PARP1 Characterization as a Potential Biomarker for BCR::ABL1 p190+ Acute Lymphoblastic Leukemia.

Detection of t(9;22), and consequent BCR::ABL1 fusion, is still a marker of worse prognosis for acute lymphoblastic leukemia (ALL), with resistance to tyrosine-kinase inhibitor therapy being a major obstacle in the clinical practice for this subset of patients. In this study, we investigated the effectiveness of targeting poly-ADP-ribose polymerase (PARP) in a model of BCR::ABL1 p190+ ALL, the most common isoform to afflict ALL patients, and demonstrated the use of experimental PARP inhibitor (PARPi), AZD2461, as a therapeutic option with cytotoxic capabilities similar to that of imatinib, the current gold standard in medical care. We characterized cytostatic profiles, induced cell death, and biomarker expression modulation utilizing cell models, also providing a comprehensive genome-wide analysis through an aCGH of the model used, and further validated PARP1 differential expression in samples of ALL p190+ patients from local healthcare institutions, as well as in larger cohorts of online and readily available datasets. Overall, we demonstrate the effectiveness of PARPi in the treatment of BCR::ABL1 p190+ ALL cell models and that PARP1 is differentially expressed in patient samples. We hope our findings help expand the characterization of molecular profiles in ALL settings and guide future investigations into novel biomarker detection and pharmacological choices in clinical practice.

Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo.

Introduction: Natural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary. Methods: In this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence. Results: We demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model. Conclusion: Together with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals.

Telomeric repeat-containing RNA is dysregulated in acute myeloid leukemia.

TERRA (telomeric repeat-containing RNA) is a class of long noncoding RNAs transcribed from subtelomeric and telomeric regions. TERRA binds to the subtelomeric and telomeric DNA-forming R-loops (DNA-RNA hybrids), which are involved in telomere maintenance and telomerase function, but the role of TERRA in human cells is not well characterized. Here, we comprehensively investigated for the first time TERRA expression in primary human hematopoietic cells from an exploratory cohort of patients with acute myeloid leukemia (AML), patients with acute lymphoblastic leukemia (ALL), patients with telomere biology disorder (TBD), and healthy subjects. TERRA expression was repressed in primary human hematopoietic cells, including healthy donors, patients with ALL, and patients with TBD, irrespective of their telomere length, except for AML. A second cohort comprising 88 patients with AML showed that TERRA was overexpressed in an AML subgroup also characterized by higher R-loop formation, low TERT and RNAseH2 expression, and a paucity of somatic splicing factor mutations. Telomere length did not correlate with TERRA expression levels. To assess the role of TERRA R-loops in AML, we induced R-loop depletion by increasing RNAseH1 expression in 2 AML cell lines. Decreased TERRA R-loops in AML cell lines resulted in increased chemosensitivity to cytarabine. Our findings indicate that TERRA is uniformly repressed in primary human hematopoietic cells but abnormally expressed in an AML subset with low telomerase.

Genome Editing for Engineering the Next Generation of Advanced Immune Cell Therapies.

Our current genetic engineering capacity through synthetic biology and genome editing is the foundation of a revolution in biomedical science: the use of genetically programmed cells as therapeutics. The prime example of this paradigm is the adoptive transfer of genetically engineered T cells to express tumor-specific receptors, such as chimeric antigen receptors (CARs) or engineered T-cell receptors (TCR). This approach has led to unprecedented complete remission rates in patients with otherwise incurable hematological malignancies. However, this approach is still largely ineffective against solid tumors, which comprise the vast majority of neoplasms. Also, limitations associated with the autologous nature of this therapy and shared markers between cancer cells and T cells further restrict the access to these therapies. Here, we described how cutting-edge genome editing approaches have been applied to unlock the full potential of these revolutionary therapies, thereby increasing therapeutic efficacy and patient accessibility.

Xenogeneic mesenchymal stem cell biocurative improves skin wounds healing in diabetic mice by increasing mast cells and the regenerative profile.

