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.

Comparative characterization of CD271+ and CD271- subpopulations of CD34+ human adipose-derived stromal cells.

Adipose-derived stromal/stem cells (ASCs) are promising candidates for cell-based therapies. However, the lack of markers able to unequivocally identify these cells, the differential expression of cell surface molecules among stromal progenitors from different tissues and cellular alterations caused by culture are phenomena that need to be comprehensively addressed in order to improve ASC purification and consequently refine our knowledge about their function and therapeutic efficiency. In this study, we investigated the potential of CD271, a marker used for purification of bone marrow-derived mesenchymal stem cells, on enriching ASCs from CD34+ stromal cells of human adipose tissue. Putative ASC populations were sorted based on CD271 expression (CD45- CD31- CD34+ CD271+ and CD45- CD31- CD34+ CD271- cells) and compared regarding their clonogenic efficiency, proliferation, immunophenotypic profile, and multilineage potential. To shed light on their native identity, we also interrogated the expression of key perivascular cell markers in freshly isolated cells. CD271- cells displayed twofold higher clonogenic efficiency than CD271+ cells. Upon culture, the progeny of both populations displayed similar immunophenotypic profile and in vitro adipogenic and chondrogenic potentials, while CD271+ cells produced more calcified extracellular matrix. Interestingly, uncultured freshly isolated CD271+ cells displayed higher expression of pericyte-associated markers than CD271- cells and localized in the inner region of the perivascular wall. Our results demonstrate that cells with in vitro ASC traits can be obtained from both CD271+ and CD271- stromal populations of human adipose tissue. In addition, gene expression profiling and in situ localization analyses indicate that the CD271+ population displays a pericytic phenotype.

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.

Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue.

One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.

Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b(+) myeloid cells to the lungs and facilitates B16-F10 melanoma colonization.

The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice were subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b(+) myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize "premetastatic niches" in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b(+) myeloid cells and tumor cells.

Multimodal Tracking of Hematopoietic Stem Cells from Young and Old Mice Labeled with Magnetic-Fluorescent Nanoparticles and Their Grafting by Bioluminescence in a Bone Marrow Transplant Model.

This study proposes an innovative way to evaluate the homing and tracking of hematopoietic stem cells from young and old mice labeled with SPIONNIRF-Rh conjugated with two types of fluorophores (NIRF and Rhodamine), and their grafting by bioluminescence (BLI) in a bone marrow transplant (BMT) model. In an in vitro study, we isolated bone marrow mononuclear cells (BM-MNC) from young and old mice, and analyzed the physical-chemical characteristics of SPIONNIRF-Rh, their internalization, cell viability, and the iron quantification by NIRF, ICP-MS, and MRI. The in vivo study was performed in a BMT model to evaluate the homing, tracking, and grafting of young and old BM-MNC labeled with SPIONNIRF-Rh by NIRF and BLI, as well as the hematological reconstitution for 120 days. 5FU influenced the number of cells isolated mainly in young cells. SPIONNIRF-Rh had adequate characteristics for efficient internalization into BM-MNC. The iron load quantification by NIRF, ICP-MS, and MRI was in the order of 104 SPIONNIRF-Rh/BM-MNC. In the in vivo study, the acute NIRF evaluation showed higher signal intensity in the spinal cord and abdominal region, and the BLI evaluation allowed follow-up (11-120 days), achieving a peak of intensity at 30 days, which remained stable around 108 photons/s until the end. The hematologic evaluation showed similar behavior until 30 days and the histological results confirm that iron is present in almost all tissue evaluated. Our results on BM-MNC homing and tracking in the BMT model did not show a difference in migration or grafting of cells from young or old mice, with the hemogram analysis trending to differentiation towards the myeloid lineage in mice that received cells from old animals. The cell homing by NIRF and long term cell follow-up by BLI highlighted the relevance of the multimodal nanoparticles and combined techniques for evaluation.

Noninvasive Tracking of Hematopoietic Stem Cells in a Bone Marrow Transplant Model.

The hematopoietic stem cell engraftment depends on adequate cell numbers, their homing, and the subsequent short and long-term engraftment of these cells in the niche. We performed a systematic review of the methods employed to track hematopoietic reconstitution using molecular imaging. We searched articles indexed, published prior to January 2020, in PubMed, Cochrane, and Scopus with the following keyword sequences: (Hematopoietic Stem Cell OR Hematopoietic Progenitor Cell) AND (Tracking OR Homing) AND (Transplantation). Of 2191 articles identified, only 21 articles were included in this review, after screening and eligibility assessment. The cell source was in the majority of bone marrow from mice (43%), followed by the umbilical cord from humans (33%). The labeling agent had the follow distribution between the selected studies: 14% nanoparticle, 29% radioisotope, 19% fluorophore, 19% luciferase, and 19% animal transgenic. The type of graft used in the studies was 57% allogeneic, 38% xenogeneic, and 5% autologous, being the HSC receptor: 57% mice, 9% rat, 19% fish, 5% for dog, porcine and salamander. The imaging technique used in the HSC tracking had the following distribution between studies: Positron emission tomography/single-photon emission computed tomography 29%, bioluminescence 33%, fluorescence 19%, magnetic resonance imaging 14%, and near-infrared fluorescence imaging 5%. The efficiency of the graft was evaluated in 61% of the selected studies, and before one month of implantation, the cell renewal was very low (less than 20%), but after three months, the efficiency was more than 50%, mainly in the allogeneic graft. In conclusion, our review showed an increase in using noninvasive imaging techniques in HSC tracking using the bone marrow transplant model. However, successful transplantation depends on the formation of engraftment, and the functionality of cells after the graft, aspects that are poorly explored and that have high relevance for clinical analysis.

