Fast-Proliferating Adipose Tissue Mesenchymal-Stromal-Like Cells for Therapy

Human mesenchymal stromal cells, whether from the bone marrow or adipose tissue (hASCs), are promising cell therapy agents. However, generation of abundant cells for therapy remains to be a challenge, due to the need of lengthy expansion and the risk of accumulating genomic defects during the process. We show that hASCs can be easily induced to a reversible fast-proliferating phenotype (FP-ASCs) that allows rapid generation of a clinically useful quantity of cells in <2 weeks of culture. Expanded FP-ASCs retain their finite expansion capacity and pluripotent properties. Despite the high proliferation rate, FP-ASCs show genomic stability by array-comparative genomic hybridization, and did not generate tumors when implanted for a long time in an SCID mouse model. Comparative analysis of gene expression patterns revealed a set of genes that can be used to characterize FP-ASCs and distinguish them from hASCs. As potential candidate therapeutic agents, FP-ASCs displayed high vasculogenic capacity in Matrigel assays. Moreover, application of hASCs and FP-ASCs in a fibrin scaffold over a myocardium infarct model in SCID mice showed that both cell types can differentiate to endothelial and myocardium lineages, although FP-ASCs were more potent angiogenesis inducers than hASCs, at promoting myocardium revascularization

Glioblastoma Therapy with Cytotoxic Mesenchymal Stromal Cells Optimized by Bioluminescence Imaging of Tumor and Therapeutic Cell Response

Genetically modified adipose tissue derived mesenchymal stromal cells (hAMSCs) with tumor homing capacity have been proposed for localized therapy of chemo- and radiotherapy resistant glioblastomas. We demonstrate an effective procedure to optimize glioblastoma therapy based on the use of genetically modified hAMSCs and in vivo non invasive monitoring of tumor and therapeutic cells. Glioblastoma U87 cells expressing Photinus pyralis luciferase (Pluc) were implanted in combination with hAMSCs expressing a trifunctional Renilla reniformis luciferase-red fluorescent protein-thymidine kinase reporter in the brains of SCID mice that were subsequently treated with ganciclovir (GCV). The resulting optimized therapy was effective and monitoring of tumor cells by bioluminescence imaging (BLI) showed that after 49 days GCV treatment reduced significantly the hAMSC treated tumors; by a factor of 104 relative to controls. Using a Pluc reporter regulated by an endothelial specific promoter and in vivo BLI to image hAMSC differentiation we gained insight on the therapeutic mechanism. Implanted hAMSCs homed to tumor vessels, where they differentiated to endothelial cells. We propose that the tumor killing efficiency of genetically modified hAMSCs results from their association with the tumor vascular system and should be useful vehicles to deliver localized therapy to glioblastoma surgical borders following tumor resection

Quantification of human neuromuscular function through optogenetics

© Ivyspring International Publisher. The study of human neuromuscular diseases has traditionally been performed in animal models, due to the difficulty of performing studies in human subjects. Despite the unquestioned value of animal models, inter-species differences hamper the translation of these findings to clinical trials. Tissue-engineered models of the neuromuscular junction (NMJ) allow for the recapitulation of the human physiology in tightly controlled in vitro settings. Methods: Here we report the first human patient-specific tissue-engineered model of the neuromuscular junction (NMJ) that combines stem cell technology with tissue engineering, optogenetics, microfabrication and image processing. The combination of custom-made hardware and software allows for repeated, quantitative measurements of NMJ function in a user-independent manner. Results: We demonstrate the utility of this model for basic and translational research by characterizing in real time the functional changes during physiological and pathological processes. Principal Conclusions: This system holds great potential for the study of neuromuscular diseases and drug screening, allowing for the extraction of quantitative functional data from a human, patient-specific system

Bioluminescent and micro-computed tomography imaging of bone repair induced by fibrin-binding growth factors

In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (mu CT) to evaluate bone regeneration and high-resolution mu CT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3 weeks after implantation. Results from mu CT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Fast-Proliferating Adipose Tissue Mesenchymal-Stromal-Like Cells for Therapy