Introduction: Diabetes mellitus (DM) is a chronic disease and a major cause of mortality and morbidity worldwide. The hyperglycemia caused by DM induces micro and macrovascular complications that lead, among other consequences, to chronic wounds and amputations. Cell therapy and tissue engineering constitute recent therapeutic alternatives to improve wound healing in diabetic patients. The current study aimed to analyze the effectiveness of biocuratives containing human mesenchymal stem cells (MSCs) associated with a hydrogel matrix in the wound healing process and related inflammatory cell profile in diabetic mice. Methods: Biocuratives containing MSCs were constructed by 3D bioprinting, and applied to skin wounds on the back of streptozotocin (STZ)-induced type 1 diabetic (T1D) mice. The healing process, after the application of biocuratives with or without MSCs was histologically analyzed. In parallel, genes related to growth factors, mast cells (MC), M1 and M2 macrophage profiles were evaluated by RT-PCR. Macrophages were characterized by flow cytometry, and MC by toluidine blue staining and flow cytometry. Results: Mice with T1D exhibited fewer skin MC and delayed wound healing when compared to the non-diabetic group. Treatment with the biocuratives containing MSCs accelerated wound healing and improved skin collagen deposition in diabetic mice. Increased TGF-ß gene expression and M2 macrophage-related markers were also detected in skin of diabetic mice that received MSCs-containing biocuratives. Finally, MSCs upregulated IL-33 gene expression and augmented the number of MC in the skin of diabetic mice. Conclusion: These results reveal the therapeutic potential of biocuratives containing MSCs in the healing of skin wounds in diabetic mice, providing a scientific base for future treatments in diabetic patients.

Chimeric antigen-receptor (CAR) engineered natural killer cells in a chronic myeloid leukemia (CML) blast crisis model.

During the last two decades, the introduction of tyrosine kinase inhibitors (TKIs) to the therapy has changed the natural history of CML but progression into accelerated and blast phase (AP/BP) occurs in 3-5% of cases, especially in patients resistant to several lines of TKIs. In TKI-refractory patients in advanced phases, the only curative option is hematopoietic stem cell transplantation. We and others have shown the relevance of the expression of the Interleukin-2-Receptor α subunit (IL2RA/CD25) as a biomarker of CML progression, suggesting its potential use as a therapeutic target for CAR-based therapies. Here we show the development of a CAR-NK therapy model able to target efficiently a blast crisis cell line (K562). The design of the CAR was based on the scFv of the clinically approved anti-CD25 monoclonal antibody (Basiliximab). The CAR construct was integrated into NK92 cells resulting in the generation of CD25 CAR-NK92 cells. Target K562 cells were engineered by lentiviral gene transfer of CD25. In vitro functionality experiments and in vivo leukemogenicity experiments in NSG mice transplanted by K562-CD25 cells showed the efficacy and specificity of this strategy. These proof-of-concept studies could represent a first step for further development of this technology in refractory/relapsed (R/R) CML patients in BP as well as in R/R acute myeloblastic leukemias (AML).

Chimeric antigen-receptor (CAR)-engineered natural killer (NK) cells targeting chronic myeloid Leukemia (CML) blast crisis