Cell Therapy: Effect of Locally Injected Mesenchymal Stromal Cells Derived from Bone Marrow or Adipose Tissue on Bone Regeneration of Rat Calvarial Defects.

Treatment of large bone defects is a challenging clinical situation that may be benefited from cell therapies based on regenerative medicine. This study was conducted to evaluate the effect of local injection of bone marrow-derived mesenchymal stromal cells (BM-MSCs) or adipose tissue-derived MSCs (AT-MSCs) on the regeneration of rat calvarial defects. BM-MSCs and AT-MSCs were characterized based on their expression of specific surface markers; cell viability was evaluated after injection with a 21-G needle. Defects measuring 5 mm that were created in rat calvaria were injected with BM-MSCs, AT-MSCs, or vehicle-phosphate-buffered saline (Control) 2 weeks post-defect creation. Cells were tracked by bioluminescence, and 4 weeks post-injection, the newly formed bone was evaluated by µCT, histology, nanoindentation, and gene expression of bone markers. BM-MSCs and AT-MSCs exhibited the characteristics of MSCs and maintained their viability after passing through the 21-G needle. Injection of both BM-MSCs and AT-MSCs resulted in increased bone formation compared to that in Control and with similar mechanical properties as those of native bone. The expression of genes associated with bone formation was higher in the newly formed bone induced by BM-MSCs, whereas the expression of genes involved in bone resorption was higher in the AT-MSC group. Cell therapy based on local injection of BM-MSCs or AT-MSCs is effective in delivering cells that induced a significant improvement in bone healing. Despite differences observed in molecular cues between BM-MSCs and AT-MSCs, both cells had the ability to induce bone tissue formation at comparable amounts and properties. These results may drive new cell therapy approaches toward complete bone regeneration.

Aryl hydrocarbon receptor (AHR) is a novel druggable pathway controlling malignant progenitor proliferation in chronic myeloid leukemia (CML).

Aryl Hydrocarbon Receptor (AHR) is an ubiquitous basic helix-loop-helix transcription factor, which is ligand-activated and involved in numerous biological processes including cell division, cell quiescence and inflammation. It has been shown that AHR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, loss of AHR in knockout mice is accompanied by a myeloproliferative syndrome-like disease, suggesting a role of AHR in hematopoietic stem cell (HSC) maintenance. To study the potential role of AHR pathway in CML progenitors and stem cells, we have first evaluated the expression of AHR in UT-7 cell line expressing BCR-ABL. AHR expression was highly reduced in UT-7 cell expressing BCR-ABL as compared to controls. AHR transcript levels, quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) were found to be significantly reduced compared to healthy controls (n = 15). The use of StemRegenin (SR1), an AHR antagonist, induced a marked expansion of total leukemic cells and leukemic CD34+ cells by about 4- and 10-fold respectively. SR1-treated CML CD34+ cells generated more colony-forming cells and long-term culture initiating cell (LTC-IC)-derived progenitors as compared to non-SR1-treated counterparts. Conversely, treatment of CML CD34+ cells with FICZ, a natural agonist of AHR, induced a 3-fold decrease in the number of CD34+ cells in culture after 7 days. Moreover, a 4-day FICZ treatment was sufficient to significantly reduce the clonogenic potential of CML CD34+ cells and this effect was synergized by Imatinib and Dasatinib treatments. Similarly, a 3-day FICZ treatment contributed to hinder significantly the number of LTC-IC-derived progenitors without synergistic effect with Imatinib. The analysis of molecular circuitry of AHR signaling in CML showed a transcriptional signature in CML derived CD34+ CD38- primitive cells with either low or high levels of AHR, with an upregulation of myeloid genes involved in differentiation in the "AHR low" fraction and an upregulation of genes involved in stem cell maintenance in the "AHR high" fraction. In conclusion, these findings demonstrate for the first time that down-regulation of AHR expression, a major cell cycle regulator, is involved in the myeloproliferative phenotype associated with CML. AHR agonists inhibit clonogenic and LTC-IC-derived progenitor growth and they could be used in leukemic stem cell targeting in CML.