Human mesenchymal stromal cells, whether from the bone marrow or adipose tissue (hASCs), are promising cell therapy agents. However, generation of abundant cells for therapy remains to be a challenge, due to the need of lengthy expansion and the risk of accumulating genomic defects during the process. We show that hASCs can be easily induced to a reversible fast-proliferating phenotype (FP-ASCs) that allows rapid generation of a clinically useful quantity of cells in <2 weeks of culture. Expanded FP-ASCs retain their finite expansion capacity and pluripotent properties. Despite the high proliferation rate, FP-ASCs show genomic stability by array-comparative genomic hybridization, and did not generate tumors when implanted for a long time in an SCID mouse model. Comparative analysis of gene expression patterns revealed a set of genes that can be used to characterize FP-ASCs and distinguish them from hASCs. As potential candidate therapeutic agents, FP-ASCs displayed high vasculogenic capacity in Matrigel assays. Moreover, application of hASCs and FP-ASCs in a fibrin scaffold over a myocardium infarct model in SCID mice showed that both cell types can differentiate to endothelial and myocardium lineages, although FP-ASCs were more potent angiogenesis inducers than hASCs, at promoting myocardium revascularization

Polymeric Composite Dressings Containing Calcium-Releasing Nanoparticles Accelerate Wound Healing in Diabetic Mice

[Objective] Wound healing is a complex process that involves the interaction between different cell types and bioactive factors. Impaired wound healing is characterized by a loss in synchronization of these interactions, resulting in nonhealing chronic wounds. Chronic wounds are a socioeconomic burden, one of the most prominent clinical manifestations of diabetes, however, they lack satisfactory treatment options. The objective of this study was to develop polymeric composites that deliver ions having wound healing properties and evaluate its performance using a pressure ulcer model in diabetic mice. [Approach] To develop a polymeric composite wound dressing containing ion-releasing nanoparticles for chronic wound healing. This composite was chemically and physically characterized and evaluated using a pressure ulcer wound model in diabetic (db/db) mice to explore their potential as novel wound dressing. [Results] This dressing exhibits a controlled ion release and a good in vitro bioactivity. The polymeric composite dressing treatment stimulates angiogenesis, collagen synthesis, granulation tissue formation, and accelerates wound closure of ischemic wounds created in diabetic mice. In addition, the performance of the newly designed composite is remarkably better than a commercially available dressing frequently used for the treatment of low-exuding chronic wounds. [Innovation] The developed nanoplatforms are cell- and growth factor free and control the host microenvironment resulting in enhanced wound healing. These nanoplatforms are available by cost-effective synthesis with a defined composition, offering an additional advantage in potential clinical application. [Conclusion] Based on the obtained results, these polymeric composites offer an optimum approach for chronic wound healing without adding cells or external biological factors.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO/FEDER) through the projects MAT2012-38793 and MAT2015-68906-R, the EuroNanoMed3 project nAngioDerm funded through the Spanish Ministry of Science and Innovation (ref. PCI2019-103648), the Spanish Ministry of Education, Culture, and Sports with the FPU grant (ref. AP-2012-5310), EIT Health (project EIT PoC-2016-SPAIN-03), La Caixa Banking Foundation through their CaixaImpulse Program and Caixaimpulse 2.0 Consolidate Program (Ref. LCF/TR/CN18/52210003)

Glioblastoma Bystander Cell Therapy: Improvements in Treatment and Insights into the Therapy Mechanisms