Background The use of tyrosine kinase inhibitors (TKIs) has dramatically modified the therapy of chronic myeloid leukemia (CML), generating durable remissions and prolonging survival in TKI-responders. However, progression to blast crisis (BC) still occurs especially in TKI-resistant patients and represents a clinical challenge. We and others have identified IL2RA/CD25 as a typical cell surface marker of BC-CML and reported that its overexpression is correlated with the progression of CML from CP-CML to BC-CML (Imeri et al, Cells 2023). Here we show the experimental development of a third-generation CAR-NK therapy strategy against the CD25 based on the scFV of the clinically approved monoclonal humanized antibody, Basiliximab. Methods: As NK cell model, we have used the NK92 cell line which has a well-established and clinically demonstrated NK cell activity. We have lentivirally transduced NK92 cells with the CAR construct containing a selectable gene (GFP). After FACS-sorting of GFP-positive cells, phenotypical characterization was performed by FACS. The expression of the CAR-CD25 at the surface of the cells was demonstrated an anti-Fab antibody and double-positive (Fab/GFP) cells were further purified. The functionality of the cells was evaluated using CD107a degranulation assay after 3h co-culture and ELISA for IFN-gamma release. We have in parallel engineered K562 cells expressing CD25 by lentiviral transduction (K562-CD25) as well as also a second target cell line (RAJI) using the same strategy. Annexin V staining of target K562 cells was used for in vitro cytotoxicity assessments. For in vivo assays, NSG mice were intraperitoneally (IP) injected with K562-CD25 cells expressing Luciferase at Day-3 (3.10 6 cells/mouse, n = 13). At Days 0, 3, and 7, mice were treated by IP injection of either irradiated CD25 CAR-NK92 cells (10 .10 6/mouse n=6) or irradiated Wild-type (WT)-NK92 cells (n=5). The clinical evolution of mice transplanted mice was followed weekly by luminescence (IVIS 200). Results: After cell sorting, we obtained more than 90% of double-positive NK92 CAR+/GFP+ cells. Lentiviral transduction did not affect the activatory or inhibitory signals of NK92 cells. No statistical differences were observed between CD25 CAR-NK92 and WT- NK92 cells for the expression of NKp30, NKp46, KirDl2-3, TIGIT, and DNAM. However, we observed a strong increase in the Granzyme B and Perforin in CD25 CAR-NK92 cells after co-culture with K562-CD25 as compared to WT NK92 cells (p<0.001). Importantly, we have found increased levels of degranulation after co-culture of target K562-CD25 with CD25-CAR-NK92 cells (40%) as compared to cells co-cultured with WT NK92 alone (20% ) suggesting strongly the occurrence of an additional specific effect due to CAR-CD25. IFN-gamma levels after co-culture of CAR CD25 NK92 cells were also found to be significantly increased ( 400 pg/ml) in as compared to co-cultures of target cells with WT-NK92 (200 pg/ml)(p<0.0001). Similarly,in vitro cytotoxicity assays showed induction of higher levels of apoptosis in target cells (K562-CD25 and Raji-CD25) when co-cultured with CD25 CAR-NK92 as compared to NK92 WT (p<0.0001). In in vivo experiments, we have analyzed K562-CD25 leukemia-bearing mice treated with CAR-NK92 cells (n=6) or WT-NK92 cells (n=5). These experiments analyzed at D+30 post-transplant showed stronger anti-leukemia effect of CAR-NK therapy by IVIS imaging with a survival rate of 84% for mice treated with CD25 CAR-NK92 versus 40% for those treated with WT-NK92. All control mice transplanted with K562-CD25 cells and left untreated died by D+20. Conclusion: We show here for the first time the potential use of an NK cell-mediated CAR therapy strategy targeting CD25 which has been shown to be upregulated in CML blast crisis. The experimental data show a significantly increased and selective in vitro and in vivo cytotoxicity of CD25 CAR-NK92 cells against CD25-expressing leukemia cells as compared to WT-NK92 cells. These results suggest that targeting CD25 by a CD25 CAR based on Basilixiamb's scFV might be an interesting tool in BC-CML and in all acute leukemias overexpressing CD25. In order to translate these findings to NK cells derived from induced pluripotent stem cells (iPSCs), we have produced iPSCs expressing CAR-CD25 constructs and experiments are underway to evaluate the therapeutic potential of iPSC-derived CD25 CAR-NK cells in CML blast crisis or AML models.

Xenogeneic mesenchymal stem cell biocurative improves skin wounds healing in diabetic mice by increasing mast cells and the regenerative profile

