to cut DNA at a targeted site and convert HbF to HbA during infancy, such as the suppression of BCL11A (B cell lymphoma 11A), HPFH (hereditary persistence of fetal hemoglobin) and zinc-finger nucleases. Gene therapy is progressing rapidly, with multiple clinical trials being conducted in many countries and the promise of commercial products to be available in the near future.

Diabetic Ephrin-B2-Stimulated Peripheral Blood Mononuclear Cells Enhance Poststroke Recovery in Mice

Clinical trials of cell therapy in stroke favor autologous cell transplantation. To date, feasibility studies have used bone marrow-derived mononuclear cells, but harvesting bone marrow cells is invasive thus complicating bedside treatment. We investigated the therapeutic potential of peripheral blood-derived mononuclear cells (PB-MNC) harvested from diabetic patients and stimulated by ephrin-B2 (PB-MNC+) (500,000 cells), injected intravenously 18–24 hours after induced cerebral ischemia in mice. Infarct volume, neurological deficit, neurogenesis, angiogenesis, and inflammation were investigated as were the potential mechanisms of PB-MNC+ cells in poststroke neurorepair. At D3, infarct volume was reduced by 60% and 49% compared to unstimulated PB-MNC and PBS-treated mice, respectively. Compared to PBS, injection of PB-MNC+ increased cell proliferation in the peri-infarct area and the subventricular zone, decreased microglia/macrophage cell density, and upregulated TGF-βexpression. At D14, microvessel density was decreased and functional recovery was enhanced compared to PBS-treated mice, whereas plasma levels of BDNF, a major regulator of neuroplasticity, were increased in mice treated with PB-MNC+ compared to the other two groups. Cell transcriptional analysis showed that ephrin-B2 induced phenotype switching of PB-MNC by upregulating genes controlling cell proliferation, inflammation, and angiogenesis, as confirmed by adhesion and Matrigel assays.Conclusions. This feasibility study suggests that PB-MNC+ transplantation poststroke could be a promising approach but warrants further investigation. If confirmed, this rapid, noninvasive bedside cell therapy strategy could be applied to stroke patients at the acute phase.

ID:2049 Effect of human umbilical cord-derived MSCs on the proliferation of peripheral blood T lymphocytes with different level HLA matching

Human umbilical cord-derived MSCs are very attractive sources of stem cells for the non-autologous cell transplantation therapy to treat many kinds of diseases based on their efficiency in treating graft versus host disease (GVHD), a condition that patient might suffer after receive cell-transplantation. However, the immune properties of MSCs are also critical for their efficiency in regenerative medicine applications. Thus, the mechanism of their action is now under intense investigation. A number of researches showed that MSCs exert strong anti-inflammatory and immunosuppressive effects on the main immune cell subsets through their production of various soluble factors. As a result, scientists all over the globe are changing their attention to the immunomodulatory property of MSCs, hence we are not an exception. Our research was carried out in order to strengthen our knowledge about this spectacular aspect of MSCs, especially the effect of cord-derived MSCs on the proliferation of peripheral blood T lymphocytes with different level HLA matching. Our results indicated that at passage 4 of culture, human umbilical cord-derived MSCs had no effect on the proliferation of T cells with all blood sample tested.