A preclinical model of glioblastoma (GB) bystander cell therapy using human adipose mesenchymal stromal cells (hAMSCs) is used to address the issues of cell availability, quality, and feasibility of tumor cure. We show that a fast proliferating variety of hAMSCs expressing thymidine kinase (TK) has therapeutic capacity equivalent to that of TK-expressing hAMSCs and can be used in a multiple-inoculation procedure to reduce GB tumors to a chronically inhibited state. We also show that up to 25% of unmodified hAMSCs can be tolerated in the therapeutic procedure without reducing efficacy. Moreover, mimicking a clinical situation, tumor debulking previous to cell therapy inhibits GB tumor growth. To understand these striking results at a cellular level, we used a bioluminescence imaging strategy and showed that tumor-implanted therapeutic cells do not proliferate, are unaffected by GCV, and spontaneously decrease to a stable level. Moreover, using the CLARITY procedure for tridimensional visualization of fluorescent cells in transparent brains, we find therapeutic cells forming vascular-like structures that often associate with tumor cells. In vitro experiments show that therapeutic cells exposed to GCV produce cytotoxic extracellular vesicles and suggest that a similar mechanism may be responsible for the in vivo therapeutic effectiveness of TK-expressing hAMSCs. © 2018 The Author(s)This work was funded by the Spanish Ministry of Science and Innovation (MICINN) (grant SAF2015-64927-C2-1-R), CIBER-BBN, CIBER Cardiovascular (grant CB16/11/00403), Instituto de Salud Carlos III, Red Temática de Investigación Cooperativa TerCel, and the Spanish Ministry of Economy and Competitiveness (MINECO) (grant BIO2015-66266-R). The authors specially thank Dr. Josep Roca from Delfos hospital (Dr. Roca i Noguera aesthetic surgery team) for the kind donation of liposuction for hAMSCs preparation, and to the services of cell culture (Catalonian Institute for Advanced Chemistry-Spanish National Research Council [IQAC-CISC]), animal care (IQAC-CSIC), cell sorting (Scientific and Technological Centers [CCiT]-University of Barcelona), confocal microscopy (CCiT-University of Barcelona), and Central Services for Research Support (SCAI) at the University of Málaga for their technician and specialized support.Peer reviewe

Sensitivity of PLuc-G-U87 cells, Rluc-R-tTK-hAMSC and hAMSC and co-cultures comprising different proportions of Rluc-R-tTK-hAMSCs and PLuc-U87 cells to GCV.

<p>Cells were grown during the indicated times and treated with either GCV (4 µg/ml) or PBS, as indicated. Number of viable cells was evaluated spectrophotometrically by standard 3-(4-5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS assay) (<b>A, B</b>), or by BLI (<b>C</b>), and expressed as percentage increase relative to cell number at day 0. Histograms show mean ± SEM, *p<0.05, **p<0.01 n = 4 for each group.</p

<i>In vivo</i> optimization of tumor to therapeutic cell ratio.

<p>Tumor and therapeutic cells mixed in proportions 1∶0, 1∶1, 1∶2 and 1∶4 (Pluc-G-U87∶Rluc-R-tTK-hAMSC) were implanted in the brain of SCID mice and treated at day 6 i.p. with either GCV or PBS as indicated and monitored by BLI at weekly intervals. (<b>A</b>) The graph shows <i>in vivo</i> changes in light production by Pluc expressing tumor cells resulting from GCV treatment. (<b>B</b>) Histogram showing PHC values at the end of the experiment (T = 35). Values were normalized relative to those at T = 0 according to the formula [(T5/T0)×100 = tumor variation (%)]. Data are shown as mean ± SEM, *P<0.05, compared to the 1∶0+GCV group, n = 5 for each group. (<b>C</b>) Representative pseudo-color BLI images showing evolution of tumor size during treatment of animals inoculated with the indicated proportions of Pluc-G-U87 to Rluc-R-tTK-hAMSC. Color bars represent light intensity levels from Pluc luciferase, ranging from low: blue to high: red. Luciferase images are superimposed on black and white with images of the same mouse.</p

Effect of GCV on fluorescent Rluc-R-tTK-hAMSC at tumor implantation sites.

<p>Left, representative images of HE stained brain sections; Right, representative fluorescence confocal microscope images showing implanted red fluorescent cells Rluc-R-tTK-hAMSC (arrow), and Pluc-G-U87 cells (green). (<b>A</b>) Control mouse (1∶4+PBS) at T = 0, (<b>B</b>) control mouse (1∶4+PBS) at T = 49, (<b>C</b>) treated mouse (1∶4+GCV) at day 49. Blue, Hoescht stained nuclei. Scale bar = 10 µm.</p