Introduction: Diabetes mellitus (DM) is a chronic disease and a major cause of mortality and morbidity worldwide. The hyperglycemia caused by DM induces micro and macrovascular complications that lead, among other consequences, to chronic wounds and amputations. Cell therapy and tissue engineering constitute recent therapeutic alternatives to improve wound healing in diabetic patients. The current study aimed to analyze the effectiveness of biocuratives containing human mesenchymal stem cells (MSCs) associated with a hydrogel matrix in the wound healing process and related inflammatory cell profile in diabetic mice. Methods: Biocuratives containing MSCs were constructed by 3D bioprinting, and applied to skin wounds on the back of streptozotocin (STZ)-induced type 1 diabetic (T1D) mice. The healing process, after the application of biocuratives with or without MSCs was histologically analyzed. In parallel, genes related to growth factors, mast cells (MC), M1 and M2 macrophage profiles were evaluated by RT-PCR. Macrophages were characterized by flow cytometry, and MC by toluidine blue staining and flow cytometry. Results: Mice with T1D exhibited fewer skin MC and delayed wound healing when compared to the non-diabetic group. Treatment with the biocuratives containing MSCs accelerated wound healing and improved skin collagen deposition in diabetic mice. Increased TGF-β gene expression and M2 macrophage-related markers were also detected in skin of diabetic mice that received MSCs-containing biocuratives. Finally, MSCs upregulated IL-33 gene expression and augmented the number of MC in the skin of diabetic mice. Conclusion: These results reveal the therapeutic potential of biocuratives containing MSCs in the healing of skin wounds in diabetic mice, providing a scientific base for future treatments in diabetic patients.

Combination of genetically engineered T cells and immune checkpoint blockade for the treatment of cancer.

Immune checkpoint (IC) blockade using monoclonal antibodies is currently one of the most successful immunotherapeutic interventions to treat cancer. By reinvigorating antitumor exhausted T cells, this approach can lead to durable clinical responses. However, the majority of patients either do not respond or present a short-lived response to IC blockade, in part due to a scarcity of tumor-specific T cells within the tumor microenvironment. Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) or engineered T-cell receptors (TCRs) provide the necessary tumor-specific immune cell population to target cancer cells. However, this therapy has been considerably ineffective against solid tumors in part due to IC-mediated immunosuppressive effects within the tumor microenvironment. These limitations could be overcome by associating adoptive cell transfer of genetically engineered T cells and IC blockade. In this comprehensive review, we highlight the strategies and outcomes of preclinical and clinical attempts to disrupt IC signaling in adoptive T-cell transfer against cancer. These strategies include combined administration of genetically engineered T cells and IC inhibitors, engineered T cells with intrinsic modifications to disrupt IC signaling, and the design of CARs against IC molecules. The current landscape indicates that the synergy of the fast-paced refinements of gene-editing technologies and synthetic biology and the increased comprehension of IC signaling will certainly translate into a novel and more effective immunotherapeutic approaches to treat patients with cancer.

Combination of genetically engineered T cells and immune checkpoint blockade for the treatment of cancer

Immune checkpoint (IC) blockade using monoclonal antibodies is currently one of the most successful immunotherapeutic interventions to treat cancer. By reinvigorating antitumor exhausted T cells, this approach can lead to durable clinical responses. However, the majority of patients either do not respond or present a short-lived response to IC blockade, in part due to a scarcity of tumor-specific T cells within the tumor microenvironment. Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) or engineered T-cell receptors (TCRs) provide the necessary tumor-specific immune cell population to target cancer cells. However, this therapy has been considerably ineffective against solid tumors in part due to IC-mediated immunosuppressive effects within the tumor microenvironment. These limitations could be overcome by associating adoptive cell transfer of genetically engineered T cells and IC blockade. In this comprehensive review, we highlight the strategies and outcomes of preclinical and clinical attempts to disrupt IC signaling in adoptive T-cell transfer against cancer. These strategies include combined administration of genetically engineered T cells and IC inhibitors, engineered T cells with intrinsic modifications to disrupt IC signaling, and the design of CARs against IC molecules. The current landscape indicates that the synergy of the fast-paced refinements of gene-editing technologies and synthetic biology and the increased comprehension of IC signaling will certainly translate into a novel and more effective immunotherapeutic approaches to treat patients with cancer.

Strategies to Enhance the Therapeutic Efficacy, Applicability, and Safety of Genetically Engineered Immune Cells.