CRISPR/Cas9 Mediated Gene Repair in Inherited Bleeding Disorders

[Introduction] Inherited bleeding disorders (IBD), such as coagulation factor deficiencies, Von Willebrand disease and Glanzmann thrombasthenia, are caused by various gene abnormalities of coagulation proteins, blood vessels, and platelets. IBD have been considered to be suited for gene therapy and clinical trials are ongoing. However, the safety and effectiveness of viral vectors has not been established. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, originates from the archaeal and bacterial adaptive immunity system, provides an efficient genome-editing tool in various organisms including the mammalian genome and holds potential for gene therapy. Here, we report an application of this system to gene repair using induced pluripotent stem cells (iPSCs) derived from patients of three types of IBD. [Case1] Hemophilia B (63-year-old male). Factor IX (FIX) activity was less than 1% (normal range (NR) 70-130%) and antigen level was 2.37μg/ml (average 5.0μg/ml). Molecular analysis of the FIX gene revealed an in-frame deletion in exon 2. [Case2] Factor V (FV) deficiency (55-years-old female). FV activity was less than 3% (NR 70-135%) and antigen level was less than 2% (NR 60-150%). A homozygous missense mutation was detected in FV gene of exon 14. [Case3] Factor X (FX) deficiency (4-years-old male). FX activity was less than 2.84 IU/dl (NR 50-150 IU/dl) and antigen level was 0.567 IU/dl (NR 50-150 IU/dl). A compound heterozygous missense mutation was found in FX gene of exon 6 and 8 respectively. [Methods and results] The CRISPR/Cas system comprises of a Cas9 nuclease and a sequence-specific guide RNA (gRNA). We designed gRNAs close to gene mutations. We transfected both expression vectors into HT-1080 or 293T cells, and assessed the editing activity by SURVEYOR nuclease assay. In order to repair the mutations by homology-directed repair (HDR), we prepared targeting constructs with homology arms (1.0 kbp in length) containing the corrected sequence. After introduction of Cas9, gRNA and targeting plasmid into each iPSCs generated from peripheral blood mononuclear cells (PBMCs) using Sendai virus vector expressing the Yamanaka 4-factor genes (Oct3/4, Klf4, Sox2 and c-Myc), we could obtain iPSC clones with corrected genes by HDR from all of three IBD patients. Successful HDR events were verified by PCR amplification using integration site- and targeting construct-specific primers. Locus-specific knock-in events were confirmed by Southern blot analysis. [Conclusion] We observed the cleavage of the target genome by using our designed gRNAs. Furthermore, the CRISPR/Cas system induced successful gene repair of iPSCs from three IBD patients. We are preparing hepatocytes induced from repaired iPSCs to confirm corrected coagulation factor synthesis. Gene-corrected iPSCs hold great promise as a cell source for autologous cell transplantation. Disclosures No relevant conflicts of interest to declare.

Abstract 11791: Blockade of Senescence-associated Microrna-195 in Aged Skeletal Myoblasts Facilitates Reprogramming to Produce Induced Pluripotent Stem Cells

Background: Older age is the major risk factor for heart failure, and reprogramming a patient’s own cells to produce induced pluripotent stem cells (iPSCs) is a promising strategy for autologous cell transplantation therapy. However, low reprogramming efficiency of senescent cells remains as a major pitfall. Recently, our preliminary data suggested that inhibiting senescence-associated miR-195 rejuvenated aged stem cells by reactivating anti-aging defense system. This study investigated the effects of blocking miR-195 expression on the reprogramming efficiency of old skeletal myoblasts (OSkMs). Methods and Results: MiR-195 expression was significantly higher in OSkMs isolated from aged mice (24 months) as compared to those from young mice (2 months), as examined by RT-PCR. OSkMs showed impaired expression of anti-aging factors (Tert and Sirt1) and higher expression of pro-aging markers (p53, p21, p16). Intriguingly, blocking miR-195 expression in OSkMs by transfection with anti-miR-195 significantly induced restoration of Tert and Sirt1 as well as telomere re-lengthening as examined by RT-PCR and quantitative fluorescent in situ hybridization. Luciferase assay confirmed that Sirt1 is one of the direct targets of miR-195 relevant to senescence of OSkMs. Importantly, lower reprogramming efficiency of OSkMs was significantly improved by miR-195 abrogation without altering karyotype or expression of pluripotency markers. Furthermore, iPSCs lacking miR-195 successfully differentiated into all three germ layers, indicating that deletion of miR-195 does not affect pluripotency. Notably, contraction rates were markedly higher in beating cells transfected with anti-miR-195 as compared to that with scramble (68.5±5.6 vs 47.3±2.8/min). Conclusions: Blocking age-induced miR-195 is a novel promising approach for efficient iPSCs generation from senescent cells, which has beneficial for autologous transplantation of iPSCs in elderly patients.