The field of cell therapy is leading a paradigm shift in drug development. The recent convergence of several fields, including immunology, genetics, and synthetic biology, now allows for the introduction of artificial receptors and the design of entire genetic circuitries to finely program the behavior of injected cells. A prime example of these next-generation living drugs comes in the form of T cells expressing chimeric antigen receptors (CARs), which have already demonstrated definitive evidence of therapeutic efficacy against some hematological malignancies. However, several obstacles still restrict the antitumor efficacy of and impair the widespread use of CAR-T cells. Critical challenges include limited persistence and antitumor activity in vivo, antigen escape, scarcity of suitable single markers for targeting, and therapy-related toxicity. Nevertheless, intense research activity in this field has resulted in a plethora of creative solutions to address each of these limitations. In this review, we provide a comprehensive snapshot of the current strategies used to enhance the therapeutic efficacy, applicability, and safety of genetically engineered immune cells to treat cancer.

Therapeutic Efficiency of Multiple Applications of Magnetic Hyperthermia Technique in Glioblastoma Using Aminosilane Coated Iron Oxide Nanoparticles: In Vitro and In Vivo Study.

Magnetic hyperthermia (MHT) has been shown as a promising alternative therapy for glioblastoma (GBM) treatment. This study consists of three parts: The first part evaluates the heating potential of aminosilane-coated superparamagnetic iron oxide nanoparticles (SPIONa). The second and third parts comprise the evaluation of MHT multiple applications in GBM model, either in vitro or in vivo. The obtained heating curves of SPIONa (100 nm, +20 mV) and their specific absorption rates (SAR) stablished the best therapeutic conditions for frequencies (309 kHz and 557 kHz) and magnetic field (300 Gauss), which were stablished based on three in vitro MHT application in C6 GBM cell line. The bioluminescence (BLI) signal decayed in all applications and parameters tested and 309 kHz with 300 Gauss have shown to provide the best therapeutic effect. These parameters were also established for three MHT applications in vivo, in which the decay of BLI signal correlates with reduced tumor and also with decreased tumor glucose uptake assessed by positron emission tomography (PET) images. The behavior assessment showed a slight improvement after each MHT therapy, but after three applications the motor function displayed a relevant and progressive improvement until the latest evaluation. Thus, MHT multiple applications allowed an almost total regression of the GBM tumor in vivo. However, futher evaluations after the therapy acute phase are necessary to follow the evolution or tumor total regression. BLI, positron emission tomography (PET), and spontaneous locomotion evaluation techniques were effective in longitudinally monitoring the therapeutic effects of the MHT technique.

Optimized Production of Lentiviral Vectors for CAR-T Cell.

Advances in the use of lentiviral vectors for gene therapy applications have created a need for large-scale manufacture of clinical-grade viral vectors for transfer of genetic materials. Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome of dividing and nondividing cells, resulting in long-term expression of the transgene both in vitro and in vivo. In this chapter, we present a method to transfect human cells, creating an easy platform to produce lentiviral vectors for CAR-T cell application.

Generation of Tumor Cells Expressing Firefly Luciferase (fLuc) to Evaluate the Effectiveness of CAR in a Murine Model.

Immunotherapy has been showed as a promisor treatment, in special for hematological diseases. Chimeric antigen receptor T cells (CARs) which are showing satisfactory results in early-phase cancer clinical trials can be highlighted. However, preclinical models are critical steps prior to clinical trial. In this way, a well-established preclinical model is an important key in order to confirm the proof of principle. For this purpose, in this chapter will be pointed the methods to generate tumor cells expressing firefly Luciferase. In turn, these modified cells will be used to create a subcutaneous and a systemic murine model of Burkitt's lymphoma in order to evaluate the effectiveness of CAR-T.

Titanium with nanotopography induces osteoblast differentiation through regulation of integrin αV.

Topographical modifications of titanium (Ti) at the nanoscale level generate surfaces that regulate several signaling pathways and cellular functions, which may affect the process of osseointegration. Here, we investigated the participation of integrin αV in the osteogenic capacity of Ti with nanotopography. Machined titanium discs (untreated) were submitted to treatment with H2 SO4 /H2 O2 to produce the nanotopography (nanostructured). First, the greater osteogenic capacity of the nanotopography that increased osteoblast differentiation of mesenchymal stem cells compared with untreated topography was shown. Also, the nanostructured surface increased (regulation ≥ 1.9-fold) the gene expression of 6 integrins from a custom array plate utilized to evaluate the gene expression of 84 genes correlated with cell adhesion signaling pathway, including integrin αV, which is involved in osteoblast differentiation. By silencing integrin αV in MC3T3-E1 cells cultured on nanotopography, the impairment of osteoblast differentiation induced by this surface was observed. In conclusion, it was shown that nanotopography regulates the expression of several components of the cell adhesion signaling pathway and its higher osteogenic potential is, at least in part, due to its ability to upregulate the expression of integrin αV. Together with previous data that showed the participation of integrins α1, ß1, and ß3 in the nanotopography osseoinduction activity, we have uncovered the pivotal role of this family of membrane receptors in the osteogenic potential of this surface.

Triple-modal imaging of stem-cells labeled with multimodal nanoparticles, applied in a stroke model.

BACKGROUND: Mesenchymal stem cells (MSCs) have been widely tested for their therapeutic efficacy in the ischemic brain and have been shown to provide several benefits. A major obstacle to the clinical translation of these therapies has been the inability to noninvasively monitor the best route, cell doses, and collateral effects while ensuring the survival and effective biological functioning of the transplanted stem cells. Technological advances in multimodal imaging have allowed in vivo monitoring of the biodistribution and viability of transplanted stem cells due to a combination of imaging technologies associated with multimodal nanoparticles (MNPs) using new labels and covers to achieve low toxicity and longtime residence in cells. AIM: To evaluate the sensitivity of triple-modal imaging of stem cells labeled with MNPs and applied in a stroke model. METHODS: After the isolation and immunophenotypic characterization of human bone marrow MSCs (hBM-MSCs), our team carried out lentiviral transduction of these cells for the evaluation of bioluminescent images (BLIs) in vitro and in vivo. In addition, MNPs that were previously characterized (regarding hydrodynamic size, zeta potential, and optical properties), and were used to label these cells, analyze cell viability via the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay and BLI analysis, and quantify the internalization process and iron load in different concentrations of MNPs via magnetic resonance imaging (MRI), near-infrared fluorescence (NIRF), and inductively coupled plasma-mass spectrometry (ICP-MS). In in vivo analyses, the same labeled cells were implanted in a sham group and a stroke group at different times and under different MNP concentrations (after 4 h or 6 d of cell implantation) to evaluate the sensitivity of triple-modal images. RESULTS: hBM-MSC collection and isolation after immunophenotypic characterization were demonstrated to be adequate in hBM samples. After transduction of these cells with luciferase (hBM-MSCLuc), we detected a maximum BLI intensity of 2.0 x 108 photons/s in samples of 106 hBM-MSCs. Analysis of the physicochemical characteristics of the MNPs showed an average hydrodynamic diameter of 38.2 ± 0.5 nm, zeta potential of 29.2 ± 1.9 mV and adequate colloidal stability without agglomeration over 18 h. The signal of iron load internalization in hBM-MSCLuc showed a close relationship with the corresponding MNP-labeling concentrations based on MRI, ICP-MS and NIRF. Under the highest MNP concentration, cellular viability showed a reduction of less than 10% compared to the control. Correlation analysis of the MNP load internalized into hBM-MSCLuc determined via the MRI, ICP-MS and NIRF techniques showed the same correlation coefficient of 0.99. Evaluation of the BLI, NIRF, and MRI signals in vivo and ex vivo after labeled hBM-MSCLuc were implanted into animals showed differences between different MNP concentrations and signals associated with different techniques (MRI and NIRF; 5 and 20 µg Fe/mL; P < 0.05) in the sham groups at 4 h as well as a time effect (4 h and 6 d; P < 0.001) and differences between the sham and stroke groups in all images signals (P < 0.001). CONCLUSION: This study highlighted the importance of quantifying MNPs internalized into cells and the efficacy of signal detection under the triple-image modality in a stroke model.

Image and motor behavior for monitoring tumor growth in C6 glioma model.

The primary objective of this study is to monitor tumor growth by using image techniques and behavioral testing through general and specific motor activities (spontaneous movements and gait). Our sample includes male Wistar rats, 2 months old and weighing 250-300 g, that is categorized into three groups: control, sham, and experimental. The experimental group was anesthetized; the C6 cells with luciferase expression that were suspended in a culture medium were implanted into the right frontoparietal cortex of the rats. The sham group received implant only with culture medium without cells. Images and behavioral tests were evaluated at base time and at 7, 14, 21, and 28 days after induced tumor growth analysis. The tumor volume measured by magnetic resonance imaging (MRI) and quantitative bioluminescence imaging (BLI) signal showed a correlation coefficient of r = 0.96. The MRI showed that the mean tumor volume increased by approximately 10, 26, and 49 times according to a comparison of tumor volume on the seventh day with 14, 21, and 28 days, respectively. The quantification of the BLI signal was (4.12 ± 2.01) x 10(8), (8.33 ± 3.12) x 10(8), (28.43 ± 6.32) x 10(8), and (63.02 ± 10.53) x 10(8) photons/s at the seventh, fourteenth, twenty-first, and twenty-eighth day, respectively. After 14 days of tumor induction, both behavioral tests showed significant differences between tumor and sham or control groups. Our study showed a high correlation between MRI and BLI for tumor growth monitoring with complement aspects analysis in tumor volume. In addition, functional behavioral analysis displayed sensitivity to monitor tumor growth, as well as to detect early significant changes between groups, primarily in the tumor group. The results of gait analysis were more sensitive than general motor analysis.

Production of Recombinant Factor VIII in Human Cell Lines.

Human cell lines can produce recombinant proteins much more similar to their natural counterpart, compared to other mammalian cell lines, reducing potential immunogenic reactions. Recombinant proteins produced in nonhuman cells can have in its structure glycan epitopes, such as Galα1,3-Gal (alpha-Gal) and N-glycolylneuraminic acid (Neu5Gc) residues, that are antigenic to humans and can potentially affect the efficacy of the recombinant product. Therefore, the production of recombinant factor VIII (rFVIII) in human cell lines is a new approach to avoid nonhuman glycosylation. Here, we describe a protocol to produce rFVIII in the human cell line SK-HEP, using a lentiviral vector to produce high quantities of the recombinant protein.

Pre-culture in endothelial growth medium enhances the angiogenic properties of adipose-derived stem/stromal cells.

Considerable progress has been made on the development of adipose-derived stem/stromal cells (ASCs) as pro-angiogenic therapeutic tools. However, variable clinical results highlight the need for devising strategies to enhance their therapeutic efficacy. Since ASCs proliferate and stabilize newly formed vessels during the angiogenic phase of adipose tissue formation, we hypothesized that mimicking an angiogenic milieu during culture of ASCs would enhance their capacity to support endothelial cell survival and angiogenesis. To test this, we compared the effect of an endothelial growth medium (EGM-2) and conventional media (αMEM) on the progenitor and angiogenic properties of ASCs. ASCs cultured in EGM-2 (ASC-EGM) displayed the highest clonogenic efficiency, proliferative potential and multilineage potential. After co-culture under growth factor starvation, only ASC-EGM attenuated luciferase-expressing human umbilical vein endothelial cells (HUVECluc) apoptosis and supported the formation of endothelial cords in a dose-dependent manner. These effects were recapitulated by the conditioned medium of ASC-EGM, which displayed a 100-fold higher expression of hepatocyte growth factor in comparison with ASC-αMEM. Next, HUVECluc and ASCs were co-transplanted subcutaneously into immunodeficient mice, and the survival of HUVECluc was monitored by bioluminescent imaging. After 60 days, the survival of HUVECluc transplanted alone was equivalent to that of HUVECluc co-transplanted with ASC-αMEM (15.0 ± 0.7 vs. 13.0 ± 0.5%). Strikingly, co-transplantation with ASC-EGM increased HUVECluc survival to 105.0 ± 3.5%, and the resulting organoids displayed functional vasculature with the highest human-derived vascular area. These findings demonstrate that pre-conditioning of ASCs in endothelial growth medium augment their pro-angiogenic properties and could enhance their therapeutic efficacy against ischemic diseases.

The F309S mutation increases factor VIII secretion in human cell line

OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate) on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIÄB proteins in HEK 293 cells, but the same effect was not seen for FVIIIÄB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.